Download Universitat Pompeu Fabra Molecular mechanisms involved in the process of muscle

Universitat Pompeu Fabra
Department of Experimental and Health Sciences
Doctoral Thesis
Molecular mechanisms involved in the process of muscle
wasting: human and animal studies
Thesis presented by
Clara Fermoselle Pérez
Director:
Dr. Esther Barreiro Portela
Barcelona, September 2012
A Susa, Pepe, Nora, Carlos,
Sanxo y Dolça
II
TABLE OF CONTENTS
ABBREVIATIONSVII
ACKNOWLEDGMENTSXI
ABSTRACT XIII
RESUMENXV
PREFACEXVII
1. Scientific collaborationsXVII
2. Funding 3. CommunicationsXVIII
XVII
PUBLICATIONSXXI
INTRODUCTION1
1. Skeletal muscles1
1.1. Structure and organization1
1.2. Fiber type heterogeneity
3
1.3. Muscle contractile function4
1.4. Muscle metabolism6
1.4.1. ATP synthesis
6
1.4.1.1. Generation of ATP from phosphocreatine (PCr)
6
1.4.1.2. Generation of ATP from glucose oxidation6
1.4.1.3. Generation of ATP from fat oxidation7
III
2. Skeletal muscle wasting and dysfunction7
2.1. Mechanisms involved in muscle loss8
2.1.1. Protein anabolism
8
2.1.2. Redox balance
8
2.1.3. Inflammation
10
2.1.3.1. Systemic inflammation10
2.1.3.2. Local inflammation10
2.1.4. Mitochondrial dysfunction
10
2.1.5. Ubiquitin-proteasome system
11
2.1.6. Autophagy 12
2.1.7. Apoptosis
12
12
2.1.8. Muscle growth and differentiation
2.1.9. Signaling pathways
13
2.1.9.1. NF-kB pathway13
2.1.9.2. MAPK pathway 13
2.2. Chronic conditions associated with muscle wasting13
2.2.1 COPD
13
2.2.2. Cancer
15
HYPOTHESIS17
OBJECTIVES19
METHODOLOGIES23
1. Population23
1.1. COPD patients and control subjects23
1.2. Animal models23
1.2.1. Emphysema
23
1.2.2. Lung cancer
24
2. Clinical and functional evaluation25
2.1. Human studies25
2.2. Animals25
2.2.1. Emphysema
25
2.2.2. Lung cancer
25
IV
3. Molecular biology analyses 26
RESULTS29
Study #131
Main findings in study # 133
Study #234
Main findings in study # 236
Study #338
Main findings in study # 340
Study #443
Main findings in study # 445
DISCUSSION47
STUDY LIMITATIONS55
CONCLUSIONS57
REFERENCES59
ADDENDUM75
V
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ABBREVIATIONS
ACh: Acetylcholine
AChRs: Acetylcholine receptors
ActRIIB: Activin receptor type-IIB
ADP: Adenosine diphosphate
Akt: RAC-alpha serine/threonine-protein kinase
ASK-1: Apoptosis signal-regulating kinase 1
Atg: Autophagy related gene
ATP: Adenosine triphosphate
BMI: Body mass index
C8-20S: C8 alpha-subunit of the 20S proteasome
Ca2+: Calcium
cAMP: Cyclic adenosine monophosphate
CAT: Computerized axial tomography
CI: Complex I
CII: Complex II
CIV: Complex IV
CK: Creatine kinase
CO2: Carbon dioxide
CoA: Coenzyme A
COPD: Chronic obstructive pulmonary disease
CRP: C-reactive protein
CS: Citrate synthase
CuZn-SOD: Copper zinc superoxide dismutase
VII
Abbreviations
DHE: Dihydroethidium
DNA: Deoxyribonucleic acid
E1: Ubiquitin-activating enzyme
E2: Ubiquitin-conjugating enzyme
E214k: Ubiquitin-conjugating enzyme 14k
E3: Ubiquitin-ligase enzyme
ELISA: Enzyme-linked immunosorbent assay
eNOS: Endothelial nitric oxide synthase
ERK: Extracellular-signal-regulated kinases
F-actin: Filamentous actin
FADH2: Flavin adenine dinucleotide
FEV1: Forced expiratory volume in 1 second
FFMI: Fat free mass index
FoxO: Forkhead box
FVC: Forced vial capacity
G-actin: Globular actin
GOLD: Global initiative for chronic obstructive pulmonary disease
GSH: Glutathione
GTP: Guanosine triphosphate
H2O: Water
H2O2: Hydrogen peroxide
HIV: Human immunodeficiency virus
HNE: 4-hydroxil-2-nonenal
HO•: Hydroxyl radical
IGF-1: Insulin-like growth factor 1
IL-1β: Interleukin-1 beta
IL-6: Interleukin-6
IL-8: Interleukin-8
iNOS: Inducible nitric oxide synthase
IκB: NF-κB cytoplasmic inhibitor protein
JNK: c-Jun NH2-terminal kinase
LC: Lung cancer
LC3: Ubiquitin-like protein Atg8 in mammalian cells
mRNA: Messenger ribonucleic acid
mTOR: Mammalian target of rapamycin
MAPK: Mitogen-activated protein kinases
MDA: Malonaldehyde
MEM: Minimal essential media
Mg2+: Magnesium
MLC: Myosin light chain
Mn-SOD: Manganese superoxide dismutase
VIII
Abbreviations
MRC: Mitochondrial respiratory chain
MURF-1: Muscle ring finger protein 1
MyHC: Myosin heavy chain
NAC: N-acetyl cysteine
NADH: Nicotinamide adenine dinucleotide
NADPH: Nicotinamide adenine dinucleotide phosphate
NF-κB: Nuclear factor kappa-light-chain-enhancer of activated B cells
nNOS: Neuronal nitric oxide synthase
NO•: Nitric oxide
NOSs: Nitric oxide synthases
O2: Molecular oxygen
O2•-: Superoxide anion
PCr: Phosphocreatine
PET: Positron emission tomography
PGC-1α: PPAR-alpha co-activator-1
Pi: Inorganic phosphate
PI3: Phosphatidylinositol 3-kinase
PIF: Proteolysis inducing factor
PPARs: Peroxisome proliferator-activated receptors
QMVC: Quadriceps isometric maximum voluntary contraction
RNS: Reactive nitrogen species
ROS: Reactive oxygen species
SR: Sarcoplasmic reticulum
TGF-β: Transforming growth factor β
Tn: Troponin
TNF-α: Tumor necrosis factor-alpha
TUNEL: Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling
VEGF: Vascular endothelial growth factor
IX
X
ACKNOWLEDGMENTS
Los trabajos que componen la presente tesis doctoral son fruto del trabajo que he desarrollado
a lo largo de los últimos cuatro años. Todo empezó en septiembre de 2008 cuando la Dra. Esther
Barreiro me dio la oportunidad de incorporarme a su grupo e iniciarme en el mundo de la
investigación. Gracias a esa oportunidad he podido adquirir toda una serie de conocimientos y
experiencias que me ayudarán a continuar a partir de ahora con mi carrera profesional.
No hubiese sido posible que todos estos proyectos se completasen sin la ayuda,
colaboración, apoyo y soporte de muchas personas. He tenido la suerte de poder contar con
unos excelentes compañeros de trabajo. Cisco ha vivido todo el proceso de principio a fin y sin su
ayuda y su experiencia esta tesis hoy no existiría, gracias por todas esas horas con los ratones, las
mitocondrias y el resto de experimentos que al final hemos conseguido sacar adelante. Gràcies
Ester i a la Mònica per ser tan bones companyes i persones, per haver-me ajudat sempre en el
que heu pogut i per haver-me alegrat els dies amb la vostra companyia. Gràcies Carme pel teu
suport , coneixements i ajuda al llarg d’aquests quatre anys. Gràcies Judith, Sergi (i Berta!) per
haver estat sempre allà quan us he necessitat. Gràcies a la Marina, la Lluïsa, l’Ana, la Maitane, la
Marisol, l’Alba i el Miguel Ángel per haver format part d’aquesta aventura, heu estat uns grans
companys. Gràcies als companys dels altres grups, al Jose Yélamos, la Coral i la gent de la URTEC
per haver-me ajudat sempre que ho necessitat. Gràcies a tots els companys de l’hospital, a la
Mireia, la Pilar, el Víctor Curull, i a la Lara Pijuan per haver-me donat l’oportunitat de col·laborar
en els vostres projectes i donar-me el vostre suport. Gràcies a l’Elena García Arumi per la seva
ajuda amb les activitats mitocondrials. Gracias a Luís Puente y Alberto Tejedor por ayudarnos
con los experimentos de respiración mitocondrial.
Gracias también a todos los que habéis participado en este proyecto de una forma
indirecta. Gracias Clara por estar siempre ahí, y por ser de los pocos que entienden de qué va
todo esto. Gracias a Susa, a Pepe, a Nora, a Carlos y a todos mis amigos y familia por apoyarme
siempre, sin vosotros nada de esto sería posible.
Gracias a todos por haber colaborado para que este proyecto saliese adelante, un trocito
de esta tesis es para cada uno de vosotros.
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ABSTRACT
Muscle dysfunction and muscle wasting are major systemic manifestations of chronic
conditions such as Chronic Obstructive Pulmonary Disease (COPD) and cancer. Several biological
mechanisms contribute to such a dysfunction. Our objectives were to identify cellular and
molecular mechanisms involved in the respiratory and peripheral muscle dysfunction of cachexia
models associated with chronic respiratory conditions. The diaphragm and gastrocnemius
of mice [emphysema and lung cancer (LC) models] and the vastus lateralis of severe COPD
patients were studied with their respective healthy controls. Muscle structure was analyzed in
the animal LC model and in the COPD human model. Several biological markers were studied:
proteolysis markers, signaling pathways related to proteolysis, redox balance and inflammation.
Mitochondrial respiratory chain was also explored in the LC mice model. In all three models
the cachectic subjects were identified using total body weight. In patients, fat-free mass index
was also identified. In the mouse models, muscle weights were also determined. They were
decreased in all cachectic animals compared to the controls. Muscle structure was affected in
the cachectic subjects: LC cachectic mice showed a decrease in both type I and II fibers size,
while muscle-wasted COPD patients showed a decrease in type II fiber sizes and in proportions
of type I fibers. Proportions of myofiber abnormalities were greater in both LC cachectic animals
and muscle-wasted COPD patients. Only LC cachectic mice showed higher levels of IFNγ in the
diaphragm. Oxidative stress, proteolysis markers and NFκB pathway were enhanced in the
muscles of the cachectic subjects in the three models. Mitogen-activated protein kinases (MAPK)
and forkhead box (FoxO) signaling pathways were enhanced in the muscles of the cachectic
mice. Myogenin levels were reduced in the muscles of all three models. Myostatin levels were
greater in the muscles of the cachectic mice. Mitochondrial function was depressed in both
respiratory and limb muscles of the LC cachectic mice. We conclude that enhanced protein
catabolism and mitochondrial dysfunction occurs in the muscles of these cachexia models (both
patients and animals). Major signaling pathways such as NF-kB and FoxO are involved in this
process. These findings offer future therapeutic strategies in cachexia associated with chronic
respiratory conditions.
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RESUMEN
La Enfermedad Pulmonar Obstructiva Crónica (EPOC) y el cáncer presentan importantes
manifestaciones sistémicas como son la disfunción y la pérdida de masa muscular. Diferentes
mecanismos biológicos contribuyen a dicha disfunción. Nuestros objetivos fueron los de
identificar los mecanismos celulares y moleculares implicados en la disfunción de los músculos
respiratorios y periféricos de la caquexia asociada a condiciones crónicas. Se estudiaron el
diafragma y gastrocnemio de ratones [modelos de enfisema y cáncer de pulmón (CP)] y en el
vastus lateralis de pacientes con EPOC severa, junto con sus respectivos controles. Se analizó
la estructura muscular en el modelo de CP de ratones y en el modelo de EPOC de humanos.
Se estudiaron diferentes marcadores biológicos: marcadores de proteólisis, vías de señalización
relacionadas con la proteólisis, balance redox e inflamación. Así mismo se exploró la cadena
respiratoria mitocondrial en el modelo de CP en ratones. En los tres modelos se comprobó
que los sujetos enfermos padecían caquexia mediante el análisis del peso corporal total. En los
pacientes, también se identificó el índice de masa corporal libre de grasa. En los modelos en
ratones también se determinó el peso muscular. Se encontró disminuido en todos los animales
con caquexia respecto a los controles. La estructura muscular se vio afectada en los sujetos
caquécticos: los ratones con CP caquécticos mostraron una disminución del área fibrilar en los
tipos I y II; mientras que los pacientes con EPOC caquécticos mostraron una disminución en
el área fibrilar del tipo II y en el % de tipo I. La proporción de anormalidades miofibrilares fue
superior tanto en el modelo de CP animal como en el modelo de EPOC en humanos. Tan solo
se observaron niveles superiores de IFNγ en el diafragma de los ratones con CP caquécticos.
Los niveles de estrés oxidativo, marcadores de proteólisis y la vía del Factor Nuclear κB (NFκB)
se encontraron incrementados en los músculos de los tres modelos. Se observó un incremento
de las vías de señalización de las Mitogen-activated protein kinases (MAPK) y del forkhead box
(FoxO) en los músculos de todos los ratones caquécticos. Los niveles de miogenina disminuyeron
en los tres modelos. Los niveles de miostatina fueron superiores en los músculos de todos los
ratones caquécticos. La función de la cadena respiratoria mitocondria se encontró disminuida en
los músculos respiratorios y periféricos de los ratones caquécticos con CP. Concluimos que las
diferentes vías de señalización están implicadas en este incremento del catabolismo proteico y
disfunción mitocondrial que dan lugar a la disfunción muscular observada en los tres modelos.
Estos resultados ofrecen futuras aplicaciones terapéuticas a la caquexia asociada a condiciones
crónicas respiratorias.
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XVI
PREFACE
1. Scientific collaborations
In the current PhD thesis, four different investigations have been conducted in collaboration with
researchers from other centers as outlined below.
Study # 1 was conducted in collaboration with the Centre for Applied Medical Research (CIMA)
in Pamplona (Navarra), Spain.
Study # 2 was conducted in collaboration with the Pulmonology Department at Hospital del
Mar and the Pulmonology Department (ICT) at Hospital Clinic-IDIBAPS, University of Barcelona,
Spain.
Study # 3 was conducted in collaboration with the Research Area, Institute of Oncology ‘Angel H.
Roffo’, University of Buenos Aires, Buenos Aires, Argentina, and the Department of Biomedical
Sciences, University of Padova, Dubelcco Telethon Institute, Venetian Institute of Molecular
Medicine, Padova, Italy.
Study # 4 was conducted in collaboration with the Research Area, Institute of Oncology ‘Angel H.
Roffo’, University of Buenos Aires, Buenos Aires, Argentina, and the Nephrology and Pulmonology
Departments at Hospital General Gregorio Marañón, Universidad Complutense de Madrid, Spain.
2. Funding
The research studies included in the current PhD thesis had been funded by different public
agencies: SEPAR 2008, SEPAR 2009, SEPAR 2010, MTV3-07-1010, 2009-SGR-393, FIS 06/1043,
FIS 07/0751, FIS 11/02029, CIBERES, RTICC RD06/0020/0066 (ISCIII, Ministerio de Ciencia e
Innovación), UTE-Project CIMA, SAF 2007-62719, 2005-SGR01060, 2009-SGR-393, FUCAP 2008,
FUCAP 2011 FUCAP 2012 and BIO-BRIDGE (LSHG-CT-2006-037939). E. Barreiro was a recipient of
the European Respiratory Society COPD Research Award 2008.
The binding of the present doctoral thesis has been funded by “Fundación IMIM”.
XVII
Preface
3. Communications
The preliminary results of the investigations included in the current PhD thesis have been
previously presented in the form of an abstract at several national and international conferences:
1. C Fermoselle, R Rabinovich, P Ausín, C. Coronell, J Gea, J Roca and E Barreiro. Proteolisis
y balance redox en el vasto lateral de pacientes con EPOC grave. Segundas Jornadas de
Formación del CIBERES (Abstract book, page 44), Bunyola, Mallorca, Illes Balears, España,
October 2009.
2. C Fermoselle, R Rabinovich, P Ausín, C Coronell, J Gea, J Roca, E Barreiro, on behalf of the
ENIGMA in COPD and BIO-BRIDGE projects. Proteolysis and redox balance in the vastus
lateralis of severe COPD patients. Am J Respir Crit Care Med 2010; 181: A5040.
3. C Fermoselle, R Rabinovich, P Ausín, C Coronell, J Gea, J Roca and E Barreiro. Proteolisis y
balance redox en el vasto lateral de pacientes con EPOC grave. Arch Bronconeumol 2010 (43
congreso nacional SEPAR): 119S.
4. C Fermoselle, R Rabinovich, P Ausin, C Coronell, J Gea, J Roca and E Barreiro. Severe COPD
patients exhibit increased muscle proteolysis in their vastus lateralis. Eur Respir J 2010; 36
(suppl 54): 1972.
5. C Fermoselle, R Rabinovich, P Ausín, C Coronell, J Gea, J Roca and E Barreiro. Proteolisis y
balance redox en el vasto lateral de pacientes con EPOC grave. XII International Symposium
on COPD, Barcelona, Spain, 2010.
6. C Fermoselle, G de Biurrun, D Blanco, L Montuega and E Barreiro. Mecanismos moleculares
de pérdida de masa muscular en un modelo experimental de enfisema pulmonar. Terceras
Jornadas de Formación del CIBERES (Abstract book, page 46), Bunyola, Mallorca, Illes Balears,
Spain, October 2010.
7. C Fermoselle, G de Biurrun, D Blanco, LM Montuenga and E Barreiro. Pérdida de masa
muscular mediada por miostatina en un modelo experimental de enfisema pulmonar, Arch
Bronconeumol 2011; 47 (especial congreso): 113.
8. C Fermoselle, F Sánchez, A. J Urtreger, MJ Diament, ED Bal De Kier Joffé, and E Barreiro.
Modelo de caquexia inducido por cáncer de pulmón. Cuartas Jornadas de Formación del
CIBERES (Abstract book, page 49), Bunyola, Mallorca, Illes Balears, Spain, October 2011.
9. C Fermoselle, F Sánchez, J Gea, E Barreiro. Vías de señalización y proteólisis en la caquexia
associada a cáncer de pulmón en ratones. XIII International Symposium on COPD, Barcelona,
Spain, April 2012.
XVIII
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PUBLICATIONS
Study # 1 (1)
Fermoselle C, Sanchez F, Barreiro E.
Reduction of muscle mass mediated by myostatin in an experimental model of pulmonary
emphysema.
Publication: Arch Bronconeumol 2011; 47(12): 590-598. PMID # 22056524.
Study # 2 (2)
Fermoselle C, Rabinovich R, Ausín P, Puig-Vilanova E, Coronell C, Sanchez F, Roca J, Gea J, Barreiro
E.
Does oxidative stress modulate limb muscle atrophy in severe COPD patients?
Publication: Eur Respir J 2012 (in press), PMID # 22408199
Study # 3
Fermoselle C, Urtreger A, Sanchez F, Chacon-Cabrera A, Mateu-Jimenez M, Sandri M, Diament J,
Bal de Kier Joffé E, Barreiro E.
Pharmacological strategies in lung cancer-induced cachexia: effects on muscle proteolysis,
autophagy, structure, and weakness.
Publication: Molecular Medicine. Revised version submitted.
Study # 4
Fermoselle C, García-Arumí E, Andreu A, Sanchez F, Puig-Vilanova E, Urtreger A, Bal de Kier Joffé
E, Tejedor A, Puente-Maestu L, Barreiro E.
Mitochondrial dysfunction and therapeutic approaches in respiratory and limb muscles of cancer
cachectic mice.
Publication: Submitted.
XXI
XXII
INTRODUCTION
1. Skeletal muscles
Muscle contractions are indispensable for human, as for other animals, to survive. Every human
or animal activity requires a movement. Respiratory muscles, for example, are responsible for
breathing and at least one muscle contraction is required for walking, running, catching, letting
go, talking, looking or listening. The diversity of muscle functions justifies the relevance of
skeletal muscles in human and animal lives.
1.1. Structure and organization
Striation is a major characteristic of skeletal muscles. Skeletal muscle is unusual for its particularly
high content of actin and myosin (about 80% of total protein), and for having these two proteins
arranged in a highly ordered array within the cell, enabling the controlled generation of force
and movement (3).
Thick myosin filaments are arranged so that the actin filaments can slide between them.
The unit from Z line to Z line is known as the sarcomere. Six thin filaments surround each thick
filament, so that each myosin filament may bind to any of six actin filaments (4). When examined
by electron microscopy, muscle fibers show alternate regions of high and low electron density,
the A and I bands respectively. The area in the A band where there is no overlap with thin
filaments is known as H zone, in the center of which is the region composed by thick filaments
S2 fragments (M line). The A bands are of a constant width, while I bands change as the muscle
lengthens and contracts (4).
Furthermore, there are other proteins whose function is to maintain the architecture
of the sarcomere. Proteins constituting the M line keep the myosin filaments in the correct
hexagonal arrangement for the actin filaments to slide between. Titin is a long protein that runs
from Z line to M line and could be a template on which the myosin monomers condense to make
the thick filaments. Titin also helps to provide longitudinal stability to the sarcomere. Nebulin is
another large protein found associated with actin near the Z line, it strengthen the thin filaments
and acts as a template for the actin monomers to form filaments. The Z-line structure contains
a-actinin that binds the actin filaments together, and desmin that links the Z lines of adjacent
myofibrils and serves to keep the Z lines in register. Dystrophin is another protein, the function
of which is to anchor the contractile apparatus to the surface membrane and to link with the
basement membrane (3).
1
Introduction
Myosin is found in animal and plant kingdoms. The variety found in mammalian skeletal
muscle is known as myosin II. Myosin II (540 KDa) is formed by six subunits: two heavy chains
(MyHC) (220 KDa) and four light chains (MLC) (20 KDa). The myosin molecule can be split into
two major fragments, the globular head (S1 fragment), contains the ATPase activity and is the
portion that can combine with actin. The S2 portion includes the flexible region of the molecule
and a tail, which combines with other tails, binding the myosin molecules together to form
the thick filaments. This structures act as nuclei of the contractile units. Approximately three
hundred myosin molecules are arranged in a thick filament, forming a long bipolar structure,
where myosin heads are pointing in the opposite directions in the two halves of the filament (3).
Figure 1. Myosin molecule. Adapted from Jones D et al. Structure of the muscle fiber. Elsevier Science,
1st edition, 2004 (3).
Actin (42 KDa) is a globular protein (G-actin). In vertebrates, three main groups of actin
isoforms have been identified: alpha, beta and gamma. The beta and gamma actins coexist in
most cell types as components of the cytoskeleton, and as mediators of internal cell motility.
In the skeletal muscle, alpha actin is found arranged forming long polymers, called filamentous
actin (F-actin). The polymerization of actin involves splitting adenosine triphosphate (ATP) and
binding of adenosine diphosphate (ADP). The thin filaments are formed by F-actin, tropomyosin
and three troponin (Tn) molecules: TnC, TnT and TnI. Each monomer of actin thin filament can
bind strong and specifically to a myosin globular head (S1) (3).
Figure 2. Thin filament. Adapted from Jones D et al. Structure of the muscle fiber. Elsevier Science, 1st
edition, 2004 (3).
Groups of about 200 thick and thin filaments constitute a myofibril. A membranous
complex, known as the sarcoplasmic reticulum, surrounds each myofibril. This membrane
system is a store for the uptake and release of calcium (4). The surface or plasma membrane
(sarcolemma) of the muscle fiber invaginates, forming T tubules, which run transversely across
the fiber, forming a complex branching network that contacts and surrounds every myofibril.
The invaginations of the sarcolemma occur twice in every sarcomere, approximately at the level
of the junction of the A and I bands. Where the T tubules meet the sarcoplasmic reticulum, the
two membranes run very close to each other. In electron micrographic sections, the T tubules
are often seen cut in cross-section with a portion of the sarcoplasmic reticulum on either side,
2
Introduction
this is known as a triad (3, 4).
Myofibrils are separated from adjacent myofibrils by sarcoplasmic reticulum, T tubules
and mitochondria. The number of myofibrils present in a muscle fiber varies with the size of
the muscle. There may be fifty myofibrils in a developing fetal muscle fiber, and about 2000
myofibrils in an adult muscle fiber. Bundles of muscle fibers are then arranged together to form
the anatomical muscle (3-5).
Each muscle fiber is bounded by its sarcolemma. Outside the sarcolemma is the basement
membrane, which is freely permeable and may surround more than one fiber. At the end of a
muscle fiber, the outer membranes become irregular and indented to form a close link with the
connective tissue. The connective tissue elements all come together to form the tendons, which
join the muscle to the bony skeleton (3).
Figure 3. Skeletal muscle structure. Adapted from Nelson D et al. Lehninger Principios de Bioquímica.
Omega, 3rd edition, 2001 (4).
1.2. Fiber type heterogeneity
The heterogeneity of the muscle fibers is the basis of the flexibility, which allows the same muscle
to be used for various tasks from continuous low-intensity activity (e.g., posture), to repeated
submaximal contractions (for example, locomotion), and to fast and strong maximal contractions
(jumping, kicking) (5).
Myosin has a central role as molecular motor in muscle cell physiology. There exist
3
Introduction
several MyHC isoforms, differentially distributed in various fibers, that makes MyHC the best
available marker for fiber typing (5, 6).
Fibers can be classified into four major types, with different physiological, biochemical
and metabolic characteristics. Firstly, fibers can be classified in type I and type II. Type I fibers
are characterized for being part of red muscles (high myoglobin content) and are slow-twitch
fibers, characterized by a poor developed sarcoplasmic reticulum. Type I fibers have elevated
mitochondria content, which generates oxidative metabolism and makes these fiber type fatigue
resistant. Type I fibers are also characterized for having a thick Z line, which is related to the
resistance to fatigue. Type II fibers can be classified in type II A, B and X (7-9). All type II fibers
form part of white muscles (low myoglobin content) and are fast-twitch, characterized by the
presence of a rich sarcoplasmic reticulum development (10). These fibers are also characterized
for having a glycolytic metabolism (11). Type II A fibers have high mitochondria content (oxidative
metabolism) and thick Z line, which make them fatigue resistant, but less than type I fibers. Type
II B fibers have low mitochondria content, not oxidative metabolism and thin Z line. These fibers
are fast-fatigable (11-14). Type II X fibers have intermediate resistance to fatigue, between type
II A and B (8, 9, 15).
Specialized fiber types are present in skeletal muscles from all mammalian species,
and several general aspects are common. The relative proportion of any fiber type may vary
according to species and anatomical site. For example, the diaphragm, a continuously active
respiratory muscle, is a fast muscle in rats and mice but a slow muscle in large mammals, such
as humans (9). In leg muscles, slow type 1 fibers are more abundant in the soleus muscle, in
relation to the greater postural role of posterior muscles. Finally, in many species, type II fibers
are more numerous in the forelimbs than in the hind limbs. In this respect, in humans, upper
limb muscles are faster than lower limb muscles (16, 17).
There are, however, significant variations among species, which emerged during
evolution to accommodate specific functional demands (5). Muscles from small-size animals
must be able to shorten faster than those from large-size animals. To achieve this objective, two
specializations were performed. The first was that the proportion of fast fibers (especially IIX and
IIB) in each muscle is higher in small than in big animals. For example, fast fiber proportion in
the “slow” soleus muscle is 100% in the shrew, 60% in the mouse, 20% in the rat, and virtually
0 in the rabbit. In addition, fibers expressing MyHC-IIB, which are abundant in rat and mouse
muscles, are absent in most skeletal muscles of large mammals, including human (18, 19). The
second specialization was that the maximum velocity of shortening in each specific fiber type
decreases from small to big animals (20-24). For example, slow fibers from human skeletal
muscle are slower than slow fibers from mouse muscle (21).
1.3. Muscle contractile function
The nervous system uses the ability of skeletal muscles to generate force and movement for three
main motor tasks: 1) postural joint stabilization, 2) long-lasting and repetitive activities, and 3)
fast and generally powerful actions (5). In mammals, the neuromuscular system is functionally
4
Introduction
organized into discrete units, the motor units, each consisting of a motor neuron and all the
muscle fibers that it exclusively innervates (5, 25).
In the presynaptic terminal, acetylcholine (ACh) is stored in vesicles, together with ATP.
Action potentials, arriving at the axon terminal, open the voltage-sensitive calcium channels.
The influx of calcium causes the synaptic vesicles to fuse with the presynaptic nerve membrane
and release their contents into the synaptic cleft. The release is, therefore, dependent on the
presence of external calcium, and release is depressed by high magnesium concentrations.
The postsynaptic muscle fiber membrane contains acetylcholine receptors (AChRs). Binding of
acetylcholine to the postsynaptic receptor causes a depolarization of the muscle fiber membrane.
The extent of the depolarization depends on the number of receptors binding ACh, which in
tum depends on the number of synaptic vesicles that have discharged into the synaptic cleft. If
sufficient quantity of ACh is released, it will summate and produce a large enough depolarization
to initiate an action potential. The postsynaptic muscle membrane is rich in Na+ channels, and
this ensures that depolarization leads to a large action potential, which will propagate along
the surface and T tubular membranes of the fiber. Cholinesterase, the enzyme that hydrolyses
acetylcholine, is synthesized by the muscle fiber and secreted into the synaptic cleft where it
binds to the basement membrane, which fills the cleft. Once hydrolyzed, the free choline is
transported back into the presynaptic axon terminal where it is resynthesized into ACh (25).
Action potentials initiated at the neuromuscular junction propagate along the length
of the fiber and the T tubules, which are an extension of the surface membrane, into the
interior of the muscle fiber. As the wave of depolarization passes down the T tubules there is an
interaction with the sarcoplasmic reticulum (SR) that results in the release of calcium, initiating
the interaction of actin and myosin and muscle contraction (25).
The interaction of actin and myosin in mammalian skeletal muscle is regulated by
tropomyosin and troponin complex. At rest, the tropomyosin is positioned so that it covers the
myosin binding sites on the actin monomers preventing the formation of cross-bridges. Calcium
binding to troponin C causes a change in conformation, which moves the tropomyosin so that the
actin binding sites are exposed, cross-bridges are formed and force develops. The tropomyosin
spans seven actin monomers, and one troponin complex is responsible for controlling the activity
of all seven subunits. Calcium is pumped back into the sarcoplasmic reticulum by a mechanism
that requires ATP. The calcium ATPase of the sarcoplasmic reticulum is Mg2+ dependent and
transports two Ca2+ into the SR in exchange for one H+ and at the cost of one ATP molecule (25).
The basic mechanism which leads, through cyclical interactions between myosin heads
and actin filaments to force or displacement generation, accompanied by ATP splitting, is
essentially similar in all sarcomeric myosin isoforms. Upon binding of ATP, myosin can dissociate
from actin breaking the acto-myosin “rigor complex,” and this dissociation is quickly followed
by ATP hydrolysis to Pi and ADP. Hydrolysis is accompanied by a conformational change (reverse
stroke) and then followed by myosin binding to actin and subsequent release of Pi. Release of Pi
is followed by a conformational change in the converter (inside the myosin head) amplified by
5
Introduction
the lever arm and transmitted to the actin filament as force or displacement (power stroke). The
subsequent release of ADP leads to the formation of a new acto-myosin rigor complex, which
will be in turn dissociated by ATP binding, thus starting a new cycle (5).
1.4. Muscle metabolism
Owing to the skeletal muscle metabolic needs, the energy available must be plentiful and
renewable. At is the source of energy in the skeletal muscle. During activity, ATP is consumed in
different ways (26):
1) Most of the ATP is consumed during the contractile process (detach of the myosin
crossbridges from the actin filaments, allowing the bridges to move to a new position).
2) Work of the sodium/potassium pump, which is indispensable for the maintenance of the
potential action that initiates the contractile process.
3) Work of the calcium Ca2+ pump, which returns the Ca2+ to the sarcoplasmic reticulum
after the potential action.
While the muscle is resting, ATP is also consumed in housekeeping processes (26):
1) Build and storage of phosphocreatine.
2) Phosphorylation of enzymes by protein kinases.
3) Conversion to cyclic adenosine monophosphate (cAMP) in the fiber plasmalema.
1.4.1. ATP synthesis
1.4.1.1. Generation of ATP from phosphocreatine (PCr)
For relatively brief periods of activity, ATP is generated from PCr by the action of creatine kinase
(CK). When muscles continue to contract, the production of ATP from PCr becomes inadequate,
then the fibers become totally dependent on the oxidation of glucose and fat (26).
1.4.1.2. Generation of ATP from glucose oxidation
During the contractile activity, glucose is provided by the hydrolysis of glycogen.
