Download Analysis of cell division in cardiomyocytes during development and

DISS. ETH No. 16518
Analysis of cell division in cardiomyocytes
during development and disease
A dissertation submitted to the
SWISS FEDERAL INSTITUTE OF TECHNOLOGY
ZÜRICH
for the degree of
Doctor of Natural Sciences
presented by
PREETI AHUJA
M.Sc (Hons) Microbiology, Panjab University, India
born 11.03.1975
citizen of India
accepted on the recommendation of
Prof. Dr. Jean-Claude Perriard, examiner
Dr. Elisabeth Ehler, co-examiner
Prof. Dr. Matthias Peter, co-examiner
2006
ACKNOWLEDGEMENTS
First and the foremost, I would like to express my special gratitude to my supervisors JeanClaude Perriard and Elisabeth Ehler for their whole hearted support that they conferred
through out my stay in the laboratory. I thank Jean-Claude for giving me the opportunity to
carry out my research in his laboratory and for his helpful comments and support all the way
through. Elisabeth had been extremely instrumental in shaping every aspect of my thesis work
and without her keen interest, remarkable scientific expertise and friendly supervision my
thesis would not have seen this day of light. I feel proud and privileged to have her as my
adviser. Sincerely, I can never thank her enough for her constant support and assistance.
I would like to thank Evelyne Perriard for help with cultures of cardiomyocytes and for all
those warm and friendly discussions which I had with her during this time.
During my stay in Jean-Claude’s laboratory, I came in contact with several fascinating people
with whom I shared great companionship. Many, many thanks to Irina, Jaya, Alain, Stephan,
Ruslan and Roman for creating a great atmosphere and good teamwork over the years.
I would like to express my special thanks to Jaya Krishnan for critically reviewing my
dissertation and for his support at many levels through out my work.
I am also grateful to all my collaborators: Dr. Izabela Sumara, ETH, Zürich; Dr. João Relvas,
ETH, Zürich; Prof. Matthias Peter, ETH, Zürich; Prof. William Trimble, University of
Toronto, Canada; Prof. Thierry Pedrazzini, University of Lausanne Medical School,
Switzerland and Prof. Shinji Satoh, Kyushu University, Japan.
I am also thankful to all my project granting agencies: ETH and SNF (grant number
31.63486/00), the Gebert-Rüf Foundation, the SUK Grant to the Swiss Cardiovascular
Teaching and Research Network, SCARTNet and the Swiss Foundation on Research of
Muscle Diseases to J-C Perriard.
Last but certainly not the least; I would like to let my family know how much they mean to
me and that without their support and encouragement I would have not come that far. I am
especially thankful to my husband Umesh for his continuous support and encouragement and
i
for being there and keeping up my spirits in difficult situations. I am greatly indebted to them,
for which I can never thank them enough.
I dedicate my thesis to my loving parents and to my dear husband.
ii
TABLE OF CONTENTS
ABSTRACT ............................................................................................................................. 1
ZUSAMMENFASSUNG ........................................................................................................ 3
CHAPTER 1
Introduction ............................................................................................................................. 5
1.1 The heart.......................................................................................................................... 6
1.1.1 Heart development .................................................................................................... 6
1.1.2 The contractile tissue of the heart.............................................................................. 7
1.2 Establishment of cardiac cytoarchitecture in the developing heart............................... 10
1.2.1 Myofibrillogenesis in developing cardiomyocytes in situ ...................................... 10
1.2.2 Development of cell polarity during cardiomyocyte differentiation....................... 10
1.3 Differentiation and proliferative capacity of cardiac and skeletal muscle cells............ 11
1.4 Cardiac growth during development ............................................................................ 12
1.4.1 Hyperplasia and hypertrophy .................................................................................. 12
1.4.2 Cardiomyocyte cell cycle regulation ....................................................................... 13
1.4.2.1 Basic events of mammalian cell cycle............................................................... 13
1.4.2.2 Cardiomyocyte cell cycle activity during development .................................... 15
1.5 Myofibrillar and cytoskeletal organization of cardiomyocytes undergoing mitotic
division .......................................................................................................................... 17
1.5.1 Regulation of myofibrillar organization during division ....................................... 17
1.5.2 Factors regulating myofibrillar organization during division ................................. 17
1.5.3 Ubiquitin and calpain dependent degradation pathways in muscle......................... 18
1.6 Cytokinesis and binuclearity of cardiomyocytes........................................................... 21
1.6.1 Major events contributing to cytokinesis ............................................................... 21
1.6 2 Interplay between microtubule and actin network during cytokinesis.................... 23
1.6.3 Actomyosin based contractile ring .......................................................................... 23
1.6.4 Factors regulating the formation of the contractile ring ......................................... 24
1.6.5 Rho GTPases as molecular switches in cytokinesis ................................................ 24
1.6.6 Role of septins in actomyosin ring formation ......................................................... 28
1.7 Cardiomyopathies.......................................................................................................... 29
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Table of contents
1.7 1 Classification of cardiomyopathies ......................................................................... 29
1.8 Regenerative processes in myocardium during cardiomyopathies .............................. 33
1.8.1 How do Newt and Zebrafish heart regenerate? ....................................................... 33
1.8.1.1 Plasticity in the Newt limb ............................................................................... 33
1.8.1.2 Plasticity in the Newt and Zebrafish heart ........................................................ 34
1.8.2 Reactivation of DNA synthesis in the adult mammalian heart ............................... 35
1.9 Attempts to stimulate myocardial regeneration in higher vertebrates ......................... 36
1.9.1 Promoting cell cycle re-entry in cardiomyocytes.................................................... 36
1.9.2 Cellular transplantation ........................................................................................... 37
1.9.3 Cardiac progenitor cells........................................................................................... 39
1.10 Aim of the study ......................................................................................................... 40
1.10.1 Characterization of cardiomyocyte proliferation in the embryonic heart ............. 40
1.10.2 Determination of the mechanisms that cause uncoupling of cytokinesis from
karyokinesis after birth in cardiomyocytes ........................................................... 40
1.10.3 Determination of the cytokinetic potential in cardiomyocytes during
development and disease....................................................................................... 41
CHAPTER 2
Sequential myofibrillar breakdown accompanies mitotic division of mammalian
cardiomyocytes ...................................................................................................................... 43
2.1 SUMMARY .................................................................................................................. 44
2.2 RESULTS...................................................................................................................... 45
2.2.1 Myofibrillar disassembly in cultured cardiomyocytes during cell division............ 45
2.2.2 Myofibrillar disassembly occurs in a sequential and biphasic manner................... 45
2.2.3 Myofibril disassembly also occurs in dividing cardiomyocytes in situ .................. 50
2.2.4 How is myofibril disassembly regulated? ............................................................... 54
2.3 DISCUSSION ............................................................................................................... 57
CHAPTER 3
Probing the role of septins in cardiomyocytes .................................................................... 62
3.1 SUMMARY .................................................................................................................. 63
3.2 RESULTS...................................................................................................................... 64
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Table of contents
3.2.1 Expression of septins in heart during development................................................. 64
3.2.2 Localization pattern of different septins in cultured embryonic rat cardiomyocytes
during cytokinesis .................................................................................................. 66
3.2.3 Effect of a cytokinetic inhibitor on septin localization in embryonic rat
cardiomyocytes........................................................................................................ 69
3.2.4 Effect of Cytochalasin D on SEPT2 localization in embryonic rat cardiomyocytes
during cytokinesis ................................................................................................... 69
3.2.5 Interference with different parts of the cytoskeleton has distinct effects on
SEPT2 and non muscle myosin IIB distribution in embryonic cardiomyocytes .... 72
3.3 DISCUSSION ............................................................................................................... 74
CHAPTER 4
Analysis of the cytokinetic potential of cardiomyocytes during development
and disease ............................................................................................................................. 78
4.1 SUMMARY .................................................................................................................. 79
4.2 RESULTS...................................................................................................................... 80
4.2.1 Expression and localization of several actomyosin ring associated proteins in
the heart during development.................................................................................. 80
4.2.2 Re-expression of cytokinesis associated proteins in heart during hypertrophy ...... 83
4.2.2.1 β-adrenergic stimulated and hypertension induced one-kidney, one clip
(1K1C) mouse heart .......................................................................................... 83
4.2.2.2 Hypertension-induced hypertrophy of Dahl salt sensitive rats ......................... 84
4.2.2.3 Angiotensin over-expressing mice characterized by hypertrophy .................... 86
4.2.3 Localization of upregulated cytokinetic protein markers in diseased heart ............ 88
4.2.4 Re-expression of cytokinesis associated proteins in an in-vitro model of
hypertrophy ............................................................................................................ 88
4.2.5 Subcellular distribution of RhoA in primary cultures of neonatal rat
cardiomyocytes following the treatment with hypertrophy inducing agents .......... 91
4.2.6 Binucleation in hypertrophic neonatal rat cardiomyocytes..................................... 91
4.3 DISCUSSION ............................................................................................................... 94
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Table of contents
CHAPTER 5
Additional results .................................................................................................................. 97
5.1 Role of calcium dependent calpain mediated degradation in dividing embryonic
rat cardiomyocytes ........................................................................................................ 98
5.2 Expression of calpain-1 in heart during development................................................... 98
5.3 Disassembly of myofibrils delayed after treatment with NCO-700............................ 100
5.4 Role of Cullin3 in dividing embryonic rat cardiomyocytes........................................ 101
5.5 Subcellular distribution of Cullin3 in dividing embryonic rat cardiomyocytes .......... 102
5.6 Re-expression of Cullin3 in heart during hypertrophy................................................ 104
CHAPTER 6
General discussion and outlook ......................................................................................... 106
CHAPTER 7
Material and methods ......................................................................................................... 111
7.1 Isolation and culture of embryonic (ERC) and neonatal rat cardiomyocytes
(NRC) .......................................................................................................................... 112
7.2 Fixation and staining of cultured cardiomyocytes ...................................................... 112
7.3 PFA-fixed heart whole mount preparations ................................................................ 112
7.4 Antibodies used for immunofluorescence................................................................... 113
7.5 Confocal microscopy................................................................................................... 115
7.6 SDS-PAGE and immunoblotting ................................................................................ 116
7.7 Antibodies used for immunoblotting........................................................................... 117
7.8 Drug treatment............................................................................................................. 118
7.9 Cryosections ................................................................................................................ 118
7.10 Immunofluorescence of cryosections ....................................................................... 118
7.11 Densitometric analysis .............................................................................................. 118
7.12 Binucleation .............................................................................................................. 119
7.13 Statistical analysis ..................................................................................................... 119
7.14 Hypertrophic samples................................................................................................ 119
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CHAPTER 8
References ............................................................................................................................ 120
Publications.......................................................................................................................... 144
Curriculum vitae ................................................................................................................. 145
vii
ABSTRACT
The heart is the first functional organ in the embryo. Embryonic cardiomyocytes are able to
divide in their differentiated state (Ahuja et al., 2004), unlike skeletal muscle cells where
proliferation and differentiation are mutually exclusive processes (Zak, 1974). In the early
postnatal period the cardiomyocytes stop dividing and growth is only achieved by increase in
cell volume. However, in some species cardiomyocytes can undergo an additional incomplete
mitosis where karyokinesis takes place in the absence of cytokinesis leading to binucleation.
The co-existence of cell division and the terminal differentiated traits like contractile
cytoarchitecture, in the embryonic cardiomyocytes presents an interesting biological puzzle
that remains to be solved. Past attempts to understand cytoskeletal and myofibrillar
organization during division provided only limited insight into the proliferative events in the
embryonic heart.
The work presented here tries to answer the following key questions related to mitotic
division and the cytoskeleton of embryonic cardiomyocytes during division: (i) Does the
myofibrillar cytoarchitecture of cardiomyocytes survive division? (ii) What factors regulate
the organization of the myofibrils during division? (iii) Which kind of signals are responsible
for progression to cytokinesis in embryonic cardiomyocytes and why does cytokinesis stop
after birth? Analyses of triple-stained specimen of cultured cardiomyocytes and of whole
mount preparations of embryonic mouse hearts by confocal microscopy revealed that, for
differentiated cardiomyocytes to go through cell division, disassembly of the myofibrils has to
take place. This disassembly occurs in two steps with Z-disk and thin filament associated
proteins disintegrating before the disassembly of the M-bands and the thick filaments. After
cytokinesis a rapid reassembly of the myofibrillar proteins to their mature cross-striated
pattern can be observed. At present, it is not quite clear whether the myofibrils are only
disassembled and the sarcomeric proteins are recycled again after cytokinesis or protein
degradation of myofibrillar components takes place as well. However, results showed a
drastic upregulation of ubiquitin expression, which spreads throughout the cytoplasm in
dividing cardiomyocytes suggesting that protein degradation is important during this process.