Glucose oxidation is performed on four consecutive steps:
a) Glycolisis: each molecule of glucose is transformed in 2 molecules of pyruvate, two ATP
and two NADH molecules are produced. This step can be performed without molecular
oxygen (O2) (anaerobic conditions) (27).
b) Oxidative decarboxylation: this reaction is performed by pyruvate dehydrogenase.
Pyruvate loses a carboxyl group like a CO2 molecule, and then is combined with coenzyme
A (CoA), generating Acetyl CoA. Two NADH molecules are produced for each molecule
of glucose (28).
c) Citric acid cycle: citrate is produced by the combination of acetyl CoA and oxoloacetate.
After eight consecutive steps, citrate is oxidized to form oxoloacetate and CO2. Six NADH,
6
Introduction
two FADH2 and two GTP molecules are produced for each molecule of glucose (28).
d) Electron transfer and oxidative phosphorylation: the electrons transported by NADH
and FADH2 arrive to the mitochondrial respiratory chain, which is located in the inner
mitochondrial membrane. Electrons pass along the respiratory chain, and the energy is
used to pump protons across the inner mitochondrial membrane to the intermembrane
space, where the protons concentration becomes higher than in the matrix. Protons
tend then to diffuse back, across the membrane, to the matrix. This reentry of protons
stimulates ATP synthase (located in the matrix) and ATP is generated. At the end of the
respiratory chain, electrons are accepted by O2 and H2O is produced. For each NADH
molecule, 2.5 ATP molecules are produced; and for each FADH2 molecule, 1.5 ATP
molecules are produced (26, 29).
For each molecule of glucose, after its complete oxidation, 32 molecules of ATP are gained.
1.4.1.3. Generation of ATP from fat oxidation
The fat available for the muscle fiber is composed by fatty acids and triglycerides (composed by
glycerol and fatty acids). Fatty acids are transformed to fatty acyl CoA molecules in the cytosol
and taken into the mitochondrion intermembrane space. Fatty acyl CoA molecules are then taken
into the mitochondrion matrix space through carnitin-mediated transport, and by β-oxidation
are transformed in acetyl CoA molecules. These acetyl CoA molecules can enter into the citric
acid cycle together with the acetyl CoA molecules generated in the glucose metabolism and
continue to their complete oxidation to CO2 and H2O (26, 30).
2. Skeletal muscle wasting and dysfunction
Muscle dysfunction is defined as the presence of low muscle mass and poor muscle function
(strength and/or endurance). Muscle dysfunction can be a consequence of aging, and it may also
result from prolonged periods of rest or as a consequence of a sedentary lifestyle (31). Moreover,
muscle dysfunction accompanies many chronic illnesses such as chronic heart failure, chronic
obstructive pulmonary disease (COPD), chronic kidney disease, cancer, human immunodeficiency
virus (HIV), sepsis, immune disorders, and dystrophies (32, 33). Muscle dysfunction represents
a clinical feature of muscle wasting. Muscle wasting is an inevitable part of aging, where it is
known as sarcopenia. Muscle wasting related to chronic illnesses is known as cachexia (31).
The definition of cachexia had been controversial. The lack of a definition accepted by
clinicians and researchers had limited the identification and treatment of cachectic patients
as well as the development and approval of potential therapeutic agents. In 2008, a definition
of cachexia emerged from the Cachexia Consensus Conference that took place in Washington
DC: “Cachexia, is a complex metabolic syndrome associated with underlying illness and
characterized by loss of muscle with or without loss of fat mass” (34). Diagnostic criteria for
cachexia identification in adults were established as follows (34):
Weight loss of at least 5% in 12 months or less in the presence of an underlying illness
7
Introduction
[in cases where weight loss cannot be documented, a body mass index (BMI) <20.0 kg/m2 is
sufficient], together with three of the following criteria:
- Decreased muscle strength.
- Fatigue.
- Anorexia.
- Low fat-free mass index.
- Abnormal biochemistry:
a) Increased inflammatory markers C-reactive protein (CRP) (>5.0 mg/l), IL-6
>4.0 pg/ml).
b) Anemia (<12 g/dl).
c) Low serum albumin (<3.2 g/dl).
In 2011, Fearon et al published the review Definition and classification of cancer cachexia: an
international consensus (35). The consented definition was: “Cancer cachexia is defined as
a multifactorial syndrome characterized by an ongoing loss of skeletal muscle mass (with or
without loss of fat mass) that cannot be fully reversed by conventional nutritional support and
leads to progressive functional impairment. The pathophysiology is characterized by a negative
protein and energy balance driven by a variable combination of reduced food intake and
abnormal metabolism.” The established criteria for the specific diagnosis of cancer cachexia is
one of the three:
1) Weight loss >5% over past 6 months (in absence of simple starvation).
2) BMI <20 and any degree of weight loss >2%.
3) Appendicular skeletal muscle index consistent with sarcopenia (males <7.26 Kg/m and
females <5.45 Kg/m) and any degree of weight loss >2%.
2.1. Mechanisms involved in muscle loss
2.1.1. Protein anabolism
Low concentrations of anabolic hormones are important contributors to cachexia syndrome.
Cancer patients show low levels of testosterone before chemotherapy (36), and reduced levels
of insulin-like growth factor 1 (IGF-1) and testosterone have been also observed in COPD patients
(37, 38).
The PI3/Akt pathway is the major controller of muscle size. This pathway promotes
protein synthesis, activating S6 kinase via mTOR. PI3/Akt also inhibits protein degradation: Akt1
phosphorilates FoxO transcription factors and prevent them from inducing transcription of E3
ligases or autophagy genes (32).
2.1.2. Redox balance
Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are highly reactive molecules,
normally produced at relatively low levels inside skeletal muscle fibers (39). ROS may be found
8
Introduction
as superoxide anion (O2•-), hydrogen peroxide (H2O2) or hydroxyl radicals (HO•) (40). ROS
constitutive levels play a positive role in maintaining skeletal muscle contractility (39). The
mitochondria are the major site of intracellular ROS production, resulting from the respiratory
chain (41), but there exist other sources of ROS and RNS. Under hypoxic or ischemic conditions,
xanthine dehydrogenase is converted to xanthine oxidase, which reduces O2 to O2-• and H2O2
(42). Neutrophil- and macrophage-derived NADPH oxidases are likely to generate pathological
levels of ROS that causes oxidative stresses in skeletal muscles
RNS are derived from nitric oxide (NO•), which is formed from L-arginine by nitric oxide
synthases (NOSs). Three NOS isoforms have been identified: neuronal (nNOS), endothelial
(eNOS) and inducible by pro-inflammatory cytokines (iNOS) (43). In normal skeletal muscles,
nNOS is expressed closed to the sarcolemma, eNOS is expressed inside the mitochondria and
iNOS is induced in the cytosol (44). Constitutive NO• production in the skeletal muscle modulates
glucose transport and insulin-mediated skeletal muscle vasodilation (45).
In order to maintain the constitutive levels of ROS and RNS inside the cell, there exist
different antioxidant mechanisms to neutralize the excess of reactive species. These antioxidants
can be enzymatic, like catalase or super oxide dismutase (SOD), or no enzymatic, like ascorbic
acid and α-tocopherol (46).
Increased production of ROS and RNS, to levels significantly greater than those that
can be neutralized by intracellular antioxidant defenses, leads to the development of a state
of oxidative stress (39). Excessive levels of ROS and RNS can generate deleterious effects in the
cell. These effects include the peroxidation of membrane phospholipids, which provokes the
fragmentation of fatty acids, resulting in the production of malonaldehyde (MDA) and 4-hydroxy2-nonenal (HNE) (40). The excess of ROS causes the generation of carbonyls (ketones and
aldehydes), formed as a result of oxidation of the amino acids arginine, lysine, threonine and
proline (47). The excess of RNS causes the nitration of tyrosine residues by NO• (48).
Redox imbalance has consistently been shown to be involved in the muscle dysfunction
of severe COPD patients, influencing the skeletal muscle contractile performance by targeting
several enzymes involved in mitochondrial respiration (49), glycolysis (50, 51), CK (52), Ca++ATPase
(53) and several myofibrillar proteins including actin, myosin heavy and light chain, tropomyosin,
actin and actinin (54-56).
Consistent evidence indicates that increased oxidative stress is also involved in muscle
wasting in cancer cachexia. Gomes-Marcondes and Tisdale (57) showed that muscle wasting
in cancer cachexia is associated with increased levels of malondialdehyde in gastrocnemius
muscles. Barreiro et al. (58, 59) showed that in the muscles of tumor bearing animals, oxidative
and nitrosative stress was increased when compared with that in control animals. Also, protein
levels of the antioxidant enzymes were similar in the muscles of tumor bearing rats and control
animals, suggesting that oxidative stress results from increased ROS production and inefficient
antioxidant activity. Increased oxidative stress is also detected in cancer patients (60), Mantovani
et al. (61) found that ROS levels were significantly higher and the activities of antioxidant
9
Introduction
enzymes, were significantly lower in patients than in controls.
Oxidative and nitrosative stress may induce the activation of different pathways that are
thought to be implicated in muscle wasting. On the one hand, ROS upregulate the ubiquitin–
proteasome system, via nuclear factor-kB (NF-kB) activation (57). On the other hand, tyrosine
nitration also generates increased degradation by the proteasome of the modified proteins (62).
Oxidative stress also promotes skeletal muscle apoptosis (63).
2.1.3. Inflammation
2.1.3.1. Systemic inflammation
Systemic inflammation is a common mechanism involved in all the different types of cachexia.
In stable COPD patients, elevated serum levels of C-reactive protein, fibrinogen, circulating
leukocytes and pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α),
interleukin-8 (IL-8), IL6 and soluble TNF receptors have been observed (64, 65).
As to cancer cachexia, elevated serum levels of TNF-α have been found in cachectic
pancreatic adenocarcinoma patients (66), IL-6 was also involved in the cachectic state of
murine colon adenocarcinoma tumor-bearing mice (67), endogenous production of IFNγ was
found in cachectic mice bearing Lewis lung adenocarcinoma (68). Others cytokines, such as
transforming growth factor β (TGF-β) or IL1 have also been suggested as mediators of cancer
cachexia (69). Many pro-inflammatory cytokines can adversely influence skeletal muscle growth
and contractile performance: TNF-α promotes muscle wasting by enhancing the activity of the
ubiquitin proteasome pathway (70), also inducing apoptosis (71) and inhibiting mitochondrial
biogenesis (72).
2.1.3.2. Local inflammation
The results obtained in relation to the pro-inflammatory cytokines present in the limb muscles
are more controversial. On the one hand, studies from our group (73) did not find differences
in protein levels of the proinflammatory cytokines IL6, IL1β, IFNγ and TGF-β in the quadriceps
levels between severe COPD and controls. On the other hand, Montes de Oca et al. (74) found
increased TNF-α levels in the quadriceps of severe COPD patients compared to controls. The
reasons of this contradictory results could be due to the different clinical characteristics of
the patients, exacerbations or a relative low number of patients. In relation to the respiratory
muscles, TNF-α levels were foun(75)d to be increased in the intercostal muscles of COPD patients
compared to controls.
2.1.4. Mitochondrial dysfunction
Mitochondria are the organelles responsible of the energy generation in the cell. The
mitochondrial enzymes catalyze the oxidation of organic nutrients by O2. The chemical energy
released from these oxidations is used to generate ATP, the major energy-transporting molecule
in the cells (76).
Because of their important role in energy production, mitochondria are important
10
Introduction
regulators of muscle mass loss in wasting diseases. Ushmorov et al (77) reported that skeletal
muscles from tumor-bearing mice had an abnormally low mitochondrial respiratory chain
activity. White et al (78) demonstrated in 2011 that cancer-induced muscle mass loss coincides
with a reduction in the expression of proteins related to oxidative metabolism and mitochondrial
biogenesis in both red and white mouse hind limb muscles. Recently, Julienne et al (79) have
shown that cachectic rats with peritoneal carcinosis exhibited muscle mitochondrial oxidative
capacities reduced due to a decrease in complex IV activity.
Likewise, mitochondrial dysfunction has been also reported in COPD cachectic patients.
Rabinovich et al in 2007 (80) found that mitochondrial respiratory function was impaired in
cachectic COPD patients, compared with both normal weight COPD patients and controls. Gosker
et al in 2007 (81) also showed that mitochondrial number and fractional area were reduced in the
vastus lateralis of COPD patients compared with controls. Puente-Maestu et al (82, 83) shown
that the production of H2O2 by isolated mitochondria from the vastus lateralis was significantly
higher in COPD patients than in controls, that complex III was the main mitochondrial site of
ROS production and that ROS production in vitro was significantly related to skeletal muscle
oxidative stress induced in vivo by exercise. Additionally, it has also been reported that prolonged
mechanical ventilation causes diaphragmatic weakness resulting from both fiber atrophy
and contractile dysfunction. Oxidative stress is thought to be one of the major causes of this
dysfunction. The origin of the oxidative stress was found in the mitochondrial reactive oxygen
species emission (84, 85).
Several factors have been shown to be involved in mitochondrial biogenesis such as
the peroxisome proliferator-activated receptors (PPARs) transcription factors family, and their
modulator PPAR-alpha co-activator-1 (PGC-1α) (86). PGC-1α expression has been shown to
correspond with alterations in muscle mass: rat gastrocnemius muscle catabolism with diabetes,
uremia, and tumor implantation is associated with a reduction in PGC-1α mRNA expression (87).
2.1.5. Ubiquitin-proteasome system
In normal skeletal muscles, protein degradation prevents the accumulation of unnecessary and
defective proteins and allows recycling of amino acids. Defective proteins are generally degraded
by ATP systems (ubiquitin-proteasome system).
The ubiquitin-proteasome system is the main mechanism of protein degradation within cells.
To be degraded by this system, proteins first have to be marked with poly-ubiquitin, then the
marked proteins are degraded by the 26s proteasome complex (88). The 26s proteasome
complex is composed of 19s (regulatory) and 20s (catalytic) (89). The ubiquitination of proteins
is regulated by three enzymes: E1 (ubiquitin-activating), E2 (ubiquitin-conjugating) (E214K) and
E3 (ubiquitin-ligase) (Atrogin-1 and MURF-1) (90). Different studies conclude that the ubiquitinproteasome system could be the major responsible of excessive protein degradation in cancer
cachectic patients and animal models, with preferential degradation of myofibrillar proteins (9196). It has been reported an increased expression of Atrogin-1 and MURF-1 in different models
of cancer cachexia (97, 98).
11
Introduction
An increase in protein degradation by the ubiquitin-proteasome system has also been
shown in the limb muscles of cachectic COPD patients (38), and in the diaphragm (99).
Atrogin-1 and MURF-1 are controlled by a complex upstream signaling network, FoxO
transcription factors are involved in this regulation (100). Increased expression of FoxO1 and
FoxO3 has been reported in the quadriceps of COPD patients (101, 102).
2.1.6. Autophagy
Autophagy is a different system that recycles the amino acids from membrane and extracellular
proteins through a lysosomal pathway (103). Tree major mechanisms of autophagy have
been described: microautophagy (destroys only a small portion of the cytoplasm, proteins
and glycogen), chaperone-mediated autophagy (soluble proteins are degraded) and
macroautophagy (a portion of cytoplasm, including organelles, is fused with a lysosome) (104).
Macroautophagy is the most prevalent form of autophagy, and further will be referred as
autophagy.
Autophagy is constitutively active at baseline levels in the skeletal muscle, controlling
muscle mass (105). More than 30 autophagy related (Atg) genes have been identified that
regulate autophagy induction and autophagosome formation (106). No differences in the levels
of Beclin-1 and LC3 transcripts were reported in the vastus lateralis muscle between COPD
patients and control subjects (107). In contrast, a significant increase in the Atg7, Atg12, Beclin-1
proteins and ratio of LC3-II to LC3-I was observed in the skeletal muscles of endotoxin-induced
cachexia in mice (108).
2.1.7. Apoptosis
Cells undergoing apoptosis are characterized by cytoplasmic and nuclear condensation, cleavage
of the chromosomal DNA, violent blebbing of the plasma membrane, packaging of the cellular
contents into membrane enclosed vesicles (apoptotic bodies), and elimination of these vesicles
by heterophagocytosis. These phenomena are catalyzed by a group of enzymes called caspases
(109). A study showed that skeletal muscle of cachectic tumor-bearing animals presented high
DNA fragmentation levels (110). In 2002, Agustí et al. (111) described an increase in the numbers
of apoptotic nuclei in quadriceps of severe COPD patients, especially of those exhibiting more
muscle waste.
2.1.8. Muscle growth and differentiation
Myostatin is also known as “growth and differentiation factor 8”. Different studies indicate that
Myostatin inhibits cell cycle progression and reduces the levels of myogenic regulator factors
(112, 113). Myostatin appears to inhibit the differentiation of myoblast into myotubes, this
seems to be consequence from the inhibition of myogenin (112). Myogenin is necessary for
the myoblast differentiation (114). Increased myostatin signaling has been observed in different
types of cancer (115, 116). An upregulation of myostatin has also been detected in cachexia
related to COPD (107, 117).
12
Introduction
2.1.9. Signaling pathways
2.1.9.1. NF-kB pathway
NF-kB pathway is an important factor that participates in the generation of cachexia and muscle
atrophy (118). NF-kB is activated when its inhibitor, Ik-Bα, is degraded after phosphorylation, the
NF-kB activation is mediated by several cytokines (119). NF-kB activation is related to oxidative
stress (120). NF-kB can bind directly to MURF-1 promoter and then induce the ubiquitinproteasome protein degradation (121).
2.1.9.2. MAPK pathway
The MAPK family of proteins is composed of four distinct signaling modules in skeletal muscle: 1)
extracellular signal regulated kinases (ERK) 1 and 2 (ERK1/2); 2) p38 MAPK; 3) c-Jun NH2 -terminal
kinases (JNK); and 4) ERK5 or big MAPK. These MAPK branches are stimulated by cytokines,
growth factors, and cellular stress (122, 123).
The proteolysis-inducing factor (PIF) is produced by cachexia-inducing tumors and
initiates protein catabolism in skeletal muscle, Smith et al (124) demonstrated that PIF induces
proteasome expression through the MAPK pathway.
2.2. Chronic conditions associated with muscle wasting
2.2.1 COPD
COPD is defined, by the 2012 Global initiative for chronic obstructive pulmonary disease (GOLD)
guideline, as a common preventable and treatable disease characterized by persistent airflow
limitation that is usually progressive and associated with an enhanced inflammatory response in
the airways and the lungs to noxious particles or gases. Exacerbations and comorbidities contribute
to the overall severity in individual patients. The chronic airflow limitation characteristic of COPD
is caused by a mixture of small airways disease (obstructive bronchiolitis) and parenchymal
destruction (emphysema), the relative contributions of which vary from person to person (125).
COPD is the result of cumulative exposures over decades, it is often directly related to tobacco
smoking, although air pollution is also a major COPD risk factor (burning of wood and other
biomass fuels) (126).
A clinical diagnosis of COPD should be considered in any patient who has dyspnea,
chronic cough or sputum production, and /or a history of exposure to risk factors for the disease.
Spirometry is required to make the diagnosis, the presence of a post-bronchodilator FEV1/FVC <
0.70 confirms the presence of persistent airflow limitation and thus of COPD. Table 1 shows the
classification of airflow limitation severity in COPD is based on post-bronchodilator FEV1 (125).
13
Introduction
In patients with FEV1/FVC < 0.70
GOLD 1 Mild
FEV1 ≥ 80% predicted
GOLD 2 Moderate
50% ≤ FEV1 < 80% predicted
GOLD 3 Severe
30% ≤ FEV1 < 50% predicted
GOLD 4 Very severe FEV1 < 30% predicted
Table 1. COPD classification
COPD prevalence has risen in developing countries, owing to increased smoking rates and
reductions in other causes of death, particularly from severe infections. Worldwide prevalence of
COPD GOLD stage 2 or higher in adults aged 40 years and older is 9–10% (125, 127). The Burden
of Obstructive Lung Disease initiative investigated the prevalence of COPD around the world
and showed important differences between countries: prevalence ranged from 9% in Reykjavik,
Iceland, to 22% in Cape Town, South Africa, for men, and from 4% in Hannover, Germany, to 17%
in Cape Town for women. In 2007, in Spain, COPD prevalence in the population between 40 to
69 years was 4.5% (125). COPD imposes a significant economic burden. In the European Union,
the total direct costs of respiratory diseases are estimated to be about 6% of the total health
care budget, with COPD alone accounting for 56% (38.6 billion Euros) of this cost of respiratory
disease (128).
Although COPD is a lung disease, it is associated with systemic manifestations and
comorbid conditions. The most common comorbidities are ischaemic heart disease, diabetes,
skeletal muscle wasting, dysfunction, osteoporosis, depression, and lung cancer (129). These
comorbidities affect health resources, increase the risks of admission to hospital and death, and
account for more than 50% of use of health-care resources for COPD (130).
Dysfunction of respiratory and limb muscles is one of the most important systemic
manifestations of COPD patients. It has been demonstrated that the strength and endurance
of both muscle groups is reduced in COPD patients compared to healthy controls (131). Muscle
dysfunction influences the symptoms and prognosis of the disease, while reducing exercise
capacity and quality of life in these patients (132, 133).
The limb muscles are a very heterogeneous group, and have high variability of structural
phenotypes and functions. In COPD, the most affected are the lower limb muscles (134-136). The
reduction of physical activity, which in turn is partly derived from the airflow primary limitation
generated by the respiratory condition, is the main factor that causes this dysfunction in the
lower limb muscles (137).
Immobilization may lead to muscle atrophy, small fiber-fiber phenotype and a
disadvantaged aerobic metabolism: reduced proportion of type I fibers, reduced capillary
density, reduced enzymatic capacity in oxidative pathways and low myoglobin levels (138, 139).
Respiratory muscles can be classified into inspiratory and expiratory muscles. Inspiratory
muscles ensure an appropriate level of ventilation to facilitate pulmonary gas exchange. Their
dysfunction may result in hypoxemia and hypercapnia in the patients. Dysfunction of the
14
Introduction
expiratory muscles will give difficulties upon exertion, coughing and attempts to expectorate
secretions from the airways (33).
There is increasing evidence that the degree of skeletal muscle dysfunction in COPD
patients is not homogenous between respiratory and limb muscles. This difference is due mainly
to the different functions performed by the two muscle groups (140). On the one hand, lower
limb muscles, as previously mentioned, are in a chronically underloaded state due to chronic
inactivity and disuse. On the other hand, respiratory muscles, particularly the diaphragm, are
in a chronically overloaded state due to increased work of breathing brought on by airflow
obstruction and hyperinflation (41).
These different loading patterns are the likely cause of biochemical and structural
adaptations that take place in the respiratory muscles but that are not present in lower limb
muscles of COPD patients. It was reported that the proportion of type I fibers was increased
in the diaphragms of patients with severe COPD compared to control diaphragms (141). These
differences were only observed in severe COPD. No differences in the proportion of type I fibers
in the diaphragm were observed in moderate COPD patients compared to controls (142). It
was concluded that the diaphragm fiber type switching to type I fibers was dependent on the
COPD severity (143). Other adaptive changes have been observed in the diaphragms of CODP
patients: increased mitochondria density, increased oxidative capacity and increased myosin
ATPase activity (142, 144). Despite all these compensatory mechanisms, diaphragm strength and
endurance are reduced in COPD patients (131, 145). This dysfunction was traditionally attributed
to the hyperinsuflation-induced shortening of the diaphragmatic length relationship (146).
However in 2003, Levine et al. demonstrated that the strength of a single diaphragm fiber of
severe COPD patients was reduced compared to controls (143). In 2005 Ottenheim et al showed
that this strength reduction was also observed in mild and moderated COPD patients (147).
2.2.2. Cancer
When cachexia develops as a consequence of cancer, it is known as cancer cachexia. As mentioned
above, cancer cachexia has its specific definition and diagnostic criteria. Cancer cachexia is usually
shown by patients with an advanced cancer experience (148). However cachexia may also be
present early in the progression of cancer, indicating the importance of its earlier diagnosis and
treatment (149). In the United States, it has been estimated that cancer cachexia affects the 30%
of all individuals with cachexia (more than 1.3 million people) (150). The prevalence of cancer
cachexia varies depending on the type of malignancy, with the greatest frequency of weight loss
(∼50%–85% of patients) observed in gastrointestinal, pancreatic, lung, and colorectal cancers at
diagnosis and before initiation of chemotherapy (151, 152).
Cancer cachexia can be classified into three different stages depending on their clinical
relevance: precachexia, cachexia and refractory cachexia (35). Cancer cachexia has several
implications in the patients’ lives: it is also associated with reduced physical function (153),
reduced tolerance to anticancer therapy (154), and reduced survival (155).
15
16
HYPOTHESIS
Muscle wasting is an important systemic manifestation of chronic diseases such as COPD and
lung cancer. Several studies have tried to elucidate which are the molecular mechanisms involved
in the process of muscle mass loss. Proteasome, myostatin and autophagy have been proposed
as possible mechanisms to enhancing muscle protein breakdown. Different pathways such as
NF-κB and MAPK are thought to signal/regulate the activity of the proteasome, myostatin and
autophagy systems. Likewise, oxidative stress has also been proposed to be involved in cachexia:
i) by directly damaging proteins, lipids and DNA, thus affecting the cellular homeostasis; ii) it
may regulate the pathways that generate muscle loss, acting as a second messenger and thus,
enhancing the proteasome and autophagy system activity.
On this basis, we hypothesized that the ubiquitin-proteasome, myostatin, and autophagy
systems could be participating in the muscle loss observed in several experimental models of
cachexia as well as in patients with COPD and muscle loss. Also, it was hypothesized that signaling
mechanisms such as MAPK and NF-κB may be involved in that process of muscle wasting, and
that mitochondrial respiratory chain dysfunction could also be another contributor.
17
18
OBJECTIVES
Study # 1: Reduction of muscle mass mediated by myostatin in an experimental model of
pulmonary emphysema.
In this investigation we explored two different objectives:
a) To identify molecular mechanisms that may be involved in the process of muscle mass loss
of respiratory and peripheral muscles in animals with a major chronic lung disease such as
emphysema by measuring the following markers:
1) Oxidative stress.
2) Proteolytic systems, including myostatin.
3) Signaling pathways.
4) Muscle proteins susceptible to be degraded (actin, myosin, and CK).
b) To establish an animal model of emphysema in which respiratory and peripheral muscles
were affected.
Study # 2: Does oxidative stress modulate limb muscle atrophy in severe COPD patients?
The objectives of the present investigation were to assess the following potentially interrelated
molecular events within the limb muscles of a population of severe COPD patients exhibiting
different degrees of body composition:
1) Redox balance: protein oxidation, O2•- content in myonuclei and muscle fiber compartments,
antioxidant enzymes, and the antioxidant glutathione (GSH).
2) Levels of redox-sensitive signaling pathways, total protein ubiquitination and markers of the
ubiquitin-proteasome system.
3) Levels of molecular inflammation.
4) Content of key contractile and functional proteins, such as MyHC, actin, CK, and carbonic
anhydrase-3.
5) Muscle structural abnormalities.
19
Objectives
Study # 3: Pharmacological strategies in lung cancer-induced cachexia: effects on muscle
proteolysis, autophagy, structure, and weakness.
The objectives of this investigation were to assess in respiratory and limb muscles from lung
cancer (LC) cachectic mice receiving concomitant treatment with MAPK, NF-κB, or proteasome
inhibitors:
1) Body and muscle weights.
2) Limb muscle force.
3) Oxidative stress and inflammation.
4) Proteolysis markers.
5) Signaling pathways.
6) Autophagy.
7) Muscle proteins susceptible to be degraded (actin, myosin, and CK).
8) Myostatin and myogenin content.
9) Muscle structural abnormalities.
Study # 4: Mitochondrial dysfunction and therapeutic approaches in respiratory and limb
muscles of cancer cachectic mice.
The objectives of this investigation were to assess in respiratory and limb muscles from lung
cancer (LC) cachectic mice receiving concomitant treatment with MAPK, NF-κB, proteasome
inhibitors, or N-acetyl cysteine (NAC):
1) Body and muscle weights.
2) Limb muscle force.
3) Activities of citrate synthase (CS) complexes I, II and IV from the mitochondrial respiratory
chain (MRC).
4) Oxygen consumption of mitochondrial complexes I and IV from the MRC.
20
Objectives
21
22
METHODOLOGIES
The different methodologies employed in the four studies are briefly outlined below. Specific
details of the different methodologies are described in the online data supplement of the
manuscripts.
1. Population
1.1. COPD patients and control subjects
Twenty-nine stable Caucasian male severe COPD patients were recruited from the COPD clinics
at Hospital del Mar and Hospital Clinic (Barcelona). Patients were further subdivided into those
exhibiting low weight and reduced muscle mass [BMI ≤ 21 kg/m2 and fat-free mass index (FFMI)
≤ 18 kg/m2, muscle-wasted patients, n=18] and normal weight and muscle mass (BMI > 21 kg/
m2 and FFMI > 18 kg/m2, non-wasted patients, n=11). Additionally, 10 healthy male sedentary
control subjects were also recruited from the general population. Muscle samples were obtained
from the vastus lateralis of severe COPD patients and controls using the open muscle biopsy
technique.
1.2. Animal models
1.2.1. Emphysema
In the current thesis, one of the studies was aimed to identify molecular mechanisms that may
be involved in muscle dysfunction in respiratory and peripheral muscles of animals with a major
chronic lung disease such as emphysema. The emphysema was induced using a previously
validated methodology (156, 157). Male A/J mice, 2 months old (body weight 21–23 g), were used.
To induce the emphysema, the animals received a single instillation (oropharyngeal aspiration)
23
Methodologies
of elastase-high purity (EC134GI, Elastin Products Company) (0.15 mg/100 g of weight). A control
group was performed by the instillation of normal saline. At the beginning of the study, the
mice were randomly assigned to two groups: 1) mice with emphysema, sacrificed at 34 weeks
of disease progression (n=6), and 2) control group, also sacrificed at 34 weeks following the
single instillation of saline (n=7). Throughout the 34-week study period, all animals from both
groups received food and water ad libitum and remained under routine housing environment
conditions. They also maintained a level of physical activity characteristic for this type of animal
throughout the entire duration of the study. All animals from both groups were sacrificed at
week 34. Lungs, diaphragm and gastrocnemius muscles were then obtained.
1.2.2. Lung cancer
In the context of the current thesis, one of the included investigations has addressed the question
about whether several pharmacological strategies could attenuate the effects of cachexia using
an animal mouse model of lung cancer. For this purpose, we developed a model of LC in mice,
in which methodologies previously published were followed (158, 159). Female BALB/c mice, 2
months old (weight ~20g), were used. LC was induced by the subcutaneous inoculation of LP07
viable cells, LP07 is a cell line derived from the P07 lung tumor, spontaneously arisen in a Balb/c
mouse. A number of 4·105 LP07 cells resuspended in 0.2 mL minimal essential media (MEM)
were inoculated in the left flank (day 1). A control group was performed by the inoculation of
MEM in the left flank. All groups (n=10/group) were studied for a period of one month.
Animals were randomly assigned to the following groups: 1) control; 2) LC cachexia
group; 3) LC cachectic mice receiving concomitant treatment with NAC 3mmol/kg/24h, oral
administration using a 22G, 25 mm needle (gavage); 4) LC cachectic mice receiving concomitant
treatment with the proteasome inhibitor Bortezomib (Velcade, Millenium Pharmaceuticals,
Cambridge, MA), 0.15 mg/Kg, 0.1 mL/6 days, intravenous injection into the tail vein; 5) LC cachectic
mice receiving concomitant treatment with sulfasalazine (Pfizer, Madrid, Spain), 200 mg/Kg, 0.3
mL/48h, intraperitoneal injection; and 6) LC cachectic mice receiving concomitant treatment
with the MAPK inhibitor U0126 (a highly selective inhibitor of ERK1 and ERK2 proteins, Selleck
chemicals, Houston, TX), 30 mg/Kg, 0.1 mL/48h, intraperitoneal injection. All pharmacological
therapies were administered on day 15 after the inoculation of the LP07 cells up until the end of
the study period (day 30). All the animals were sacrificed at day 30. Lungs, subcutaneous tumor,
diaphragm and gastrocnemius muscles were obtained.
On day 30 after inoculation, animals presented a subcutaneous tumor in the place of the
cells inoculation, lung adenocarcinomas and developed cachexia in 100% of the cases.
24
Methodologies
Figure 4. LC cachectic mouse (left) and control mouse (right). Adapted from Fermoselle et al.
Pharmacological strategies in lung cancer-induced cachexia: effects on muscle proteolysis, autophagy,
structure, and weakness (study #3).