The observation that the myofibrils have to be disassembled for cytokinesis to occur might
provide also a simple mechanistic explanation, why cardiomyocytes cease to divide after
birth. With the hypertrophic growth that is caused by the increased workload on the heart after
birth, this disassembly-reassembly process might be simply too costly from an energetic point
1
Abstract
of view. In addition, too many cytoskeletal elements in the form of myofibrils might
physically impede cell division as well.
Another interesting finding of this work was the analysis of the cytokinetic potential of
cardiomyocytes during development. There are several key players known that are important
for cytokinesis such as various small GTPases like RhoA, Rac1, Cdc42, septins along with
their downstream effectors like ROCK I, ROCK II and Citron Kinase. The contractile ring has
been investigated in postnatal cardiomyocytes from rat (Li et al., 1997a; Li et al., 1997b).
However, these localization studies were restricted only to F-actin and non-muscle myosin
and none of the other proteins involved in cytokinesis was analyzed. We demonstrate here for
the first time that cardiomyocytes show a developmental stage specific expression of all the
proteins such as RhoA, Cdc42, Rac1, ROCK-I, ROCK-II, p-cofilin that are coupled with the
formation of the actomyosin ring with high levels being only expressed at stages when
cytokinesis still occurs (i.e. embryonic and perinatal). This suggests that downregulation of
many regulatory and cytoskeletal components involved in cytokinesis may also be responsible
for uncoupling of cytokinesis from karyokinesis in cardiomyocytes after birth. Also we found
out that when stressed myocardium tries to adapt to the increased work load during
pathological hypertrophy there is indeed an upregulation of karyokinetic and cytokinetic
proteins, despite the fact that the mitotic cycle is usually not resumed under these conditions.
The failure to undergo complete division could be due to the presence of stable, highly
ordered and functional sarcomeres in the adult myocardium or could be due to the absence of
degradation mechanisms (ubiquitin and/or calcium dependent) which facilitate division of
embryonic cardiomyocytes by disintegrating the myofibrils.
The developmentally regulated cessation of cytokinesis makes cardiomyocytes an interesting
model system to investigate the factors involved in cell division.
2
ZUSAMMENFASSUNG
Das Herz ist das erste Organ, das während der Embryonalentwicklung funktionell wird. Kurz
nach der Geburt hören die Kardiomyozyten damit auf, sich zu teilen und das Myokard
Wachstum kann nur durch die Vergrösserung des Zellvolumens erreicht werden. In manchen
Spezies findet noch eine unvollständige Mitose statt, bei der es zu Kernteilungen ohne
Zellteilung kommt, wodurch zweikernige Zellen entstehen. Wie das Nebeneinander von
Zellteilung und hochgradiger Differenzierung mit kontraktiler Zytoarchitektur in embryonalen
Kardiomyozyten genau funktioniert, ist eine noch ungelöste Frage. Bisherige Untersuchungen
am Zytoskelett und der Anordnung der Myofibrillen während der Zellteilung im embryonalen
Herzen haben nur bedingt aufschlussreiche Ergebnisse gebracht.
Die hier vorgelegte Arbeit hat folgende Fragestellungen zum Thema der Zellteilung in
differenzierten, embryonalen Kardiomyozyten: (i) Bleiben die Myofibrillen während der
Zellteilung intakt? (ii) Welche Faktoren regulieren die Organisation von Myofibrillen
während der Zellteilung? (iii) Welche Signalwege sind für die erfolgreiche Zytokinese in
embryonalen Kardiomyozyten verantwortlich und warum gibt es nach der Geburt keine
Zytokinese mehr in Herzzellen? Die Analyse von dreifach-immungefärbten Präparaten von
Total präparaten kultivierten Kardiomyozyten und von ganzen embryonalen Mäuseherzen im
konfokalen Mikroskop ergab, dass die Myofibrillen ab gebaut werden müssen, damit sich die
Herzzellen teilen können. Dieser Prozess läuft in zwei Phasen ab: zuerst werden die ZScheiben und die dünnen Filamente entfernt, danach die M-Banden und die dicken Filamente.
Nach der Zytokinese werden die Proteine der Myofibrillen rasant wieder zu quergestreiften
Strukturen zusammen gebaut. Ob die Proteine der Myofibrillen während diesem Prozess auch
vollständig degradiert werden, oder ob sie nach der Zytokinese wiederverwendet werden, ist
momentan noch unklar. Die beobachtete Hochregulierung der Expression von Ubiquitin in
sich teilenden Herzzellen könnte darauf hindeuten, dass der Proteinabbau für diesen Prozess
eine Rolle spielt.
Die Tatsache, dass die Myofibrillen auseinandergebaut werden müssen, damit es zur
Zellteilung kommen kann, könnte auf eine einfache Erklärung, warum sich Herzzellen nach
der Geburt nicht mehr teilen. Das hypertrophe Wachstum der Herzzellen im Fötus und
aufgrund der erhöhten Belastung nach der Geburt könnte dazu führen, dass dieser Abbau
einfach zu aufwendig wird und energetisch für die Zelle zu Kost spielig ist. Eine zusätzliche
physische Behinderung könnte von der vermehrten Menge an Zytoskelett in Form der
Myofibrillen kommen.
3
Zusammenfassung
Zusätzlich wurde in dieser Arbeit die Fähigkeit der Kardiomyozyten zur Zytokinese während
der gesamten Entwicklung untersucht. Von mehreren Proteinen ist bekannt, dass sie für die
Zytokinese wichtig sind. Dazu gehören kleine GTPasen wie RhoA, Rac1, Cdc42 und Septin
Proteine und ihre “downstream” Effektoren wie ROCK I, ROCK II und Citron Kinase. Bis
jetzt wurde der kontraktile Ring, der für die Zytokinese gebraucht wird, in Herzzellen nur im
neugeborenen Stadium untersucht (Li et al., 1997a; Li et al., 1997b). Die Untersuchungen
beschränkten sich auf F-Aktin und die nicht-muskuläre Isoform von Myosin und es wurden
keine anderen Proteine analysiert, die für die Zytokinese wichtig sind. Wir zeigen hier zum
ersten Mal, dass Kardiomyozyten sich durch eine regulierte Expression der Proteine
auszeichnen, die mit dem Aufbau des kontraktilen Rings zusammen hängen. RhoA, Cdc42,
Rac1, ROCK I, ROCK II, p-Cofilin zeigen eine hohe Expression in Herzzellen in
Entwicklungsstadien in denen sich die Kardiomyozyten noch teilen, also embryonal und
perinatal. Das könnte bedeuten, dass die Entkopplung von Kernteilung ohne darauffolgende
Zellteilung in der postnatalen Phase darauf beruht, dass die gesamte Proteinmaschinerie, die
zur Zytokinese benötigt wird, nicht mehr in ausreichenden Mengen in der Zelle vorhanden ist.
Zusätzlich konnten wir feststellen, dass der Herzmuskel auf vermehrten Stress bei der
krankhaften Hypertrophie mit einer Hochregulierung der Expression von diesen Proteinen
reagiert, die mit Kernteilung und Zellteilung zu tun haben, obwohl es im Endeffekt dann doch
zu keiner Zellteilung kommt. Mehrere Faktoren könnten der Grund sein, warum die
Zellteilung in adulten Herzzellen nicht mehr möglich ist. Die Masse an hochgeordneten
Myofibrillen im Zytoplasma könnte diese verhindern oder es ist auch denkbar, das
Abbaumechanismen (vermittelt über Ubiquitin oder über Calpain), die im embryonalen
Herzen den Abbau der Myofibrillen regulieren, nicht mehr angeschaltet werden können.
Die entwicklungsstadienspezifische Hemmung der Zytokinese, die in dieser Arbeit
beschrieben wird, bedeutet, dass Kardiomyozyten verschiedener Altersstufen ein interessantes
Studienobjekt zur Analyse verschiedener Faktoren sein könnten, die für die Zytokinese eine
Rolle spielen.
4
CHAPTER 1
Introduction
5
Chapter 1
Introduction
1 Introduction
1.1 The Heart
The body requires oxygen and nutrients to carry on the process of life. A network of arteries
and veins transport oxygen-rich blood to the body and carry oxygen-poor blood back to the
lungs. At the centre of this continuous process is the heart, a beating muscle about the size of
the fist in adult humans. The heart pumps approximately 5 litres of blood every minute, and
each heartbeat circulates blood to both the lungs and the body. This is possible because of the
heart’s complex internal structure.
1.1.1 Heart development
The heart is the first functional organ in the developing embryo and arises from cardiac
progenitor cells derived from the embryonic mesoderm in the so called heart field region
(Fishman and Chien, 1997). The heart field initially forms as a crescent shaped structure in
the anterior part of the embryo that later develops into a linear tube (Figure 1.1), (Viragh et
al., 1989; DeRuiter et al., 1992). The tubular heart undergoes segmentation along the anteriorposterior axis, followed by rightward looping. This process results in the formation of the
right and left ventricles, the atrioventricular canal, the sinoatrial, and the outflow tract
segments (Olson and Srivastava, 1996; Srivastava and Olson, 2000). Subsequently, the
ventral side of the heart tube rotates and forms the outer curvature of the heart, with the dorsal
side becoming the inner curvature (Christoffels et al., 2000). These outer and inner curvatures
play critical roles in the morphogenesis of the four chambered heart, as the individual
chambers balloon out from the outer curvature due to the rapid proliferation of resident
myocardial cells. These morphogenetic changes are accompanied by the expression of
chamber specific genes exclusively in the ventral side and the outer curvature of the heart
tube. In contrast, myocardial cells residing in the inner curvature are thought to be relatively
undifferentiated, which allows this region to participate in the alignment of the right atrium
with the right ventricle and the left ventricle with the outflow tract. This process results in the
separation of the pulmonary and systemic circulations (Christoffels et al., 2000). All these
developmental processes are characterised by several transcription factors that are required for
cardiac development, morphogenesis, and/or chamber specification like Nkx2.5 (Lyons et al.,
1995), GATA4 (Kuo et al., 1997; Molkentin et al., 1997), MEF2 (Lin et al., 1997), d and
6
Chapter 1
Introduction
eHAND (Srivastava et al., 1995), Tbx5 (Hatcher et al., 2001) and HRT (Nakagawa et al.,
1999).
Figure 1.1: Summary of mouse heart development. Five major stages of heart development are shown: (1)
cardiac crescent formation at embryonic day (E) 7.5; (2) formation of the linear heart tube at E8; (3) looping and
the initiation of chamber morphogenesis at E8.5 to E9.5; (4) chamber formation; and (5) chamber maturation and
septation and valve formation. See text for further details. ao indicates aorta; a, atrium; la, left atrium; lv, left
ventricle; ra, right atrium; rv, right ventricle; ot, outflow tract; sv, sinus venosa; and pa, pulmonary artery
(adapted from Bruneau, 2002).
1.1.2 The contractile tissue of the heart
The contractile tissue of the heart (Figure 1.2 (a)) is composed of individual cells, the
cardiomyocytes (Figure 1.2 (b)). These cells are the most active cells in the body, contracting
constantly about 3 billion times or more in an average human lifespan to pump 7000 liters of
blood per day along 100000 miles of blood vessels (Severs, 2000). To ensure proper function,
two multiprotein cytoskeletal complexes have to be assembled correctly, the myofibrils and
the intercalated discs. The myofibrils consist of actin and myosin filaments that are organized
in a paracristalline fashion to ensure maximal contractile force. Their basic unit is the
sarcomere, which is defined as the region between two neighbouring Z-discs, where the thin
(actin) filaments are inserted (Figure 1.3). The thick (myosin) filaments are located in the
middle of the sarcomere and held in place by another transverse structure, the M-band. A
third filament system, the elastic (titin) filaments, seems to serve as a template for myofibril
assembly as well as an elastic spring for the central positioning of the thick filaments during
contraction (Figure 1.3a), (for review see Clark et al., 2002; Tskhovrebova and Trinick,
2003). According to the currently accepted theory, muscle contraction is caused by the sliding
of thin and thick filaments past each other, resulting in a shortening of the sarcomere (i.e. the
distance between two adjacent Z-discs). Simultaneous shortening of many sarcomeres thus
leads to the development of a macromolecular force, resulting in muscle contraction (Figure
1.4) (Huxley and Simmons, 1971). In contrast to skeletal muscle in heart the antagonistic
force of contraction is produced possibly by the extracellular matrix and the blood flowing
from the atria into the ventricles.
7
Chapter 1
Introduction
The second multiprotein cytoskeletal complex characteristic for cardiomyocytes is formed by
the intercalated disc at the cell-cell contacts, which consists of three separate junctions; the
adherens junctions, the desmosomes and the gap junctions (Figure 1.5). It is a specialized site
for different types of cell-cell contacts that ensures intercellular communication, mechanical
integration and force transmission between neighbouring cardiomyocytes to guarantee
optimal contractile work of the cardiac tissue (for review see Perriard et al., 2003).
Figure 1.2: (a) The contractile tissue of the heart is composed of individual cells, (b) the cardiomyocytes, whose
cytoskeleton is formed by the assembly of myofibrils and intercalated discs shown in green (β-catenin) and red
(myomesin) respectively (from Ehler E, King`s College, London, UK).
Figure 1.3: (a) Schematic representation of the sarcomeric multi-protein complex. Three different filament
systems, the thick (myosin) filaments, blue; the thin (actin) filaments, yellow; and the elastic (titin) filament,
green are shown. The transverse structures are the Z-disc, black and the M-band, red which cross connects the
filament systems (adapted from Agarkova and Perriard, 2005). (b) Corresponding electron micrograph of a
skeletal sarcomere. The sarcomeric borders are delineated by the Z-discs (Z) in the middle of the I-band. The Mband (M) is seen as an electron-dense transverse band in the middle of the dark A-band.
8
Chapter 1
Introduction
Figure 1.4: Muscle contraction is produced by the sliding of actin filaments on myosin filaments. Each giant
titin molecule extends from the Z-disc to the M-band, a distance over 1 µm. Part of each titin molecule is closely
associated with myosin molecules in the thick filament; the rest of the molecule is elastic and changes length as
the muscle contracts and relaxes (adapted from Alberts, 1994).
Figure 1.5: The intercalated disc (ICD) is composed of three types of junctions visible in the electron
microscope. Adherens junctions (Fascia adherens, FA) and desmosomes (D) are easily identified under electron
microscope as electron dense material along the cellular membrane. Gap junctions (Nexus, N) bring the plasma
membrane in close apposition (adapted from Wheater et al., 1987).
9
Chapter 1
Introduction
1.2 Establishment of cardiac cytoarchitecture in the developing heart
1.2.1 Myofibrillogenesis in developing cardiomyocytes in situ
Cardiac myofibrillogenesis is the process of expression and integration of sarcomeric
proteins, which results in the formation of regular structures known as myofibrils in the
cytoplasm of cardiomyocytes. Studies with chicken explants (Imanaka-Yoshida, 1997) and
whole mount preparations using immunohistochemistry and confocal microscopy showed
sequential assembly of sarcomeric subunits. In the eight somite stage chicken heart, before the
start of beating, the first organized complexes can be distinguished which are composed of
alpha-actinin, F-actin and the N-terminus of titin (Ehler et al., 1999). Thick filament proteins
as well as M-band proteins are distributed in a completely diffuse fashion at this time. While
N-terminal (Z-disc) epitopes of titin can be visualized in a distinct pattern already at the 8
somite stage, C-terminal (M-band) epitopes show a slight delay in their alignment.
Nevertheless they become concentrated soon enough so that titin can fulfill its role as an
organizing molecule of the sarcomere. When the degree of organization of the M-band protein
myomesin and that of the A-band protein MyBP-C is compared, it turns out that the
myomesin striations precede double bands of MyBP-C (Ehler et al., 1999). Thus there is an
existence of a basic framework for myofibril assembly, which consists of alpha-actinin at the
Z-disc, myomesin at the M-band with titin spanning in between. Alpha-actinin serves together
with titin for the anchorage of the thin filaments, whereas myomesin helps to integrate the
thick filaments with the titin filaments (Ehler et al., 1999; Ehler et al., 2004).
1.2.2 Development of cell polarity during cardiomyocyte differentiation
The overall shape of a cardiomyocte changes during differentiation from polygonal in the
embryonic heart to rod shaped in the adult heart. This shape change is accompanied by a
restriction of cell-cell contact proteins to the bipolar ends, where they form specific types of
cell-cell contact, the intercalated discs (Hirschy et al., 2006). During the progressive
differentiation of cardiomyocytes a gradual increase is observed in the size, number and
complexity of organization of myofibrils from being a loose meshwork to myofibrils that are
packed tightly together and are aligned strictly in parallel (for review see Rumyantsev, 1977;
Pasumarthi and Field, 2002; Olson and Schneider, 2003). The organization of different cellcell contact proteins during embryonic heart development has been studied and it has been
shown that restriction of the intercalated disc components to the bipolar ends is a slow process
10
Chapter 1
Introduction
and is achieved for adherens junctions and desmosomes only after birth, for gap junctions
even later (Hirschy et al., 2006). This suggests that the changes affecting adherens junctions
and desmosomes during development are probably coordinated because both junctions
provide together the mechanical coupling which is essential for the work of the
cardiomyocytes, as suggested previously (Perriard et al., 2003).
1.3 Differentiation and proliferative capacity of cardiac and skeletal muscle
cells
Cardiac myogenesis has been much less studied in respect of interrelationship between
proliferation and differentiation compared to skeletal myogenesis. Differentiation of cardiac
muscle cells is different from that of skeletal muscle in two main features: first the synthesis
of DNA and the synthesis of cell specific proteins are not mutually exclusive (Manasek, 1968;
Rumyantsev, 1977). In the first phase of cardiac development, there is a very active
proliferation of undifferentiated myogenic cells. These cells gradually initiate synthesis of
myofibrillar proteins but continue to divide mitotically. As development progresses, the
number of dividing cells decreases and synthesis of muscle proteins is accentuated (Zak,
1974). The term myoblast thus has a different meaning for skeletal and heart muscle. A
myoblast in skeletal muscle is a cell withdrawn from the mitotic cycle and committed to
synthesis of myofibrillar proteins. The cardiac myoblast, on the other hand, is a dividing cell
simultaneously synthesizing cell specific proteins (Weinstein and Hay, 1970; Rumyantsev,
1972).
The second difference is in the regenerative capacity of skeletal and cardiac muscle. Mature
fibers in skeletal muscle have a population of undifferentiated cells which are wedged
between the plasma membrane and the basement membrane of the muscle fiber. These cells
are referred to as satellite cells (Mauro, 1961). The available evidence indicates that they are
myogenic in nature (Moss and Leblond, 1971; Collins and Partridge, 2005). Unlike skeletal
muscle, myocardium is very rarely a subject to neoplastic transformation, which is strongly
suggestive of a stable inhibition of DNA synthesis and mitosis of the cardiomyocytes and of
the absence of a pool of undifferentiated myogenic cells (Rumyantsev, 1977; Olson and
Schneider, 2003).
11
Chapter 1
Introduction
1.4 Cardiac growth during development
1.4.1 Hyperplasia and hypertrophy
During embryogenesis heart mass mainly increases by cell division of cardiomyocytes, a
process that is called hyperplasia (Manasek, 1968; Weinstein and Hay, 1970; Chacko, 1972).
After birth cell division ceases in mammalian heart and growth is mainly by increase in the
cell size, i.e. hypertrophy (Figure 1.6) (Oparil et al., 1984; Klug et al., 1995). At the
morphological level this transition from hyperplasia to hypertrophy after birth is marked by
binucleation, where the cardiomyocytes completely loose their ability to finish cytokinesis
(Katzberg et al., 1977; Ferrans and Rodriguez, 1987; Li et al., 1996). In rodents for example,
the accumulation of binucleated cardiomyocytes starts around day four and by the third
postnatal week 85-90% of the cardiomyocytes are binucleated (Clubb and Bishop, 1984; Li et
al., 1996; Soonpaa et al., 1996), in pigs the number of nuclei can even reach up to 32
(Grabner and Pfitzer, 1974). In humans, the withdrawal of ventricular cardiomyocytes from
the cell cycle occurs within the first few weeks of life (Zak, 1974) and as opposed to other
species, the nuclei of cardiomyocytes in man and monkeys attain a higher ploidy level.
Polyploidization in humans has been reported to be associated with aging and increased heart
weight (Ferrans and Rodriguez, 1987). At present the cause for uncoupling of karyokinesis
from cytokinesis is not known.
In contrast, cardiomyocytes from lower vertebrates are capable to divide postnatally (Nag et
al., 1979; Oberpriller et al., 1995; Matz et al., 1998; Bettencourt-Dias et al., 2003). In adult
newt and zebrafish, heart tissue can even be regenerated after injury (Matz et al., 1998; Poss
et al., 2002; Scott and Stainier, 2002; Keating, 2004; Raya et al., 2004). However, in
mammals the general consensus is that cardiomyocytes in adults are postmitotic and do not
retain any proliferation potential, (for review see MacLellan and Schneider, 2000;
Swynghedauw, 2003; von Harsdorf et al., 2004). This view is supported in part by clinical
observations, as myocardial regeneration has not been reported in diseases or injuries that lead
to cardiomyocyte loss. Besides, primary myocardial tumors are rarely observed in adults
(Soonpaa and Field, 1998).
12
Chapter 1
Introduction
Figure 1.6: Schematic representation of heart development at the cellular level. Prenatal heart growth is due to
hyperplasia (cell division) with a switch shortly after birth to the hypertrophic phase, where heart growth is due
to cell volume increase that ends in adulthood (from M.C. Schaub, University of Zürich, Switzerland).
1.4.2 Cardiomyocyte cell cycle regulation
1.4.2.1 Basic events of the mammalian cell cycle
The two most important events during the passage through cell cycle are the S phase (Ssynthesis) and the M phase (M-mitosis), when chromosomes are replicated and segregated
into the dividing cells, respectively (Figure 1.7(a)) (Nurse, 1994). The interval between the
completion of M phase and the beginning of S phase, called the G1 phase (G-gap), and the
interval between the end of S phase and the beginning of M phase, called the G2 phase, forms
the links necessary to fulfil the image of the cell cycle as a circle. To ensure proper
progression through each phase, cells have developed a series of orchestrated events that are
governed by various molecular regulators such as cyclins, cyclin-dependent kinases (CDKs),
CDK activators, CDK inhibitors (CKIs) and members of the retinoblastoma protein family
(Heichman and Roberts, 1994; King et al., 1994). Different cyclin-CDK complexes are
required for distinct cell cycle events and their activities are regulated by CAKs
13
Chapter 1
Introduction
(cyclinH/CDK7) and CKIs (p15, p16, p18, p19, p21, p27 and p57) in positive and negative
manners, respectively (Hunter and Pines, 1994).
Figure 1.7: (a) Four successive phases of a standard eukaryotic cell cycle. During interphase the cell grows
continuously; during M phase it divides. DNA replication is confined to the part of interphase known as S phase.
G1 phase is the gap between M phase and S phase; G2 is the gap between S phase and M phase (Alberts, 1994).
(b) The principal events typical of animal cell division. For explanations see text (adapted from Nigg, 2001).
In a typical somatic cell cycle, the M phase comprises karyokinesis (nuclear division) and
cytokinesis (cytoplasmic division). The main purpose of mitosis is to segregate sister
chromatids into two nascent cells, such that each daughter cell inherits one complete set of
chromosomes. Mitosis is usually divided into five distinct stages: prophase, prometaphase,
metaphase, anaphase and telophase. During 'prophase', interphase chromatin condenses into
well-defined chromosomes and previously duplicated centrosomes migrate apart, thereby
defining the poles of the future spindle apparatus. Concomitantly, centrosomes begin
14
Chapter 1
Introduction
nucleating highly dynamic microtubules that probe space in all directions, and the nuclear
envelope breaks down. During 'prometaphase', microtubules are captured by kinetochores
(specialized proteinaceous structures associated with centromere DNA on mitotic
chromosomes). Although monopolar attachments of chromosomes are unstable, the eventual
interaction of paired sister chromatids with microtubules emanating from opposite poles
results in a stable, bipolar attachment. Chromosomes then congress to an equatorial plane, the
metaphase plate, where they continue to oscillate throughout 'metaphase', suggesting that a
balance of forces keeps them under tension. After all the chromosomes have undergone a
proper bipolar attachment, a sudden loss in sister-chromatid cohesion triggers the onset of
'anaphase'. Sister chromatids are then pulled towards the poles (anaphase A) and the poles
themselves separate further towards the cell cortex (anaphase B). Once the chromosomes have
arrived at the poles, nuclear envelopes reform around the daughter chromosomes, and
chromatin decondensation begins 'telophase'. Finally, an actomyosin-based contractile ring is
formed and 'cytokinesis' is completed. The figure (Figure 1.7(b)) summarizes the stages of the
M phase. It also indicates where the major checkpoints exert quality control over mitotic
progression and where mitotic kinases are thought to act (for review see Nigg, 2001).