2. Clinical and functional evaluation
2.1. Human studies
Anthropometrical evaluation included BMI and determination of the FFMI by bioelectrical
impedance. Lung function was evaluated through determination of spirometric values, static
lung volumes, and diffusion capacity, using standard procedures and reference values by Roca et
al (160). Quadriceps muscle strength was evaluated by isometric maximum voluntary contraction
(QMVC) of the dominant lower limb.
2.2. Animals
2.2.1. Emphysema
Histological confirmation of the presence of emphysema in the lungs of the diseased mice was
performed. Lung compliance was also evaluated in all mice. Prior to their sacrifice (week 34),
computerized axial tomography (CAT scan) was done on all the mice for radiographic confirmation
of the presence of emphysema in the diseased animals. All animals were weighed on day 0 and
immediately prior to their sacrifice (week 34).
2.2.2. Lung cancer
Body weight and food intake were determined every day during the entire duration of the study
(30 days). Limb strength was determined on days 0 and 30 using a strength grip meter (Bioseb,
Chaville, France). In the LC cachexia group of mice, tumor progression was determined using
positron emission tomography (PET) on days 13 and 20.
25
Methodologies
3. Molecular biology analyses
A brief description of the different cellular and molecular biology techniques employed in the
four investigations follows.
• 1 dimension electrophoresis and immunoblotting. Protein levels of markers of proteolysis,
signaling pathways, redox balance, muscle proteins, and carbonylated MyHC were
determined (161).
• Detection of O2•- within myonuclei. The presence of O2•- was detected using the fluorescent
probe dihydroethidium (DHE) on 3-micrometer muscle paraffin-embedded sections (162).
• Detection of O2•- radicals in muscle compartments. Superoxide anion levels were detected in
cytosolic, membrane, and mitochondria compartments (161).
• Reduced GSH in muscles. GSH content was measured using the Glutathione Assay (Northwest
Life Sciences Specialties, Vancouver, WA, USA) (163).
• Cytokine Enzyme-linked Immunosorbent Assay (ELISA). Protein levels of the cytokines tumor
necrosis factor (TNF)-alpha (RayBiotech, Norcross, GA, USA), interferon-gamma (IFNγ)
(RayBiotech), vascular endothelial growth factor (VEGF) (RayBiotech), interleukin 1β (IL-1β)
and IL-6 were determined (eBiosience, Bender MedSystems, Vienna) (73).
• Immunohistochemistry. On 3-micrometer muscle paraffin-embedded sections, and using
specific antibodies, MyHC-I and –II isoforms were determined (161).
• Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. On
3-micrometer muscle paraffin-embedded sections, apoptotic nuclei were identified using
the TUNEL assay (In Situ Cell Death Detection Kit, POD, Roche Applied Science, Mannheim,
Germany) in diaphragms and gastrocnemius (164).
• Hematoxylin and eosin staining. On 3-micrometer paraffin-embedded sections, muscle
abnormalities were determined (165). Also, a qualitative evaluation of the increase of
interalveolar spaces in the lungs of the mice with emphysema was performed (156).
• Protein catabolism. Protein degradation was explored on the basis of the rate of production
of free tyrosine from tissue proteins in diaphragm and gastrocnemius (166).
• Luciferase reporter gene assay. NF-κB gene expression was determined in gastrocnemius
(108).
• Mitochondrial enzymatic activities. Activities of citrate synthase (CS) complexes I, II and IV
from the mitochondrial respiratory chain were determined in gastrocnemius and diaphragm
(167).
• Mitochondrial oxygen consumption. Using a Clark-type electrode, oxygen consumption of
mitochondrial complexes I and IV was determined in gastrocnemius and diaphragm (167).
26
Methodologies
27
28
RESULTS
Results contained in the current PhD thesis have partly been published in the form of research
papers in Journals of the area of Respiratory Medicine and Pathophysiology. Study #1 and #2
have been published in the Journals Archivos de Bronconeumología and European Respiratory
Journal, respectively. As to Study #3, the revised version has been submitted to the Molecular
Medicine Journal. Study #4 has recently been submitted to another journal.
Study # 1 (1)
Fermoselle C, Sanchez F, Barreiro E.
Reduction of muscle mass mediated by myostatin in an experimental model of pulmonary
emphysema.
Publication: Arch Bronconeumol 2011; 47(12): 590-598. PMID # 22056524.
Study # 2 (2)
Fermoselle C, Rabinovich R, Ausín P, Puig-Vilanova E, Coronell C, Sanchez F, Roca J, Gea J, Barreiro
E.
Does oxidative stress modulate limb muscle atrophy in severe COPD patients?
Publication: Eur Respir J 2012 (in press), PMID # 22408199.
Study # 3
Fermoselle C, Urtreger A, Sanchez F, Chacon-Cabrera A, Mateu-Jimenez M, Sandri M, Diament J,
Bal de Kier Joffé E, Barreiro E.
Pharmacological strategies in lung cancer-induced cachexia: effects on muscle proteolysis,
autophagy, structure, and weakness.
Publication: Molecular Medicine, Revised version submitted.
Study # 4
Fermoselle C, García-Arumí E, Andreu A, Sanchez F, Puig-Vilanova E, Urtreger A, Bal de Kier Joffé
E, Tejedor A, Puente-Maestu L, Barreiro E.
Mitochondrial dysfunction and therapeutic approaches in respiratory and limb muscles of cancer
cachectic mice.
Publication: Submitted.
29
Results
30
Results
Study #1 (1)
Reduction of muscle mass mediated by myostatin in an experimental
model of pulmonary emphysema.
31
Results
32
Results
Main findings in study # 1
Muscle and body weight
• Total body weight gain was lower in the mice with emphysema compared to controls.
• Diaphragm and gastrocnemius weights were lower in the mice with emphysema compared
to the control group.
Proteins susceptible to be degraded in muscles
• MyHC protein levels were reduced in the diaphragm of the emphysema group compared to
the control animals.
• No differences were observed for actin and CK protein content in the diaphragm and
gastrocnemius of the emphysema mice, compared to control rodents.
Redox balance
• Total levels of carbonylated proteins and MDA adducts were higher in the diaphragm of the
emphysema mice compared to the controls.
• No differences were observed for nitrated proteins in the diaphragm and gastrocnemius of
the emphysema rodents compared to the control animals.
• Mn-SOD protein levels were reduced and peroxiredoxin II protein levels were increased, in
the diaphragm of the emphysema group compared to the control rodents.
• Peroxiredoxin III protein levels were lower in the gastrocnemius of the emphysema animals
compared to the controls.
• Glutathione peroxidase-1 protein levels did not differ in the diaphragm and gastrocnemius
of the emphysema mice compared to the control group.
Proteolytic systems
• Levels of ubiquitinated proteins, C8-20S proteasome subunit, Atrogin1, E214k and MURF-1
protein levels did not differ in the diaphragm and gastrocnemius of the emphysema group
compared to control animals.
Signaling pathways
• ERK1/2 protein levels were higher in the diaphragm, and JNK protein levels were higher in the
gastrocnemius of the emphysema group compared to the controls.
• p38 protein levels did not differ in the diaphragm and gastrocnemius of the emphysema
rodents compared to the control animals.
• Fox-O1 protein levels were higher in the diaphragm of the emphysema mice compared to
control group.
• NF-κB p50 protein levels were higher in the gastrocnemius of the emphysema rodents
compared to the control animals.
• Myostatin protein levels were higher in both the diaphragm and gastrocnemius of the
emphysema mice compared to controls.
• Myogenin protein levels were lower in the diaphragm of the emphysema animals compared
to the control group.
33
Results
Fermoselle C, Sanchez F, Barreiro E. Reduction of muscle mass mediated by myostatin in an
experimental model of pulmonary emphysema. Arch Bronconeumo. 2011; 47(12): 590-598.
34
Results
Results
Study #2 (2)
Does oxidative stress modulate limb muscle atrophy in severe COPD
patients?
34
Results
35
Results
Main findings in study # 2
Muscle mass and function
• BMI and FFMI were decreased in muscle-wasted severe COPD patients compared to the
healthy control group.
• Muscle-wasted COPD patients exhibited a more severe lung disease than non-wasted COPD
patients.
• All severe COPD patients (wasted and non-wasted) exhibited reduced quadriceps muscle
force and exercise capacity compared to the healthy individuals.
• Airway obstruction (FEV1) and FFMI significantly correlated among all COPD patients (wasted
and non-wasted).
Muscle structure
• Proportion of type I fibers and size of type II were decreased in muscle-wasted patients
compared to the healthy subjects.
• Levels of abnormal muscle were higher in all COPD patients (wasted and non-wasted)
compared to the healthy control group.
Proteins susceptible to be degraded
• Protein levels of MyHC and CK were reduced in muscle-wasted patients compared to the
healthy control group.
• Levels of carbonylated MyHC were greater in all COPD patients (wasted and non-wasted)
compared to the healthy individuals.
• Levels of carbonic anhydrase 3 were decreased in all COPD patients (wasted and non-wasted)
compared to the healthy subjects.
• Levels of actin did not differ between the three groups.
36
Results
Redox Balance
• Levels of nuclear O2•- were higher in all COPD patients (wasted and non-wasted) compared
to the healthy subjects.
• Levels of O2•- were increased in the membrane and cytosol of all COPD patients (wasted and
non-wasted) compared to the control group.
• Mitochondrial O2•- levels were only increased in muscle-wasted patients compared to the
healthy controls.
• Total protein carbonylation was increased in all COPD patients (wasted and non-wasted) and
correlated with O2•- levels within the membrane and cytosolic fractions compared to the
healthy control group.
• Mn-superoxide dismutase (SOD) and CuZn-SOD protein contents were higher in musclewasted patients compared to the healthy individuals.
• Protein content of catalase, glutathione peroxidase I, peroxiredoxin II and III, and GSH did
not differ between the three groups.
Inflammation
• INFγ, TNF-α and VEGF levels did not differ between the three groups.
Proteolytic systems
• Total protein ubiquitination, Atrogin1 and E214k levels were higher in COPD muscle-wasted
patients compared to the healthy control group.
• Levels of proteasome subunit 20S did not differ between the three groups.
Signaling pathways
• Fox-O1 protein levels were higher in COPD muscle-wasted patients compared to the healthy
subjects, and correlated with ubiquitination among all COPD patients (wasted and nonwasted).
• Protein levels of JNK, ERK1/2 and p38 did not differ between the three groups.
• NF-κB p65 protein content was higher in COPD muscle-wasted patients compared to the
healthy controls, and correlated significantly with total protein ubiquitination among all
COPD patients (wasted and non-wasted).
• NF-κB p50 levels did not differ between the three groups.
• Myogenin levels were reduced in all COPD patients (wasted and non wasted) compared to
the healthy controls, and correlated with FFMI.
• Myostatin levels did not differ between the three groups.
37
Results
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Online Depository
DOES OXIDATIVE STRESS MODULATE LIMB MUSCLE ATROPHY IN SEVERE
COPD PATIENTS?
Clara Fermoselle, Roberto Rabinovich, Pilar Ausin, Ester Puig-Vilanova, Carlos Coronell,
Josep Roca, Joaquim Gea, Esther Barreiro
1
Results
METHODS
Human study subjects
This is a hospital-based study in which a group of 29 stable male severe COPD patients
(1) were recruited from the COPD clinics at Hospital del Mar and Hospital Clinic (Barcelona).
Severe COPD patients were further subdivided into those exhibiting low weight and reduced
muscle mass [body mass index (BMI) ≤ 21 kg/m2 and fat-free mass index (FFMI) ≤ 18 kg/m2,
muscle-wasted patients, n=18] and patients with normal weight and muscle mass (BMI > 21
kg/m2 and FFMI > 18 kg/m2, non-wasted, n=11) in accordance with previously published
studies (2,3). Additionally, 10 healthy male sedentary control subjects were also recruited from
the general population (patients’ relatives or friends). All 3 groups of individuals were
Caucasian. In the present investigation, 15 COPD patients (8 non-wasted and 7 muscle-wasted)
and 7 sedentary control subjects were also involved in another previously published study aimed
at investigating the effects of chronic exercise on redox balance in the peripheral muscles of
patients with severe COPD patients(4).
Exclusion criteria for COPD patients and control subjects included chronic respiratory
or cardiovascular disorders, limiting osteoarticular condition, chronic metabolic diseases,
suspected para-neoplastic or myopathic syndromes, and/or treatment with drugs known to alter
muscle structure and/or function including oral corticosteroids. COPD patients were exsmokers
and healthy controls were all nonsmokers.
COPD patients and healthy controls were qualified as sedentary after being specifically
inquired about whether they were conducting any regular outdoor physical activity, going
regularly to the gymnasium, or participating in any specific training program.
The current investigation was designed in accordance with both the ethical standards on
human experimentation in our institutions and the World Medical Association guidelines
(Helsinki Declaration of 1975, as revised in 1983) for research on human beings. Approval was
obtained from the institutional Ethics Committees on Human Investigation (Hospital del Mar
and Hospital Clinic, Barcelona). Informed written consent was obtained from all individuals.
Anthropometrical and Functional Assessment
Anthropometrical evaluation included BMI and determination of the FFMI by
bioelectrical impedance(2,5). Lung function was evaluated through determination of spirometric
values, static lung volumes, and diffusion capacity using standard procedures and reference
values by Roca et al(6-8).
Quadriceps muscle strength was evaluated in both patients and controls by isometric
2
Results
maximum voluntary contraction (QMVC) of the dominant lower limb as formerly described
(9,10). Patients were seated with both trunk and thigh fixed on a rigid support of an exercise
platform (Domyos HGH 050, Decathlon, Lille, France). The highest value from three brief
reproducible maneuvers (<5% variability among them) was accepted as the MVC.
Muscle biopsies
Muscle samples were obtained from the quadriceps muscle (vastus lateralis) of severe
COPD patients and control subjects using the open muscle biopsy technique, as described
previously (4,11-14). Samples were 80 mg size in average. Muscle specimens were immediately
frozen in liquid nitrogen and stored at –80ºC for further analysis or immersed in an alcoholformol bath for 2h to be thereafter embedded in paraffin. All subjects were prevented from
doing any potentially exhausting physical exercise 10 to 14 days before coming to the hospital
to undergo the surgical procedures.
Muscle biology analyses
All muscle biology analyses were conducted blind in the same laboratory by the same
investigators, at Hospital del Mar-IMIM (Barcelona).
Detection of reactive carbonyls in muscle proteins. Changes in protein carbonylation in crude
muscle homogenates were detected using the commercially available Oxyblot kit (Chemicon
International Inc., Temecula, CA, USA). Carbonyl groups in the protein side chains were
derivatized to 2,4-dinitrophenylhydrazone (DNP) by reaction with 2,4-dinitrophenylhydrazine
(DNPH) according to the manufacturer’s instructions. Briefly, 15 µg of protein were used per
derivatization reaction. Proteins were then denatured by addition of 12% SDS. The samples
were subsequently derivatized by adding 10 µl of 1X DNPH solution and incubated for 20
minutes. Finally, 7.5 µl of neutralization solution and 2-mercaptoethanol were added to the
sample mixture. The specificity of reactive carbonyl measurements was confirmed by avoiding
the derivatization process and by omission of the primary antibody, and incubation of the
membranes only with secondary antibody [Goat anti-rabbit IgG, horseradish peroxidase (HRP)conjugated, from the Oxyblot kit, dilution: 1/300]. DNP-derivatized proteins were loaded onto
12% tris-glycine sodium dodecylsulfate polyacrylamide gels (SDS-PAGE) and separated by
electrophoresis.
Immunoblotting of 1D electrophoresis. Protein levels of the different molecular markers
analyzed in the study were explored by means of immunoblotting procedures as previously
described (4,11-17). Briefly, frozen muscle samples from the vastus lateralis of both
musclewasted and non-wasted COPD patients and healthy control subjects were homogenized
3
Results
in a buffer containing HEPES 50 mM, NaCl 150 mM, NaF 100 mM, Na pyrophosphate 10 mM,
EDTA 5 mM, Triton-X 0.5%, leupeptin 2 µg/ml, PMSF 100 µg/ml, aprotinin 2 µg/ml and
pepstatin A 10 µg/ml. Myofibrillar proteins were also isolated in order to identify levels of actin
and myosin heavy chain (MyHC)(18). Protein levels in crude homogenates were
spectrophotometrically determined with the Bradford technique using triplicates in each case
and bovine serum albumin (BSA) as the standard (Bio-Rad protein reagent, Bio-Rad Inc.,
Hercules, CA, USA). The final protein concentration in each sample was calculated from at
least two Bradford measurements that were almost identical. Equal amounts of total protein
from crude muscle homogenates were always loaded onto the gels, as well as identical simple
volumes/lanes.
For the purpose of comparisons among the different groups of COPD patients and
healthy controls, muscle sample specimens were always run together and kept in the same
order. Proteins were then separated by electrophoresis, transferred to polyvinylidene difluoride
(PVDF) membranes, blocked with non-fat milk and incubated overnight with selective
antibodies. Protein content of markers of proteolysis, signaling pathways of muscle atrophy, and
redox balance were explored using specific antibodies: protein carbonylation (anti-2,4- DNP
moiety antibody, Oxyblot kit, Chemicon International Inc., Temecula, CA, USA, dilution:
1/150), Mn-superoxide dismutase (SOD) and CuZn-SOD (anti-Mn-SOD and CuZn-SOD
antibodies, Santa Cruz), catalase (anti-catalase antibody, Calbiochem, Darmstadt, Germany),
glutathione peroxidase-I (anti-glutathione peroxidase-I antibody, AB Frontier, Seoul, Korea),
peroxiredoxins-II and III (anti-peroxiredoxins-II and III antibodies, AB Frontier), MyHC (antiMyHC antibody, clone A4.1025, Upstate-Millipore, Temecula, CA, USA), actin (anti-alphasarcomeric actin antibody, clone 5C5, Sigma Sigma-Aldrich, St. Louis, MO, USA), creatine
kinase (anti-creatine kinase antibody, Santa Cruz), and carbonic anhydrase-3 (anti-carbonic
anhydrase-3 antibody, Santa Cruz), total ubiquitinated proteins (anti-ubiquitin proteins, Boston
Biochem, Cambridge, MA, USA), 20S proteasome subunit C8 (anti-C8 antibody, Biomol,
Plymouth Meeting, PA, USA), ubiquitin-conjugating E214k (anti-E214K antibody, Boston
Biochem), ubiquitin-ligase atrogin-1 (anti-atrogin-1 antibody, Santa Cruz), ubiquitin-ligase
muscle ring finger (MURF)-1 (anti-MURF-1 antibody, Everest Biotech, Oxfordshire, UK),
transcription factor fork-head box O (FoxO, anti-FoxO antibody, Santa Cruz), mitogen activated
kinase (MAPK) c-Jun terminal (JNK, anti-JNK antibody, Santa Cruz), MAPK extracellular
kinase (ERK1/2, anti-ERK1/2 antibody, Santa Cruz), MAPK p38 (anti-p38 antibody, Santa
Cruz), nuclear factor (NF)-kB p50 (anti-p50 antibody, Santa Cruz), NF-kB p65 (anti-p65
antibody, Santa Cruz), myostatin (anti-myostatin antibody, Bethyl, Montgomery, TX, USA),
and myogenin (anti-myogenin antibody, Santa Cruz),
4
Results
Specific proteins from all samples were detected with horseradish peroxidase (HRP)conjugated secondary antibodies and a chemiluminescence kit. For each of the antigens,
samples from the different groups were always detected in the same picture under identical
exposure times. The specificity of the different antibodies was confirmed by omission of the
primary antibody, and incubation of the membranes only with secondary antibodies.
PVDF membranes were scanned with the Molecular Imager Chemidoc XRS System
(Bio–Rad Laboratories, Hercules, CA, USA) using the software Quantity One version 4.6.5
(Bio–Rad Laboratories). Optical densities of specific proteins were quantified using the
software Image Lab version 2.0.1 (Bio-Rad Laboratories). Values of total reactive carbonyl
groups in a given sample were calculated by addition of optical densities (arbitrary units) of
individual protein bands in each case. Final optical densities obtained in each specific group of
subjects corresponded to the mean values of the different samples (lanes) of each of the antigens
studied. In order to validate equal protein loading among various lanes, SDS-PAGE gels were
stained with Coomassie Blue.
Identification of carbonylated proteins. 1-D electrophoresis. In order to specifically identify
carbonylation of proteins such as MyHC, crude muscle homogenates from control subjects,
non-wasted and wasted COPD patients were subjected to 1D electrophoresis following
methodologies described above. Gels underwent electrophoretical transfer to a PVDF
membrane and immunoblotting with anti-DNP antibody. As mentioned above, PVDF
membranes were scanned with the Molecular Imager Chemidoc XRS System (Bio–Rad
Laboratories) using the software Quantity One version 4.6.5 (Bio–Rad Laboratories). Optical
densities of the carbonylated MyHC protein band were quantified using the software Image Lab
version 2.0.1 (Bio-Rad Laboratories). Proteomic analyses were conducted on the MyHC protein
band in order to confirm that the modified protein was indeed MyHC.
Muscle fiber counts and morphometry. On 3-micrometer muscle paraffin-embedded sections
from both patients and controls, MyHC-I and –II isoforms were identified using anti-MyHC-I
(clone MHC, Biogenesis Inc., Poole, England, UK) and anti-MyHC-II antibodies (clone MY32, Sigma, Saint Louis, MO), respectively, as published elsewhere(14-17). The crosssectional
area, mean least diameter, and proportions of type I and type II fibers were assessed using a
light microscope (Olympus, Series BX50F3, Olympus Optical Co., Hamburg, Germany)
coupled with an image-digitizing camera (Pixera Studio, version 1.0.4, Pixera Corporation, Los
Gatos, CA, USA) and a morphometry program (NIH Image, version 1.60, Scion Corporation,
Frederick, MD, USA). At least 100 fibers were measured and counted in each muscle specimen
from cachectic and noncachectic severe patients and healthy controls.
5
Results
Muscle structure abnormalities. The area fraction of normal and abnormal muscle was
evaluated on 3-micrometer paraffin-embedded sections of the vastus lateralis of all COPD
patients and healthy controls following previously published methodologies(19). Briefly,
normal and abnormal tissue was quantified using computer-assisted point counting in all the
limb muscle sections, previously stained with hematoxylin-eosin. A grid of 63 pointintercepts (7
x 9 rectangular pattern), built by means of the software Imaging Cell-B (Olympus Corporation,
Tokyo, Japan), was superimposed onto the image of the muscle cross section at a magnification
of x400 under the light microscope (Olympus BX 61, Olympus Corporation) using an image
digitizing camera (Olympus DP 71, Olympus Corporation). Each point-intercept was assigned
to a specific category and entered into the software. Categories for point counting were defined
as follows: 1) normal muscle, 2) internal nucleus, 3) inflammatory cell, 4) lipofuscin, 5)
abnormal viable, 6) inflamed/necrotic, 7) vessel, and 0) no count. The area fraction for each
category was defined as the percentage of points that fell on each of these traits relative to the
total number of points superimposed on all viable fields (all features except for categories 0 and
7) of each cross section. It follows that the area fraction of normal muscle was equivalent to the
proportion of points falling in category 1, while the area fraction of abnormal muscle was
determined by calculating the proportion of points in categories 2, 3, 4, 5, and 6.
Statistical Analysis
Results are presented as mean (SD). Comparisons of physiological and biological
variables among healthy controls, muscle-wasted and non-wasted severe COPD patients were
analyzed using one-way analysis of variance. Tukey’s post hoc analysis was used to adjust for
multiple comparisons. Correlations between physiological and biological variables were
explored using the Pearson’s correlation coefficient. The sample size chosen was based on
previous studies, where very similar approaches were employed (4,11-17,20-23).
6
Results
Reference List
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Rabe KF, Hurd S, Anzueto A, Barnes PJ, Buist SA, Calverley P et al. Global strategy
for the diagnosis, management, and prevention of chronic obstructive pulmonary disease:
GOLD executive summary. Am J Respir Crit Care Med 2007; 176(6):532- 555.
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Coin A, Sergi G, Minicuci N, Giannini S, Barbiero E, Manzato E et al. Fat-free mass
and fat mass reference values by dual-energy X-ray absorptiometry (DEXA) in a 20- 80 yearold Italian population. Clin Nutr 2008; 27(1):87-94.
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Montes dO, Loeb E, Torres SH, De Sanctis J, Hernandez N, Talamo C. Peripheral
muscle alterations in non-COPD smokers. Chest 2008; 133(1):13-18.
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Barreiro E, Rabinovich R, Marin-Corral J, Barbera JA, Gea J, Roca J. Chronic
endurance exercise induces quadriceps nitrosative stress in patients with severe COPD.
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Steiner MC, Barton RL, Singh SJ, Morgan MD. Bedside methods versus dual energy X-
ray absorptiometry for body composition measurement in COPD. Eur Respir J 2002;
19(4):626-631.
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Roca J, Sanchis J, Agusti-Vidal A, Segarra F, Navajas D, Rodriguez-Roisin R et al.
Spirometric reference values from a Mediterranean population. Bull Eur Physiopathol Respir
1986; 22(3):217-224.
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Roca J, Rodriguez-Roisin R, Cobo E, Burgos F, Perez J, Clausen JL. Single-breath
carbon monoxide diffusing capacity prediction equations from a Mediterranean population.
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Prediction equations for plethysmographic lung volumes. Respir Med 1998; 92(3):454-460.
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Coronell C, Orozco-Levi M, Mendez R, Ramirez-Sarmiento A, Galdiz JB, Gea J.
Relevance of assessing quadriceps endurance in patients with COPD. Eur Respir J 2004;
24(1):129-136.
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Swallow EB, Reyes D, Hopkinson NS, Man WD, Porcher R, Cetti EJ et al. Quadriceps
strength predicts mortality in patients with moderate to severe chronic obstructive pulmonary
disease. Thorax 2007; 62(2):115-120.
(11)
Barreiro E, Gea J, Corominas JM, Hussain SN. Nitric oxide synthases and protein
oxidation in the quadriceps femoris of patients with chronic obstructive pulmonary disease.
Am J Respir Cell Mol Biol 2003; 29(6):771-778.
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Barreiro E, Gea J, Matar G, Hussain SN. Expression and carbonylation of creatine
kinase in the quadriceps femoris muscles of patients with chronic obstructive pulmonary
disease. Am J Respir Cell Mol Biol 2005; 33(6):636-642.
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Barreiro E, Schols AM, Polkey MI, Galdiz JB, Gosker HR, Swallow EB et al. Cytokine
profile in quadriceps muscles of patients with severe COPD. Thorax 2008; 63(2):100-107.
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Barreiro E, Peinado VI, Galdiz JB, Ferrer E, Marin-Corral J, Sanchez F et al. Cigarette
smoke-induced oxidative stress: A role in chronic obstructive pulmonary disease skeletal
muscle dysfunction. Am J Respir Crit Care Med 2010; 182(4):477-488.
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Barreiro E, de la PB, Minguella J, Corominas JM, Serrano S, Hussain SN et al.
Oxidative stress and respiratory muscle dysfunction in severe chronic obstructive pulmonary
disease. Am J Respir Crit Care Med 2005; 171(10):1116-1124.
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Oxidised proteins and superoxide anion production in the diaphragm of severe COPD
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Redox balance and carbonylated proteins in limb and heart muscles of cachectic rats. Antioxid
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Myopathological features in skeletal muscle of patients with chronic obstructive pulmonary
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Koechlin C, Couillard A, Simar D, Cristol JP, Bellet H, Hayot M et al. Does oxidative
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Rabinovich RA, Bastos R, Ardite E, Llinas L, Orozco-Levi M, Gea J et al.
Mitochondrial dysfunction in COPD patients with low body mass index. Eur Respir J 2007;
29(4):643-650.
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Study #3
Pharmacological strategies in lung cancer-induced cachexia: effects on
muscle proteolysis, autophagy, structure, and weakness.
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Main findings in study # 3
In LC cachectic mice, compared to controls and in the different treatment groups, compared to
non-treated LC cachectic rodents:
Body and muscle mass weight and muscle function
• LC cachectic mice exhibited a reduction in body weight gain and diaphragm and gastrocnemius
weight compared to control animals.
• LC groups treated with NF-κB and MAPK inhibitors exhibited an increase in body weight
compared to non-treated LC cachectic rodents.
• Limb strength was reduced in the LC cachectic mice compared to the control animals.
• Limb strength reduction was corrected by treatment with NF-κB and MAPK inhibitors
compared to non-treated LC cachectic group.
• Food intake was similar in all groups.
Muscle structure
• Sizes of types I and II fibers were reduced in the LC cachectic animals compared to the control
mice.
• Fiber proportions did not differ between groups in both muscles.
• The LC animals treated with the proteasome and MAPK inhibitors showed an increase in
type I and II fibers size compared to non-treated LC cachectic rodents.
• Muscle abnormalities were higher in both muscles in the LC cachectic mice compared to the
control group.
• In LC rodents, treated with the MAPK inhibitor, muscle abnormalities were reduced in the
gastrocnemius, while cellular inflammation diminished in the respiratory muscle compared
to non-treated cachectic animals.
Subcutaneous tumor
• All inhibitors reduced subcutaneous tumor weight: proteasome (18%), NF-κB (27%) and
MAPK (60%), compared to LC cachectic mice.
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Proteins susceptible to be degraded
• MyHC protein levels were reduced in both muscles in the LC cachectic mice compared to the
control animals.
• MyHC protein levels were increased in the LC animals treated with NF-κB and MAPK
inhibitors, compared to non-treated LC cachectic rodents.
• CK protein levels were only reduced in gastrocnemius in the LC cachectic group compared to
the control animals.
• CK protein levels were increased by all the three treatments, compared to the non-treated
LC cachectic group.
• Actin levels and did not differ between the groups.
Redox Balance
• Total protein carbonylation was increased in both muscles in the LC cachectic mice compared
to the control animals.
• Total protein carbonylation was reduced by all the three treatments compared to the nontreated LC cachectic rodents.
• HNE protein adducts were increased in gastrocnemius in the LC cachectic mice compared to
the control animals.
• HNE-protein adduct levels did not differ in any of the muscles in response to any of the
inhibitors.
• Mn-SOD levels were decreased in both muscles in the LC cachectic animals compared to the
control rodents.
• CuZn-SOD and catalase levels did not differ between the groups.
• Treatment with NF-κB and MAPK inhibitors increased the Mn-SOD levels in the gastrocnemius
compared to the non-treated LC cachectic rodents.
Inflammation
• INF-γ levels were increased in the diaphragm in the LC cachectic mice compared to the
control animals.
• INF-γ levels were reduced by MAPK inhibitor compared to the non-treated LC cachectic
rodents.
• TNF-α, IL-6 and IL-1β did not differ between LC cachectic mice and the control group.
• Proteasome inhibitor reduced INF-γ, TNF-α, IL-6 and IL-1β in gastrocnemius compared to the
non-treated LC cachectic animals.
• MAPK inhibitor reduced TNF-α and IL-6 in diaphragm compared to the non-treated LC
cachectic rodents.
Proteolytic systems
• Protein catabolism was increased in both muscles in the LC cachectic mice compared to the
control animals.
• Protein catabolism was reduced in both muscles by all treatments compared to the nontreated LC cachectic animals.
• E214k protein levels were increased in the gastrocnemius in the LC cachectic mice compared
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to the control rodents.
• E214k protein levels were reduced by the treatment with NF-κB and MAPK inhibitors
compared to the non-treated LC cachectic animals.
• Levels of atrogin-1 and MURF did not differ between the groups.
• Protein levels of proteasome C8-20S subunit did not differ between LC cachectic mice and
the control group.
• Proteasome C8-20S protein levels were reduced in both muscles by proteasome inhibitor
treatment compared to the non-treated LC cachectic rodents.
• Protein ubiquitination was higher in both muscles of the LC cachectic mice compared to the
control animals.
• Protein ubiquitination was reduced by all treatments in gastrocnemius, and in diaphragm
only by proteasome inhibitor, compared to the non-treated LC cachectic animals.
Signaling pathways
• Total Fox-O1 levels did not differ in the LC cachectic group compared to the control rodents.
•
p-Fox-O1 levels were higher in the gastrocnemius of the LC cachectic mice compared to the
control animals.
• Total Fox-O1 levels were reduced in the diaphragm by proteasome inhibitor treatment
compared to the non-treated LC cachectic group.
• Calpain protein levels were increased in the diaphragm in the LC cachectic mice compared
to the control animals.
• Calpain protein levels were reduced by NF-κB and MAPK inhibitors treatments compared to
the non-treated LC cachectic group.
• Levels of p38 were higher in both muscles in the LC cachectic mice compared to the controls
• p38 protrein levels were reduced by MAPK inhibitor compared to the non.treated LC
cachectic animals.
• Levels of ASK-1, ERK1/2, pERK2, JNK and p-p38 did not differ between LC cachectic mice and
the control group.