1.4.2.2 Cardiomyocyte cell cycle activity during development
Cell division is one of the key factors contributing to cardiac growth during early embryonic
development. At the embryonic stage of heart, high levels of cyclins involved in G1, S, G2
and M-phase like D1, D2, D3, A, B1 and E in cardiomyocytes are expressed at both mRNA
and protein levels (Yoshizumi et al., 1995; Brooks et al., 1997; Kang and Koh, 1997; Flink et
al., 1998). Additionally, PCNA and the cyclin-dependent kinases Cdc2, Cdk2,Cdk4 and Cdk6
are highly expressed and their associated kinase activities are also present (Yoshizumi et al.,
1995; Brooks et al., 1997; Kang and Koh, 1997; Flink et al., 1998). The protein expression
profiles of cyclins D1, D2, D3, A, B1 and E and their associated kinases become
progressively and significantly downregulated in postnatal cardiomyocytes compared to the
levels observed in the embryonic stage. Moreover, the protein levels of cyclin A, B, D1, E
and Cdc2 become even undetectable by immunoblotting in adult cardiomyocytes (Yoshizumi
et al., 1995; Brooks et al., 1997; Kang and Koh, 1997; Flink et al., 1998). The kinase Plk1,
involved particularly in cytokinesis, was demonstrated to be downregulated on both the
transcriptional and translational level during heart development (Georgescu et al., 1997). The
downregulation in the expression of cyclins and cyclin-dependent kinases during normal
development of cardiomyocytes have been shown to be concomitant with the specific
15
Chapter 1
Introduction
upregulation of the cyclin-dependent kinase inhibitor molecules p21 and p27 (Li and Brooks,
1997; Burton et al., 1999). Expression at the transcriptional level of the third member of the
Cip/Kip family, p57, has been reported (Matsuoka et al., 1995), but the protein is detectable
only at the early stages in rat, however, it persists throughout life in man (Burton et al., 1999).
A review of the expression patterns of these proteins is provided in Table 1.1.
Gene
Cyc D1
Cyc D2
Cyc D3
CDK4
p16
p18
p21
p27
p57
Cyc E
Cyc A
CDK2
RB
p107
p130
E2F1
p53
p193
PCNA
Cyc B
cdc2
Cell Cycle Activity
Cdk cofactor, positive regulator of restriction point transit
Cdk cofactor, positive regulator of restriction point transit
Cdk cofactor, positive regulator of restriction point transit
Phosphorylates RB, promotes restriction point transit
Inhibits Cdk 4 and 6; blocks restriction point and G1/S transit
Inhibits Cdk 4 and 6; blocks restriction point and G1/S transit
Inhibits Cdk 2, 4, and 6; blocks restriction point and G1/S transit
Inhibits Cdk 2, 4, and 6; blocks restriction point and G1/S transit
Inhibits Cdk 2, 4, and 6; blocks restriction point and G1/S transit
Cdk cofactor, positive regulator of G1/S transit
Cdk cofactor, positive regulator of S and G2/M transit
Promotes G1/S transit when complexed with Cyc E
Regulates G1/S transit by inhibiting E2F family member activity
Regulates G1/S transit by inhibiting E2F family member activity
Regulates G1/S transit by inhibiting E2F family member activity
Transcription factor for cell cycle genes, inhibited by RB family
Tumor suppressor that promotes cell cycle arrest or apoptosis
BH3-only proapoptosis protein induces apoptosis at G1/S
Required for DNA synthesis during S-phase and repair
Cdk cofactor, positive regulator of G2/M transit
Promotes G2/M transit when complexed with Cyc B
Expression Level
EMB
NEO
AD
++
++
++
++
ND
++
+
+
++
++
++
++
+
++
+
ND
ND
++
++
++
++
+
+
+
+
++
+
+
+
+
+
+
+
+
++
+
ND
+
+
+
+
ND
++
+
+
+
+/ND
-
Table 1.1: Expression patterns of cell cycle regulatory proteins during cardiac development as determined by
western blot (adapted from Pasumarthi et al., 2002). ++ refers to relatively stronger expression than +, - refers to
not detected and ND, not determined.
16
Chapter 1
Introduction
1.5 Myofibrillar and cytoskeletal organization of cardiomyocytes
undergoing mitotic division
1.5.1 Regulation of myofibrillar organization during division
It is difficult to imagine, how embryonic cardiomyocytes can handle the highly dynamic
processes of cell division and contraction of the myofibrils at the same time. Most cell types
disassemble their cytoskeletal filaments before entering mitosis and microtubules reorganize
to form the spindle apparatus, while actin and myosin make up the contractile ring that leads
to cytokinesis (for review see Straight and Field, 2000; Sanger and Sanger, 2000; Nigg,
2001). So far, comparatively little is known on how cardiomyocytes manage to divide and
beat in the developing heart. In the newt heart, where regeneration is possible also in the adult
(Oberpriller and Oberpriller, 1974), differences in the proliferative potential of adult
cardiomyocytes were detected (Bettencourt-Dias et al., 2003). Slowed proliferation was
ascribed to cells from the future cardiac conduction system versus ventricular cardiomyocytes
in the murine embryonic heart (Sedmera et al., 2003). There are few reports in the literature
that describe seemingly intact myofibrils next to condensed chromosomes (Manasek, 1968;
Kelly and Chacko, 1976), while others claim that myofibrils have to disassemble before cell
division can occur (Goode, 1975; Rumyantsev, 1977; Kaneko et al., 1984). However, most of
these studies were performed by electron microscopy, therefore presenting only a restricted
view and a correlative study of proliferative events in the entire embryonic heart together with
the investigation of cardiomyocyte cytoarchitecture is missing so far. One of the most
conclusive studies carried out up to now that deals with the fate of myofibrils in dividing
cardiomyocytes demonstrated by live birefringence microscopy that the cross-striated pattern
was completely lost in newt cardiomyocytes undergoing cytokinesis, suggesting that
myofibril disassembly has to occur before cardiomyocytes can divide (Kaneko et al., 1984).
1.5.2 Factors regulating myofibrillar organization during division
The disorganization of myofibrils during mitosis in newt was explained as being a special
kind of cellular adaptation allowing chromosomal movement and cytokinesis (Kaneko et al.,
1984). However, the factors that regulate myofibril disassembly during mitosis could not be
identified. The existence of a putative Z-disc degradation factor was postulated thirty years
ago (Rumyantsev, 1977), however, its molecular nature is still unclear.
17
Chapter 1
Introduction
Myofibril disassembly as it occurs during myopathies or muscle wasting is also characterized
by degradation of Z-disc and I-band material before the rest of the sarcomere is affected
(Taylor et al., 1995). During this process, degradation of myofibrils seems to be mainly
regulated by the activity of muscle-specific calpain, which can associate with the I-band
region of titin (Kinbara et al., 1998) and thereafter by the ubiquitin-proteasome pathway
(Hasselgren and Fischer, 2001).
1.5.3 Ubiquitin and calpain dependent degradation pathways in muscle
A number of catabolic disease states, including sepsis, severe injury, cancer, AIDS and
diabetes are characterised by muscle wasting, mainly reflecting increased breakdown of
myofibrillar proteins (Mitch and Goldberg, 1996; Hasselgren and Fischer, 2001). Intracellular
protein breakdown is regulated by a number of proteolytic pathways that can be divided into
lysosomal and nonlysosomal mechanisms. Among the nonlysosomal mechanisms, the
ubiquitin-dependent and calcium-dependent pathways are particularly important for
regulation of muscle protein breakdown (Attaix et al., 1998).
Ubiquitin-proteasome proteolytic pathway
Most muscle proteins are degraded by the ubiquitin-proteasome dependent proteolytic
pathway. Proteins degraded by this mechanism are first conjugated to multiple molecules of
ubiquitin, which is a 76-amino acid, 8.5 kDa residue, highly conserved and present in all
eukaryotic cells (Ciehanover et al., 1978). Proteins targeted for degradation by the ubiquitindependent mechanism are conjugated to polyubiquitin, which enables them to be recognized
by the large 26S proteolytic complex. This complex consists of the 19S cap complex and the
20S proteasome. The function of the 19S complex is to recognize, bind and unfold
ubiquitinated substrates that are then funnelled through the 20S proteasome. Ubiquitination of
proteins is regulated by different enzymes: ubiquitin activating enzyme E1, which activates
ubiquitin to a high energy thiolester at its C-terminal Gly; ubiquitin conjugating enzyme E2,
which transfers ubiquitin in its high energy state from E1 to a member of the E3 ubiquitinprotein ligase family, to which the substrate protein is specifically bound. This E3 enzyme
catalyzes the last step in the conjugation process, i.e. covalent attachment of ubiquitin to the
substrate, wherein the ubiquitin is transferred to an NH2 group of an internal Lys residue of
the protein substrate to generate an isopeptide bond (Haas and Siepmann, 1997). The structure
of the ubiquitin system is hierarchical in that a single E1 carries out activation of ubiquitin for
all modifications, whereas substrate and tissue specificity are accounted for by different E2s
18
Chapter 1
Introduction
and E3s. Individual E2s work in concert with specific E3s, which in turn have substrate
specificity. A simplified scheme of the ubiquitin-proteasome pathway and the different steps
involved in the ubiquitination of proteins is depicted in (Figure 1.8) (for review see Schwartz
and Ciechanover, 1999). As illustrated in Figure1.8 ubiquitin is released from the substrate
protein after its degradation, and because ubiquitin is a stable protein, it can be recycled in the
proteolytic pathway. The de-ubiquitination process is highly regulated by several deubiquitinating enzymes that actually make up the largest known family in the ubiquitin
system (Wilkinson, 2000). Recent studies suggest that de-ubiquitination may regulate the
catabolic rate of ubiquitinated proteins. This process therefore offers an additional point of
regulation of the ubiquitin-proteasome pathway.
Figure 1.8: The ubiquitin-proteasome system: sequence of events in the degradation of a protein via the
ubiquitin-proteasome pathway. For explanation see text.
Calcium dependent, calpain mediated proteolytic pathway
Although the ubiquitin-proteasome pathway is upregulated in septic muscle, it is unclear how
the myofibrillar proteins are ubiquitinated and become substrates for the 26S proteasome
(Hasselgren, 1999). It has been suggested that a calcium-dependent, calpain-mediated process
releases myofilaments from Z-disc during sepsis. Calpains constitute a family consisting of at
least 14 members that may be ubiquitous enzymes, such as µ- and m-calpain, or tissue
specific proteins, such as muscle specific calpain 3, (p94) (Goll et al., 2003). Support for a
role of calpains in muscle wasting caused by sepsis was provided in studies in which it was
found that the gene expression of µ-, m-calpain and p94 was increased in muscle of septic rats
19
Chapter 1
Introduction
(Bhattacharyya et al., 1991; Williams et al., 1999). A model has been proposed in which
increased calpain activity provides an early, and perhaps rate limiting step in muscle wasting,
accounting for degradation of Z-disc associated proteins and release of actin and myosin from
the myofibrils which are then subsequently ubiquitinated and degraded by the proteasome.
This suggests that the proteasome acts as a “cleanup” mechanism disposing of proteins
released by the action of calpain on myofilaments (Figure 1.9) (Hasselgren et al., 2005).
Figure 1.9: Calcium-calpain dependent release of myofilaments from the myofibrils which may be an early, and
perhaps rate-limiting, component of sepsis-induced muscle wasting. In this model, myofilaments (actin and
myosin) released from the myofibrils are ubiquitinated and subsequently degraded by the 26S proteasome
(Hasselgren et al., 2005).
The role of ubiquitin and calcium-dependent pathways in cardiomyocytes and particularly in
dividing cardiomyocytes remains unknown. At present it is not clear whether the myofibrils in
dividing cardiomyocytes remain intact or gets disassembled and whether protein degradation
plays any role in the process.
20
Chapter 1
Introduction
1.6 Cytokinesis and binuclearity of cardiomyocytes
Binucleation and terminal differentiation in cardiomyocytes occur shortly after birth in
rodents and are concurrent with the irreversible withdrawal from the cell cycle (Rumyantsev,
1977; Clubb and Bishop, 1984). By the third postnatal week in rodents the number of
binucleated cardiomyocytes increases by 80-90% (Clubb and Bishop, 1984; Soonpaa et al.,
1996). A gradual decrease in radiolabeled thymidine incorporation, coincident with the
appearance of binucleated cells, suggests that binucleation in cardiomyocytes arises as a
consequence of nuclear mitotic division without cytoplasmic separation i.e. karyokinesis
without cytokinesis (Rumyantsev, 1977; Soonpaa et al., 1996). At present the mechanism
behind uncoupling of karyokinesis from cytokinesis is not known. This block in cytokinesis in
cardiomyocytes is important from two aspects: first, if the expansion of differentiated
cardiomyocytes for transplantation experiments is intended, this block has to be overcome.
Second, if the factors that cause this block could be identified, they might be an interesting
tool to introduce a similar block when uncontrolled cell division is not required.
1.6.1 Major events contributing to cytokinesis
Cytokinesis is the final step of cell division. It is responsible for equal partitioning and
separation of the cytoplasm between daughter cells to complete mitosis. There are four major
events contributing to cytokinesis (Figure 1.10) which include (a) determination of the
division site, (b) cleavage furrow formation followed by ingression of membrane, (c)
midbody formation, and (d) cell separation (Glotzer, 2005). Much of the information on
factors that promote cytokinesis has been gained from studies on yeast (Field and Kellogg,
1999), however; recently these studies have also been extended to higher eukaryotes. In
eukaryotic cells, microtubules specify the position of the cleavage furrow (McCollum and
Gould, 2001). In late mitosis, microtubules bundle into antiparallel interdigitating arrays in
the central region of the mitotic spindle to form the spindle midzone. The spindle midzone has
been shown to directly contribute to signalling to achieve the actomyosin based contractile
ring assembly (Figure 1.10(a)), (Cao and Wang, 1996; Wheatley and Wang, 1996; Eckley et
al., 1997). Two major classes of proteins from the spindle midzone appear important for that:
first chromosomal passenger proteins,
21
Chapter 1
Introduction
Figure 1.10: The subprocesses and the structures that mediate cytokinesis (Glotzer, 2001). For explanation see
text. Four major events contributing to cytokinesis which include (a) determination of the division site, (b)
cleavage furrow formation followed by ingression of membrane, (c) midbody formation, and finally (d) cell
separation.
which localize initially to chromosomes and centromeres and subsequently to the midzone
and furrow (Adams et al., 2001). The major chromosomal passengers include the inner
centromere protein (INCENP), the Aurora-B/Ip11 kinase and Bir1/Survivin (Cooke et al.,
1987; Bischoff and Plowman, 1999; Silke and Vaux, 2001). A second class of proteins
includes microtubule motor proteins. The major motor proteins associated with the spindle
midzone are the microtubule motor protein kinesins, specifically, those of the CHO1/MKLP1,
KLP3A families and the polo kinase (Lee et al., 1995; Williams et al., 1995). It has been
proposed that these proteins provide structural support for interdigitating microtubules at the
midzone region, which may provide a platform to localize furrow components to the middle
of the cell. Another alternative could be that motors help delivering the necessary molecules
to build the cleavage furrow (for a review see Guertin et al., 2002). After the position of the
division plane has been established, the next key step is the assembly of the contractile
ring/cleavage furrow (Figure 1.10(b)). The furrow contains actin, myosin and other proteins
that are organized into a contractile ring called the actomyosin ring. The ring then ingresses
generating a membrane barrier between the cytoplasmic contents of each daughter cell (Wolf
et al., 1999; Guertin et al., 2002). The ingressing furrow constricts components of the spindle
midzone into a focused structure called the midbody (Figure 1.10(c)). In the final cytokinetic
event, called abscission, the furrow seals, generating two daughter cells (Figure 1.10(d)), (for
review see Satterwhite and Pollard, 1992; Fishkind and Wang, 1995; Glotzer, 1997; Oegema
and Mitchison, 1997).
22
Chapter 1
Introduction
1.6.2 Interplay between microtubule and actin network during cytokinesis
New developments suggest that the mitotic spindle and contractile ring are intimately linked
during cytokinesis. It is believed that the interaction between the microtubule and actin
cytoskeleton systems during cytokinesis could serve to position the cleavage furrow and
initiate ingression (Mabuchi, 1986; Rappaport, 1986; Bray and White, 1988). It has been
suggested that spindle microtubules may influence actin organization in the contractile ring,
perhaps in part by affecting cortical flow of actin filaments to and from the equator. Canman
and Bement (Canman and Bement, 1997) found that increased microtubule polymerization
inhibited cortical actin assembly during cortical flow, whereas microtubule depolymerization
increased cortical flow. These results imply that microtubules or their associated proteins
might inhibit cortical flow of actomyosin. At present it is not known whether the continued
presence of spindle microtubules is required for maintenance of the contractile ring
throughout cytokinesis. It would be interesting to study the nature of this cross-talk between
these two cytoskeletal networks.
1.6.3 Actomyosin based contractile ring
As the name suggests both actin and myosin are essential components of the actomyosin ring.
Concentration of actin at the position of the future cleavage furrow is one of the first signs of
cytokinesis (Marks et al., 1986; Alfa and Hyams, 1990; Cao and Wang, 1990; Momany and
Hamer, 1997; Wolf et al., 1999). Following actin, myosin II also assembles at the specified
cleavage furrow plane, which is thought to provide the necessary force to constrict the
cytoplasm of the dividing cells (Satterwhite and Pollard, 1992; Wolf et al., 1999). Type II
myosin consists of a dimer of two heavy chains, each having two associated proteins, called
the essential light chain (ELC) and the regulatory chain (RLC). Myosin heavy chain has a
head domain, which has the motor and actin binding domains, and a long coiled-coiled
domain involved in dimerization. In muscle, myosin forms long bipolar filaments which
crosslink actin filaments and pull them together to bring about muscle constraction (Spudich,
2001). The force-producing ability of myosin II is regulated by phosphorylation, particularly
through modification of the associated light chains (Trotter and Adelstein, 1979). Recently, it
was shown that myosin II regulatory light chain phosphorylation, is required for the initial
recruitment to the furrow, whereas the assembly of parallel, unbranched actin filaments, is
required for maintaining myosin II exclusively at the equatorial cortex (Dean et al., 2005).
23
Chapter 1
Introduction
Cardiomyocytes contain muscle myosin II (β and α) and non-muscle myosin IIB. During
cytokinesis, myosin IIB concentrates in the cleavage furrow, but muscle myosin II is excluded
from the central region of dividing cardiomyocytes (Conrad et al., 1995). It was shown that
ablation of non-muscle myosin heavy chain IIB results in defects of cytokinesis in
cardiomyocytes (Takeda et al., 2003). Apart from actin and myosin a number of substantial
advances in recent years have identified other possible key factors regulating the formation of
the contractile ring.
1.6.4 Factors regulating the formation of the contractile ring
A successful strategy to study cytokinesis has been to focus on the regulation of the
mechanical components responsible for contraction of the cleavage furrow-namely the
actomyosin cytoskeleton. Actin has been postulated to act as scaffolding onto which the rest
of the cytokinesis machinery assembles. Apart from actin and myosin other possible key
players such as small GTPases like RhoA and its effectors ROCK I and ROCK II, citron
kinase, formin-homology proteins, GTPase Cdc42, Rac and septins have been identified in
regulating the formation of the contractile ring (for review see Wolf et al., 1999; Glotzer,
2001; Guertin et al., 2002; Glotzer, 2005).
1.6.5 Rho GTPases as molecular switches in cytokinesis
GTPases are molecular switches that use a simple biochemical strategy to control complex
cellular processes (for review see Etienne-Manneville and Hall, 2002). They cycle between
two conformational states: one bound to GTP (‘active’ state), the other bound to GDP
(‘inactive’ state), and they hydrolyse GTP to GDP (Figure 1.11). In the ‘active’ (GTP) state,
GTPases recognize target proteins and generate a response until GTP hydrolysis returns the
switch to the 'inactive' state. In the active state, they interact with one of over 60 target
proteins (effectors). The cycle is highly regulated by three classes of protein: in mammalian
cells, around 60 guanine nucleotide exchange factors (GEFs) catalyse nucleotide exchange
and mediate activation; more than 70 GTPase-activating proteins (GAPs) stimulate GTP
hydrolysis, leading to inactivation; and four guanine nucleotide exchange inhibitors (GDIs)
extract the inactive GTPase from membranes (Schmidt and Hall, 2002; Moon and Zheng,
2003).
24
Chapter 1
Introduction
Figure 1.11: The Rho GTPase cycle. Rho GTPases cycle between an active (GTP-bound) and an inactive (GDPbound) conformation. In the active state, they interact with one of over 60 target proteins (effectors). The cycle is
highly regulated by three classes of protein: in mammalian cells, around 60 guanine nucleotide exchange factors
(GEFs) catalyse nucleotide exchange and mediate activation; more than 70 GTPase-activating proteins (GAPs)
stimulate GTP hydrolysis, leading to inactivation; and four guanine nucleotide exchange inhibitors (GDIs)
extract the inactive GTPase from membranes (Etienne-Manneville and Hall, 2002).
Small GTPases of the Rho family are most prominent in carrying out essential functions of
cytokinesis. Members of the Rho GTP family, namely Rho, Rac and Cdc42, mediate distinct
actin-based cytoskeletal changes, despite the extensive crosstalk among all three signalling
partners (Hall, 1998). Rho induces stress fibre formation, and potentiates myosin activity
through phosphorylation of the myosin regulatory light chain (Amano et al., 1996; Kimura et
al., 1996; Amano et al., 1997). On the other hand, activated Rac produces membrane ruffling
and lamellipodia formation, whereas Cdc42 induces filopodia (Lamarche and Hall, 1994).
How these proteins regulate the state of the actin cytoskeleton is currently under intense
study.
The role of Rho GTPases in cytokinesis is emphasized by the fact that inactivation of Rho
GTPase in animal cells inhibits cytokinesis by disrupting normal assembly of actin filaments
and triggering disassembly of the contractile ring (Kishi et al., 1993; Mabuchi et al., 1993;
Larochelle et al., 1996; Moorman et al., 1996; Drechsel et al., 1997).
RhoA GTPase is the most prominent of all and is known to play an important role in a wide
spectrum of cell activities, including cytoskeletal organization, focal adhesion, cell division
(cytokinesis in particular), cell migration, apoptosis and cardiomyocyte hypertrophy
(Yanazume et al., 2002). In numerous cell types, inactivation of RhoA leads to a profound
defect in cytokinesis, and in most cases cleavage furrow formation is completely blocked
(Mabuchi, 1983; Kishi et al., 1993; Drechsel et al., 1997). It has been shown that Rho has a
role in regulating the assembly of actin containing structures within the cortex during
25
Chapter 1
Introduction
cytokinesis (Drechsel et al., 1997). There are several known effectors of RhoA GTPases,
some of which have been implicated in regulating cytokinesis, (Figure 1.12), (Madaule et al.,
1998; Yasui et al., 1998; Kosako et al., 2000; Eda et al., 2001). The activation of RhoA
requires the guanine nucleotide exchange factor (GEF) ECT2 in mammals (Tatsumoto et al.,
1999) or Pebble in drosophila (Prokopenko et al., 1999) for cytokinesis to occur.
Figure 1.12: Role of Rho GTPases and their downstream effectors in cytokinesis (adapted from Glotzer, 2001
and Kinoshita and Noda, 2001). For explanation see text.
Rho-kinase/ROK/ROCK is one of the targets for RhoA GTPase (for review see Riento and
Ridley, 2003) and is known to phosphorylate myosin light chain at the cleavage furrow
(Kosako et al., 2000) and at the same time, also to inhibit myosin phosphatase, thereby
inhibiting the dephosphorylation of the myosin light chain (Matsumura et al., 2001) (Figure
1.12). Both the activities lead to an increase in myosin light chain phosphorylation thereby
26
Chapter 1
Introduction
activating contractile ring formation for cytokinesis (Amano et al., 1996). A synthetic
compound named Y-27632 (Ishizaki et al., 2000) has been shown to be a specific inhibitor of
the ROCK family of kinases (Uehata et al., 1997). This compound has been widely used to
identify and evaluate the involvement and roles of ROCK kinases in a variety of systems,
including platelet activation (Klages et al., 1999), aortic smooth muscle contraction by
various stimuli (Ben-Ze'ev and Geiger, 1998), hypertrophy of cardiomyocytes (Kuwahara et
al., 1999), wound healing (Nobes and Hall, 1999), tumor invasion (Itoh et al., 1999), cell
transformation (Sahai et al., 1999), and cytokinesis (Kosako et al., 2000). However, the
mechanisms of kinase inhibition and cell permeation of Y-27632 as well as its specificity
have not been fully addressed (Narumiya et al., 2000; Riento and Ridley, 2003).