• ERK1/2 and pERK1/2 levels were reduced by MAPK inhibitor in both muscles compared to
the non-treated LC cachectic animals.
• Total NF-κB p50, p- NF-κB p50 and total NF- κB p65 were higher in both muscles in the LC
cachectic mice compared to the control group.
• In both muscles, total NF-κB p50 levels were reduced by all treatments compared to the nontreated cachectic animals.
•
p-NF-κB p50, total NF-κB p65 and p NF-κB p65 levels were reduced by proteasome and NFκB inhibitors compared to the non-treated LC cachectic rodents.
• IκB and p-IκB were reduced in both muscles in the LC cachectic mice compared to the control
animals.
• IκB and p-IκB were increased by all treatments in both muscles compared to the non-treated
LC cachectic rodents.
• Transcriptional activity of NF-κB was increased in the gastrocnemius in the LC cachectic mice
compared to the control animals.
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• Transcriptional activity of NF-κB was reduced by proteasome and NF-κB inhibitors compared
to the non-treated LC cachectic group.
• In both muscles, myostatin levels were increased in the LC cachectic mice compared to the
control group.
• Myostatin levels were reduced by all treatments in both muscles compared to non-treated
LC cachectic animals.
• Myogenin was reduced in both muscles in the LC cachectic mice compared to the control
group.
• Myogenin levels were increased by NF-κB and MAPK inhibitors compared to the non-treated
LC cachectic animals.
Autophagy markers
• The levels of the autophagy marker LC3-II/LC3-I were increased in both muscles in the LC
• cachectic mice compared to the control group.
• LC3-II/LC3-I levels were reduced by all treatments compared to the non-treated LC cachectic
animals.
• The levels of the autophagy markers p62 and beclin-1 levels did not differ between all the
groups.
Apoptosis
• Apoptosis (TUNEL positive nuclei) levels were higher in both muscles in the LC cachectic
mice compared to the control animals.
• Apoptosis (TUNEL positive nuclei) levels were reduced by MAPK inhibitor compared to the
non-treated LC cachectic animals.
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PHARMACOLOGICAL STRATEGIES IN LUNG CANCER-INDUCED CACHEXIA:
EFFECTS ON MUSCLE PROTEOLYSIS, AUTOPHAGY, STRUCTURE, AND
WEAKNESS
Clara Fermoselle1,2, Alejandro J. Urtreger3, Francisco Sanchez1,2, Alba Chacon-Cabrera1,2,
Mercè Mateu-Jimenez1,2, Marco Sandri4, Miriam J. Diament3, Elisa D. Bal de Kier Joffé3,
Esther Barreiro1,2
1
Lung Cancer Research Group, IMIM-Hospital del Mar, Parc de Salut Mar, Health and
Experimental Sciences Department (CEXS), Universitat Pompeu Fabra (UPF), Parc de Recerca
Biomèdica de Barcelona (PRBB), Barcelona, Spain.
2
Centro de Investigación en Red de Enfermedades Respiratorias (CIBERES), Instituto de Salud
Carlos III (ISCIII), Bunyola, Majorca, Balearic Islands, Spain.
3
Research Area, Institute of Oncology ‘Angel H. Roffo’, University of Buenos Aires, Buenos
Aires, Argentina.
4
Department of Biomedical Sciences, University of Padova, Dubelcco Telethon Institute,
Venetian Institute of Molecular Medicine, Padova, Italy.
Corresponding author: Dr. Esther Barreiro, Pulmonology Department-URMAR, IMIMHospital del Mar, PRBB, C/ Dr. Aiguader, 88, Barcelona, E-08003 Spain, Telephone: (+34) 93
316 0385, Fax: (+34) 93 316 0410, e-mail: [email protected]
Running title: muscle biology, structure, and function in cachexia
Key words: cancer-induced cachexia in mice; diaphragm and gastrocnemius; ubiquitinproteasome system; autophagy; oxidative stress; muscle structure and function; proteasome,
NF-κB, and MAPK inhibitors
GRANTS: This study has been supported by CIBERES; FIS 11/02029; 2009-SGR-393;
SEPAR 2010; FUCAP 2011; FUCAP 2012; and Marató TV3 (MTV3-07-1010) (Spain). Dr.
Esther Barreiro was a recipient of the ERS COPD Research Award 2008.
“The authors disclose no potential conflicts of interest." Word count: 5,057
ABSTRACT
Muscle wasting and cachexia are important systemic manifestations of highly prevalent
conditions including cancer. Inflammation, oxidative stress, autophagy, ubiquitin-proteasome
system, nuclear factor (NF)-κB, and mitogen activated protein kinases (MAPK) are involved in
the pathophysiology of cancer cachexia. Currently available treatment is limited and data
demonstrating effectiveness in in vivo models are lacking. Our objectives were to explore in
respiratory and limb muscles of lung cancer (LC) cachectic mice whether proteasome, NF-κB,
and MAPK inhibitors improve muscle mass and function loss through several molecular
mechanisms. Body and muscle weights, limb muscle force, protein degradation and the
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ubiquitin-proteasome system, signaling pathways, oxidative stress and inflammation,
autophagy, contractile and functional proteins, myostatin and myogenin, and muscle structure
were evaluated in the diaphragm and gastrocnemius of LC (LP07 adenocarcinoma) bearing
cachectic mice (BALB/c), with and without concomitant treatment with NF-κB (sulfasalazine),
MAPK (U0126), and proteasome (bortezomib) inhibitors. Compared to control animals, in both
respiratory and limb muscles of LC cachectic mice: muscle proteolysis, ubiquitinated proteins,
autophagy, myostatin, protein oxidation, FoxO-1, NF-κB and MAPK signaling pathways, and
muscle abnormalities were increased, while myosin, creatine kinase, myogenin, and slow- and
fast-twitch muscle fiber size were decreased. Pharmacological inhibition of NF-κB and MAPK,
but not the proteasome system, induced in the cachectic animals, a substantial restoration of
muscle mass and force through a decrease in muscle protein oxidation and catabolism,
myostatin, and autophagy, together with a greater content of myogenin, and contractile and
functional proteins. These findings offer new therapeutic strategies in cancer-induced cachexia.
Word count: 247
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INTRODUCTION
Muscle wasting and cachexia are important systemic manifestations of highly prevalent
conditions including cancer. The prevalence of cachexia varies across diseases, but in advanced
malignancies it ranges from 60 to 80% (1), being greater in patients with lung and
gastrointestinal tumors than in other solid or hematologic malignancies (2). Cachexia,
characterized by muscle mass loss and body composition alterations, has a negative impact on
the patients’ quality of life(3).
Although the etiology of cancer-induced cachexia remains to be understood, several
cellular and molecular mechanisms have been proposed such as systemic inflammation(4),
oxidative stress (5-8), metabolic disturbances and nutritional abnormalities(5, 6, 9).
Furthermore, mitogen-activated protein kinases (MAPK) and nuclear factor (NF)-κB, which are
central regulators of gene expression, redox balance, and metabolism, have also been shown to
play a major role in adaptive or maladaptive responses to cellular stress within skeletal
muscles(10). MAPK activation also seems to mediate oxidative stress-induced muscle
atrophy(11). Interestingly, MAPK signaling was also shown to be involved in enhanced
expression of proteasome via proteolysis-inducing factor in C2C12 myotubes (12). However,
studies demonstrating the potential beneficial effects of MAPK inhibition in cancer-induced
cachexia in vivo are lacking.
NF-κB was also shown to be involved in the process of muscle wasting under several
conditions such as sepsis(13), cancer cachexia(14, 15), and chronic obstructive pulmonary
disease (COPD) (15, 16). In fact, the double NF-κB and AP-1 inhibitor SP100030 elicited an
improvement in muscle weights and markers of the ubiquitin-proteasome system(15).
Sulfasalazine, a well-known drug used for the treatment of patients with inflammatory bowel
disease and rheumatoid arthritis, is a potent and specific inhibitor of NF-κB. It inhibits its
translocation into the nucleus, without affecting the activation of the AP-1 transcription
factor(17). Furthermore, that inhibition is accompanied by a blockade of the degradation of its
inhibitory subunit IκB(17). Sulfasalazine was also shown to restore tissue injury and function in
several experimental models(18, 19). However, the potential beneficial effects of NF-κB
inhibition using sulfasalazine on cancer-induced cachexia are still unknown.
The ubiquitin-proteasome system plays a major role in conditions characterized by muscle
wasting and weakness(19-24). Specifically, bortezomib, the first proteasome inhibitor approved
for use in patients, was demonstrated to significantly restore diaphragm muscle contractile
function and myosin heavy chain content (MyHC) following coronary heart failure (23)and
elastase-induced emphysema(24). In another investigation(25), however, bortezomib failed to
improve the endotoxin-induced diaphragm dysfunction, while partially restoring enhanced
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protein catabolism. Whether inhibition of the proteasome restores muscle mass and function in
cancer cachexia remains to be identified.
As treatment options for cachexia are extremely limited and in vivo studies demonstrating
the effectiveness of several potential anti-cachectic agents are clearly lacking, the current
investigation was specifically designed to explore whether inhibitors of MAPK, NF-κB, and the
proteasome exert beneficial effects on muscle mass loss and force generation in cancer
cachectic mice. Moreover, research conducted so far has exclusively focused on the evaluation
of limb muscles. On this basis, our objectives were to assess in respiratory and limb muscles
from lung cancer (LC) cachectic mice receiving concomitant treatment with either MAPK, NFκB, or proteasome inhibitors: 1) body and muscle weights, 2) limb muscle force, 3) proteolysis
markers, 4) signaling pathways, 5) oxidative stress and inflammation, 6) autophagy, 7)
contractile and functional proteins, 8) myostatin and myogenin levels, and 9) muscle structural
alterations.
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METHODS
(See the online supplementary material for additional information).
Animal experiments
Tumor. LP07 is a cell line derived from the transplantable P07 lung tumor that appeared
spontaneously in the lung of a BALB/c mouse(26). The LP07 cell line was obtained in vitro,
after successive passages of a P07 primary culture, by some of us(27). LP07 cell line shares
identical characteristics regarding lung tumor incidence and histology, and cachexia with its
parental P07 tumor(26-28). It was also consistently demonstrated that one month after tumor
transplantation, all animals developed lung metastasis, spleen enlargement, and cachexia
without affecting any other organs(26-28). Female BALB/c mice, 2 months old (weight ~20g),
were obtained from Harlan Interfauna Ibérica SL (Barcelona, Spain).
Experimental design and Ethics. In all experimental groups (except for control rodents), LP07
viable cells (4·105) resuspended in 0.2 mL minimal essential media (MEM) were
subcutaneously inoculated in the left flank of the mice (day 1). All groups (n=10/group) were
studied for a period of one month. Animals were randomly assigned to the following groups: 1)
control, inoculation of 0.2 mL MEM in the left flank; 2) LC cachexia group, inoculation of
LP07 cells; 3) LC cachectic mice received concomitant treatment with the proteasome inhibitor
Bortezomib (Velcade, Millenium Pharmaceuticals, Cambridge, MA), 0.15 mg/Kg, 0.1 mL/6
days, intravenous injection into the tail vein (LC cachectic-bortezomib group)(29); 4) LC
cachectic mice received concomitant treatment with sulfasalazine (Pfizer, Madrid, Spain), 200
mg/Kg, 0.3 mL/48h, intraperitoneal injection (LC cachectic-NF-κB inhibitor group)(19); and 5)
LC cachectic mice received concomitant treatment with the MAPK inhibitor U0126 (a highly
selective inhibitor of ERK1 and ERK2 proteins, Selleck chemicals, Houston, TX), 30 mg/Kg,
0.1 mL/48h, intraperitoneal injection (LC cachectic-MAPK inhibitor group)(30). All
pharmacological therapies were administered on day 15 post-inoculation of the LP07 cells up
until the end of the study period. In order to assess the level of NF-κB transcription and ex-vivo
protein degradation using the luciferase reporter and the tyrosine release assays, respectively
(see below), a second batch of mice experiments was also conducted on identical experimental
groups (N=10/group). One hind-limb and half of the diaphragm were used for each type of these
experiments.
All animal experiments were conducted in the animal facilities at Parc de Recerca Biomèdica
de Barcelona (PRBB, Spain). This controlled study was designed in accordance with the ethical
standards on animal experimentation (EU 609/86 CEE, Real Decreto 1201/05 BOE 252, Spain)
at PRBB and the Helsinki convention for the use and care of animals. Ethical approval was
obtained by the Animal Research Committee at PRBB and Catalan Government (Animal
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welfare department).
In vivo measurements and sample collection from mice
Body weight and food intake were determined every day during the entire duration of the study.
Limb strength was determined on days 0 and 30 using a strength grip meter (Bioseb, Chaville,
France) following previously published methodologies(31). In the LC cachexia group of mice,
tumor progression was determined using positron emission tomography (PET) on days 13 and
20. Mice from all the experimental groups were always sacrificed on day 30 post-inoculation of
LP07 cells or MEM (control animals). Each mouse was inoculated intraperitoneally with 0.1
mL sodium pentobarbital (60 mg/Kg). Diaphragm and gastrocnemius muscles and the
subcutaneous tumor were obtained from all animals. The weight of both muscles and tumor
were determined in each animal using a high-precision scale. Frozen tissues were used for
immunoblotting and enzyme-linked immunosorbent assay (ELISA) techniques, while paraffinembedded tissues were used for the assessment of muscle structure abnormalities and fiber type
morphometry.
Muscle biology analyses
All muscle biology analyses were conducted blind in the same laboratory by the same
investigators, at Hospital del Mar-IMIM (Barcelona).
Immunoblotting of 1D electrophoresis. Protein levels of the different molecular markers
analyzed in the study were explored by means of immunoblotting procedures as previously
described(7, 21, 32, 33). Protein content of markers of proteolysis, signaling pathways of
muscle atrophy, redox balance, and different muscle proteins were identified using specific
primary antibodies (See detailed information in the online supplementary material).
Protein catabolism. Protein degradation was explored on the basis of the rate of production of
free tyrosine from tissue proteins as previously described(34). As muscles cannot synthesize or
degrade this amino acid, its accumulation reflects the net degradation of proteins. The results
were expressed as nmol of tyrosine/mg of muscle/2 hours of incubation.
Luciferase reporter gene assay. On day 23 of the animal protocol (7 days exactly before
sacrifice) all mice were injected in the right gastrocnemius muscle (for obvious reasons the
diaphragm was not used for this purpose) with a mixture of 15 µg of control plasmid pRL-TK
vector, which contains a cDNA encoding renilla luciferase (Promega Corporation, Madison,
WI, USA) and 20 µg of reporter plasmid pNF-κB-Luc, which contains the firefly luciferase
gene (Clonetech, Mountain View, CA, USA). The procedures followed have already been
published(11).
Cytokine Enzyme-linked Immunosorbent Assay (ELISA). Protein levels of the cytokines tumor
necrosis factor (TNF)-alpha, interferon-gamma, interleukin (IL)-6 and IL-1beta were quantified
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in the muscles of all study animals (diaphragms and gastrocnemius muscles) using specific
sandwich ELISA kits eBiosience, Bender MedSystems, Vienna, Austria) following previously
published methodologies(21, 35).
Muscle fiber counts and morphometry. On 3-micrometer muscle paraffin-embedded sections
from diaphragms and gastrocnemius muscles of all study groups, morphometrical analyses were
performed as previously reported(7, 21, 35).
Muscle structure abnormalities. The area fraction of normal and abnormal muscle was
evaluated on 3-micrometer paraffin-embedded sections of the diaphragm and gastrocnemius of
all study groups following previously published methodologies(Figure E1)(21).
Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. In
muscle paraffin-embedded sections, apoptotic nuclei were identified using the TUNEL assay (In
Situ Cell Death Detection Kit, POD, Roche Applied Science, Mannheim, Germany) in both
diaphragms and gastrocnemius muscles from all study groups following precisely the
manufacturer’s instructions and previously published studies(36).
Statistical Analysis
Results are presented as mean (SD). Comparisons of physiological and biological variables
among the different study groups were analyzed using one-way analysis of variance. For the
purpose of the study two different sets of comparisons were made: i) control and LC cachectic
mice, and on the other hand ii) LC cachectic and LC cachectic-bortezomib, iii) LC cachectic
and LC cachectic-NF-κB inhibitor; and iv) LC cachectic and LC cachectic-MAPK inhibitor.
Tukey’s post hoc analysis was used to adjust for multiple comparisons. Statistical significance
corresponding to these two different sets of comparisons is being specifically indicated in both
Figures and Tables. A level of significance of P≤ 0.05 was established.
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RESULTS
Physiological characteristics
As shown in Table 1, at the end of the study period, LC cachectic mice exhibited a reduction in
body weight that was not observed in control animals. Food intake was similar among the study
groups (3 g/24h). Diaphragm and gastrocnemius muscle weights and limb strength were
significantly reduced in the LC cachectic mice compared to control rodents (Table 1). LC
cachectic mice treated with the NF-κB and MAPK inhibitors exhibited a significantly smaller
reduction in weight gain, a significant improvement in diaphragm and gastrocnemius weight
loss, and a significant recovery of muscle strength gain compared to the non-treated cachectic
animals (Table 1). The proteasome inhibitor did not induce any significant effects on body or
muscle weights, or limb muscle strength in the LC cachectic mice (Table 1). Importantly, the
weight of the subcutaneous tumor was significantly reduced in the cachectic rodents treated
with the inhibitors of the proteasome (18%), NF-κB (27%), and MAPK (60%) pathways
compared to non-treated cachectic mice (Table 1).
Muscle proteolysis
Tyrosine release. Protein degradation, as measured by the release of the amino acid tyrosine,
was increased in both diaphragm and gastrocnemius muscles of LC cachectic mice compared to
controls (Figure 1A). Interestingly, treatment of cachectic mice with bortezomib, NF-κB, and
MAPK inhibitor induced a significant reduction in tyrosine release levels in both respiratory
and limb muscles (Figure 1A).
Proteolytic systems. The diaphragm muscle, but not gastrocnemius, of LC cachectic mice
exhibited a significant increase in protein levels of calpain compared to controls (Table 2). The
inhibitors of NF-κB and MAPK pathways elicited a significant reduction in calpain content
only in the diaphragm of cachectic mice, while bortezomib did not induce any significant effect
in any muscle (Table 2). Levels of the ubiquitin-conjugating enzyme E214κ were increased only
in the gastrocnemius of cachectic mice compared to controls (Figure 1B). Importantly,
inhibitors of proteasome, NF-κB, and MAPK pathways significantly decreased levels of E214k
in the gastrocnemius, but not the diaphragm, of cachectic mice (Figure 1B). Levels of the E3
ligases atrogin-1 and MURF-1 did not differ in any muscle of the study groups (Figure 1C, top
and bottom panels, respectively). Protein content of 20S proteasome subunit C8 did not differ
between LC cachectic and control mice (Figure 1D, top panel). As expected, bortezomib
induced a significant reduction, especially in gastrocnemius, in protein content of subunit C8
among cachectic animals, while NF-κB and MAPK inhibitors did not elicit any significant
modification (Figure 1D, top panel). Protein ubiquitination levels were significantly greater in
both respiratory and limb muscles of LC cachectic mice than in controls (Figure 1 D, bottom
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panel). In cachectic mice, bortezomib induced a significant reduction in protein ubiquination in
both diaphragm and gastrocnemius, whereas NF-κB and MAPK inhibitors elicited a decrease in
that proteolytic marker only in limb muscles (Figure 1D, bottom panel).
Signaling pathways of muscle proteolysis
FoxO pathway. While total content of FoxO-1 did not differ in any muscle between cachectic
and control mice, content of p-FoxO-1 was significantly greater in gastrocnemius of cachectic
rodents than in controls (Table 2). Bortezomib induced a reduction in total FoxO-1 content, but
not p-FoxO-1, only in cachectic diaphragms, whereas NF-κB and MAPK inhibitors did not
elicit any modification in muscle content of either total or active FoxO-1 (Table 2).
MAPK pathway. Muscle levels of MAPK subfamilies ASK1, ERK1/2, p-ERK1/2, JNK, and pp38 did not differ between LC cachectic and control mice (Table 2).However, levels of total
p38 were significantly greater in both diaphragm and gastrocnemius in cachectic mice than in
controls (Table 2). Interestingly, treatment of cachectic animals with MAPK inhibitor elicited a
significant reduction in protein levels of ERK1/2, p-ERK1/2, and total p38 in respiratory and
limb muscles (Table 2).
NF-κB pathway. In respiratory and limb muscles, protein content of p50 and p-p50 was
significantly increased in cachectic animals compared to controls (top and bottom panels,
Figure 2A). A significant decrease was observed in total p50 in both diaphragm and
gastrocnemius of cachectic mice in response to proteasome, NF-κB, and MAPK inhibitors,
while p-p50 levels were only reduced in both muscles among animals treated with the NF-κB
inhibitor (top and bottom panels, Figure 2A). Total protein content of p65, but not p-p65, were
increased in diaphragms and gastrocnemius of cachectic mice compared to controls (top and
bottom panels, Figure 2B). A significant reduction was detected in protein content of p65 in
both respiratory and limb muscles of cachectic rodents in response to proteasome and NF-κB
inhibitors, while p-p65 protein levels were decreased only in response to NF-κB inhibitor in
those muscles (Figure 2B, top and bottom panels, respectively). Protein levels of total IκB and
p-IκB were significantly diminished in both diaphragms and gastrocnemius muscles in
cachectic mice compared to controls (top and bottom panels, Figure 2C). Interestingly,
treatment of cachectic rodents with proteasome, NF-κB, and MAPK inhibitors elicited an
increase in both total IκB and p-IκB markers in respiratory and limb muscles (top and bottom
panels, Figure 2C). Transcriptional activity of NF-κB was increased in cachectic mice
compared to controls (Figure 2D). Importantly, among cachectic rodents, treatment with both
proteasome and NF-κB inhibitors elicited a significant decline in NF-κB transcriptional activity,
especially the latter, while no differences were observed in response to MAPK inhibition
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(Figure 2D).
Muscle growth and differentiation. In cachectic mice, protein levels of myostatin were
increased in both diaphragms and gastrocnemius compared to control rodents (Figure 3A).
Importantly, proteasome, NF-κB, and MAPK inhibitors elicited a significant reduction in
myostatin levels in muscles of cachectic animals (Figure 3A). Nevertheless, compared to
controls, protein content of myogenin was significantly reduced within respiratory and limb
muscles of cachectic rodents, while treatment with either NF-κB or MAPK inhibitors, but not
bortezomib, elicited a rise in myogenin content in their muscles (Figure 3A).
Contractile and functional muscle proteins
Protein levels of contractile MyHC were reduced in diaphragm and gastrocnemius of cachectic
mice compared to controls (Figure 3B, top panel). Interestingly, NF-κB and MAPK inhibitors,
but not bortezomib, elicited an improvement in MyHC protein content in respiratory and limb
muscles among cachectic rodents (Figure 3B, top panel). Protein content of skeletal muscle
actin did not differ among any of the study groups (Figure 3B, bottom panel). Compared to
controls, creatine kinase protein content was significantly reduced only in gastrocnemius of
tumor-bearing animals (Figure 3C, top panel). Treatment of these mice with proteasome, NFκB, and MAPK inhibitors induced a rise in creatine kinase content only in limb muscles (Figure
3C, top panel). No differences were detected in carbonic anhydrase-3 muscle levels among the
study groups (Figure 3C, bottom panel).
Muscle inflammation and redox balance
Inflammatory cytokines. ELISA levels of interferon-gamma were only significantly increased in
diaphragms of cachectic mice compared to controls (Table 2). TNF-alpha, IL-6, and IL-1beta
ELISA levels did not differ between cachectic and control mice (Table 2). Treatment with
bortezomib elicited a significant decline in protein levels of interferon-gamma, TNF-alpha, IL6, and IL-1beta in gastrocnemius of cachectic rodents (Table 2). Protein content of the studied
cytokines was not different in any muscles of cachectic animals in response to NF-κB inhibition
(Table 2). Diaphragm levels of interferon-gamma, TNF-alpha, and IL-6 were decreased among
cachectic rodents treated with MAPK inhibitor (Table 2).
Oxidative stress markers. Compared to controls, protein carbonylation levels were significantly
increased in both diaphragms and gastrocnemius muscles of cachectic mice (Table 2).
Treatment of these rodents with proteasome, NF-κB, and MAPK inhibitors elicited a reduction
in total protein carbonylation in respiratory and limb muscles (Table 2). HNE-protein adduct
levels were increased only in gastrocnemius of tumor-bearing mice compared to controls, and
treatment with proteasome, NF-κB, and MAPK inhibitors did not induce any significant effect
on their muscles (Table 2). Muscle protein nitration levels did not differ among the study
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groups (Table 2).
Antioxidant enzymes. Compared to control rodents, protein levels of Mn-SOD were decreased in
diaphragms and gastrocnemius of tumor-bearing mice, and treatment with proteasome, NF-κB,
and MAPK inhibitors elicited a significant rise in Mn-SOD content only in gastrocnemius
(Table 2). Muscle protein content of CuZn-SOD and catalase did not differ among study groups
(Table 2).
Muscle autophagy
Muscle protein levels of the autophagy system p62 and beclin-1 did not differ among the study
groups (Figures 4A and 4B). Protein levels of LC3 as measured by the ratio of LC3-II to LC3-I,
however, were significantly increased in diaphragm and gastrocnemius of cachectic mice
compared to controls (Figure 4C), and treatment with proteasome, NF-κB, or MAPK inhibitors
elicited a significant decline in LC3II/LC in diaphragms and gastrocnemius of tumor-bearing
mice (Figure 4C).
Muscle structure
Fiber type composition. Proportions of type I and type II fibers were not different in any muscle
among the study groups (Table 3). Compared to controls, the size of slow- and fast-twitch fibers
was significantly diminished in both diaphragm and gastrocnemius of cachectic mice (Table 3
and Figures E2A and E2B). In these animals, treatment with either proteasome or MAPK
inhibitors, but not NF-κB inhibitor, elicited a significant improvement in sizes of both type I
and II fibers (Table 3 and Figures E3A and E3B).
Muscle abnormalities. Compared to control mice, proportions of abnormal muscle were greater
in both diaphragms and gatrocnemius of cachectic animals (Table 3). In these animals,
treatment with MAPK inhibitor elicited a significant reduction in total muscle abnormalities
only in the limb muscle, while inducing a decrease in inflammatory cell proportions in both
muscles (Table 3). Compared to controls, TUNEL-stained nuclei counts were increased in both
diaphragms and gastrocnemius of cachectic mice (Table 3 and Figures E3A and E3B).
Importantly, only treatment of these animals with MAPK inhibitor induced a significant
decrease in proportions of TUNEL-stained nuclei in their diaphragms (Table 3 and Figures E3A
and E3B).
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DISCUSSION
In BALB/c mice, subcutaneous inoculation of LP07 cells induced lung metastases, together
with a severe cachexia that was accompanied by a significant decline in the diaphragm and limb
muscle mass and strength generation(27). As far as we are concerned, this is the first study in
which a major respiratory muscle, the diaphragm, was analyzed concomitantly with limb
muscles. In line with the study hypothesis, the main findings in the investigation were that
compared to control animals, in both respiratory and limb muscles of LC cachectic mice: 1)
muscle proteolysis was enhanced; 2) total amounts of ubiquitinated proteins and autophagy
were increased; 3) calpain levels were higher only in the diaphragm; 4) myostatin levels were
greater, while those of myogenin were reduced; 5) protein creatine kinase levels were decreased
only in the gastrocnemius; 6) levels of contractile MyHC were reduced, while those of actin
were not modified; 7) the size of slow- and fast-twitch muscle fibers was decreased in both
muscles, while proportions of muscle abnormalities and TUNEL-stained nuclei were increased;
8) protein oxidation levels were greater, whereas inflammatory cytokine levels were not
modified; and 9) signaling pathways such as FoxO-1, NF-κB and MAPK were increased.
Treatment of LC cachectic mice with the pharmacological inhibitors induced several
modifications that are discussed below.
Pharmacological proteasome inhibition in cancer cachectic muscles
Proteasome, an energy-dependent proteolytic system, is a key modulator of cellular processes
including NF-κB signaling and is also involved in cell metabolism and DNA repair. Among the
different pharmacological proteasome inhibitors described so far, bortezomib is currently the
only agent approved for clinical use in patients with cancer(37). Proteasome also seems to play
a major role in the muscle mass loss and dysfunction process associated with several chronic
conditions(23-25) and severe infections(22).
In the current investigation, although cachectic mice treated with bortezomib exhibited a
significantly lower tumor size compared to non-treated tumor-bearing rodents, body and muscle
weights together with limb muscle force did not improve. In fact, only an improvement in the
size of slow-twitch fibers, but not type II, was observed in either respiratory or limb muscles in
response to proteasome inhibition. Besides, no significant modifications in contractile MyHC or
actin contents were detected in any muscle. These findings are in agreement with those reported
in a previous study conducted in endotoxemic rats (25), in which treatment with several
proteasome inhibitors prevented muscle proteolysis, but not diaphragm muscle loss or force
generation. Nevertheless, the present findings and those previously reported(25), are counter to
those demonstrated in other two investigations, in which contractile diaphragm function was
shown to improve in response to bortezomib in rats bearing congestive heart failure(23) and
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emphysema(24).
One likely explanation to account for differences encountered among studies(23-25) is that
different proteolytic systems other than the proteasome may participate in reduced body weight,
and decreased muscle mass and weakness under several experimental conditions. In line with
this, it was demonstrated (38, 39) that other proteases such as caspase and calpain (not modified
by bortezomib in any muscle) need to be initially activated within the myocytes in order to
break down the contractile myofilaments under muscle wasting conditions. Another explanation
accounting for the failure of bortezomib to improving muscle mass or function in the cachectic
mice is the lack of effects of the proteasome inhibitor on muscle protein synthesis. This pathway
is likely to be reduced in the muscles of the cachectic mice, as was shown to occur in other
models of cancer-induced cachexia (40). However, the present investigation was not designed to
explore protein anabolism in the mouse cachectic muscles.
Pharmacological NF-κB inhibition in cancer cachectic muscles
NF-κB modulates the expression of cytokines, growth factors, and a wide variety of genes that
regulate physiological and pathological conditions. NF-κB is one of the most relevant signaling
pathways leading to skeletal muscle loss. It is composed by a family of five members (p65, RelB, c-Rel, p50, and p52), which are all expressed within skeletal muscles. NF-κB dimers, which
are most frequently composed of the members RelA (p65) and NF-κB1 (p50), are sequestered
in an active cytoplasmic complex by binding to its inhibitory subunit, IκB. Following the
stimulus, IκB becomes phosphorylated by specific kinases. This phosphorylation entails
ubiquitination and fast degradation of IκB by the proteasome. Free NF-κB dimers translocate to
the nucleus and activate target genes. Sulfasalazine was shown to be a powerful and specific
NF-κB inhibitor by essentially inhibiting NF-κB-dependent transcription in several models(1719).
In the current investigation, it has been clearly demonstrated that NF-κB pathway was
upregulated in both respiratory and limb muscles of LC cachectic mice. In those muscles,
sulfasalazine elicited a significant decline in protein levels of total and phosphorylated p65 and
p50 members, while inducing a substantial upregulation of IκB and p-IκB among the cachectic
rodents. Moreover, in the present experimental model, these findings were further confirmed by
experiments performed to specifically assess NF-κB transcriptional activity. In this regard, NFκB activity was increased in diaphragm and gastrocnemius muscles of cachectic mice, whereas
the activity was specifically downregulated in response to both proteasome and NF-κB
inhibition, especially the latter. These are interesting findings which suggest that NF-κB plays a
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crucial role in cancer-induced cachexia signaling in this experimental model of cancer cachexia.
Importantly, treatment of the cachectic mice with sulfasalazine also elicited other
interesting modifications in muscles of the cachectic rodents. In this respect, a substantial
improvement was seen in body and muscle weights, limb muscle strength, and tumor size (27%
decrease), together with a reduction in muscle protein degradation and oxidation, E214κ and
protein ubiquitination levels, myostatin content, and autophagy as measured by LC3-II/LC3-I
levels. Additionally, in muscles of tumor-bearing mice treated with sulfasalazine, a significant
increase in myogenin, contractile MyHC, and creatine kinase content, was also observed in
response to NF-κB inhibition. No significant modifications, however, were seen in muscle fiber
sizes or cytokine content, whose levels were, indeed, similar to those observed in the nontreated cachectic and control animals. This is somehow counter to previous reports (41), in
which NF-κB and TNF-alpha were shown to signal muscle atrophy.
Body and muscle weight and strength improvement in response to NF-κB inhibition was
likely to be achieved through the contribution of several factors such as reduced levels of
protein degradation, myostatin, and autophagy, and increased myogenin content, which may
have led, in turn, to higher content of contractile MyHC in the cachectic muscles. Myostatin,
which is almost exclusively expressed in skeletal muscles, is a potent negative regulator of
muscle mass. The increase in myostatin levels observed in the cachectic muscles are in line with
previous reports, in which significant increases in myostatin content were shown in diaphragm
and gastrocnemius of emphysema cachectic mice (33), in muscles of cachectic COPD
patients(21, 42-44), and in cancer patients(45). Myogenin plays an important role in skeletal
muscle differentiation, maintenance and repair, regulating muscle metabolism and energy
utilization. Levels of myogenin were decreased in muscles of the cachectic mice, while NF-κB
restored those levels in both muscles. These findings are in keeping with a previous
investigation(21, 42), in which myogenin levels were also decreased in cachectic severe COPD
patients. Another aspect that deserves attention is the smaller size of the subcutaneous tumor in
response to NF-κB inhibition among the LC cachectic mice. However, the present investigation
was not designed to disentangle the potential relationships between tumor size and the degree of
cachexia in the animals.
Pharmacological MAPK inhibition in cancer cachectic muscles
MAPK cascade leads to the activation of protein kinases and transcription factors through
phosphorylation, resulting in signal transduction, hence playing a key role in cell signaling. In
the current study, both total and phosphorylated protein content of the best characterized MAPK
subfamilies ERK1/2, p38, JNK, and ASK1 was explored in the muscles of all groups of mice.
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Total p38, but not p-p38, levels were increased in both respiratory and limb muscles of tumorbearing rodents. Importantly, MAPK inhibition elicited a significant improvement in body and
muscle weight gain, limb strength, proteolysis and protein ubiquitination levels, especially in
the gastrocnemius. These findings are line with a recent investigation in which muscle mass loss
was restored by inhibiting ERK activity in colon cancer mice(46).
MAPK inhibition was confirmed by the significant decrease in protein content of both
total and phosphorylated ERK1/2 in respiratory and limb cachectic muscles. Additionally,
MAPK inhibitor elicited a significant decrease in p38 protein content in diaphragm and limb
muscles of tumor-bearing rodents. Importantly, inhibition of MAPK pathway also induced other
interesting findings in the cachectic animals such as an increase in myogenin content together
with a reduction in myostatin levels, a substantial rise in contractile MyHC in both diaphragm
and gastrocnemius, an increase in creatine kinase protein in the limb muscle, and a decrease in
protein oxidation and autophagy levels in both muscles. Interestingly, from a structural
standpoint, cachectic mice treated with MAPK inhibitor also exhibited larger slow-twitch
muscle fibers in diaphragm and gastrocnemius along with a decrease in the percentage of
muscle structural abnormalities.
Taken together, these findings suggest that MAPK pathway controls muscle proteolysis
through the ubiquitin-proteasome and autophagy systems in both respiratory and limb muscles
of cancer cachectic mice. Other mechanisms involved in the regulation of muscle mass such as
myostatin and myogenin were also modulated by the effect of MAPK inhibitor in the cachectic
muscles. Finally, an interesting finding in the current study is the substantial reduction in tumor
size (60%) observed in tumor-bearing mice treated with the MAPK inhibitor. As
abovementioned, a significant reduction in tumor size might have also contributed to
attenuating the cachectic effects in the muscles of the treated mice. We do not believe, however,
this has been a relevant contributor in the investigation for the following reasons. The effects of
NF-κB inhibition on the size of the subcutaneous tumor were smaller compared to those elicited
by MAPK inhibitor and relatively similar to those elicited by bortezomib, while a greater
modulation of body weight and muscle mass and force development was achieved in response
to NF-κB than to MAPK or proteasome inhibitions. Future studies may be designed in order to
specifically address to what extent tumor size and modulation by pharmacological agents
contribute to muscle wasting and weakness in cancer-induced cachexia.
Finally, another relevant finding in the investigation was the increased protein oxidation
levels encountered in respiratory and limb cachectic muscles. Inhibition of proteasome, NF-κB,
and MAPK activities elicited a significant decline in protein carbonylation in diaphragm and
gastrocnemius muscles. Indeed, NF-κB and MAPK activation seem to play a relevant role in the
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transcriptional regulation of redox balance within muscles, while also mediating oxidative
stress-induced muscle atrophy (11, 47).
Conclusions
We conclude from the present experimental model of LC-induced cachexia that NF-κB and
MAPK are predominant signaling pathways. Pharmacological inhibition of NF-κB and MAPK,
but not the proteasome system, induced in the cachectic animals, a substantial restoration of
muscle mass and force through a decrease in muscle protein oxidation and catabolism,
myostatin, and autophagy, together with a greater content of contractile and functional proteins.
These findings offer new therapeutic strategies in cancer-induced cachexia.
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ACKNOWLEDGMENTS
This study has been supported by CIBERES; FIS 11/02029; 2009-SGR-393; SEPAR 2010;
FUCAP 2011; FUCAP 2012; and Marató TV3 (MTV3-07-1010) (Spain). Dr. Esther Barreiro
was a recipient of the ERS COPD Research Award 2008.
The authors are thankful to Dr. Xavier Mateu for his help and advice with the pharmacological
inhibitors, Dr. Juan Martin-Caballero for his assistance with part of the animal experiments, and
Mrs. Mònica Vilà-Ubach and Ariadna Estivill-Pérez for their technical support with part of the
molecular biology experiments.
DISCLOSURE
“The authors declare they have no competing interests as defined by Molecular Medicine, or
other interests that might be perceived to influence the results and discussion reported in the
paper”.
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FIGURE LEGENDS
Figure 1:
A) Mean values and standard deviation of tyrosine release (nmol/mg/2h) in diaphragm
(white bars) and gastrocnemius (black bars) muscles (expressed in Mean values and
standard deviation). Tyrosine release levels were significantly greater in diaphragm and
gastrocnemius of lung cancer (LC) cachectic mice than in the controls. Furthermore, a
significant decrease in tyrosine release levels was detected in both diaphragms and limb
muscles of LC cachectic animals treated with proteasome, NF-κB, and MAPK
inhibitors compared to LC cachectic rodents without any of these treatments. Statistical
significance is represented as follows: i) †††: p<0.001 and ††: p<0.01 levels in muscles
between LC cachectic and control mice and ii) **: p<0.01 and *: p<0.05 levels in
muscles between any group of LC cachectic mice treated with each of the inhibitors and
the LC cachectic animals without any pharmacological treatment. The dashed line
separates both types of comparisons between the groups.
B) Mean values and standard deviation of the ubiquitin conjugating enzyme E214κ in
diaphragm (white bars) and gastrocnemius (black bars) muscles, as measured by optical
densities in arbitrary units (OD, a.u.). E214κ protein levels were significantly greater in
gastrocnemius of lung cancer (LC) cachectic mice than in the controls. Furthermore, a
significant decrease in E214κ protein levels was detected only in limb muscles of LC
cachectic animals treated with proteasome, NF-κB, and MAPK inhibitors compared to
LC cachectic rodents without any of these treatments. Statistical significance is
represented as follows: i) n.s.: non significant and †: p<0.05 levels in muscles between
LC cachectic and control mice and ii) n.s.: non significant, ***: p<0.001, **: p<0.01
and *: p<0.05 levels in muscles between any group of LC cachectic mice treated with
each of the inhibitors and the LC cachectic animals without any pharmacological
treatment. The dashed line separates both types of comparisons between the groups.
C) Mean values and standard deviation of the ubiquitin protein ligases atrogin-1 (top panel)
and MURF-1 (bottom panel) in diaphragm (white bars) and gastrocnemius (black bars)
muscles as measured by optical densities in arbitrary units (OD, a.u.). Atrogin-1 and
MURF-1 protein levels did not significantly differ in any muscle of lung cancer (LC)
cachectic mice than in the controls. Furthermore, no significant differences in atrogin-1
or MURF-1 protein levels were detected in any of the muscles of the LC cachectic
animals treated with proteasome, NF-κB, and MAPK inhibitors compared to LC
cachectic rodents without any of these treatments. Statistical significance is represented
as follows: i) n.s.: non significant levels in muscles among groups. The dashed line
21
Results
separates both types of comparisons between the groups.
D) Mean values and standard deviation of 20S proteasome subunit C8 (top panel) and total
ubiquitinated proteins (bottom panel) in diaphragm (white bars) and gastrocnemius
(black bars) muscles as measured by optical densities in arbitrary units (OD, a.u.).
Subunit C8 protein levels did not significantly differ in any muscle of lung cancer (LC)
cachectic mice than in the controls. Furthermore, no significant differences in protein
levels of the subunit C8 were detected in any of the muscles of the LC cachectic
animals treated with either NF-κB or MAPK inhibitors, except for the proteasome
inhibitor, compared to LC cachectic rodents without any of these treatments. Levels of
total ubiquitinated proteins (bottom panel) were significantly greater in diaphragm and
gastrocnemius of lung cancer (LC) cachectic mice than in the controls. Furthermore, a
significant decrease in tyrosine release levels was detected in limb muscles, but not the
diaphragm, of LC cachectic animals treated with proteasome, NF-κB, and MAPK
inhibitors compared to LC cachectic rodents without any of these treatments. Statistical
significance is represented as follows: i) n.s.: non significant, †††: p<0.001, and †:
p<0.05 levels in muscles between LC cachectic and control mice and ii) n.s.: non
significant, ***: p<0.001, **: p<0.01, and *: p<0.05 levels in muscles between any
group of LC cachectic mice treated with each of the inhibitors and the LC cachectic
animals without any pharmacological treatment. The dashed line separates both types of
comparisons between the groups.
Figure 2:
A) Mean values and standard deviation of NF-κB p50 (top panel) and NF-κB p-p50
(bottom panel) in diaphragm (white bars) and gastrocnemius (black bars) muscles as
measured by optical densities in arbitrary units (OD, a.u.). NF-κB p50 protein levels
were significantly increased in both diaphragm and gastrocnemius muscles of lung
cancer (LC) cachectic mice than in the controls. Furthermore, a significant decrease in
NF-κB p50 levels was detected in both diaphragms and limb muscles of LC cachectic
animals
treated
with
proteasome,
NF-κB, and MAPK inhibitors compared to LC cachectic rodents without any of these
treatments (top panel). NF-κB p-p50 protein levels were significantly increased in both
diaphragm and gastrocnemius muscles of lung cancer (LC) cachectic mice than in the
controls. Furthermore, a significant decrease in NF-κB p-p50 levels was only detected
in both diaphragms and limb muscles of LC cachectic animals treated with NF-κB
inhibitor compared to LC cachectic rodents without any of these treatments (bottom
panel). Statistical significance is represented as follows: i) †: p<0.05 levels in muscles
22
Results
between LC cachectic and control mice and ii) n.s.: non significant, ***: p<0.001, **:
p<0.01, and *: p<0.05 levels in muscles between any group of LC cachectic mice
treated with each of the inhibitors and the LC cachectic animals without any
pharmacological treatment. The dashed line separates both types of comparisons
between the groups.
B) Mean values and standard deviation of NF-κB p65 (top panel) and NF-κB p-p65
(bottom panel) in diaphragm (white bars) and gastrocnemius (black bars) muscles as
measured by optical densities in arbitrary units (OD, a.u.). NF-κB p65 protein levels
were significantly increased in both diaphragm and gastrocnemius muscles of lung
cancer (LC) cachectic mice than in the controls. Furthermore, a significant decrease in
NF-κB p65 levels was detected in both diaphragms and limb muscles of LC cachectic
animals treated with proteasome and NF-κB inhibitors compared to LC cachectic
rodents without any of these treatments (top panel). NF-κB p-p65 protein levels were
not significantly different in diaphragm or gastrocnemius muscles of lung cancer (LC)
cachectic mice than in the controls. Furthermore, a significant decrease in NF-κB p-p65
levels was only detected in both diaphragms and limb muscles of LC cachectic animals
treated with NF-κB inhibitor compared to LC cachectic rodents without any of these
treatments (bottom panel). Statistical significance is represented as follows: i) †: p<0.05
levels and n.s.: non significant in muscles between LC cachectic and control mice and
ii) n.s.: non significant, ***: p<0.001, and *: p<0.05 levels in muscles between any
group of LC cachectic mice treated with each of the inhibitors and the LC cachectic
animals without any pharmacological treatment. The dashed line separates both types of
comparisons between the groups.
C) Mean values and standard deviation IκBα (top panel) and p-IκBα (bottom panel) in
diaphragm (white bars) and gastrocnemius (black bars) muscles as measured by optical
densities in arbitrary units (OD, a.u.). IκBα protein levels were significantly decreased
in both diaphragm and gastrocnemius muscles of lung cancer (LC) cachectic mice than
in the controls. Furthermore, a significant increase in IκBα levels was detected in both
diaphragms and limb muscles of LC cachectic animals treated with proteasome, NF-κB,
and MAPK inhibitors compared to LC cachectic rodents without any of these treatments
(top panel). Protein levels of p-IκBα were significantly decreased in both diaphragm
and gastrocnemius muscles of lung cancer (LC) cachectic mice than in the controls.
Furthermore, a significant increase in p-IκBα levels was detected in both diaphragms
and limb muscles of LC cachectic animals treated with proteasome, NF-κB, and MAPK
(only gastrocnemius) inhibitors compared to LC cachectic rodents without any of these
23
Results
treatments (bottom panel). Statistical significance is represented as follows: i) ††:
p<0.01 and †: p<0.05 levels in muscles between LC cachectic and control mice and ii)
***: p<0.001, **: p<0.01, *: p<0.05, and n.s.: non significant, levels in muscles
between any group of LC cachectic mice treated with each of the inhibitors and the LC
cachectic animals without any pharmacological treatment. The dashed line separates
both types of comparisons between the groups.
D) Mean values and standard deviation of NF-κB transcriptional activity expressed as the
ratio to control gastrocnemius muscles as measured by optical densities in arbitrary
units (OD, a.u.). NF-κB transcription levels were significantly increased in the limb
muscle of lung cancer (LC) cachectic mice than in the controls. Furthermore, a
significant decrease in NF-κB transcription was detected in muscles of LC cachectic
animals treated with proteasome and NF-κB inhibitors, but not MAPK, compared to LC
cachectic rodents without any of these treatments. Statistical significance is represented
as follows: i) ††: p<0.01 levels in muscles between LC cachectic and control mice and
ii) *: p<0.05 and n.s.: non significant levels in muscles between any group of LC
cachectic mice treated with each of the inhibitors and the LC cachectic animals without
any pharmacological treatment. The dashed line separates both types of comparisons
between the groups.
Figure 3:
A) Mean values and standard deviation of myostatin (top panel) and myogenin (bottom
panel) in diaphragm (white bars) and gastrocnemius (black bars) muscles. Myostatin
protein levels were significantly increased in both diaphragm and gastrocnemius
muscles of lung cancer (LC) cachectic mice than in the controls. Furthermore, a
significant decrease in myostatin levels was detected in both diaphragms and limb
muscles of LC cachectic animals treated with proteasome, NF-κB, and MAPK
inhibitors compared to LC cachectic rodents without any of these treatments (top
panel). Protein levels of myogenin were significantly decreased in both diaphragm and
gastrocnemius muscles of lung cancer (LC) cachectic mice than in the controls.
Furthermore, a significant increase in myogenin levels was detected in both diaphragms
and limb muscles of LC cachectic animals treated with NF-κB and MAPK inhibitors,
but not bortezomib, compared to LC cachectic rodents without any of these treatments
(bottom panel). Statistical significance is represented as follows: i) ††: p<0.01 and †:
p<0.05 levels in muscles between LC cachectic and control mice and ii) ***: p<0.001,
**: p<0.01, *: p<0.05, and n.s.: non significant levels in muscles between any group of
LC cachectic mice treated with each of the inhibitors and the LC cachectic animals
24
Results
without any pharmacological treatment. The dashed line separates both types of
comparisons between the groups.
B) Mean values and standard deviation of myosin heavy chain (MyHC) (top panel) and
actin (bottom panel) levels in diaphragm (white bars) and gastrocnemius (black bars)
muscles as measured by optical densities in arbitrary units (OD, a.u.). MyHC protein
levels were significantly decreased in both diaphragm and gastrocnemius muscles of
lung cancer (LC) cachectic mice than in the controls. Furthermore, a significant
increase in MyHC levels was detected in both diaphragms and limb muscles of LC
cachectic animals treated with NF-κB and MAPK inhibitors, but not bortezomib,
compared to LC cachectic rodents without any of these treatments (top panel). Protein
levels of actin did not significantly differ among the study groups (bottom panel).
Statistical significance is represented as follows: i) ††: p<0.01 and †: p<0.05 levels in
muscles between LC cachectic and control mice and ii) ***: p<0.001, *: p<0.05, and
n.s.: non significant levels in muscles between any group of LC cachectic mice treated
with each of the inhibitors and the LC cachectic animals without any pharmacological
treatment. The dashed line separates both types of comparisons between the groups.
C) Mean values and standard deviation of creatine kinase (top panel) and carbonic
anhydrase-3 (bottom panel) in diaphragm (white bars) and gastrocnemius (black bars)
muscles as measured by optical densities in arbitrary units (OD, a.u.). Creatine kinase
protein levels were significantly decreased in gastrocnemius of lung cancer (LC)
cachectic mice than in the controls. Furthermore, a significant increase in creatine
kinase protein levels was detected in limb muscles of LC cachectic animals treated with
proteasome, NF-κB, and MAPK inhibitors compared to LC cachectic rodents without
any of these treatments (top panel). Protein levels of carbonic anhydrase-3 did not
significantly differ among the study groups (bottom panel). Statistical significance is
represented as follows: i) n.s.: non significant and †††: p<0.001 levels in muscles
between LC cachectic and control mice and ii) ***: p<0.001, *: p<0.05, and n.s.: non
significant levels in muscles between any group of LC cachectic mice treated with each
of the inhibitors and the LC cachectic animals without any pharmacological treatment.
The dashed line separates both types of comparisons between the groups.
Figure 4:
A) Mean values and standard deviation of the autophagy protein p62 in diaphragm (white
bars) and gastrocnemius (black bars) muscles as measured by optical densities in
arbitrary units (OD, a.u.). Protein levels of p62 did not significantly differ among the
study groups. Statistical significance is represented as follows: i) n.s.: non significant
25
Results
levels in muscles between LC cachectic and control mice and ii) n.s.: non significant
levels in muscles between any group of LC cachectic mice treated with each of the
inhibitors and the LC cachectic animals without any pharmacological treatment. The
dashed line separates both types of comparisons between the groups.
B) Mean values and standard deviation of the autophagy protein beclin-1 in diaphragm
(white bars) and gastrocnemius (black bars) muscles as measured by optical densities in
arbitrary units (OD, a.u.). Protein levels of beclin-1 did not significantly differ among
the study groups. Statistical significance is represented as follows: i) n.s.: non
significant levels in muscles between LC cachectic and control mice and ii) n.s.: non
significant levels in muscles between any group of LC cachectic mice treated with each
of the inhibitors and the LC cachectic animals without any pharmacological treatment.
The dashed line separates both types of comparisons between the groups.
C) Mean values and standard deviation of the autophagy protein LC3-II/LC3-I in
diaphragm (white bars) and gastrocnemius (black bars) muscles as measured by optical
densities in arbitrary units (OD, a.u.). LC3-II/LC3-I protein levels were significantly
increased in both diaphragm and gastrocnemius muscles of lung cancer (LC) cachectic
mice than in the controls. Furthermore, a significant decrease in LC3-II/LC3-I levels
was detected in both diaphragms and limb muscles of LC cachectic animals treated with
proteasome, NF-κB, and MAPK inhibitors compared to LC cachectic rodents without
any of these treatments (top panel). Statistical significance is represented as follows: i)
†: p<0.05 levels in muscles between LC cachectic and control mice and ii) **: p<0.01,
*: p<0.05, and n.s.: non significant levels in muscles between any group of LC
cachectic mice treated with each of the inhibitors and the LC cachectic animals without
any pharmacological treatment. The dashed line separates both types of comparisons
between the groups.
26
Results
Table 1. Physiological characteristics in all groups at the end of the study period.
Lung cancer cachexia
Control
LC
cachexia
Body weight gain (%)
+8.97 (2.72)
Diaphragm weight (g)
Gastrocnemius weight (g)
Limb strength gain (%)
Sc. Tumor weight (g)
p
Proteasome
inhibitor
p
NFκβ
inhibitor
p
MAPK
inhibitor
p
-7.69 (12.89)
†††
-11.72 (8.14)
n.s.
+0.42 (7.26)
**
-0.30 (5.59)
**
0.089 (0.009)
0.067 (0.012)
†††
0.071 (0.01)
n.s.
0.08 (0.007)
***
0.076 (0.009)
*
0.114 (0.008)
0.087 (0.016)
†††
0.086 (0.009)
n.s.
0.101 (0.01)
***
0.094 (0.009)
*
+10.27 (19.59 )
-11.7 (15.37)
†††
-12.2 (24.04)
n.s.
+3.04 (12.9)
**
+0.49 (9.27)
***
1.42 (0.59)
-
1.17 (0.56)
*
1.03 (0.31)
**
0.57 (0.32)
***
-
Variables are presented as mean (SD).
Definition of abbreviations: LC, Lung Cancer; NF-κB, Nuclear Factor-κB; MAPK, Mitogenactivated protein kinases; Sc., subcutaneous.
Statistical significance: †††, p≤0.001 between LC cachectic and control mice; n.s., nonsignificant,* p≤0.05, **,
p≤0.01 and ***, p≤0.001 between any of the treated mouse groups with cachexia and LC
cachexia only animals.
27
27
Results
Table 2. Molecular markers in respiratory and limb muscles of all mice from the study
groups.
Muscle
Control
LC cachexia
p
Proteasome
inhibitor
p
Lung cancer cachexia
NFκβ
p
inhibitor
MAPK
inhibitor
p
Muscle proteolysis & signaling
Calpain µ, OD
(a.u.)
Diaphragm
0.57 (0.28)
1.19 (0.49)
†
1.19 (0.49)
n.s.
0.64 (0.34)
*
0.59 (0.28)
*
Gastrocnemius
0.55 (0.18)
0.53 (0.2)
n.s.
0.77 (0.25)
n.s.
0.85 (0.14)
n.s.
0.5 (0.25)
n.s.
FOXO1, OD
(a.u.)
Diaphragm
1.38 (0.7)
1.27 (0.3)
n.s.
0.74 (0.5)
*
1.33 (0.16)
n.s.
1.61 (0.23)
n.s.
Gastrocnemius
1.36 (0.4)
1.47 (0.65)
n.s.
1.44 (0.44)
n.s.
1.44 (0.29)
n.s.
1.27 (0.27)
n.s.
pFOXO1, OD
(a.u.)
Diaphragm
0.4 (0.024)
0.31 (0.05)
n.s.
0.34 (0.04)
n.s.
0.31 (0.028)
n.s.
0.31 (0.03)
n.s.
Gastrocnemius
0.27 (0.24)
0.88 (0.21)
†††
0.68 (0.12)
n.s.
0.94 (0.15)
n.s.
0.59 (0.4)
n.s.
ASK1, OD
(a.u.)
Diaphragm
1.07 (0.08)
1.05 (0.07)
n.s.
1.07 (0.06)
n.s.
1.05 (0.07)
n.s.
1.09 (0.07)
n.s.
Gastrocnemius
0.2 (0.1)
0.8 (0.6)
n.s.
1.1 (0.3)
n.s.
1.3 (0.2)
*
0.9 (0.2)
n.s.
Diaphragm
1.18 (0.5)
1.44 (0.29)
n.s.
1.55 (0.37)
n.s.
1.64 (0.35)
n.s.
0.60 (0.11)
***
Gastrocnemius
3.2 (0.99)
3.49 (0.67)
n.s.
4.49 (1.04)
n.s.
5.72 (1.27)
**
0.98
***
pERK1/2, OD
(a.u.)
Diaphragm
0.18 (0.09)
0.19 (0.04)
n.s.
0.18 (0.09)
n.s.
0.25 (0.15)
n.s.
0.08 (0.02)
***
Gastrocnemius
0.51 (0.14)
0.64 (0.07)
n.s.
0.49 (0.14)
n.s.
0.79 (0.26)
n.s.
0.50 (0.11)
*
JNK-1, OD
(a.u.)
Diaphragm
3.5 (0.78)
3.75 (1.46)
n.s.
3.45 (0.72)
n.s.
3.73 (0.8)
n.s.
4.3 (0.95)
n.s.
Gastrocnemius
1.63 (0.49)
1.5 (0.5)
n.s.
1.25 (0.11)
**
1.68 (0.84)
n.s.
1.73 (0.67)
n.s.
Diaphragm
0.41 (0.2)
0.68 (0.12)
†
0.46 (0.16)
n.s.
0.43 (0.102)
n.s.
0.33 (0.04)
***
Gastrocnemius
1.9 (1.6)
4.7 (1.3)
††
4.5 (0.9)
n.s.
4.4 (0.9)
n.s.
3.1 (0.5)
*
Diaphragm
0.69 (0.4)
0.67 (0.4)
n.s.
0.96 (0.59)
n.s.
0.37 (0.07)
n.s.
0.56 (0.19)
n.s.
Gastrocnemius
0.27 (0.12)
0.36 (0.07)
n.s.
0.46 (0.1)
n.s.
0.32 (0.07)
n.s.
0.35 (0.03)
n.s.
ERK1/2 (a.u.)
p38, OD (a.u.)
p-p38, OD
(a.u.)
Muscle redox balance & inflammation
IFNγ , OD
(pg/mL)
Diaphragm
68.7 (35.1)
113.6 (22.9)
†
79.8 (70.6)
n.s.
117.3 (55.9)
n.s.
48.8 (16.3)
***
Gastrocnemius
16.3 (8.4)
22.2 (17.2)
n.s.
4.9 (3.4)
*
38.7 (10.4)
n.s.
27.1 (16.3)
n.s.
TNF α , OD
(pg/mL)
Diaphragm
36.4 (23.4)
53.2 (23.8)
n.s.
36.1 (31)
n.s.
63.7 (33.9)
n.s.
21.8 (11.3)
*
Gastrocnemius
12.6 (6.8)
17.5 (11.6)
n.s.
4.9 (1.9)
*.
22.8 (5.4)
n.s.
18.9 (9.9)
n.s.
IL-6, OD
(pg/mL)
Diaphragm
1444.5 (623.1)
1806 (554.5)
n.s.
1644 (1089.7)
n.s.
1962 (709.5)
n.s.
983 (314)
**
Gastrocnemius
388.7 (211.5)
404.9 (256.3)
n.s.
144.4 (60.2)
*
539.3 (139.9)
n.s.
456 (318.9)
n.s.
IL-1β, OD
(pg/mL)
Diaphragm
57.4 (30.3)
80.9 (34.6)
n.s.
54 (32.1)
n.s.
80.1 (34.6)
n.s.
49.9 (37.9)
n.s.
Gastrocnemius
9.1 (4.9)
12.9 (9.9)
n.s.
3.1 (0.54)
*
19.5 (4.2)
n.s.
19.9 (8.7)
n.s.
Protein
carbonylation,
OD (a.u.)
HNE-protein
adducts, OD
(a.u.)
Protein
nitration, OD
(a.u.)
Diaphragm
3.8 (1.8)
7.05 (2.2)
†
4.2 (1.2)
*
4.7 (1.2)
*
3.9 (1.3)
**
Gastrocnemius
2.9 (0.6)
3.8 (0.29)
†
2.6 (0.9)
*
2.05 (0.16)
***
2.05 (0.16)
***
Diaphragm
4.8 (1.8)
4.05 (0.43)
n.s
5.2 (1.6)
n.s
3.62 (0.5)
n.s
0.67 (0.8)
n.s
Gastrocnemius
2.48 (1.76)
5.25 (1.28)
†††
5.6 (1.4)
n.s.
4.7 (0.6)
n.s.
4.9 (1.6)
n.s.
Diaphragm
1.56 (0.76)
1.4 (0.67)
n.s.
1.29 (0.7)
n.s.
2.03 (0.9)
n.s.
1.45 (0.5)
n.s.
Gastrocnemius
5.71 (1.24)
5.5 (0.43)
n.s.
5.48 (1.18)
n.s.
5.1 (1.4)
n.s.
5.7 (0.76)
n.s.
MnSOD, OD
(a.u.)
Diaphragm
5.33 (1.24)
3.35 (1.5)
†
3.65 (0.92)
n.s.
3.13 (0.92)
n.s.
3.78 (2.52)
n.s.
Gastrocnemius
2.36 (0.49)
1.29 (0.66)
†
2.52 (0.21)
**
2.7 (0.55)
**
2.6 (0.94)
*
CuZnSOD, OD
(a.u.)
Diaphragm
0.74 (0.55)
0.76 (0.13)
n.s.
1.18 (0.7)
n.s.
0.86 (0.17)
n.s.
1.09 (0.65)
n.s.
Gastrocnemius
0.43 (0.09)
0.33 (0.06)
n.s.
0.403 (0.07)
n.s.
0.28 (0.04)
n.s.
0.41 (0.15)
n.s.
Catalase, OD
(a.u.)
Diaphragm
0.99 (0.5)
1.74 (1.43)
n.s.
1.29 (0.52)
n.s.
1.45 (0.65)
n.s.
1.19 (1.44)
n.s.
Gastrocnemius
0.99 (0.24)
0.74 (0.07)
n.s.
0.9 (0.4)
n.s.
0.92 (0.17)
n.s.
0.68 (0.18)
n.s.
28
28
Results
Variables are presented as mean (SD).
Definition of abbreviations: LC, Lung Cancer; NF-κB, Nuclear Factor-κB; MAPK, Mitogenactivated protein kinases; OD, optical densities; a.u.; arbitrary units; FOXO-1, Forkhead box
protein O1; p-FOXO1, phosphorylated FoxO-1; ASK1, Apoptosis signal-regulating kinase-1;
ERK1/2, Mitogen-activated protein kinase-1/2; p-ERK2, phosphorylated-ERK-1/2; JNK, c-Jun
N-terminal kinase; p38, p38 mitogen-activated protein kinase; p-p38, phosphorylated-p38; pg,
picograms; mL, milliliter; IFN, interferon; TNF, tumor necrosis factor; IL, interleukin; HNE, 4Hydroxynonenal; NT, nitrotyrosine; Mn, manganese; CuZn, copper zinc.
Statistical significance: n.s., non-significant, †, p≤0.05, ††≤0.01 and †††, p≤0.001 between LC
cachectic and control animals; n.s., non-significant; * p≤0.05, **, p≤0.01 and ***, p≤0.001
between any of the treated mouse groups with cachexia and LC cachexia only animals.
29
Results
Table 3. Structure and cellular markers in respiratory and limb muscles of all animals.
Proteasome
inhibitor
Lung cancer cachexia
NFκβ
p
p
inhibitor
MAPK
inhibitor
p
n.s.
6.64 (1.89)
n.s.
n.s.
14.5 (2.97)
n.s.
91.98 (1.02)
n.s.
93.4 (1.89)
n.s.
n.s.
85.7 (2.35)
n.s.
85.5 (2.97)
n.s.
320.8 (66)
**
237 (83.71)
n.s.
280 (28.9)
**
†††
824.6 (88.7)
**
754.7 (98.8)
n.s.
792.5 (102)
*
297.1 (68)
†
319.9 (66.5)
n.s.
297.1 (68)
n.s.
291 (35.3)
n.s.
723.5 (143.9)
†
764 (93.8)
n.s.
742.9 (121.5)
n.s.
793 (91.3)
n.s.
Muscle
Control
LC cachexia
Type I fibers
(%)
Diaphragm
9.04 (2.05)
8.22 (2.61)
n.s.
8.59 (1.37)
n.s.
8.12 (1.02)
Gastrocnemius
13.4 (2.74)
13.17 (2.61)
n.s.
11.94 (2.97)
n.s.
14.3 (2.35)
Type II fibers
(%)
Diaphragm
90.96 (2.05)
91.78 (2.61)
n.s.
91.41 (1.37)
n.s.
Gastrocnemius
86.6 (2.74)
86.83 (2.98)
n.s.
88.06 (2.97)
Type I fibers
2
area (µm )
Diaphragm
326.1 (66.59)
233.4 (33.54)
††
Gastrocnemius
951.7 (109.7)
658.8 (122.6)
Type II fibers
2
area (µm )
Diaphragm
382.5 (82.7)
Gastrocnemius
919.2 (135.1)
p
Fiber type composition
Muscle abnormalities
Abnormal
fraction (%)
Diaphragm
0.08 (0.01)
0.14 (0.04)
†††
0.15 (0.03)
n.s.
0.12 (0.03)
n.s.
0.13 (0.03)
n.s.
Gastrocnemius
0.04 (0.01)
0.08 (0.02)
†††
0.08 (0.02)
n.s.
0.07 (0.01)
n.s.