Citron kinase, also activated by RhoA has been implicated in cytokinesis, too (Madaule et
al., 1998; Di Cunto et al., 2000; Eda et al., 2001), (Figure 1.12). It has been shown that
overexpression of citron kinase results in the formation of multinucleate cells and that a
kinase active mutant causes abnormal constriction during cytokinesis (Madaule et al., 1998).
However, a targeted knockout of citron kinase in the mouse leads to a surprisingly mild
phenotype. Mice lacking citron kinase are born with expected frequency and show defects
mainly in the brain and not in the majority of the other tissues (Di Cunto et al., 2000). The
downstream targets of citron kinase that are involved in cytokinesis are currently unknown;
however, it is believed that citron kinase also phosphorylates myosin light chain at the
cleavage furrow (Kinoshita and Noda, 2001).
LIM kinase, another substrate of ROCK, indirectly contributes to assembly of the contractile
ring by stabilizing filamentous actin. One factor that destabilizes filamentous actin is
cofilin/ADF (Moon and Drubin, 1995). Cofilin is inactivated by phosphorylation (Moon and
Drubin, 1995; Moriyama et al., 1996). ROCK phosphorylates LIM kinase (Maekawa et al.,
1999), which in turn phosphorylates cofilin making it inactive (Figure 1.12). This helps in
stabilizing pre-existing actin filaments for the formation of the contractile ring (Maekawa et
al., 1999; Glotzer, 2005).
The Rho family GTPase Cdc42 also plays a role in the ingression of the cleavage furrow
(Drechsel et al., 1997; Prokopenko et al., 2000). In cultured mammalian cells, inhibition of
Cdc42 leads to a delay in cell cycle progression, failure of chromosomes to bi-orient on the
metaphase plate and to the formation of multinucleated cells (Yasuda et al., 2004). It has been
suggested that there is an interplay between RhoA and Cdc42, both of which affect actin
27
Chapter 1
Introduction
reorganization during interphase and play distinct roles in regulating cytokinesis (Figure
1.12), (Drechsel et al., 1997). The downstream targets of Cdc42 involved in cytokinesis are
currently unknown (Wolf et al., 1999).
The involvement of another Rho family member, Rac1, has been suggested to be important
for maintaining proper cortical tension during cytokinesis (Gerald et al., 1998; Wolf et al.,
1999).
1.6.6 Role of septins in the formation of the actomyosin ring
Among these regulators of cytokinesis, septins are members of another family of cytoskeletal
GTPases that has been shown to be important for cell division (for review see Kinoshita,
2003; Longtine and Bi, 2003; Hall and Russell, 2004; Martinez and Ware, 2004). However, it
is still not certain which step in cytokinesis is dependent on septin function. Septins might be
functioning in the selection of the site for furrowing, organizing the actin-myosin ring at that
site, regulating the contraction of the ring or the attachment of the actin-myosin ring to the
plasma membrane (Finger, 2005; Versele and Thorner, 2005). Alternatively, septins might be
involved in the addition of new plasma membrane that presumably must accompany furrow
formation, by associating with vesicles (Joo et al., 2005). Septins were first discovered in
budding yeast Saccharomyces cerevisiae, where they seem to function as scaffold proteins for
the assembly of other ring components (DeMarini et al., 1997). In the budding yeast,
temperature sensitive mutations in septins (Cdc3p, Cdc10p, Cdc11p and Cdc12p) result in
cells that fail to form “neck filaments” beneath the cleavage furrow (Byers and Goetsch,
1976) and eventually lead to multinucleated cells (Hartwell, 1971). Despite considerable
diversity in cytokinesis among different organisms, septins play a significant role also in the
cytokinetic machinery of mammals (Kinoshita and Noda, 2001). A general role for septins in
cytokinesis of higher eukaryotes was first suggested when it was discovered that the peanut
mutation in Drosophila resulted in multinucleated syncytia within imaginal discs. This
phenotypic effect was related to a mutation in a septin homologue, which apparently disrupted
cytokinesis (Neufeld and Rubin, 1994). It has been proposed that septins may have additional
functions in mammals, at sites where actin dynamics, cell surface organization and vesicle
fusion processes are taking place (Beites et al., 1999). Recently it was shown in yeast that
formation of the septin ring depends on the Cdc42 Rho-GTPase which involves cycles of
GTP loading and hydrolysis by Cdc42 (Gladfelter et al., 2002) and may be the mechanism
behind septin assembly and/or to recruit molecules to the septin scaffold during cytokinesis
(Figure 1.12).
28
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Introduction
Most cell cycle regulatory proteins have been shown to be downregulated in adult
cardiomyocytes (Pasumarthi and Field, 2002). At present little is known about the expression
of proteins that are associated with cytokinesis in cardiomyocytes during development with
the exception of Polo-like kinase, which was shown to be downregulated in the adult heart
(Georgescu et al., 1997). To find out whether neonatal rodent cardiomyocytes stop dividing
due to a lack of the entire cytokinesis machinery or due to the downregulation of an individual
regulatory component, it is important to investigate the eventual differences in expression
levels or activity of all these proteins associated with the formation of the contractile ring at
different stages in the developing heart.
1.7 Cardiomyopathies
Cardiac dysfunction may have devastating physiological consequences for the organism.
Heart disease is the predominant cause of disability and death in industrialized nations
accounting for about 40% of all postnatal deaths (Schoen, 1999; Seidman and Seidman,
2001). In Switzerland, pathological cardiac hypertrophy and heart failure is one of the ten
most common causes of hospitalization in patients older than 65 and the second most
common in those over 80 years of age (Ess et al., 2002). As these two groups represent 17%
of the Swiss population (Ess et al., 2002), understanding the causes and consequences of
pathways leading to cardiac hypertrophy is of prime importance for the development of novel
cost-effective and beneficial therapeutic strategies.
1.7.1 Classification of cardiomyopathies
Cardiomyopathy is any type of heart disease in which the heart’s function is compromised. As
a result, the heart muscle’s ability to pump blood is usually weakened. This condition is
generally progressive and may lead to heart failure. Cardiomyopathies can be classified in a
number of ways. They may be classified by anatomical abnormalities, by when the condition
is noticed, or by how the heart is affected. Although there is some overlap, cardiomyopathies
are often classified as ischemic (resulting from a lack of oxygen) and nonischemic.
Ischemic cardiomyopathy is a chronic disorder caused by either recurrent heart attacks or
coronary artery disease (CAD) - a disease in which there is hardening of the arteries on the
29
Chapter 1
Introduction
surface of the heart. CAD often leads to episodes of cardiac ischemia, in which the heart
muscle does not receive enough oxygen-rich blood. Recurrent ischemia may also lead to
fibrosis and weakening of the heart, resulting in ischemic cardiomyopathy. The ischemic heart
becomes stunned, stiff and non contractile, when the stress on the heart exceeds the blood
supply.
Nonischemic cardiomyopathy is a less common, progressive disease and frequently occurs
in young people. There are two main types of nonischemic cardiomyopathies: i) Dilated
cardiomyopathy and ii) Hypertrophic cardiomyopathy.
Dilated cardiomyopathy (DCM) involves dilation or enlargement of the heart’s ventricles
together with the thinning of the chamber walls and is usually accompanied by an increase in
cardiac mass. This dilation reduces the pumping ability of the heart (systolic function) and
therefore results in a reduced ejection fraction, often leading to heart failure (Figure 1.13).
Hypertrophic cardiomyopathy (HCM) or concentric hypertrophy involves abnormal
thickening of the heart walls, predominantly in the left ventricular wall and the septum. The
thickening reduces the size of the pumping chamber and prevents the heart from properly
relaxing between beats (diastolic function) and therefore properly filling with blood (Figure
1.13).
30
Chapter 1
Introduction
Figure 1.13: Defects in sarcomeric or cytoskeletal components lead either to hypertrophic cardiomypathy
(HCM) or dilated cardiomyopathy (DCM), respectively (Miller et al., 2004). For explanation see text.
In broad terms, there are two forms of cardiac hypertrophy, physiological, as occurs during
embryonic or postnatal heart growth or even as a response to exercise, and pathological, as
occurs in response to abnormal stress (for review see Olson and Schneider, 2003). Stress
signals that induce hypertrophy include hypertension, pressure overload, endocrine disorders,
myocardial infarction and contractile dysfunction from inherited mutations in sarcomeric or
cytoskeletal proteins. Pathological hypertrophic cardiomyopathy frequently progresses to
dilated cardiomyopathy which may be due, at least in part, to activation of apoptotic pathways
(for a review, see Kang and Izumo, 2003). Under certain pathological conditions myocardial
hypertrophy can be associated with an increased risk of secondary cardiac diseases such as
infarction and heart failure (Hunter and Chien, 1999).
The hypertrophic response is characterised by an increase in myocyte size, accumulation of
contractile proteins, and by induction of immediate early transcription factors such as Fos,
Myc, and Jun (Parker and Schneider, 1991), followed by characteristic changes in cardiacspecific gene expression (Komuro and Yazaki, 1993; Sadoshima and Izumo, 1997). These
changes have often been referred to as reactivation of a fetal gene program, given the reexpression of several genes not normally expressed in the adult ventricle but seen in the
embryonic and neonatal heart. Common examples are the re-expression of atrial natriuretic
factor and genes for fetal isoforms of contractile proteins, such as skeletal α-actin, atrial
myosin light chain-2, and β-myosin heavy chain in rodents and α-myosin heavy chain in
humans. This can be accompanied by downregulation of genes normally expressed at higher
31
Chapter 1
Introduction
levels in the adult than in the embryonic ventricle, such as α-myosin heavy chain and the
sarcoplasmic reticulum calcium pump, SERCA2a. Intrinsic changes in the cardiomyocyte
mechanical performance, myocyte loss due to apoptosis and myocyte encasement by fibrosis
have been postulated to mediate the eventual decline in myocardial function that occurs with
the transition from hypertrophy to failure. Thus, investigators have been intrigued by
extracellular signals and their cytoplasmic mediators, which together might explain these
diverse aspects of hypertrophy (Komuro and Yazaki, 1993; Sadoshima and Izumo, 1997).
Multiple candidate pathways have been identified including adrenergic signals, peptide
growth factors and cytokines. The best studied are the G protein-coupled receptors and their
signalling pathways (for review see Frey and Olson, 2003). Myocardial over-expression of
their downstream effectors Gαq (Adams et al., 1998) or PKC (Wakasaki et al., 1997) results
in pathological hypertrophy associated with contractile dysfunction and sometimes heart
failure. Members of the mitogen-activated protein kinase (MAPK) signalling cascades have
also been shown to be important regulators of cardiac hypertrophy (for review see Sugden and
Clerk, 1998). On the basis of sequence homology, MAPKs can be divided into three major
subfamilies: extracellularly responsive kinases (ERKs), c-Jun N-terminal kinases (JNKs) and
p38 MAPKs. Surprisingly, despite similar effects in vitro, over-expression of these signalling
molecules in vivo in the mouse heart produces very distinct phenotypes (for review see Frey
and Olson, 2003). It was shown that transgenic over-expression of MEK1, a MAPK kinase
that activates ERK1/2, results in compensatory concentric hypertrophy (Bueno et al., 2000): a
phenotype in contrast to the decompensated eccentric (chamber dilation) hypertrophy
observed in MEK5/ERK5 over-expressing mice (Nicol et al., 2001).
Many secreted factors are also induced with hypertrophy, including TGFβ (Takahashi et al.,
1994), insulin like growth factor-1 (Donohue et al., 1994), angiotensinogen (Baker et al.,
1990), the precursor of angiotensin-II, endothelin-1 (Yorikane et al., 1993) and cardiotrophin1 (Ishikawa et al., 1996) and can act on cardiomyocytes directly, evoking transcriptional
responses at least partially similar to those induced by load itself (for review see MacLellan
and Schneider, 2000).
32
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Introduction
1.8 Regenerative processes in myocardium during cardiomyopathies
Vertebrates respond to injury through activation of committed progenitor cells or stem cells
(e.g. bone marrow) or through proliferation of differentiated cells (liver or endothelial cells),
(for review see Michalopoulos and DeFrances, 1997; Gage, 1998). In skeletal muscle,
committed progenitor cells (satellite cells) located below the basal lamina are induced to
proliferate in response to injury (Mauro, 1961). Skeletal muscle development or repair occurs
along an orderly pathway, with commitment of stem cells to myogenic lineage (myoblasts),
proliferation of myoblasts, and fusion of myoblasts to form myotubes. In contrast to skeletal
muscle, a population of myosatellite-type cells, a potential source of regeneration, is not
formed during cardiomyogenesis. The failure of the adult mammalian myocardium to
reactivate the cell cycle accounts for the major difficulty in restoring the function to the
damaged heart (Armstrong et al., 2000; Olson and Schneider, 2003). Induction of DNA
synthesis as a response to stress or other factors has been described (Ferrans and Rodriguez,
1987; Capasso et al., 1992) however cytoplasmic division or cytokinesis does not occur,
resulting in binucleation or polyploidy (Erokhina et al., 1997; Soonpaa and Field, 1998;
MacLellan and Schneider, 2000; Pasumarthi et al., 2005).