0.05 (0.01)
***
Inflamatory
cells (%)
Diaphragm
0.037 (0.006)
0.065 (0.015)
†††
0.052 (0.01)
*
0.047 (0.02)
*
0.05 (0.01)
**
Gastrocnemius
0.02 (0.009)
0.05 (0.02)
†††
0.05 (0.01)
n.s.
0.035 (0.006)
**
0.03 (0.01)
***
Internal
nuclei (%)
Diaphragm
0.04 (0.01)
0.07 (0.03)
†
0.06 (0.03)
n.s.
0.06 (0.02)
n.s.
0.08 (0.01)
n.s.
Gastrocnemius
0.016 (0.07)
0.024 (0.008)
†
0.03 (0.009)
*
0.03 (0.01)
*
0.02 (0.01)
n.s.
TUNELstained
nuclei (%)
Diaphragm
46.08 (8.55)
67.85 (10.62)
†††
60.9 (17.93)
n.s.
64.1 (11.86)
n.s.
60.2 (6.57)
*
Gastrocnemius
49.71 (12.93)
69.88 (12.62)
†††
70.77 (9.74)
n.s.
64.09 (10.4)
n.s.
70.7 (9.47)
n.s.
Variables are presented as mean (SD).
Definition of abbreviations: LC, Lung Cancer; NF-κB, Nuclear Factor-κB; MAPK,
Mitogenactivated protein kinases; µm; micrometer.
Statistical significance: n.s., non-significant, †, p≤0.05, ††≤0.01 and †††, p≤0.001 between LC
cachectic and control mice; n.s., non-significant, * p≤0.05, **, p≤0.01 and ***, p≤0.001
between any of the treated mouse groups with cachexia and LC cachexia only animals.
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Online supplementary material
PHARMACOLOGICAL STRATEGIES IN LUNG CANCER-INDUCED CACHEXIA:
EFFECTS ON MUSCLE PROTEOLYSIS, AUTOPHAGY, STRUCTURE, AND
WEAKNESS
Clara Fermoselle, Alejandro J. Urtreger, Francisco Sánchez, Alba Chacon-Cabrera, Mercè
Mateu-Jimenez, Marco Sandri, Miriam J. Diament, Elisa D. Bal de Kier Joffé, Esther Barreiro
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METHODS
Animal experiments
Tumor. LP07 is a cell line derived from the transplantable P07 lung tumor that appeared
spontaneously in the lung of a BALB/c mouse (1). The LP07 cell line was obtained in vitro,
after successive passages of a P07 primary culture, by some of us (2). LP07 cell line shares
identical characteristics regarding lung tumor incidence and histology, and cachexia with its
parental P07 tumor (1-3). It has also been consistently demonstrated that one month after tumor
transplantation, all animals developed lung metastasis, spleen enlargement, and cachexia
without affecting any other organs (1-3).
Mice. Female BALB/c mice, 2 months old (weight ~20g), were obtained from Harlan Interfauna
Ibérica SL (Barcelona, Spain). Mice were housed under a 12:12 h light-dark cycle with ad
limitum access to water and food.
Experimental design and Ethics. In all experimental groups (except for control rodents), LP07
viable cells (4·105) resuspended in 0.2 mL minimal essential media (MEM) were
subcutaneously inoculated in the left flank of female BALB/c mice on day 1. All groups were
studied for a period of one month. Animals were randomly assigned to the following groups
(N=10 mice in all groups): 1) control, inoculation of 0.2 mL MEM in the left flank; 2) LC
cachexia group, inoculation of LP07 cells; 3) LC cachectic mice received concomitant treatment
with the proteasome inhibitor Bortezomib (Velcade, Millenium Pharmaceuticals, Cambridge,
MA), 0.15 mg/Kg, 0.1 mL/6 days, intravenous injection into the tail vein (LC cachecticbortezomib group) (2, 4); 4) LC cachectic mice received concomitant treatment with the NF-κB
inhibitor sulfasalazine (Pfizer, Madrid, Spain), 200 mg/Kg, 0.3 mL/48h, intraperitoneal
injection (LC cachectic-NF-κB inhibitor group) (5); and 5) LC cachectic mice received
concomitant treatment with the MAPK inhibitor U0126 (a highly selective inhibitor of ERK1
and ERK2 proteins, Selleck chemicals, Houston, TX), 30 mg/Kg, 0.1 mL/48h, intraperitoneal
injection (LC cachectic-MAPK inhibitor group) (6). All pharmacological therapies were
administered on day 15 post-inoculation of the LP07 cells up until the end of the study period.
In order to assess the level of NF-κB transcription and ex-vivo protein degradation using the
luciferase reporter and the tyrosine release assays, respectively (see below), a second batch of
mice experiments was also conducted on identical experimental groups (N=10/group). One
hind-limb and half of the diaphragm were used for each type of these experiments.
All animal experiments were conducted in the animal facilities at Parc de Recerca Biomèdica
de Barcelona (PRBB, Spain). This controlled study was designed in accordance with the ethical
standards on animal experimentation (EU 609/86 CEE, Real Decreto 1201/05 BOE 252, Spain)
at PRBB and the Helsinki convention for the use and care of animals. Ethical approval was
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obtained by the Animal Research Committee at PRBB and Catalan Government (Animal
welfare department).
In vivo measurements in the mice
Body weight and food intake were determined every day during the entire duration of the study.
Limb strength was determined at day 0 and 30 using a strength grip meter (Bioseb, Chaville,
France) following previously published methodologies (7). In the LC cachexia group of mice,
tumor progression was determined using positron emission tomography (PET) at days 13 and
20.
Sacrifice and sample collection
Mice from all the experimental groups were always sacrificed on day 30 post-inoculation of
LP07 cells or MEM (control animals). Each mouse was inoculated intraperitoneally with 0.1
mL sodium pentobarbital (60 mg/Kg). In all cases, the pedal and blink reflexes were evaluated
in order to verify total anesthetic depth. At this time, the following samples were obtained from
all animals: diaphragm and gastrocnemius muscles and the subcutaneous tumor. The weight of
both muscles and tumor were determined in each animal using a high-precision scale. Frozen
tissues were used for immunoblotting and enzyme-linked immunosorbent assay (ELISA)
techniques, while paraffin-embedded tissues were used for the assessment of muscle structure
abnormalities and fiber type morphometry.
Muscle biology analyses
All muscle biology analyses were conducted blind in the same laboratory by the same
investigators, at Hospital del Mar-IMIM (Barcelona).
Immunoblotting of 1D electrophoresis. Protein levels of the different molecular markers
analyzed in the study were explored by means of immunoblotting procedures as previously
described (8-12). Briefly, frozen muscle samples from the diaphragm and gastrocnemius
muscles of all mouse experimental groups were homogenized in a buffer containing HEPES 50
mM, NaCl 150 mM, NaF 100 mM, Na pyrophosphate 10 mM, EDTA 5 mM, Triton-X 0.5%,
leupeptin 2 µg/ml, PMSF 100 µg/ml, aprotinin 2 µg/ml and pepstatin A 10 µg/ml. Myofibrillar
proteins were also isolated in order to identify levels of actin and myosin heavy chain (MyHC)
(11, 12). The entire procedures were always conducted at 4ºC. Protein levels in crude
homogenates were spectrophotometrically determined with the Bradford method (13) using
triplicates in each case and bovine serum albumin (BSA) as the standard (Bio-Rad protein
reagent, Bio-Rad Inc., Hercules, CA, USA). The final protein concentration in each sample was
calculated from at least two Bradford measurements that were almost identical. Equal amounts
of total protein (ranging from 5 to 20 micrograms, depending on the antigen and antibody) from
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crude muscle homogenates were always loaded onto the gels, as well as identical sample
volumes/lanes. For the purpose of comparisons among the different groups of experimental and
control rodents, muscle sample specimens were always run together and kept in the same order.
Two independent sets of immunoblots were conducted in which diaphragm and gastrocnemius
muscle specimens were run separately.
Four fresh 10-well mini-gels were always loaded for each of the antigens. Experimental
and control mice were unevenly distributed across gels in order to attenuate eventual loading
problems in a specific area of the gels which might lead to misinterpretation of the
measurements. Fresh gels were specifically loaded for each of the antigens in muscle specimens
of all mice. Antigens were not identified from stripped membranes in any case.
Proteins were then separated by electrophoresis, transferred to polyvinylidene difluoride
(PVDF) membranes, blocked with non-fat milk and incubated overnight with selective primary
antibodies. Protein content of markers of proteolysis, signaling pathways of muscle atrophy,
redox balance, and different muscle proteins were identified using specific primary antibodies
(11): Actin (anti-alpha-sarcomeric actin antibody, clone 5C5, Sigma-Aldrich, St. Louis, MO,
USA), myosin heavy chain (anti-MyHC antibody, clone A4.1025, Upstate-Millipore, Temecula,
CA, USA), carbonic anhydrase-3 (anti-carbonic anhydrase-3 antibody, Santa Cruz
Biotechnology, Santa Cruz, CA, USA), creatine kinase (anti-creatine kinase antibody, Santa
Cruz), total ubiquitinated proteins (anti-ubiquitinated proteins antibody, Boston Biochem,
Cambridge, MA, USA), 20S proteasome subunit C8 (anti-C8 antibody, Biomol, Plymouth
Meeting, PA, USA) ubiquitin conjugating enzyme E214k (anti-E214K antibody, Boston Biochem),
ubiquitin-ligase atrogin-1 (anti-atrogin-1 antibody, Santa Cruz), ubiquitin-ligase muscle ring
finger (MURF)-1 (anti-MURF 1 antibody, Everest Biotech, Oxfordshire, UK), calpain (anticalpain 1 large subunit µ-type antibody, Cell Signaling, Boston, MA, USA), nuclear factor
(NF)-κB p50 (anti-p50 antibody, Santa Cruz), phospho-NF-κB p50 (anti-p-p50 antibody, Santa
Cruz), NF-κB p65 (anti-p65 antibody, Santa Cruz), phospho-NF-κB p65 (anti-p-p65 antibody,
Santa Cruz), inhibitor of κB-α (Iκβ-α) (anti-IκB-α antibody, Santa Cruz), phospho- IκB-α (antip-IκB-α antibody, Santa Cruz), myogenin (anti-myogenin antibody, Santa Cruz), myostatin
(anti-myostatin antibody, Bethyl, Montgomery, TX, USA), 4-hydroxy-2-nonenal-protein
adducts (anti-hydroxynonenal antibody, MyBiosource, San Diego, CA, USA), total protein
carbonylation (anti-2,4- DNP moiety antibody, Oxyblot kit, Chemicon International Inc.,
Temecula, CA, USA), apoptosis signal-regulating kinase 1 (ASK1) (anti-ASK1 antibody, Santa
Cruz), MAPK extracellular kinase (ERK1/2) (anti-ERK1/2 antibody, Santa Cruz), phosphoERK1/2 (anti-p-ERK1/2 antibody, Santa Cruz), MAPK c-Jun terminal (JNK) (anti-JNK
antibody, Santa Cruz), MAPK p38 (anti-p38 antibody, Santa Cruz), phospho-MAPK p38 (anti-
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p-p38 antibody, Santa Cruz), transcription factor fork-head box O (FoxO)-1 (anti-FoxO-1
antibody, Santa Cruz), phospho-FoxO (anti-p-FoxO antibody, Santa Cruz), total protein
nitration
(anti-3-nitrotyrosine antibody, Invitrogen, Eugene, Oregon, USA), catalase (anti-
catalase antibody, Calbiochem, Darmstadt, Germany), Mn-superoxide dismutase (SOD) (antiMn-SOD antibody, Santa Cruz), CuZn-SOD (CuZn-SOD antibody, Santa Cruz), p-62 (antip62/SQSTM1 antibody, Sigma-Aldrich), Beclin-1 (anti-Beclin 1 antibody, Santa Cruz), LC3B
(anti-LC3B antibody, Cell Signaling). Antigens from all samples were detected with horseradish
peroxidase (HRP)-conjugated secondary antibodies and a chemiluminescence kit. For each of
the antigens, samples from the different groups were always detected in the same picture under
identical exposure times. The specificity of the different antibodies was confirmed by omission
of the primary antibody, and incubation of the membranes only with secondary antibodies.
PVDF membranes were scanned with the Molecular Imager Chemidoc XRS System (Bio–
Rad Laboratories, Hercules, CA, USA) using the software Quantity One version 4.6.5 (Bio–Rad
Laboratories). Optical densities of specific proteins were quantified using the software Image
Lab version 2.0.1 (Bio-Rad Laboratories). Values of total reactive carbonyl groups, HNEprotein adducts, protein tyrosine nitration, and total protein ubiquitination in a given sample
were calculated by addition of optical densities (arbitrary units) of individual protein bands in
each case. Final optical densities obtained in each specific group of subjects corresponded to the
mean values of the different samples (lanes) of each of the antigens studied. In order to validate
equal protein loading among various lanes, SDS-PAGE gels were stained with Coomassie Blue
.
Protein catabolism. Protein degradation was explored on the basis of the rate of production of
free tyrosine from tissue proteins as previously described (14, 15). As muscles cannot
synthesize or degrade this amino acid, its accumulation reflects the net degradation of proteins.
All incubations were performed at 35ºC in a 95% air-5% CO2 mixture. Briefly, whole excised
muscles were placed in individual tissue chambers containing 3 mL of TKH1 buffer (127.8 mM
NaCl, 4.7 mM KCl, 2.4 mM MgSO4·7H2O, 1.2 mM KH2PO4, 2.5 mM CaCl2·2H2O, 20 mM
Hepes, 170 µM L-leucine, 100 µM L-isoleucione, 200 µM L-valine and 0.5 M glucose) and
were preincubated for 15 minutes. After the preincubation, the buffer was extracted and 3 mL of
TKH2 (TKH1 supplemented with 500 mM cicloheximide) buffer were added to the same
chamber, and samples were then incubated for 15 minutes. Thereafter, the buffer was extracted
and replaced with 4mL of fresh TKH2, and samples were then incubated for 2 hours.
Immediately afterwards, the buffer was recovered and stored at -20ºC until the tyrosine release
measurements were performed as described as follows: 1.4 mL of sample, blank (127.8 mM
NaCl, 4.7 mM KCl, 2.4 mM MgSO4·7H2O, 1.2 mM KH2PO4, 2.5 mM CaCl2·2H2O and 20 mM
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HEPES) or standards (tyrosine in blank from 0 to 2.5 µg/mL) were combined with 250 µL of
30% TCA in a centrifuge tube and centrifuged at 2500 rpm for 15 minutes, supernatant was
recovered and placed on a new tube. Supernatant was combined with 300 µL of 1-nitroso-2naphthol 0.1% in 95% ethanol and 300 µL of nitric acid mixture, and incubated for 30 minutes
at 55ºC and then 15 minutes at room temperature. Four mL of ethylene dichloride were added to
the tubes and were then shaken. The tubes were then centrifuged at 1500 rpm for 4 minutes.
Four-hundred µL of supernatant were transferred to a 96-well black microplate and
measurements of tyrosine fluorescence was performed at 570 nm, resulting from its activation
at 460 nm using a fluorometer (Infinite M200, TECAN, Männedorf, Switzerland). The results
were expressed as nmol of tyrosine/mg of muscle/2 hours of incubation.
Luciferase reporter gene assay. On day 23 of the animal protocol (7 days exactly before
sacrifice) all mice were injected in the right gastrocnemius muscle (for obvious reasons the
diaphragm was not used for this purpose) with a mixture of 15 µg of control plasmid pRL-TK
vector, which contains a cDNA encoding renilla luciferase (Promega Corporation, Madison,
WI, USA) and 20 µg of reporter plasmid pNF-κB-Luc, which contains the firefly luciferase
gene (Clonetech, Mountain View, CA, USA). After the intramuscular injection of the plasmids,
a mechanical massage was performed in the muscle in order to improve the gene transfer. The
procedures followed have already been published (16). Briefly, during sacrifice, the right
gastrocnemius was obtained to be immediately homogenized using a polytron homogenizer and
6 volumes (w/v) of passive lysis buffer (Dual Assay, Promega). The homogenate was stored on
ice for 30 minutes and then centrifuged at 12,000 g at 4ºC for 10 minutes, the supernatants were
then transferred to a new tube and stored at -80ºC. The Luciferase Assay was performed using
the Dual Assay kit (Promega) and a luminometer (Lumat LB 9507, Berthold Technologies
GmbH, Bad Wildbad, Germany). Protein concentration was measured in all samples using the
Bradford methodology (13). Measurements were conducted as follows: 50 µL of each sample,
containing 150 µg of protein, were placed on a luminometer tube, 50 µL of the luciferase assay
reagent (LAR II, Promega) were added to the tube and the luminometer performed a first
measurement during 4 seconds (raw light units, RLU, firefly emission), then 50 µL of the Stop
& Glo solution (Promega) were added to the same tube and the luminometer performed a
second measurement during 4 seconds (RLU, renilla emission). The results were expressed as
firefly RLUs/renilla RLUs.
Cytokine Enzyme-linked Immunosorbent Assay (ELISA). Protein levels of the cytokines tumor
necrosis factor (TNF)-alpha, interferon-gamma, interleukin (IL)-6 and IL-1beta were quantified
in the muscles of all study animals (diaphragms and gastrocnemius muscles) using specific
sandwich ELISA kits eBiosience, Bender MedSystems, Vienna, Austria) following previously
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published methodologies (8, 11, 17). Frozen muscle sample specimens were homogenized and
protein concentration calculated as described above. For all the sample specimens equal
amounts of total protein from muscle homogenates were always loaded in triplicates (15 µg in
200 µL total volume each singlet for all the triplicates of all the study samples) onto the ELISA
plates. All samples were incubated with the specific primary antibodies and were always run
together in each assay. Before commencing the assay, samples and reagents were equilibrated to
room temperature. A standard curve was always run with each assay run. Standards (200 µL)
were performed as indicated by the manufacturer’s instructions. Protocol was also followed
according to the corresponding manufacturer’s instructions for each cytokine. Absorbances
were read at 450 nm using as a reference filter that of 655 nm. Intra-assay coefficients of
variation for the different cytokines and studies ranged from 4.5% to 10%. Inter-assay
coefficients of variation for the same cytokines ranged from 8% to 12%.
Muscle fiber counts and morphometry. On 3-micrometer muscle paraffin-embedded sections
from diaphragms and gastrocnemius muscles of all study groups, MyHC-I and –II isoforms
were identified using anti-MyHC-I (clone MHC, Biogenesis Inc., Poole, England, UK) and antiMyHC-II antibodies (clone MY-32, Sigma, Saint Louis, MO), respectively, as published
elsewhere (8, 11, 17). The cross-sectional area, mean least diameter, and proportions of type I
and type II fibers were assessed using a light microscope (Olympus, Series BX50F3, Olympus
Optical Co., Hamburg, Germany) coupled with an image-digitizing camera (Pixera Studio,
version 1.0.4, Pixera Corporation, Los Gatos, CA, USA) and a morphometry program (NIH
Image, version 1.60, Scion Corporation, Frederick, MD, USA). At least 100 fibers were
measured and counted in each type of muscle specimen from all groups of mice.
Muscle structure abnormalities. The area fraction of normal and abnormal muscle was
evaluated on 3-micrometer paraffin-embedded sections of the diaphragm and gastrocnemius of
all study groups muscles following previously published methodologies (11). Briefly, normal
and abnormal tissue was quantified using computer-assisted point counting in all the limb
muscle sections, previously stained with hematoxylin-eosin. A grid of 63 point-intercepts (7 x 9
rectangular pattern), built by means of the software Imaging Cell-B (Olympus Corporation),
was superimposed onto the image of the muscle cross section at a magnification of x400 under
the light microscope (Olympus BX 61, Olympus Corporation) using an image digitizing camera
(Olympus DP 71, Olympus Corporation). Each point-intercept was assigned to a specific
category and entered into the software. Categories for point counting were defined as follows:
1) normal muscle, 2) internal nucleus, 3) inflammatory cell, 4) lipofuscin, 5) abnormal viable, 6)
inflamed/necrotic, 7) vessel, and 0) no count. The area fraction for each category was defined as
the percentage of points that fell on each of these traits relative to the total number of points
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superimposed on all viable fields (all features except for categories 0 and 7) of each cross
section. It follows that the area fraction of normal muscle was equivalent to the proportion of
points falling in category 1, while the area fraction of abnormal muscle was determined by
calculating the proportion of points in categories 2, 3, 4, 5, and 6 (Figure E1).
Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. In
muscle paraffin-embedded sections, apoptotic nuclei were identified using the TUNEL assay (In
Situ Cell Death Detection Kit, POD, Roche Applied Science, Mannheim, Germany, lot number
12469600) in both diaphragms and gastrocnemius muscles from all study groups following
precisely the manufacturer’s instructions and previously published studies (18). In brief, this
assay is based on the principal that during apoptotis nuclei genomic DNA may yield doublestranded, low molecular weight fragments as well as single strand breaks (nicks) in high
molecular weight DNA. These DNA strand breaks can be identified by labeling 3’-OH termini
with modified nucleotides in an enzymatic reaction. In this assay, deoxynucleotidyl transferase,
which catalyzes the polymerization of nucleotides to free 3’-OH DNA ends, is used to label
DNA strand breaks. Briefly, muscle sections were fixed and permeabilized. Subsequently, they
were incubated with the TUNEL reaction mixture that contains TdT and fluorescein-dUTP.
During this incubation period, TdT catalyzed the addition of fluorescein-dUTP at free 3'-OH
groups in single- and double-stranded DNA. After washing, the label incorporated at the
damaged sites of the DNA was marked by an anti-fluorescein antibody conjugated with the
reporter enzyme peroxidase. After washing to remove unbound enzyme conjugate, the
peroxidase retained in the immune complex was visualized by a substrate reaction. Negative
control experiments in which addition of the reaction mixture was avoided, were also
conducted. Apoptotic nuclei were brown while negative nuclei were blue (hematoxylin
counterstaining). TUNEL positive nuclei were those clearly located within the muscle fiber
boundary in each section. In each muscle cross-section, the TUNEL-positive nuclei and the total
number of nuclei were counted blind by 2 independent observers, who were previously trained
for that purpose. On this basis, in each muscle preparation, altered fibers were expressed as the
percentage of the TUNEL-positive nuclei from the total number of counted nuclei following
previously published methodologies (18). A minimum amount of 300 nuclei were counted in
each muscle preparation. Final results correspond to the mean values of the counts provided by
the 2 independent observers (correlation coefficient 95%). Negative control experiments, in
which the TUNEL reaction mixture was omitted, were also conducted. Moreover, rat testicles
were used as positive controls in these experiments.
Statistical Analysis
Results are presented as mean (SD). Comparisons of physiological and biological variables
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among the different study groups were analyzed using one-way analysis of variance. For the
purpose of the study two different sets of comparisons were made: i) control and LC cachectic
mice, and on the other hand ii) LC cachectic and LC cachectic-bortezomib, iii) LC cachectic
and LC cachectic-NF-κB inhibitor; and iv) LC cachectic and LC cachectic-MAPK inhibitor.
Tukey’s post hoc analysis was used to adjust for multiple comparisons. Statistical significance
corresponding to these two different sets of comparisons is being specifically indicated in both
Figures and Tables. Correlations between physiological and biological variables were explored
using the Pearson’s correlation coefficient. The sample size chosen was based on previous
studies, where very similar approaches were employed (8-12, 19-22) and on assumptions of
80% power to detect an improvement of more than 20% in measured outcomes at a level of
significance of P≤ 0.05. In most of the biological variables, mean difference between groups
was initially estimated at a minimum of 20-25% and standard deviation was approximately 2530% of the mean value for each of the variables.
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FIGURE LEGENDS
Figure E1: Representative examples of 4 grids of 63 point-intercepts extracted from a
gastrocnemius of a cachectic mouse (x 400), in which all different categories for point counting
were identified. Those categories were defined using a specific number as defined in the
Methods section above.
Figure E2A: Representative examples obtained from the diaphragm muscle of the different
study groups of animals. Myofibers positively stained with the anti-MyHC type II antibody are
stained in brown color (x 400).
Figure E2B: Representative examples obtained from the gastrocnemius muscle of the different
study groups of animals. Myofibers positively stained with the anti-MyHC type II antibody are
stained in brown color (x 400).
Figure E3A: Representative examples of nuclei positively (arrows) and negatively stained for
the TUNEL assay in the diaphragm muscles of the different study groups of mice (x 400).
Negative nuclei appear in blue (hematoxylin counterstaining), while TUNEL-positive nuclei are
brown.
Figure E3B: Representative examples of nuclei positively (arrows) and negatively stained for
the TUNEL assay in the gastrocnemius muscles of the different study groups of mice (x 400).
Negative nuclei appear in blue (hematoxylin counterstaining), while TUNEL-positive nuclei are
brown.
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(9) Barreiro E, Del Puerto-Nevado L, Puig-Vilanova E, Perez-Rial S, Sanchez F,
Martinez-Galan L, et al. Cigarette smoke-induced oxidative stress in skeletal
muscles of mice. Respir Physiol Neurobiol 2012.
(10) Fermoselle C, Sanchez F, Barreiro E. [Reduction of muscle mass mediated by
myostatin in an experimental model of pulmonary emphysema]. Arch
Bronconeumol 2011;47:590-8.
(11) Fermoselle C, Rabinovich R, Ausin P, Puig-Vilanova E, Coronell C, Sanchez F,
et al. Does oxidative stress modulate limb muscle atrophy in severe copd
patients? Eur Respir J 2012.
(12) Marin-Corral J, Fontes CC, Pascual-Guardia S, Sanchez F, Olivan M, Argiles
JM, et al. Redox balance and carbonylated proteins in limb and heart muscles of
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cachectic rats. Antioxid Redox Signal 2010;12:365-80.
(13) Bradford MM. A rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein-dye binding. Anal
Biochem 1976;72:248-54.
(14) Furuno K, Goodman MN, Goldberg AL. Role of different proteolytic systems in
the degradation of muscle proteins during denervation atrophy. J Biol Chem
1990;265:8550-7.
(15) Tischler ME, Desautels M, Goldberg AL. Does leucine, leucyl-tRNA, or some
metabolite of leucine regulate protein synthesis and degradation in skeletal and
cardiac muscle? J Biol Chem 1982;257:1613-21.
(16) McClung JM, Judge AR, Powers SK, Yan Z. p38 MAPK links oxidative stress
to autophagy-related gene expression in cachectic muscle wasting. Am J Physiol
Cell Physiol 2010;298:C542-C549.
(17) Barreiro E, Schols AM, Polkey MI, Galdiz JB, Gosker HR, Swallow EB, et al.
Cytokine profile in quadriceps muscles of patients with severe COPD. Thorax
2008;63:100-7.
(18) Barreiro E, Ferrer D, Sanchez F, Minguella J, Marin-Corral J, Martinez-Llorens
J, et al. Inflammatory cells and apoptosis in respiratory and limb muscles of
patients with COPD. J Appl Physiol 2011;111:808-17.
(19) Barreiro E, Garcia-Martinez C, Mas S, Ametller E, Gea J, Argiles JM, et al.
UCP3 overexpression neutralizes oxidative stress rather than nitrosative stress in
mouse myotubes. FEBS Lett 2009;583:350-6.
(20) Penna F, Costamagna D, Fanzani A, Bonelli G, Baccino FM, Costelli P. Muscle
wasting and impaired myogenesis in tumor bearing mice are prevented by ERK
inhibition. PLoS One 2010;5:e13604.
(21) Supinski GS, Callahan LA. Caspase activation contributes to endotoxin-induced
diaphragm weakness. J Appl Physiol 2006;100:1770-7.
(22) Supinski GS, Vanags J, Callahan LA. Effect of proteasome inhibitors on
endotoxin-induced diaphragm dysfunction. Am J Physiol Lung Cell Mol Physiol
2009;296:L994-L1001.
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Results
Study #4
Mitochondrial dysfunction and therapeutic approaches in respiratory
and limb muscles of cancer cachectic mice.
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44
Results
Main findings in study # 4
In the LC cachectic mice compared to controls, and in the different treatment groups compared
to non-treated LC cachectic rodents:
Body and muscle mass weight and muscle function
• LC cachectic mice exhibited a reduction in body weight gain and diaphragm and gastrocnemius
weight compared to control animals.
• LC groups treated with NF-κB and MAPK inhibitors exhibited an increase in body weight
compared to non-treated LC cachectic rodents.
• Limb strength was reduced in the LC cachectic mice compared to the control animals.
• Limb strength reduction was corrected by treatment with NF-κB and MAPK inhibitors
compared to non-treated LC cachectic group.
• Food intake was similar in all groups.
MRC enzymes activity
• CS activity did not differ in both muscles in the LC cachectic mice compared to the control
group.
• All treatments decreased the CS activity in gastrocnemius and increased it in the diaphragm
compared to the non-treated LC cachectic animals.
• Complex I activity was decreased in both muscles in the LC cachectic mice compared to the
control group.
• Complex I activity was increased in the diaphragm by proteasome, NF-κB and MAPK
inhibitors; and increased in gastrocnemius by MAPK inhibitor compared to the non-treated
LC cachectic rodents.
• The ratio of CI/CS was decreased in both muscles in the LC cachectic mice compared to the
control animals.
• The ratio of CI/CS was increased by NF-κB and MAPK inhibitors compared to the non-treated
LC cachectic group.
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Results
• Complex II activity was decreased in both muscles in the LC cachectic mice compared to the
control animals.
• Complex II activity was increased in the diaphragm by proteasome and NF-κB inhibitors, and
in the gastrocnemius by MAPK inhibitor compared to the non-treated LC cachectic animals.
• The ratio of CII/CS was decreased in both muscles in the LC cachectic mice compared to the
control rodents.
• The ratio of CII/CS was increased in gastrocnemius by NF-κB and MAPK inhibitors compared
to the non-treated LC cachectic group.
• Complex IV activity was decreased in the diaphragm in the LC cachectic mice compared to
the control animals.
• Complex IV activity was increased in diaphragm by all treatments and, in gastrocnemius by
MAPK inhibitor compared to the non-treated LC cachectic animals.
• The ratio of CIV/CS was decreased in the diaphragm in the LC cachectic mice compared to
the control group.
• The ratio of CIV/CS was increased in both muscles by all treatments compared to the nontreated LC cachectic animals.
MRC oxygen consumption
• Complex I oxygen consumption (state 3) was decreased in both muscles in the LC cachectic
mice compared to the control animals.
• All the treatments increased Complex I oxygen consumption compared to the non-treated
LC cachectic rodents.
• State 4 did not differ in both muscles between all the groups.
• Respiratory control index (state3/state4) did not differ in both muscles between LC cachectic
mice and the control group.
• In the diaphragm State 4 was increased by all treatments and in gastrocnemius it was
increased by NF-κB and MAPK inhibitors compared to the non-treated LC cachectic animals.
• Complex IV oxygen consumption was decreased in both muscles in the LC cachectic mice
compared to the control rodents.
• Complex IV oxygen consumption was increased with all treatments in both muscles compared
to the non-treated LC cachectic animals.
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Results
MITOCHONDRIAL DYSFUNCTION AND THERAPEUTIC APPROACHES IN
RESPIRATORY AND LIMB MUSCLES OF CANCER CACHECTIC MICE
1,2
1,2
Clara Fermoselle,
3,4
Elena García-Arumí,
3,4
Antoni L. Andreu,
5
1,2
Francisco Sanchez,
5
Ester Puig-Vilanova, Alejandro J. Urtreger, Elisa D. Bal de Kier Joffé, 6Alberto
Tejedor, 7Luís Puente-Maestu, 1,2Esther Barreiro
1
Pulmonology Department-Lung Cancer Group, IMIM-Hospital del Mar, Health and
Experimental Sciences Department, Universitat Pompeu Fabra, Barcelona Biomedical Research
Park (PRBB), Barcelona, Spain.
2
Centro de Investigación en Red de Enfermedades Respiratorias (CIBERES), Instituto de Salud
Carlos III (ISCIII), Bunyola, Majorca, Balearic Islands, Spain.
3
Unitat de Patologia Neuromuscular i Mitocondrial, Hospital Universitari Vall d'Hebron
Institut de Recerca (VHIR), Universitat Autònoma de Barcelona.
4
Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER),
ISCIII, Barcelona, Spain
5
Research Area, Institute of Oncology ‘Angel H. Roffo’, University of Buenos Aires, Buenos
Aires, Argentina.
6
Servicio de Nefrología, Hospital General Gregorio Marañón, Universidad Complutense de
Madrid, Madrid, Spain.
7
Servicio de Neumología, Hospital General Gregorio Marañón, Universidad Complutense de
Madrid, Madrid, Spain.
Corresponding author: Dr.
Esther Barreiro, URMAR, IMIM-Hospital del Mar, C/ Dr.