1.8.1 How do Newt and Zebrafish hearts regenerate?
Amphibians, such as newt, are the only adult vertebrates capable of regenerating their organs.
This ability to regenerate large sections of the body plan is widespread in Metazoan
phylogeny, and the discovery was an important aspect of the emergence of experimental
biology in the eighteenth century (for review see Dinsmore, 1992). Although the processes of
tissue restoration unfold in a different way in the heart, limbs and tail of the adult newt, the
outcome depends on the plasticity of the differentiated cells that remain after the tissue
removal. Recently, a similar observation was made in the teleost fish with Zebrafish’s
regeneration of heart and other organs like fins, retina and spinal cord (Poss et al., 2002).
1.8.1.1 Plasticity in the newt limb
The animal heals the wound, cells beneath the epidermis dedifferentiate, and regeneration
occurs by local formation of a growth zone (blastema) that proliferates to form a new organ
(Brockes, 1994; Brockes, 1997). Newt myotubes lack reserve cells, and the regeneration
involves reversal from the differentiated state instead of recruitment of satellite cells. It has
33
Chapter 1
Introduction
been suggested that the environment of the growth zone leads to a destabilization of the
differentiated state (Brockes, 1997).
1.8.1.2 Plasticity in the newt and zebrafish heart
After the removal of the apical region of the ventricle, the heart in both newt and zebrafish
seals by contraction around the clot. Newt and zebrafish adult cardiomyocytes re-enter the
cell cycle and divide in a zone that surrounds the clot (Figure 1.14), (Oberpriller and
Oberpriller, 1974; Oberpriller et al., 1995; Poss et al., 2002). If the animal is injected with
tritiated thymidine, to identify those cells that are in S-phase, approximately 10% of the
cardiomyocytes in this region are labelled in a one-day period. In similar experiments with the
adult mammalian heart, very few cells label after injury (Soonpaa and Field, 1998).
Although both, regeneration of the heart and limb involves re-entry into the cell cycle, the
cardiomyocyte is the only example to retain the differentiated state during regeneration, a
feature that is also observed in culture, (for review see Brockes and Kumar, 2002) whereas in
other tissues including the newt limb and zebrafish fin, the differentiated cells adjacent to the
wound site first dedifferentiate to form a blastema which then gives rise to a fully formed
organ (Scott and Stainier, 2002).
However, no similar response to injury has been observed in the mammalian hearts. Perhaps
the ability to regenerate tissue in several organs, including the heart, has been lost or
diminished through evolution in parallel with the emergence of increased complexity of
patterning and function. Similar to newt and zebrafish, a unique regenerative response was
also documented in the MRL mouse strain after a cryoinjury to the myocardium (Leferovich
et al., 2001). Currently it is not clear whether this phenotype observed in MRL mice is
reflective of actual cardiomyocyte cell division or due to a wound healing response which is
also observed in other tissues of these mice (Heber-Katz et al., 2004). Defining the molecular
basis of cardiac regeneration in these unique model systems may provide fundamental
insights into cardiomyocyte regeneration.
34
Chapter 1
Introduction
Figure 1.14: Regenerating hearts and limbs. Following surgical removal of a portion of the zebrafish ventricle,
cardiomyocytes (green cells) adjacent to the wound site (bright red) undergo proliferation, presumably due to
signals (arrows) emanating from the wound. In contrast to the heart, the newt limb is composed of various
differentiated cell types (indicated by cells of different colours). Following injury, cells adjacent to the wound
epithelium dedifferentiate to form a blastema (green). These cells proliferate and subsequently differentiate to reform a properly patterned limb (Scott and Stainier, 2002).
1.8.2 Reactivation of DNA synthesis in the adult mammalian heart during disease
Mammalian cardiomyocytes retain the ability to undergo partial cell cycle reactivation
following hypertrophic stimulation. It was demonstrated by Rumyantsev that 9% of the
myocytes in the ventricles after ventricular infarction respond by undergoing DNA synthesis
around the perinecrotic area (Rumyantsev, 1974; Rumyantsev, 1977). The increase in the
activities of the G1/S phase cyclin-Cdk complexes has been described, followed by
35
Chapter 1
Introduction
progression of cardiomyocytes through the G1/S transition during development of pressure
overload-induced LVH in rats (Li et al., 1998). Capasso et al. showed that ventricular loading
is coupled with DNA synthesis in adult cardiomyocytes after acute and chronic myocardial
infarction in rats (Capasso et al., 1992). Similarly, it was found that ventricular failure
occurring after acute myocardial infarction (Reiss et al., 1996) or conditions of global
ischemia (Reiss et al., 1993) upregulates the mRNA levels of PCNA and p-histone-H3
protein. In humans, it has been repeatedly found that after myocardial injury the ploidy level
and number of nuclei per myocyte increases (Beltrami et al., 1997; Herget et al., 1997). The
percentage of cardiomyocytes expressing PCNA increases in the diseased human hearts
particularly in response to hypertrophy (Arbustini et al., 1993). Thus, it seems that the heart is
endowed with a programme that protects against continuing proliferation of contracting
cardiomyocytes. Nevertheless, the genetic programme for reinitiating DNA synthesis exists in
post-mitotic cardiomyocytes.
1.9 Attempts to stimulate myocardial regeneration in higher vertebrates
The definitive therapeutic goal in treating patients with heart failure would be either to
preserve the number of pre-existing cells or to increase the number of functionally active
force generating cells. Several approaches to accomplish this are currently under
development. These include genetic manipulation of key cell cycle regulators to promote cell
cycle progression, the exogenous grafting of skeletal myoblasts, fetal and neonatal
cardiomyocytes and embryonic and mesenchymal stem cells into the infarcted myocardium
(for review see Pasumarthi and Field, 2002; Reffelmann and Kloner, 2003; Dimmeler et al.,
2005; Laflamme and Murry, 2005; Smits et al., 2005). Results of all these attempts to
regenerate the diseased heart have been summarised in brief below.
1.9.1 Promoting cell cycle re-entry in cardiomyocytes
Proliferating cells express high levels and activity of cell cycle promoting factors such as
cyclins D1, E, A and B, Cdk2, Cdk4/6, and Cdc2 as well as E2F family members and low
levels of the Cdk inhibitors p21 and p27 (Brooks et al., 1997; Poolman and Brooks, 1998).
Strategies to induce cardiomyocyte proliferation have therefore been directed towards the
genetic manipulation of cell cycle regulatory factors to promote cell cycle progression. For
example, constitutive expression of c-myc, a transcription factor that is known to effect
36
Chapter 1
Introduction
transcription of cell cycle genes in the heart, resulted in increased nuclei per myocyte and
hypertrophy during fetal development (Jackson et al., 1990; Perez-Roger et al., 1999). In
another study, transgenic hearts that expressed high levels of Cdk2 mRNA showed
significantly increased levels of Cdk4 and cyclins A, D3, and E and augmented DNA
synthesis in the adult animal (Liao et al., 2001). Over-expression of all three D-type cyclins
(cyc D1, D2 or D3) in adult transgenic mice could stimulate DNA synthesis but did not result
in a sustained myocyte proliferation under both normal and diseased conditions (Pasumarthi
et al., 2005). Postnatal cardiomyocytes entering into mitosis have been demonstrated in cyclin
A2 overexpressing mice but with no evidence of completing the division (Chaudhry et al.,
2004). Viral proteins such as adenovirus E1A, SV40 large T antigen (SV40) bind to and
inactivate Rb and p107, promote G1 exit of non proliferating cells. In postnatal
cardiomyocytes, E1A expression results in partial cell cycle reactivation with cells arresting
in G2/M (Kirshenbaum and Schneider, 1995). However, a compounding effect of reactivating
DNA synthesis with E1A or SV40 was the increased incidence of apoptosis.
Although most of these studies were able to demonstrate that re-entry of cardiomyocytes into
the cell cycle there was no true evidence of cytokinesis (for review see Dowell et al., 2003;
Regula et al., 2004; von Harsdorf et al., 2004).
1.9.2 Cellular transplantation
Fetal and neonatal cardiomyocytes
Cell transplantation has emerged as a potential therapy for heart diseases and the most
appropriate choice for transplantation are cardiomyocytes themselves to the damaged heart as
the implanted cells should possess the same electrical and mechanical characteristics as native
cardiomyocytes and should enhance the cardiac performance by directly contributing to
contraction. The first studies employed the use of fetal cardiomyocytes in the infarcted and
failing myocardium (Leor et al., 1996) and the results demonstrated that the cardiac
environment was favourable to the engrafted fetal cardiomyocytes (Watanabe et al., 1998).
However, fetal and neonatal cardiomyocytes seem to be highly sensitive to ischemic injury
and their therapeutic use might ultimately require additional interventions. Moreover, human
cardiomyocytes are difficult to obtain and are limited with respect to their ability to be
amplified in culture. The use of fetal cardiomyocytes may also face ethical and political
difficulties in human application (for review see Reinlib and Field, 2000).
37
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Introduction
Skeletal myoblasts
Cell based cardiac repair began with the transplantation of autologous skeletal muscle satellite
cells, progenitor cells that normally mediate regeneration of skeletal muscle (Koh et al., 1993;
Chiu et al., 1995; Taylor et al., 1998). In many species such as dogs, rabbits and rats
engraftment of skeletal myoblasts successfully colonised injured cardiac tissue (Murry et al.,
1996; Taylor et al., 1998; Pouzet et al., 2000). Survival has been shown for 12 weeks after the
transplantation into normal hearts (Reinecke et al., 2002), up to 18 weeks in cryoinjured
myocardium (Chiu, 1999), and Pouzet et al. reported survival along with improved cardiac
performance over a period of 1 year after transplantation (Pouzet et al., 2000). While the
possibility of amplifying satellite cells in vitro, and potentially continuing proliferation after
transplantation are desirable advantages of skeletal myoblasts, the central question whether
they can function as cardiomyocytes, including adaptation to chronic workload and
integration into the host has not been answered so far (for review see Laflamme and Murry,
2005).
Stem cells
Stem cells possess a great potential for regenerative medicine particularly if there are
difficulties obtaining sufficient numbers of donor cells. Embryonic stem cells, which are
totipotent cells derived from the inner cell mass of blastocytes, can differentiate into true
cardiomyocytes, making them in principle an unlimited source of transplantable cells for
cardiac repair although immunological and ethical constraints exist (for review see Smits et
al., 2005).
Somatic stem cells are an attractive option to explore for transplantation as they are
autologous, but their differentiation potential is more restricted than that of embryonic stem
cells. Currently, the major sources of somatic cells used for basic research and in clinical
trials originate from the bone marrow which includes hematopoietic stem cells (HSCs) and
mesenchymal stem cells (MSCs). To date, only mesenchymal stem cells seem to form
cardiomyocytes, and only a small percentage of this population will do so in vitro or in vivo
(for review see Smits et al., 2005).
38
Chapter 1
Introduction
1.9.3 Cardiac progenitor cells
The dogma that the heart is a postmitotic non-regenerating organ has recently been
challenged. Studies have reported the existence of resident cardiac stem cells (Beltrami et al.,
2003; Oh et al., 2003; Laugwitz et al., 2005). Sylvia Evans and coworkers showed that a
subpopulation of cells in the anterior pharynx expresses the homeobox gene islet-1 (isl1) (Cai
et al., 2003). Expression of isl1 is lost when these cells differentiate into cardiomyocytes.
Interestingly, some isl1+ cells can be identified in the mature hearts of newborn rodents and
humans where they remain undifferentiated (Laugwitz et al., 2005). However, it remains to be
determined whether isl1+ cells exist in the adult heart beyond the early postnatal period.
Moreover, their ability to be engrafted in the heart to regenerate myocardium, to electrically
couple, and to contribute to cardiac work remains to be tested (for review see Parmacek and
Epstein, 2005).