Aiguader, 88, E-08003 Barcelona, Spain, Telephone: (+34) 93 316 0385, Fax: (+34) 93 316
0410, e-mail: [email protected]. Requests for reprints: Dr. Esther Barreiro
RUNNING TITLE: Mitochondrial respiratory chain dysfunction in cachexia
WORD COUNT: 4,744
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ABSTRACT
Abnormalities in mitochondrial content and function have been reported in muscle wasting
conditions including COPD, cardiac cachexia, mechanical ventilation, and muscles of interleukin
(IL)-6-induced cachexia. It remains an open question to elucidate whether mitochondrial
respiratory chain (MRC) function is altered in muscles of cancer-induced cachexia and whether
therapeutic approaches could attenuate such a dysfunction. Our objective was to specifically
explore MRC function in respiratory and limb muscles of a model of lung cancer (LC) cachectic
mice, which concomitantly received treatment with either MAPK, NF-κB, or proteasome
inhibitors, or NAC. We evaluated complex I, II, and IV enzyme activities and mitochondrial
respiratory chain (MRC) oxygen consumption using polarographic measurements in diaphragm
and gastrocnemius of cachectic mice bearing the LP07 lung tumor with and without treatment
with N-acetylcysteine (NAC), bortezomib, and selective inhibitors of nuclear factor (NF)-κB and
mitogen-activated protein kinases (MAPK). Whole body and muscle weights and limb muscle
force were also assessed in all rodents. Novel findings in the investigation are that in respiratory
and limb cachectic muscles, complexes I, II, and IV enzyme activity and oxygen consumption by
the MRC were significantly decreased compared to control animals. We conclude that both
respiratory and limb muscles of cancer cachectic mice exhibit a significant depression of the
MRC complexes and oxygen consumption by the MRC. Concomitant treatment with the specific
inhibitors of NF-κB and MAPK signaling pathways restore MRC function in both types of
muscles, while also significantly improving whole body and muscle weights and force in the
mice.
Word count: 248
KEY WORDS: lung cancer cachexia, mitochondrial respiratory chain function, diaphragm,
gastrocnemius, pharmacological strategies
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INTRODUCTION
Muscle wasting and cachexia are common systemic manifestations of acute and chronic
conditions including sepsis, respiratory and cardiovascular disease, and cancer, especially those
of the lung and gastrointestinal tract. Although the prevalence of cachexia may vary widely, in
advanced malignancies, it may range from 60 to 80%, thus having a great impact on the patient’s
quality of life.
Several molecular and cellular mechanisms have been proposed to contribute to the
etiology of cancer-induced cachexia such as inflammation (1), oxidative stress (4; 5; 27), insulin
resistance, and other metabolic disturbances (13). The mitochondria play a major role in meeting
the cellular requirements of energy through oxidative phosphorylation of different substrates.
Additionally, mitochondrial content and function are important modulators of physiological
muscle mass maintenance and loss in disease. Abnormalities in muscle mitochondrial content and
function have been reported in several muscle wasting conditions including limb muscles of
chronic obstructive pulmonary disease (COPD) patients (17; 31-33), skeletal muscle in cardiac
cachexia (10), the diaphragm in mechanical ventilation (19), and limb muscles of interleukin
(IL)-6-induced cachexia (46).
Additionally, mitochondrial respiratory chain (MRC) dysfunction may lead to enhanced
reactive oxygen species (ROS) production as shown in muscles of COPD patients(31; 32), in the
respiratory muscles of rats exposed to mechanical ventilation(19), and in human lung cancer cells
(24). Nonetheless, ROS synthesized within the mitochondria may also participate in signal
transduction pathways involved in different adaptive responses including hypoxia (22), muscle
differentiation (23), and adaptation to ischemia-reperfusion in cardiomyocytes (8). Despite the
relevance of mitochondria oxidative metabolism as a source of energy production, knowledge on
potential alterations of muscle MRC in cancer-induced cachexia is still at its infancy.
Mitogen-activated protein kinases (MAPK) and nuclear factor (NF)-κB are central
regulators of gene expression, redox balance, and metabolism. Importantly, these molecules also
play a relevant role in oxidative stress-mediated muscle wasting and atrophy in several models of
cachexia (28; 39) and in tumor cell growth (16). In this regard, NF-κB was shown to enhance
mitochondrial ROS production, which in turn, led to mitochondrial and cardiac dysfunction in
diabetic mice (26).
The ubiquitin-proteasome system plays a major role in conditions characterized by
muscle wasting and weakness (14; 40; 43; 44). Bortezomib, the first proteasome inhibitor used in
patients, was shown to restore muscle function and mass in several models (43; 44), while it did
not induce any beneficial effects on sepsis-induced cachexia (40). Bortezomib also exerted
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important anti-tumor activities in cancer cell lines via oxidative stress-mediated apoptosis (24).
The antioxidant N-acetyl cysteine (NAC) was shown to directly scavenge ROS in skeletal muscle
fibers through targeting different cellular structures including mitochondrial proteins (35; 38; 45).
Importantly, NAC was also shown to interfere with MAPK activation in response to hypoxia in
cardiomyocytes (22; 45). Despite this knowledge, it remains to be elucidated whether inhibition
of MAPK, NF-κB, proteasome, and oxidative stress may have any effect on the MRC of cancer
cachectic muscles.
On this basis, the rationale for conducting the present investigation was to specifically
explore whether the MRC function is altered in the mouse diaphragm and gastrocnemius, both
predominantly fast-twitch glycolytic muscles, of cancer cachectic animals and whether several
therapeutic interventions may improve such a dysfunction. Accordingly, our main objectives
were to specifically explore MRC function in respiratory and limb muscles of a model of lung
cancer (LC) cachectic mice, which concomitantly received treatment with either MAPK, NFκB, or proteasome inhibitors, or NAC. Whole body and muscle weights and limb muscle force
were also assessed in all rodents.
MATERIALS AND METHODS
Animal experiments
Tumor. LP07 is a cell line derived from the transplantable P07 lung tumor that appeared
spontaneously in the lung of a BALB/c (11). The LP07 cell line was obtained in vitro, after
successive passages of a P07 primary culture (42). LP07 cell line shares identical characteristics
regarding lung tumor incidence and histology, and cachexia with its parental P07 tumor (11; 12;
42). It was also consistently demonstrated that one month after tumor transplantation, all
animals developed lung metastasis, spleen enlargement, and cachexia without affecting any
other organs (11; 12; 42).
Experimental groups. Female BALB/c mice, 2 months old (weight ~20g), were obtained from
Harlan Interfauna Ibérica SL (Barcelona, Spain). In all experimental groups (except for control
rodents), LP07 viable cells (4·105), resuspended in 0.2 mL minimal essential media (MEM),
were subcutaneously inoculated into the left flank of the mice (day 1). All groups (n=10/group)
were studied for a period of one month. Animals were randomly assigned to the following
groups: 1) control, inoculation of 0.2 mL MEM in the left flank; 2) LC cachexia group,
inoculation of LP07 cells; 3) LC cachectic mice receiving concomitant treatment with the
antioxidant NAC (kindly provided by Dr. Mateu, Hospital del Mar), 3mmol/kg/24h, oral
administration using a 22G, 25 mm needle (gavage); 4) LC cachectic mice receiving
concomitant treatment with the proteasome inhibitor Bortezomib (Velcade, Millenium
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Pharmaceuticals, Cambridge, MA), 0.15 mg/Kg, 0.1 mL/6 days, intravenous injection into the
tail vein (LC cachectic-bortezomib group) (25); 5) LC cachectic mice receiving concomitant
treatment with sulfasalazine (Pfizer, Madrid, Spain), 200 mg/Kg, 0.3 mL/48h, intraperitoneal
injection (LC cachectic-NF-κB inhibitor group) (30); and 6) LC cachectic mice receiving
concomitant treatment with the MAPK inhibitor U0126 (a highly selective inhibitor of ERK1
and ERK2 proteins, Selleck chemicals, Houston, TX), 30 mg/Kg, 0.1 mL/48h, intraperitoneal
injection (LC cachectic-MAPK inhibitor group) (37). All pharmacological therapies were
administered from day 15 after the inoculation of the LP07 cells up until the end of the study
period on day 30.
Ethics. All animal experiments were conducted in the animal facilities at Parc de Recerca
Biomèdica de Barcelona (PRBB, Spain). This controlled study was designed in accordance with
the ethical standards on animal experimentation (EU 609/86 CEE, Real Decreto 1201/05 BOE
252, Spain) at PRBB and the Helsinki convention for the use and care of animals. Ethical
approval was obtained by the Animal Research Committee at PRBB and Catalan Government
(Animal welfare department).
In vivo measurements and sample collection from mice
Body weight and food intake were determined every day during the entire duration of the study.
Limb strength was determined on days 0 and 30 using a strength grip meter (Bioseb, Chaville,
France) following previously published methodologies (6). In the LC cachexia group of mice,
tumor progression was determined using positron emission tomography (PET) on days 13 and
20. Mice from all the experimental groups were always sacrificed on day 30 post-inoculation of
LP07 cells or MEM (control animals). Prior to sacrifice, all mice were inoculated
intraperitoneally with 0.1 mL sodium pentobarbital (60 mg/Kg). Diaphragm and gastrocnemius
muscles and the subcutaneous tumor were obtained from all animals. The weight of both
muscles and tumor were determined in each animal using a high-precision scale. For the activity
essay measurements a first half of the diaphragm and the entire gastrocnemius of one of the
hind-limbs were immediately frozen and stored at -80ºC for further analyses in all animals.
However, in order to assess oxygen consumption using polarographic studies the second half of
the diaphragm and the entire gastrocnemius of the second hind-limb were immediately placed in
cold isolation buffer to perform the ex vivo measurements (31-33).
Homogenization procedures
Diaphragm and gastrocnemius muscles were obtained from all mice and snap-frozen to be
subsequently stored at -80ºC until further use. Muscle homogenization was always performed at
0-5ºC. Samples were homogenized using a Homogenisator Potter S (Sartorius Stedim Biotech
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GmbH, Goettingen, Germany). In general, 50 mg of whole muscle tissue were placed into the
potter containing 9 volumes (w/v) Manitol Buffer pH 7.2 [225 mM manitol, 75 mM sucrose, 10
mM Tris HCl, 0.1 mM Ethylenediaminetetraacetic acid (EDTA)], at 1000 rpm, while 3 strokes
up and down were performed. Samples were then transferred onto a new tube and centrifuged at
650 g for 20 minutes. After centrifugation, the supernatants were collected and placed into new
tubes, while the pellets were resuspended with the initial Manitol Buffer volume, pH 7.2. The
homogenization procedure was repeated again with the resuspended pellets. The supernatants
obtained from the second centrifugation were added to the first ones, thus yielding the final
sample supernatants. Protein concentrations were measured using the Bradford method (9).
Mitochondrial citrate synthase activity
The procedures employed to determine citrate synthase (CS) activity were previously published
(29). CS activity was used as a marker of mitochondrial content or density as commonly
described (29). Citrate synthase catalyzes the acetyl-coenzyme A oxaloacetate reaction resulting
citrate and coenzyme A. The latter compound can be measured using 55'-dithiobis-(2nitrobenzoic acid) (DTNB). The reaction is monitored at 412 nm. The specific activity of CS
enzyme was expressed as nmols/min/ mg protein. Intra- and inter- assay coefficients of
variation were 5.07% and 2.7%.
Mitochondrial respiratory chain enzyme activities
Mitochondrial Complex I Activity. Again all procedures employed in the current investigation
had been previously published (29).
Enzymatic determination of nicotinamide adenine
dinucleotide (NADH) ubiquinone oxido-reductase rotenone sensitive is based on the oxidation
rate of NADH in the presence of rotenone. It monitors the absorbance decrease at 340 nm in the
presence and absence of rotenone. The rotenone-resistant activity was subtracted from the total
activity of NADH ubiquinone oxido-reductase to obtain the activity sensitive to rotenone. The
specific activity of complex I was expressed as nmols/min/ mg protein. Intra- and inter- assay
coefficients of variation were 8.93% and 7.84%, respectively.
Mitochondrial Complex II Activity. Again all procedures used for measuring this complex had
been previously published (29). Succinate decylubiquinone reductase activity was measured
using 2,6-dichlorophenolindophenol (DCPIP) as the electron acceptor.
The reaction was
monitored at 600 nm. The specific activity of complex II was expressed as nmols/min/ mg
protein. Intra- and inter- assay coefficients of variation were 7.81% and 5.18%, respectively.
Mitochondrial Complex IV Activity. Again all procedures had been previously published (29).
Complex IV activity was measured using reduced cytochrome C as the substrate. Cytochrome
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C oxidation was monitored at 550 nm. The specific activity of complex IV was expressed as
nmols/min/ mg protein. Intra- and inter- assay coefficients of variation were 4.13% and 4.03%,
respectively.
Mitochondrial oxygen consumption
Methodologies employed had already been published (31-33). The gastrocnemius and
diaphragm muscles were obtained in vivo from mice and were rapidly placed into a cold tube
containing Isolation Buffer (on ice). Muscle samples, approximately 100 mg total weight, were
placed into a potter glass (Homogenisator Potter S, Sartorius Stedim Biotech GmbH) containing
4 volumes (w/v) of Isolation Buffer. Muscle specimens were homogenized at 500 rpm, applying
3 strokes with 30-second stops between each stroke in order to avoid sample heating.
Homogenates were centrifuged at 1.1g for 10 minutes. Pellets were discarded and supernatants
were centrifuged at 8.8g for another 10 more minutes. Supernatants were discarded and pellets
were softly resuspended using a finger rod in 1-ml Isolation Buffer, to be subsequently
afterwards centrifuged at 8.8g for 10 more minutes. Supernatants were discarded again and
pellets resuspended using the finger rod, in 50-µL Measurement Buffer. Mitochondrial
concentration was analyzed using Bradford procedures (9). The whole process of mitochondria
isolation was performed at 4ºC. Materials and small equipment were washed with EGTA to
inhibit reverse calcium release from uncoupled mitochondria. Furthermore, for the same
purpose, the Isolation Buffer [300 mM Manitol, 1 mM Ethylene glycol tetraacetic acid (EGTA),
10mM
Tris(hydroxymethyl)aminomethane
hydrochloride-HCl
(Trizma-HCl),
1
mM
-1
Monopotassium phosphate (KH2PO4), 1.74mg·mL phenylmethanesulfonylfluoride (PMSF),
0.2% bovine serum albumin (BSA), 10mg·L-1 amoxicillin, pH 7.4] and a part of the
Measurement Buffer (Manitol 300mM, Trizma-HCl 10mM, KH2PO4 1mM, pH 7.4) were
continuously gassed with nitrogen, which displaces molecular oxygen (O2) and keeps
mitochondria under reducing conditions (avoiding breathing).
Mitochondrial oxygen consumption (V’O2,m) was measured using a Clark-type electrode,
placed in an RC 650 6-cell respirometer attached to a 929 6-channel respirometer system
(Strathkelvin Instruments Limited, Motherwell, UK). The whole system was maintained at 37ºC
during the entire duration of the protocol. The isolated mitochondria and substrates were
introduced into the chambers through a capillary hole in the plunger using a graduated syringe
(Hamilton, Bonaduz, Switzerland). In order to quantify Complex I (NADH ubiquinone oxidoreductase) oxygen consumption (V’O2,m), 0.9 mL Measurement Buffer containing 5 mM
piruvate, 5 mM malate, and the mitochondria yields were added to the chambers (at this point,
the measured rates correspond to State 4 respiration). In order to determine State 3 respiration, 2
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mM adenosine diphosphate (ADP) were added to the chamber. Subsequently, 0.7 mM
N,N,N′,N′-tetramethyl-p-phenylenediamine (TMP) and 7 mM ascorbate were added to the
chambers in order to quantify Complex IV (Cytochrome c oxidase) oxygen consumption
(V’O2,m). V’O2,m was expressed as nmol O2/min/µg of mitochondria. The respiratory control
index (RCI) was calculated as the ratio of State 3 to State 4 respiration. Intra- and inter- assay
coefficients of variation were 8.77% and 8.72% for Complex I oxygen consumption, and 9.75%
and 9.96% for Complex IV consumption.
Statistical Analysis
Results are presented as mean (SD). Comparisons of physiological and biological variables
among the different study groups were analyzed using one-way analysis of variance. For the
purpose of the study two different sets of comparisons were made: i) control and LC cachectic
mice, and on the other hand ii) LC cachectic and LC cachectic-NAC; iii) LC cachectic and LC
cachectic-bortezomib, iv) LC cachectic and LC cachectic-NF-κB inhibitor; and v) LC cachectic
and LC cachectic-MAPK inhibitor. Tukey’s post hoc analysis was used to adjust for multiple
comparisons. Statistical significance corresponding to these two different sets of comparisons is
being specifically indicated in both Figures and Tables. The sample size chosen was based on
previous studies, where very similar approaches were employed (4; 5; 7; 27; 31-33) and on
assumptions of 80% power to detect an improvement of more than 20% in measured outcomes
at a level of significance of P≤ 0.05.
RESULTS
Physiological characteristics
As shown in Table 1, at the end of the study period, LC cachectic mice exhibited a reduction in
body weight that was not observed in control animals. Food intake was similar among the study
groups (3 g/24h). Diaphragm and gastrocnemius muscle weights and limb strength were
significantly reduced in the LC cachectic mice compared to control rodents, which showed a
significant gain in muscle force (Table 1). LC cachectic mice treated with the NF-κB and
MAPK inhibitors exhibited a significantly smaller reduction in weight gain, a significant
improvement in diaphragm and gastrocnemius weights, and a significant recovery of muscle
strength gain compared to the non-treated cachectic animals (Table 1). The proteasome
inhibitor or the antioxidant NAC did not induce any significant effects on body or muscle
weights, or limb muscle strength in the LC cachectic mice (Table 1). Compared to non-treated
tumor-bearing animals, the weight of the subcutaneous tumor was significantly reduced in the
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cachectic rodents treated with the inhibitors of the proteasome (36%), NF-κB (29%), and
MAPK (50%) pathways, but not NAC (Table 1).
Citrate synthase enzyme activity
In either diaphragm or gastrocnemius muscles, no significant differences were observed in CS
enzyme activity between LC cachectic mice and the controls (Figure 1). Interestingly, CS
activity levels were significantly increased in the diaphragms of the LC cachectic mice treated
with NAC, bortezomib, NF-κB and MAPK inhibitors compared to respiratory muscles of the
non-treated LC cachectic rodents (Figure 1). CS enzyme activity levels of CS were significantly
decreased in the gastrocnemius of LC cachectic mice treated with NAC, bortezomib, and NF-κB
and MAPK inhibitors, compared to non-treated cachectic animals (Figure 1).
Mitochondrial respiratory chain complexes: enzyme activities
Complex I enzyme activity (NADH ubiquinone oxido-reductase activity) was significantly
diminished in diaphragm and gastrocnemius of LC cachectic rodents compared to the controls
(Figure 2A). Activity of this enzyme was increased in the diaphragms of the LC cachectic mice
treated with either proteasome, NF-κB, or MAPK inhibitors, while only the last inhibitor
elicited a mild increase in complex I activity in the cachectic gastrocnemius compared to the
non-treated cachectic rodents (Figure 2A).The ratio of complex I to CS activities was
significantly decreased in both muscles of LC cachectic compared to control animals (Figure
2B). The ratio of complex I/CS activity was significantly improved in both diaphragms and
gastrocnemius of LC cachectic mice treated with NF-κB and MAPK inhibitors, but not the
antioxidant or bortezomib compared to non-treated cachectic rodents (Figure 2B).
Complex II enzyme activity (succinate ubiquinone oxido-reductase) was decreased in both
respiratory and limb muscles of LC cachectic rodents compared to the controls (Figure 3A).
While in the diaphragm, complex II activity was greater in LC cachectic rodents treated with
proteasome and NF-κB inhibitors, MAPK inhibitor elicited a significant improvement in that
activity only in the gastrocnemius compared to the non-treated cachectic mice (Figure 3A). The
ratio of complex II to CS was significantly reduced in both limb and respiratory muscles of the
cachectic rodents compared to control animals (Figure 3B). NF-κB and MAPK inhibitors
elicited a significant increase in that ratio only in the limb muscle of the cachectic rodents
compared to non-treated tumor-bearing animals (Figure 3B).
Complex IV activity (cytochrome c oxidase) was decreased in the respiratory muscle of LC
cachectic rodents compared to control animals (Figure 4A). Interestingly, while in the
diaphragm, all four inhibitors elicited a significant improvement in complex IV activity of LC
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cachectic mice, in the limb muscle, only MAPK inhibitor induced an increase in the activity of
that enzyme compared to the non-treated cachectic animals (Figure 4A). The ratio of complex
IV to CS was decreased in the diaphragm of cachectic mice compared to the controls (Figure
4B). Importantly, all pharmacological agents elicited a significant increase in complex IV/CS in
both respiratory and limb muscles of LC cachectic rodents compared to non-treated cachectic
animals (Figure 4B).
Mitochondrial respiratory chain oxygen consumption
Oxygen consumption (state 3, nmol O2/min/µg) was decreased in both diaphragm and
gastrocnemius of cachectic rodents compared to the controls (Figure 5A). Importantly,
treatment with NAC, bortezomib, and NF-κB or MAPK inhibitors elicited a significant
improvement in oxygen consumption in both respiratory and limb muscles of LC cachectic
rodents compared to non-treated cachectic animals (Figure 5A). In state 4 respiration (nmol
O2/min/µg) no significant differences were observed between cachectic and control rodents, and
the pharmacological agents did not elicit any significant effect on any muscle (Figure 5B).
Interestingly, the RCI did no differ between LC cachectic rodents and the controls (Figure 5C).
However, RCI was significantly increased in the diaphragm of cachectic mice treated with
NAC, bortezomib, and NF-κB or MAPK inhibitors, while only the last two inhibitors improved
the RCI in the gastrocnemius of the same animals compared to non-treated cachectic mice
(Figure 5C). Oxygen consumption by cytochrome c oxidase (nmol O2/min/µg) was decreased in
both respiratory and limb muscles of LC cachectic mice compared to control animals (Figure
5D). Importantly, in the cachectic rodents, oxygen consumption was increased in both types of
muscles in response to concomitant treatment with NAC, bortezomib and either NF-κB or
MAPK inhibitors compared to non-treated animals (Figure 5D).
DISCUSSION
Summary of main findings
In the current investigation, compared to control animals at the end of the 1-month study period,
LC cachectic mice exhibited a significant reduction in total body weight gain, smaller size of
their respiratory and limb muscles, and lower limb muscle performance as measured by muscle
strength. Interestingly, only treatment with either NF-κB or MAPK inhibitors elicited a
significant improvement in body weight gain, muscle mass and performance, while
simultaneously decreasing subcutaneous tumor size among the LC cachectic rodents. Despite
that bortezomib induced a significant reduction in subcutaneous tumor size in the cachectic
mice, body and muscle weights or force generation did not improve in these animals. These
findings suggest that NF-κB and MAPK are predominant pathways in the development of
10
Results
muscle mass loss and force production, while the role played by the proteasome is questionable
in that respect in this specific model of cancer-induced cachexia. Administration of the
antioxidant NAC did not induce any beneficial effects on cachexia parameters or subcutaneous
tumor size among the tumor-bearing rodents. This also indicates that oxidants are not likely to
be involved in muscle mass loss or impaired function in this experimental model of cancer
cachexia.
As expected, the activity of the MRC complexes analyzed in the present study was in
general of greater magnitude in the mouse respiratory muscle than in the limb muscle.
Importantly, novel findings in the study were that the cachectic rodents exhibited a significant
decrease in the activities of the mitochondrial respiratory chain complexes I, II, and IV, as
identified by both absolute and relative values to CS, while in the limb muscle only complex I
and II activities were significantly diminished. In general concomitant treatment of the cachectic
rodents with inhibitors of the proteasome, NF-κB or MAPK pathways elicited a significant
improvement in mitochondrial complexes I, II, and IV, especially in the diaphragm muscle.
Oxygen consumption in State 3 respiration was significantly decreased in both types of muscles
and treatment with the four different pharmacological agents induced a significant improvement
in that parameter. Likewise, complex IV oxygen consumption was also decreased in both
respiratory and limb muscles of the cachectic rodents, which showed a significant improvement
in that factor in response to concomitant treatment with the antioxidant and the three inhibitors.
The RCI was not significantly different in any of the muscles between cachectic and control
mice. Nonetheless, the four pharmacological agents induced a significant improvement in RCI
in the respiratory muscle of the tumor-bearing animals.
MRC enzyme activities in cancer cachectic muscles
In the current investigation, it has been clearly demonstrated for the first time that cancer
cachexia selectively depresses the activity of the MRC complexes I, II and IV in fast-twitch
mouse muscles. This is in line with previous studies in which muscle contractile dysfunction of
different etiology was shown to be associated with decreased MRC function in muscles of
COPD patients (31-33) and mechanical ventilation-induced atrophy of rat diaphragms (19).
Other novel findings in the study are the significant improvement observed in the activities
of the different MRC complexes analyzed, especially in the diaphragm muscle, in response to
concomitant treatment of the tumor-bearing rodents with inhibitors of NF-κB, and MAPK
pathways. These findings may suggest that these two signaling pathways may specifically
interfere, at different levels, with cellular processes involved in mitochondrial biogenesis and/or
protein synthesis, activity, and degradation of the MRC enzymes examined in the investigation.
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Results
In this respect, alternative NF-κB seems to modulate muscle oxidative metabolism, whereas
canonical NF-κB pathway was shown to reduce mitochondrial biogenesis and content (3). In a
similar fashion, p38 MAPK activation was also demonstrated to decrease mitochondrial
biogenesis in skeletal muscles of obese mice (41). Another interesting finding in the study was
that the proteasome inhibitor elicited a significant improvement in the activity of complexes I,
II, and IV, especially in the diaphragm of the LC cachectic mice. This finding indicates that
blockade of this proteolytic system, highly involved in muscle wasting conditions (2; 14; 43;
44), may restore mitochondrial protein content in muscles of the cachectic animals as shown to
occur in other models (36). Future studies will shed light into the specific contribution of these
three cellular pathways to mitochondrial biogenesis and protein synthesis and degradation.
The antioxidant NAC may act as a direct antioxidant in skeletal muscle fibers by
protecting specific cellular sites such as sarcoplasmic reticulum, sarcolemma and/or
mitochondria proteins from oxidative damage in several models (7; 20; 34; 35; 38; 45). In both
respiratory and limb muscles of the cachectic animals in the current study, NAC only elicited a
significant increase in complex IV activity as quantified by the ratio to CS, but not as absolute
values. Total mitochondrial content (CS activity) and ROS production in the muscle specimens
could play a more relevant role in complex IV activity than in the other MRC complexes.
However, these findings warrant further attention in future investigations.
Importantly, CS enzyme activity did not seem to be altered in either respiratory or limb
muscles of the LC cachectic rodents compared to control levels. Activity of CS enzyme was
used as a marker of mitochondrial content in the muscle preparations. On this basis, it would be
possible to conclude that mitochondrial content was equally preserved in both diaphragms and
gastrocnemius muscles of the cachectic and control animals. These interesting findings suggest
that the decline in MRC complex activities is likely to be due to alterations occurring in the
enzyme active sites than to a decrease in mitochondrial content within the cancer cachectic
muscles. An additional relevant finding in the study was the increase in CS activity observed in
the diaphragm of the cachectic rodents treated with any of the four pharmacological agents,
whereas the same drugs induced a significant decline in CS activity in the limb muscle of the
same mice. Differences in the activity of each type of muscle could account for this differential
pattern of response to the inhibitors administered to the cachectic animals. Potential depression
of mitochondrial biogenesis in the limb muscles elicited by the different treatments could also
partly explain those findings.
MRC oxygen consumption
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Results
State 3 respiration was significantly decreased in the respiratory and limb muscles of the
LC cachectic rodents compared to control mice. This finding has relevant implications, since it
may partly account for the metabolic derangements and energy inefficiency characteristic of
cachectic muscles (13). The current study results are also consistent with previous
investigations, in which similar findings were reported in muscles of COPD patients (31-33), in
diaphragms of mechanically ventilated rats (19), and in muscles of senescent mice (15).
Importantly, for each specific muscle, basal mitochondrial respiration (State 4) was similar
among all the study groups, irrespective of the disease status or treatment with the different
pharmacological agents. Also, State 4 respiration levels were in general of greater magnitude in
the respiratory than in the limb muscles for each of the study groups. These are relevant findings
indicating that mitochondrial integrity and stability was achieved equally in both types of
muscles of all animal groups. This is a relevant methodological feature that ensures the quality
and reliability of the results encountered in the investigation.
Oxygen consumption by MRC complex IV (cytochrome c oxidase) was also diminished in
both types of muscles of LC cachectic rodents compared to control animals. These findings are
in line with those aforementioned, reassuring the consistency of the study results. Importantly,
the antioxidant NAC and the inhibitors of the proteasome, NF-κB and MAPK elicited a
significant increase in oxygen consumption by complex IV and State 3 respiration in both
diaphragms and gastrocnemius of the tumor-bearing animals compared to the non-treated
cachectic rodents. These are novel findings suggesting that the different pathways and cellular
processes blocked by the different inhibitors are somehow involved in MRC dysfunction in
cancer cachexia.
It has been well established that the antioxidant NAC and other antioxidants influence
muscle fiber function by scavenging ROS, thereby exerting beneficial effects in several models:
during repetitive isometric contractions in both experimental animal (20; 38) and human studies
(34), in limb muscles of patients with severe COPD(21), in endotoxemic rats(45), and in the
diaphragmatic dysfunction of streptozotocin-induced diabetic rats (18). In view of these
published findings it would be possible to conclude that ROS directly synthesized within the
MRC, as shown to be the case in other conditions (15; 19; 31-33), could have a predominant
role in MRC function in muscles of cancer cachectic rodents. Indeed, the rise in oxygen
consumption detected among the cachectic muscles in response to concomitant treatment with
the antioxidant NAC points towards this conclusion. The signaling pathway NF-κB and MAPK
have also been shown to induce enhanced muscle proteolysis through a redox-sensitive
mechanism in several atrophying conditions (28), in sepsis (40), and in cardiomyocytes exposed
to hypoxia (22). On the basis of these published results, it would be possibly concluded that
13
Results
inhibition of NF-κB, MAPK, and proteasome pathways may decrease ROS synthesis by the
MRC, thus making this structure more efficient in terms of oxygen consumption in muscles of
cachectic mice. Future investigations will shed light into the specific mechanisms whereby all
these cellular processes and pathways may impair MRC function in cancer cachexia.
Conclusions
Novel findings in the investigation are that both respiratory and limb muscles of cancer
cachectic mice exhibit a significant depression of the MRC complexes and oxygen
consumption. Concomitant treatment with the specific inhibitors of NF-κB and MAPK
signaling pathways restore MRC function in both types of muscles, while also significantly
improving body and muscle weights and force. These findings indicate that these pathways are
clearly involved in MRC impairment in cancer cachectic muscles. To a lesser extent, oxidants
and the proteasome system may also contribute to MRC dysfunction in cancer cachexia.
ACKNOWLEDGEMENTS
This study has been supported by CIBERES; FIS 11/02029; 2009-SGR-393; SEPAR 2010;
FUCAP 2011; FUCAP 2012; and Marató TV3 (MTV3-07-1010) (Spain). Dr. Esther Barreiro
was a recipient of the ERS COPD Research Award 2008. The authors are thankful to Dr.
Xavier Mateu for his help and advice with the pharmacological inhibitors, Dr. Juan MartinCaballero for his assistance with part of the animal experiments, and Mrs. Mònica Vilà-Ubach
and Alba Chacón-Cabrera for their technical support with part of the laboratory experiments.
14
Results
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FIGURE LEGENDS
Figure 1:
Mean values and standard deviation of citrate synthase (CS) activity (nmol/min/mg) in
diaphragm (white bars) and gastrocnemius (black bars) muscles (expressed as mean values
and standard deviation). CS activity levels did not significantly differ between LC
cachectic rodents and the controls in any of the muscles. A significant increase in
diaphragm CS activity was observed among LC cachectic rodents in response to NAC,
proteasome, NF-κB and MAPK inhibitors, while all four pharmacological agents elicited a
significant decline in CS activity in the gastrocnemius. Statistical significance is
represented as follows: i) n.s., non-significant differences in CS levels in any muscle
between LC cachectic and control mice and ii) n.s., non-significant, *: p<0.05, *: p<0.01,
and ***: p<0.001 levels in muscles between any group of LC cachectic mice treated with
each of the inhibitors and the LC cachectic animals without any pharmacological
treatment. The dashed line separates both types of comparisons between the groups.
Figure 2:
A) Mean values and standard deviation of complex I enzyme activity (nmol/min/mg) in
diaphragm (white bars) and gastrocnemius (black bars) muscles (expressed as mean values
and standard deviation). Complex I activity levels were significantly reduced in both
diaphragm and gastrocnemius muscles among LC cachectic rodents compared to the
controls. A significant increase in diaphragm complex I activity was observed among LC
cachectic rodents in response to proteasome, NF-κB, and MAPK inhibitors, while only
MAPK inhibitor elicited a significant increase in complex I activity in the gastrocnemius.
Statistical significance is represented as follows: i) †: p<0.05 and ††: p<0.01 in complex I
activity levels in any muscle between LC cachectic and control mice and ii) n.s., nonsignificant and *: p<0.05 levels in muscles between any group of LC cachectic mice treated
with each of the inhibitors and the LC cachectic animals without any pharmacological
treatment. The dashed line separates both types of comparisons between the groups.
B) Mean values and standard deviation of the ratio of complex I enzyme activity to CS activity
in diaphragm (white bars) and gastrocnemius (black bars) muscles (expressed as mean
values and standard deviation). Complex I/CS activity levels were significantly reduced in
both diaphragm and gastrocnemius muscles among LC cachectic rodents compared to the
controls. A significant increase in diaphragm and gastrocnemius complex I/CS ratios was
observed among LC cachectic rodents in response to NF-κB, and MAPK inhibitors, while
NAC or bortezomib did not induce any significant effect on diaphragm or gastrocnemius
20
Results
muscles among the LC cachectic rodents. Statistical significance is represented as follows:
i) †: p<0.05 and ††: p<0.01 in complex I/CS ratio levels in any muscle between LC
cachectic and control mice and ii) n.s., non-significant, **: p<0.01, and *: p<0.05 levels in
muscles between any group of LC cachectic mice treated with each of the inhibitors and the
LC cachectic animals without any pharmacological treatment. The dashed line separates
both types of comparisons between the groups.
Figure 3:
A) Mean values and standard deviation of complex II enzyme activity (nmol/min/mg) in
diaphragm (white bars) and gastrocnemius (black bars) muscles (expressed as mean
values and standard deviation). Complex II activity levels were significantly reduced in
both diaphragm and gastrocnemius muscles among LC cachectic rodents compared to
the controls. A significant increase in diaphragm complex II activity was observed
among LC cachectic rodents in response to proteasome and NF-κB inhibitors, while
MAPK inhibitor elicited an increase only in the gastrocnemius. Statistical significance
is represented as follows: i) †: p<0.05 in complex II activity levels in any muscle
between LC cachectic and control mice and ii) n.s., non-significant and *: p<0.05, and
**: p<0.01 levels in muscles between any group of LC cachectic mice treated with each
of the inhibitors and the LC cachectic animals without any pharmacological treatment.
The dashed line separates both types of comparisons between the groups.
B) Mean values and standard deviation of the ratio of complex II enzyme activity to CS
activity in diaphragm (white bars) and gastrocnemius (black bars) muscles (expressed
as mean values and standard deviation). The ratio of complex II/CS was significantly
reduced in both diaphragm and gastrocnemius among LC cachectic rodents compared to
the controls. A significant increase in gastrocnemius complex II/CS ratio was observed
among LC cachectic rodents in response to NF-κB and MAPK inhibitors, compared to
non-treated cachectic mice. Statistical significance is represented as follows: i) n.s.,
non-significant †: p<0.05 in complex II/CS ratio levels in any muscle between LC
cachectic and control mice and ii) n.s., non-significant and *: p<0.05, and **: p<0.01
levels in muscles between any group of LC cachectic mice treated with each of the
inhibitors and the LC cachectic animals without any pharmacological treatment. The
dashed line separates both types of comparisons between the groups.
Figure 4:
A) Mean values and standard deviation of complex IV enzyme activity (nmol/min/mg) in
diaphragm (white bars) and gastrocnemius (black bars) muscles (expressed as mean
21
Results
values and standard deviation). Complex IV activity levels were significantly reduced
in the diaphragm of LC cachectic rodents compared to the controls. A significant
increase in diaphragm complex IV activity was observed among LC cachectic rodents
in response to NAC, bortezomib, NF-κB and MAPK inhibitors, while the last inhibitor
also elicited an increase in the gastrocnemius. Statistical significance is represented as
follows: i) n.s., non-significant and †: p<0.05 in complex II activity levels in any
muscle between LC cachectic and control mice and ii) n.s., non-significant and *:
p<0.05, **:p<0.01, and ***: p<0.001 levels in muscles between any group of LC
cachectic mice treated with each of the inhibitors and the LC cachectic animals without
any pharmacological treatment. The dashed line separates both types of comparisons
between the groups.
B) Mean values and standard deviation of complex IV/CS ratio in diaphragm (white bars)
and gastrocnemius (black bars) muscles (expressed as mean values and standard
deviation). Complex IV/CS ratios were significantly reduced in the diaphragm of LC
cachectic rodents compared to the controls. Among LC cachectic rodents, a significant
increase in complex IV/CS ratios was observed in both respiratory and limb muscles in
response to NAC, proteasome, NF-κB and MAPK inhibitors. Statistical significance is
represented as follows: i) n.s., non-significant and †: p<0.05 in complex IV/CS ratio
levels in any muscle between LC cachectic and control mice and ii) *: p<0.05,
**:p<0.01, and ***: p<0.001 levels in muscles between any group of LC cachectic mice
treated with each of the inhibitors and the LC cachectic animals without any
pharmacological treatment. The dashed line separates both types of comparisons
between the groups.
Figure 5:
A) Mean values and standard deviation of state 3 respiration (nmol O2/min/µg) in
diaphragm (white bars) and gastrocnemius (black bars) muscles (expressed as mean
values and standard deviation). State 3 levels were significantly reduced in both
diaphragm and gastrocnemius muscles among LC cachectic rodents compared to the
controls. A significant increase in state 3 was detected in both respiratory and limb
muscles among LC cachectic rodents in response to NAC, proteasome, NF-κB, and
MAPK inhibitors compared to non-treated cachectic rodents. Statistical significance is
represented as follows: i) †: p<0.05 in state 3 levels in any muscle between LC
cachectic and control mice and ii) *: p<0.05, **:p<0.01, and ***: p<0.001 levels in
muscles between any group of LC cachectic mice treated with each of the inhibitors
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Results
and the LC cachectic animals without any pharmacological treatment. The dashed line
separates both types of comparisons between the groups.
B) Mean values and standard deviation of state 4 respiration (nmol O2/min/µg) in
diaphragm (white bars) and gastrocnemius (black bars) muscles (expressed as mean
values and standard deviation). State 4 levels did not significantly differ among any of
the study groups for any of the analyzed muscles. Statistical significance is represented
as follows: i) n.s., non-significant in state 3 levels in any muscle between LC cachectic
and control mice and ii) n.s., non-significant levels in muscles between any group of
LC cachectic mice treated with each of the inhibitors and the LC cachectic animals
without any pharmacological treatment. The dashed line separates both types of
comparisons between the groups.
C) Mean values and standard deviation of the respiratory control index (RCI, state 3/state
4) in diaphragm (white bars) and gastrocnemius (black bars) muscles (expressed as
mean values and standard deviation). RCI levels did not significantly differ between LC
cachectic mice and the controls. RCI levels were significantly greater in the diaphragms
of cachectic rodents concomitantly treated with NAC, bortezomib, and either NF-κB or
MAPK inhibitors, while an increase in RCI was observed in the gastrocnemius of the
cachectic animals only treated with NF-κB or MAPK inhibitors, but not NAC or
bortezomib. Statistical significance is represented as follows: i) n.s., non-significant in
RCI levels in any muscle between LC cachectic and control mice and ii) n.s., nonsignificant and *: p<0.05 and **: p<0.01 RCI levels in muscles between any group of
LC cachectic mice treated with each of the inhibitors and the LC cachectic animals
without any pharmacological treatment. The dashed line separates both types of
comparisons between the groups.
D) Mean values and standard deviation of complex IV oxygen consumption (nmol
O2/min/µg) in diaphragm (white bars) and gastrocnemius (black bars) muscles
(expressed as mean values and standard deviation). Complex IV oxygen consumption
levels were significantly reduced in both diaphragm and gastrocnemius muscles among
LC cachectic rodents compared to the controls. A significant increase in complex IV
oxygen consumption was detected in both respiratory and limb muscles among LC
cachectic rodents in response to NAC, proteasome, NF-κB, and MAPK inhibitors
compared to non-treated cachectic rodents. Statistical significance is represented as
follows: i) †: p<0.05 in complex IV oxygen consumption levels in any muscle between
LC cachectic and control mice and ii) *: p<0.05, **:p<0.01, and ***: p<0.001 levels of
23
Results
complex IV oxygen consumption in muscles between any group of LC cachectic mice
treated with each of the inhibitors and the LC cachectic animals without any
pharmacological treatment. The dashed line separates both types of comparisons
between the groups.
24
Results
25
Results
26
Results
27
Results
28
Results
29
Results
30
Results
Table 1. Physiological characteristics in all groups of animals at the end of the
study period.
Lung cancer cachexia
Control
LC cachexia
Antioxidant
Proteasome
inhibitor
†††
-10.03 (11.9)
-9.72 (9.81)
-0.11 (6.43)
0.079 (0.01)
Body weight gain (%)
+9.5 (3.9)
-5.74 (8.61)
Diaphragm weight (g)
0.087 (0.01)
0.06 (0.01)
†††
0.067 (0.01)
0.07 (0.01)
Gastrocnemius weight (g)
0.12 (0.008)
0.09 (0.01)
†††
0.09 (0.014)
0.085 (0.01)
Limb strength gain (%)
+10.1 (14.6)
-9.04 (9.3)
†††
-6.96 (14.4)
-12.8 (21.4)
NA
1.68 (0.47)
†††
1.48 (0.45)
1.08 (0.6)
Sc. Tumor weight (g)
***
NF-κB
inhibitor
MAPK
inhibitor
*
***
*
0.098 (0.01)
+5.12 (13.6)
1.19 (0.41)
***
***
-0.24 (5.04)
*
0.074 (0.012)
0.098 (0.01)
+3.47 (12.7)
0.84 (0.57)
Variables are presented as mean (SD).
Definition of abbreviations: LC, lung cancer; NF-κB, nuclear factor-κB; MAPK, Mitogenactivated protein kinases; Sc., subcutaneous, NA, not applicable.
Statistical significance:
†††,
*
p≤0.001 between LC cachectic and control mice; * p≤0.05 and ***,
p≤0.001 between any of the treated mouse groups with cachexia and LC cachexia only animals.
31
*
***
***
DISCUSSION
In the present PhD thesis, three different models of cachexia have been employed using two
different approaches: i) human studies conducted on patients with severe COPD and cachexia
and ii) animal experiments of cachexia: lung emphysema and lung cancer-induced cachexia. The
discussion section has been organized in such a way that results encountered in each specific
investigation are being interpreted and discussed independently together with a general
discussion, in which an attempt to link the main findings of each study has been made, thus
providing a general overview.
Muscle mass and function
In all three models a loss of body weight was observed. In the mouse models, we could also
report a decrease in the gastrocnemius and diaphragm muscle weights, thus demonstrating
that the body weight loss was not only a consequence of reduced fat content,which was totally
absent in both animal models, but also due to a loss of protein content. This reduction in muscle
mass is a major cause of the muscle dysfunction observed in many chronic diseases associated
with cachexia that leads to reduced exercise capacity of the patients, a poor quality of life
and increased mortality risk (155, 168-170). Both severe COPD patients and LC cachectic mice
showed a decrease in limb strength (it was not possible to obtain this information from the mice
with emphysema). These results are in agreement with previous studies COPD patients (170)
and in animal cachexia models (171), in which a reduction of limb strength was also observed. In
the case of the COPD study, compared to control subjects, both groups of severe COPD patients
exhibited reduced quadriceps muscle force and exercise capacity, being the latter dramatically
impaired in the muscle-wasted patients. These results are probably due to the important effect
of hypoxia in the muscle atrophy (172), in fact, muscle mass (FFMI) was shown to be directly
associated with disease severity as measured by FEV1.
Muscle structure
COPD muscle-wasted patients showed a decrease in the proportion of slow-twitch fibers, leading
to a less resistant phenotype. These results are in agreement with previous investigations in COPD
patients with abnormal body composition (139), in which this decrease of slow-twitch fibers was
also observed. LC cachexia mice did not show any differences on the fiber type proportions in
diaphragm or gastrocnemius. A previous study from our group performed with cachectic rats
showed similar results (59). This lack of change in the fiber distribution in the LC cachexia mice
could be due to the animal model is semi-acute (30 days). COPD patients, representing a chronic
model, showed a change in their fiber type distribution. Probably the fibers in the LC cachexia
47
Discussion
mice may require more than thirty days to change their distribution.
COPD muscle-wasted patients showed a reduction in the fast-twitch fibers area size. These
results are consistent with a previous study with COPD patients (139) in which COPD patients
showed a decrease in the fibers type I, IIa and IIb area size. In LC cachectic mice, a decrease in the
size of both slow- and fast-twitch fiber types in both diaphragm and gastrocnemius muscles was
observed. All these findings are signs of muscle atrophy and are in line with earlier investigations
from our group (59) and other investigators (173).
All severe COPD patients exhibited increased levels of muscle abnormalities. This
parameter was measured by the identification of internal nuclei, inflammatory cells, lipofuscin,
abnormal viable cells and inflamed/necrotic cells. Again this fact seems to be closely related to
the COPD severity. LC cachexia mice showed increased abnormal muscle levels and apoptotic
nuclei in both gastrocnemius and diaphragm muscles, as previous studies have reported before
in cachexia animal models (174).
Key muscle proteins content
As described in the introduction section above, myosin is the most abundant protein of the
skeletal muscle and is essential for its correct functioning. A decrease in myosin content was
observed in the quadriceps of muscle-wasted COPD patients, the diaphragm of emphysematous
mice, and in both the diaphragm and gastrocnemius of LC cachectic mice. Actin levels, however,
did not differ between disease and control subjects in any of the models. These results are in
agreement with previous publications of our group that reported a decrease of myosin levels in
the diaphragms of COPD patients, but no differences in actin content(161). The levels of other
key muscle proteins, which were previously shown to be targeted by oxidants and proteolytic
systems (99, 175), have also been determined: carbonic anhydrase III levels were decreased in all
COPD patients. CK levels were decreased in all COPD patients and in the gastrocnemius of the LC
cachexia mice. These results are in agreement with a previous study of our group (52).
Redox balance
There exist many studies in which oxidative stress have been proposed as a major mechanism
involved in the wasting of the skeletal muscle observed in cachexia, both in COPD (50, 51, 54-56)
and in cancer cachexia models (57, 176-178). In the muscles of our different models, an increase
in the levels of oxidants was observed, which was in line with the previous studies (50, 51, 5457, 176-178). Interestingly, in the muscle-wasted COPD patients, it was observed an increase in
the mitochondrial O2•- levels, what is in line with previous studies that propose that the origin of
this oxidative stress could be in the mitochondria (83, 179). It is interesting also to comment the
fact that increased protein carbonylation (including myosin), nuclear, membrane and cytosolic
O2•- was detected in all the severe COPD patients, and not only in the muscle-wasted. These
findings lead to the point that this phenomenon could be highly related to the COPD severity.
The fact that only muscle-wasted COPD patients showed an increased O2•- production in the
mitochondria could be a sign indicating that oxidants from the mitochondria are the trigger that
48
Discussion
are involved in muscle-wasting in cachexia. In the case of the LC cachexia mice, all the treatments
reduced the levels of protein carbonylation, which in the case of the NF-κB and MAPK inhibitors
means that oxidative stress could have an important role in muscle wasting.
The antioxidant mechanisms are more controversial. In the case of the muscle-wasted
COPD patients, an increase of both Mn- and CuZn-SOD was detected. The emphysematous
mice showed in their diaphragms a decrease in Mn-SOD and an increase in peroxiredoxin II,
and in the gastrocnemius, a decrease of peroxiredoxin III. Finally, the LC cachectic mice showed
a decrease in both diaphragm and gastrocnemius of Mn-SOD (which was corrected by NF-κB
and MAPK inhibitors). Regarding the fact that oxidative stress coming from the mitochondria
seems to be the one implicated in the muscle wasting, mitochondrial antioxidant systems (MnSOD and peroxiredoxin III) seem to be important. On one hand, in both the gastrocnemius and
diaphragm of emphysematous and LC cachexia mice the mitochondrial antioxidant systems are
decreased, this could explain in part the increased oxidative stress observed in these models. On
the other, Mn-SOD protein levels are increased in the quadriceps of the muscle-wasted COPD
patients, this could be explained as a compensatory mechanism of the cell to try to counter the
increased levels of O2•- produced in the mitochondria, this results are in line with those observed
in previous studies (180, 181).
The reduced Mn-SOD levels observed in the LC cachexia mice were increased in the
gastrocnemius muscle by the NF-κB and MAPK inhibitors, indicating that these mechanism could
be important to reestablish the muscle mass. Oxidative stress in known to directly damage
proteins, membranes and DNA, but it also has an important role in the activation of different
pathways that are supposed to be involved the muscle-wasting mechanisms, like the NF-κB
pathway (57). The fact that the two treatments that reported physiological benefits are reducing
the oxidative stress levels and increasing the mitochondrial antioxidants could indicate that
oxidative stress has an important role in the process of muscle wasting.
Inflammation
Levels of different markers of molecular inflammation were studied in the skeletal muscles of
the COPD patients and the LC cachexia mice. IFN-γ, TNF-α and VEGF levels did not show any
differences in the quadriceps of the severe COPD patients, this results are consistent with a
previous study of our group (73) in which there were no differences for IL-6, IL-1β, INF-γ and
TGF-β in the quadriceps of severe COPD patients, and even the levels of TNF-α were found to
be lower compared to the control group. In the LC cachexia mice it was observed an increase
of INF-γ levels in the diaphragm (decreased by the MAPK treatment), but no differences were
observed for TNF-α, IL-6 or IL-1β in the diaphragm or gastrocnemius of these mice. Little is known
about the local production of cytokines in cachectic skeletal muscle, but even tough cytokines
are thought to play an important systemic role in the tumor derived cachexia (69), another study
showed no differences for systemic levels of TNF-α, IFN-γ and IL-6 in lung cancer and colorectal
cancer patients (182). From our results we could say that inflammatory cytokines don’t seem to
have a very important role in the skeletal muscle wasting.
49
Discussion
Proteolysis
Both diaphragm and gastrocnemius muscles of LC cachexia mice showed an increase of the
protein degradation measured by the release of tyrosine. This measure couldn’t be performed
in the other two models, but the fact that muscle-wasted COPD patients showed a decrease in
myosin content, and that the muscles of emphysematous mice had a reduced weight compared to
controls, makes us think that a protein loss is also taking place in these cases. Several studies have
tried to elucidate which are the mechanisms responsible of this reduction in protein content, but
are still unclear. As the total protein content is the result from the balance of proteins synthesis
(anabolism) and degradation (catabolism), a malfunction of one, ore both, sides of the balance
could result in the imbalance. Different studies have shown the importance of anabolism in the
cachexia syndrome (171, 183), and others have demonstrated how catabolism is also involved
(99, 100, 102, 110, 184, 185). Despite the relevance of the study of anabolism in muscle wasting,
the investigations included in the current PhD thesis have exclusively focused on the assessment
of the proteolytic systems involved in the cachexia syndrome.
Most of the studies published so far have focused on proteasome as the main mechanism
involved in cachexia. Our results in the different models show that though proteasome system
seems to be increased (increased ubiquitinated proteins and E3 ligases in muscle-wasted
COPD patients and LC cachexia mice), it doesn’t exist a clear increase in the protein levels of
the 20S subunit of the proteasome in any of the models and even the emphysematous mice
didn’t show any difference related to the proteasome system. These results are not in line with
previous published works (99, 110). Interestingly, we have reported that the LC cachexia mice
treated with the proteasome inhibitor showed no improvement in body and muscles weight, or
limbs strength. These results are in line with those reported in a previous study (186), in which
treatment with proteasome inhibitors did not prevent diaphragm muscle loss and strength in
endotoxemic rats. Likewise these results are not in agreement with those reported in other two
studies, in which diaphragm function improved in response to bortezomib in emphysematous
and congestive heart failure rats (187, 188).
In view of the results we concluded that proteasome does not seem to be the main
responsible mechanism for the protein loss observed in our models. Its inhibition, although
generating an increase in the area size of slow-twitch fibers, did not solve the problem of protein
loss in the cachectic animals. These findings lead us to the point that some other mechanisms
involved either in protein anabolism or catabolism could have more relevance.
Autophagy is a catabolic process that involves the degradation of cytoplasmatic
components through the lysosomal pathway. In this thesis, it was only possible to study
autophagy in the LC cachexia mouse model. In the LC cachectic mice, an increase of autophagy
levels was observed. There exist a low number of studies about autophagy in cachexia models.
Our results are in line with a study in which autophagy levels were increased in an animal model
of endotoxin-cachexia (108). All the different treatments decreased the autophagy levels in
the LC cachexia mice. Again, on the one hand, proteasome inhibitor is blocking this pathway
50
Discussion
without physiological benefits. On the other, NF-κB and MAPK inhibitors seem to be improving
the noxious effects of cachexia.
Signaling pathways involved in cachexia
Myostatin inhibits cell cycle progression and reduces levels of myogenic factors, controlling
myoblastic proliferation and differentiation during myogenesis (112, 113). In all of the models,
either myostatin levels were shown to be increased or levels of myogenic factors were decreased.
These results are in agreement with previous studies in cancer cachexia models (189, 190) and
in muscle-wasted COPD patients (191). In all our models increased FoxO1 protein levels were
observed, which is in line with a previous study showing activation of FoxO1 signaling pathway
in a myostatin-induced cachexia model (192). It seems that FoxO1 is regulates myostatin mRNA
and its promoters (193), what seems to be happening in our cachexia models.
Interestingly, FoxO transcription factors are also associated with the transcription of
Atrogin1 and MURF1, the E3 ligases involved in the ubiquitin-proteasome system (100), this
could explain why in some of our models we detected an increase in the expression of E3 ligases,
but we did not appreciate a direct increase in the ubiquitin-proteasome system. It has been
demonstrated that the attenuation of the myostatin signaling improves the muscle wasting in
different cachexia models (191, 194), in these models, the myostatin signaling inhibition was
performed by the blockage of the specific myostatin receptor ActRIIB. ActRIIB ligands also
include growth and differentiation factor-11 and activins (195), and so the protective effect of
myostatin signaling through the inhibition of its receptor could be due to the inhibition all of
these different targets.
LC cachexia mice shown an increase in the myostatin and a decrease in the myogenin
levels, all the different inhibitors improved these events. In the case of the proteasome inhibitor,
maybe this effect was not enough to compensate the muscle loss. In the case of cachexia mice
elicited an improvement in the body and muscles weigh and in the strength, thus this could
make us think that this reduction in myostatin and increase in the myogenin levels could be
contributing, together with other mechanisms, to the reduction of the muscle-wasting effects
of cachexia.
NF-κB pathway was shown to have increased protein levels in all the cachexia models,
these results are in line with previous studies in which there was an enhanced NF-κB signaling
in the skeletal muscle of different cachexia models (106, 196). In the gastrocnemius of the LC
cachexia mice it was also observed an increased transcription of NF-κB, which was reduced with
the proteasome and NF-κB inhibitors. In these mice it was also observed that the levels of the
NF-κB inhibitors IκB were reduced in both the diaphragm and the gastrocnemius muscles, what
could be the cause of the increased expression of NF-κB, all the treatments increased the levels
of the inhibitors.
In the LC cachexia mice, the inhibition of the NF-κB pathway with sulfasalazine reported
important physiological improvements, what indicates that this pathway is implicated in an
important way in the wasting of the skeletal muscles observed in cachexia.
51
Discussion
MAPK cascade play a key role in signaling, it acts phosphorylating protein kinases and
transcription factors, what leads to their activation. In the present thesis, JNK, ERK2, p-ERK1/2,
p38 and p-p38 protein levels were analyzed in the different cachexia models. In the quadriceps
of the COPD patients, no differences were observed for the MAPK levels, suggesting that in
this specific cachexia model, MAPK would not have an important role in the muscle wasting
generation. In the emphysema mice, an increase of ERK2 was observed in the diaphragm and
in the gastrocnemius it was detected an increase of JNK. Finally, in the LC cachexia mice, it was
detected and increase in the p38 levels in both diaphragm and gastrocnemius. The results of
these two last models suggest that MAPK could have an implication in the generation of muscle
wasting.
LC cachexia mice treated with MAPK inhibitor showed a reduction of the p38 levels in both
muscles and reported important physiological improvements. As we have commented before,
this inhibition generated also a muscle structure and composition important improvement
(increase of the size of type I and II fibers, reduction of muscle abnormalities and reduction
of apoptosis). All these results are in line with a previous study in which muscle mass loss was
restored by inhibiting ERK activity in colon cancer mice (197). These important benefits suggest
that the MAPK pathways, implicated in many different cellular mechanisms, are modulating the
loss of protein content in this cachexia model.
Mitochondrial dysfunction
The mitochondria are the major organelles responsible of the ATP production in the cell. As we
have commented in the “oxidative stress” section, mitochondria seem to be the main source of
O2•- in the wasted muscles (82, 83). For this reason we thought that it would be interesting to
study the functionality of the mitochondria respiratory chain in the LC cachexia mice.
In was detected a clear reduction in the activity of the MRC complexes I, II and IV in both
diaphragm and gastrocnemius muscles. These results are in line with previous studies in which
muscle dysfunction leaded to a decreased MRC enzymes activity in the skeletal muscles of COPD
patients (82, 83, 179).
NF-κB and MAPK inhibitors generated an improvement in the activity of all the
complexes, indicating that these pathways could be implicated in the mitochondrial biogenesis
or in the dysfunction of the studied MRC enzymes. In a previous study it was shown that the
NF- κB pathway was implicated in the reduction of mitochondrial biogenesis and content (198),
p38 MAPK, which was shown to be increased in our LC cachexia mice, was demonstrated to be
implicated in the decrease of mitochondrial biogenesis in the muscles of obese mice (199).
The proteasome inhibitor induced an improvement of the activity of complexes I,
II and IV in the diaphragm of LC cachexia mice. As we have commented in previous sections,
proteasome inhibitor has had potential beneficial effects in the LC cachexia treated mice, but
at the end this inhibition did not elicit a clear physiological improvement. Proteasome pathway
has been shown to be partially increased in the LC cachexia mice, and it could be implicated in
the loss of mitochondria content or function, thus its inhibition could report an increase in the
52
Discussion
activity of the enzymes.
The treatment of LC cachexia mice with the antioxidant NAC only reported an
improvement in the activity of complex IV as quantified by ratio to CS, but not as absolute values.
The CS activity was used to quantify the total mitochondrial content, there were no
differences between the LC cachexia mice and the control group for the CS activity. This indicates
that all the dysfunction observed in the different complexes were due to the specific enzymes
and not to a decrease in the mitochondria content. The four treatments elicited different effects
in both muscles. On the one hand, in the diaphragm it was shown an increase of CS activity. On
the other, gastrocnemius presented a decrease of the activity. These differences could be due to
the different activity performed by the both muscles.
A decrease in the oxygen consumption of complexes I (state 3) and IV was observed in
diaphragm and gastrocnemius of the LC cachexia mice compared to the control mice. These
results are consistent to the previous commented about the activities and are in line with
those reported in the previous studies in COPD patients (82, 83, 179). The decreased oxygen
consumptions were increased by all the treatments, thus it indicates that all the inhibited
pathways (proteasome, NF-κB and MAPK) and oxidative stress are implicated in the dysfunction
of the MRC. Further investigations would be necessary to elucidate how these different markers
are involved in the mitochondria dysfunction.
Subcutaneous tumor
In the LC cachexia mice, as it was brief commented in the methodology section, the inoculation
of the LP07 cells was performed subcutaneously in the left flank. As a consequence of this
type of inoculation, at the end of the thirty days of study all the inoculated mice developed
a subcutaneous tumor in the place of inoculation. It has been very interesting to see how the
different treatments showed a reduction in the tumor weights, the MAPK inhibitor elicited the
biggest reduction (60%), followed by the NF-κB inhibitor (27%) and the proteasome inhibitor
(18%). The present work was focused on the study of the skeletal muscles of these cachectic
mice, and because of this at the present we cannot explain the mechanisms that generate these
reductions in the tumor size. Studies are already being performed in order to study which are
the molecular mechanisms involved in the subcutaneous tumor of the LC cachexia mice and in
their treatments.
To summarize, the three models have shown a decrease in the body weight that can be
attributable to a loss of muscle mass. Muscle-wasted subjects also elicited a reduced muscle
function and an affected muscle structure. Despite previous studies, proteasome does not seem
to be the main mechanism responsible of muscle wasting. Some other mechanisms, such as
autophagy and the myostatin-FoxO1 pathway, have been implicated in the three models. NF-κB
and MAPK pathways, and oxidative stress were increased in the muscle wasting subjects and
they seem to have a great implication in the modulation of the muscle wasting.
53
54
STUDY LIMITATIONS
The main limitations encountered in the four studies are summarized as follows.
Limitations in study #1
First, due to the lack of histological preparations (because of the small size of muscle biopsies)
it was impossible to analyze the structural characteristics of the muscles. In this study, priority
was given to the analysis of the molecular mechanisms involved in the muscle mass loss. In
futures studies, it would be interesting to describe structural changes that may be present in the
respiratory and peripheral muscles in mice with emphysema.
Limitations in study #2
A major limitation in this investigation was that patients with muscle wasting were also those
showing worse lung function. As we have described in the introduction section of the current PhD
thesis, weight loss and increasing severity of COPD are interrelated phenomena. This association
is linked to poor prognosis of the disease. In fact, progressive disease severity and nutritional
depletion have already been shown to be associated. Future studies will help to elucidate the
direct contribution of the lung disease to the weight loss.
Limitations in study #3
We focused mainly our investigations on the study of the proteolytic mechanisms leading to
muscle loss and dysfunction. In future studies it would be very interesting to assess to what
extent anabolism may also be affected in the cachexia model of LC employed in this investigation.
It will also be of relevance to explore whether the different treatments used in this study, which
have been shown to improve the physiological parameters and body and muscle weights, may
also influence the anabolic pathways.
Limitations in study #4
A limitation in this study was that ROS were not measured in the mitochondrial preparations. It
is likely that the beneficial effects induced by the different inhibitors may have been mediated,
at least to some extent, by a reduction in ROS synthesis by the MRC. In future investigations, ROS
levels specifically synthesized by the MRC will be identified.
55
56
CONCLUSIONS
From the different studies, we could conclude that molecular mechanisms involved in the
generation of cachexia in our three models are not so different, despite the fact that different
conditions were studied. All the three models showed decreased muscle mass, muscle
dysfunction and similar modifications in the structure. From the studied pathways, NF-κB and
MAPK have shown to be main in the process of muscle wasting. The physiological improvements
obtained from their inhibition in the LC cachexia model corroborate their important implication.
Further studies about the implication of these two pathways should be performed to try to
elucidate their role in the muscle wasting process.
The ubiquitin-proteasome system was shown not to be the main proteolytic system. LC
cachexia mice did not show physiological benefits from its inhibition. Other catabolic systems,
such as autophagy and myostatin showed increased levels, and myogenin showed decreased
levels. These mechanisms may have important roles in the process of muscle wasting in the
three models. It would be interesting to further study these catabolic systems in the cachexia
models.
Future investigations could be performed using the LC cachexia model together with
the inhibition of myostatin and autophagy systems. Oxidative stress was demonstrated to be
increased in all the cachexia models, and muscle-wasted COPD patients showed that its main
origin could be the mitochondria.
Finally, the study of the functionality of the mitochondria in the LC cachectic mice have
elucidated that there exists a dysfunction in the activity of the MRC enzymes as well and of the
oxygen consumption, and this dysfunction could be the origin of the increased O2•- observed
in the mitochondria. New investigations could be carried out to further the origin of oxidative
stress.
57
58
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ADDENDUM
Apart from the four studies that compose the current PhD thesis, during the four years that I
have been working in the Molecular Mechanisms of Lung Cancer Predisposition group, under
the supervision of Dr. Esther Barreiro, I had the opportunity to participate in other studies.
The study that follows has recently been submitted to a Respiratory Medicine Journal.
We decided not to include that study in the PhD thesis because its contents were far beyond
the scope of the current PhD thesis.
Barreiro E, Fermoselle C, Sánchez-Font A, Pijuan L, Gea J. Curull V.
Molecular events within the normal bronchial epithelium of patients with lung cancer and
COPD.
Publication: Submitted.
Finally, it should be mentioned that in the last four years I have also participated in other
ongoing investigations conducted in the same research group. However, we are still finishing
the molecular and statistical analyses in hopes that they will ready for submission next year.
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