Piero Anversa and colleagues reported the discovery of a resident population of cardiac stem
cells (Beltrami et al., 2003). These cells are negative for blood lineage markers CD34, CD45,
CD20, CD45RO and CD8 (Lin−) and positive for c-kit, the receptor for stem cell factor
(SCF). In the adult rat myocardium, Lin− c-kit
pos
cells are relatively rare but more prevalent
than the isl1+ progenitors described above. c-kit positive clones also differentiate into smooth
muscle cells and endothelial cells, indicative of their possible pluripotency. In humans,
significant myocardial regeneration is not observed following myocardial infarction,
suggesting that if c-kit positive cardiac stem cells exist, they are either nonresponsive or
inhibited from migrating and differentiating in response to acute myocardial infarction.
Alternatively, the resident population of cardiac stem cells becomes senescent over time as
most myocardial infarctions occur in older patients (for review see Parmacek and Epstein,
2005).
Schneider and colleagues at the same time also reported a resident population of cardiac
progenitors that copurifies with the nonmyocyte fraction and is characterized by expression of
stem cell antigen 1(Sca-1+) (Oh et al., 2003). However, it remains to be determined whether
the subpopulation of cardiac Sca-1+ cells exists with restricted developmental potential to
differentiate at high frequency into cardiac progenitors or cardiomyocytes (for review see
Parmacek and Epstein, 2005).
These groups suggested that under normal conditions cardiac stem cells are quiescent and
able to revive during cardiac hypertrophy or after myocardial infarction, but it seems even if
39
Chapter 1
Introduction
progenitor properties of these cells exist, the turnover is too slow to repair the damaged
myocardium (for review see Laflamme and Murry, 2005).
1.10 Aim of the study
1.10.1 Characterization of cardiomyocyte proliferation in the embryonic heart
The first part of the project deals with the characterization of cardiomyocyte proliferation in
the embryonic heart as well as the fate of myofibrils in proliferating cardiomyocytes. We also
tried to investigate the lineage relationship of the overtly differentiated cardiac phenotype and
the capacity to divide, in other words to try to define a precursor cardiomyocyte population
with high proliferation potential. In order to determine whether differentiated cardiomyocytes
are capable of undergoing cell division, we used high resolution confocal microscopy on
triple stained specimen of cultured embryonic cardiomyocytes as well as by analysis of
dividing cardiomyocytes in situ by using whole mount preparations of embryonic hearts.
Using this method we were able to determine the state of myofibrillar organization in
cardiomyocytes that had been ascertained as undergoing division by virtue of their expression
of proliferation markers. The factors that regulate myofibril disassembly during mitosis were
also looked into. The existence of a putative Z-disc degradation factor was postulated thirty
years ago (Rumyantsev, 1977), however, its molecular nature is still unclear. Numerous
proteins that are either integral components or transiently associated with the Z-disc have been
identified in the recent years (Faulkner et al., 2001), but for none of them has a direct
involvement in the organization of cardiomyocyte proliferation been shown so far.
1.10.2 Determination of the mechanisms that cause uncoupling of cytokinesis from
karyokinesis after birth in cardiomyocytes
The second part aimed at elucidating the mechanisms that cause the uncoupling of the events
of karyokinesis and cytokinesis, which is typical for postnatal cardiomyocytes. Most cell
cycle regulatory proteins have been shown to be downregulated in adult cardiomyocytes
(Pasumarthi and Field, 2002), but at present little is known about the expression of proteins
that are associated with cytokinesis in cardiomyocytes during development with the exception
of Polo-like kinase, which was shown to be downregulated in the adult heart (Georgescu et
al., 1997). To find out whether neonatal rodent cardiomyocytes stop dividing due to a lack of
the entire cytokinesis machinery or due to the downregulation of an individual regulatory
40
Chapter 1
Introduction
component, it is important to investigate the eventual differences in expression levels or
activity of all these proteins in different stages in the developing heart. We examined the
expression pattern of septins, the GTP-binding proteins that have been shown to be involved
in cytokinesis from yeast to vertebrates in the heart during development and their localization
in dividing cardiomyocytes. We wanted to determine whether septin expression patterns can
be correlated to the cessation of cytokinesis during heart development. In addition we studied
any effects on septin localization in cultured cardiomyocytes after interfering with the ROCK
signaling pathway, with the integrity of the actin cytoskeleton or the activity of myosin.
1.10.3 Determination of the cytokinetic potential of cardiomyocytes during development
and disease
The third part aims at determining the cytokinetic potential of cardiomyocytes in the adult and
diseased heart despite the fact that mitotis is usually not resumed under these conditions. In
this part we set out to investigate (i) the expression pattern of Rho GTPases; (RhoA, Cdc42,
Rac1) and their direct modulators like ROCK I, ROCKII, p-cofilin that are responsible for the
formation of the cleavage furrow, in embryonic heart, (ii) whether these signals are repressed
in neonatal and adult heart, which might account for the uncoupling of karyokinesis from
cytokinesis after birth and (iii) and whether there is re-expression of these markers
responsible for furrow formation again in adult and diseased heart even though mitosis is
usually not resumed under these conditions.
To answer these interesting questions, expression and localization of actomyosin based
contractile ring components were checked at various developmental stages of the heart. In
addition, to determine whether cardiomyocytes make an attempt to go through complete cell
division during the diseased state, different mouse and rat models of pathological hypertrophy
were probed for re-expression of karyokinetic and cytokinetic markers. To test this wellestablished in vitro and in vivo mouse and rat models like β-adrenergic stimulated and
hypertension induced one-kidney, one clip (1K1C) mouse heart, hypertension-induced
hypertrophy of Dahl salt sensitive rats and angiotensin over-expressing mice characterized by
hypertrophy were investigated. For an in vitro model, primary cultures of neonatal rat
cardiomyocytes grown in serum free medium containing hypertrophy inducing agents such as
phenylephrine, isoproterenol and norepinephrine were investigated. These results should
provide important information on the process of cytokinesis in cardiomyocytes in general and
on the influence of the highly specialised cardiac cytoskeleton, namely the myofibrils, on this
process in particular. Due to the intrinsic changes in their cytokinetic behaviour before and
41
Chapter 1
Introduction
after birth, cardiomyocytes might provide an interesting model system to dissect the role of
different components involved in the process of cytokinesis. Understanding these correlations
is not only important for cardiomyocyte biology but may become essential in the study of
neoplastic transformation.
42
CHAPTER 2
Sequential myofibrillar breakdown accompanies
mitotic division of mammalian cardiomyocytes
Preeti Ahuja1, Evelyne Perriard1, Jean-Claude Perriard1 and Elisabeth Ehler2,3
1
2
Institute of Cell Biology, ETH Zurich-Honggerberg, CH-8093 Zurich, Switzerland
The Randall Division of Cell and Molecular Biophysics, King’s College London, London
SE1 1UL, UK
3
The Cardiovascular Division, King’s College London
Journal of Cell Science 117(15): 3295-3306 (2004)
43
Chapter 2
Myofibril disassembly in dividing cardiomyocytes
2.1 SUMMMARY
The contractile tissue of the heart is composed of individual cardiomyocytes. During
mammalian embryonic development, heart growth is achieved by cell division while at the
same time the heart is already exerting its essential pumping activity. There is still some
debate whether the proliferative activity is carried out by a less differentiated, stem cell-like
type of cardiomyocytes or whether embryonic cardiomyocytes are able to perform both of
these completely different dynamic tasks, contraction and cell division. Our analysis of triplestained specimen of cultured embryonic cardiomyocytes and of whole mount preparations of
embryonic mouse hearts by confocal microscopy revealed that differentiated cardiomyocytes
are indeed able to proliferate. However, to go through cell division, a disassembly of the
contractile elements, the myofibrils, has to take place. This disassembly occurs in two steps
with Z-disc and thin (actin) filament associated proteins getting disassembled before
disassembly of the M-bands and the thick (myosin) filaments happens. After cytokinesis
reassembly of the myofibrillar proteins to their mature cross-striated pattern can be seen.
Another interesting observation was that the cell-cell contacts remain seemingly intact during
division, probably reflecting the requirement of intact integration sites of the individual cells
in the contractile tissue.
Our results suggest that embryonic cardiomyocytes have developed an interesting strategy to
deal with their major cytoskeletal elements, the myofibrils, during mitosis. The complex
disassembly-reassembly process might also provide a mechanistic explanation, why
cardiomyocytes cede to divide postnatally.
44
Chapter 2
Myofibril disassembly in dividing cardiomyocytes
2.2 RESULTS
2.2.1 Myofibrillar disassembly in cultured cardiomyocytes during cell division
To study the changes taking place in the cytoskeleton during cell division we first investigated
primary cultures of embryonic cardiomyocytes by triple immunofluorescence. Dividing cells
were unambiguously identified by staining with an antibody specific for phosphorylated
histone H3 (Figure 2.1 B, E, H, K, N). Phosphorylation of Serine 10 in histone H3 is a
prerequisite for the condensation of chromosomes during mitosis (Wei et al., 1998). The
behaviour of the microtubular cytoskeleton shows no difference in dividing cardiomyocytes
compared to other cultured cells (Figure 2.1 C, F, I, L, O). The microtubules are assembled to
a spindle (Figure 2.1 F, I, L), which serves for segregation of the chromosomes and finally
concentrated in the midzone region before the cells pinch off each other (Figure 2.1 O,
arrow). A dramatic difference can be observed when the organization of the myofibrils is
observed during cell division (Figure 2.1 A, D, G, J, M). Sarcomeric alpha-actinin, which is a
component of the Z-disc, is localized in a cross-striated pattern in interphase cardiomyocytes
as well as in cardiomyocytes in early prophase when the chromosomes are not yet condensed
(right hand cell in Figure 2.1 A). In metaphase however, the localization of alpha-actinin
becomes completely diffuse and stays that way during telophase (Figure 2.1 D, G, J, left hand
cell in A). Only in late cytokinesis as demonstrated in Figure 2.1 M, is the cross-striated
localization pattern of alpha-actinin re-established. These results suggest that the myofibrils
undergo a disassembly-reassembly cycle during cell division in cultured cardiomyocytes.
2.2.2 Myofibrillar disassembly occurs in sequential and biphasic manner
To find out whether all components of the sarcomere are affected in a similar way during
division we stained cultured cardiomyocytes for alpha-actinin, a Z-disc and an M-band
epitope of titin, cardiac alpha-actin, sarcomeric myosin heavy chain, myosin binding proteinC and for the M-band protein myomesin. In dividing cardiomyocytes in metaphase, as
identified by the visualization of condensed chromosomes with the staining for
45
Chapter 2
Myofibril disassembly in dividing cardiomyocytes
phosphorylated histone H3 (Figure 2.2 B, D, F, H, J, L, N), interesting differences in the
organization can be observed. While Z-disc associated proteins like alpha-actinin and also Z-
Figure 2.1: Single confocal sections of immunostained cultured embryonic cardiomyocytes at different cell
division stages showing the degree of disassembly of the myofibrils with an antibody against the Z-disc protein
alpha-actinin (A, D, G, J, M). The spindle apparatus is visualized by staining for tubulin (C, F, I, L, O), while
dividing cells can be identified by staining with an antibody that specifically recognizes phosphorylated histone
H3 (B, E, H, K, N). In prophase, when the nuclear membrane is still intact, clear cross-striations can be seen for
alpha-actinin (panel A, right hand cell). During metaphase, when the condensed chromosomes are arranged in
the middle of the cell, the signal for alpha-actinin gets diffuse (panel D) and stays like that during early anaphase
(panel G), when the chromosomes start to be pulled towards the poles, telophase (panel J) and early cytokinesis
(panel A, left hand cell), when the signal for phosphorylated histone H3 disappears. Reassembly of alpha- actinin
to a cross-striated pattern starts in late cytokinesis (panel M). The arrow in O indicates, where the daughter cells
are being pinched off. Bar represents 10 µm.
46
Chapter 2
Myofibril disassembly in dividing cardiomyocytes
disc epitopes of titin attain already a diffuse localization pattern at this stage (Figure 2.2 A,
C), the organization of the thick filaments, as indicated by the localization of sarcomeric
myosin heavy chain (Figure 2.2 G), myosin binding protein-C (Figure 2.2 I) and of the Mband with myomesin (Figure 2.2 K) and an M-band epitope of titin (Figure 2.2 M) remains
remarkably intact despite the presence of condensed chromosomes in these cells. Due to the
different states of contraction, it is very difficult to identify well separated I-bands in cultured
cardiomyocytes; nevertheless the localization of cardiac actin appears more diffuse in
metaphase cardiomyocytes as well (Figure 2.2 E). Therefore there is an interesting delay in
the way different parts of the sarcomere are disassembled during cell division with Z-discs
and thin filaments attaining a diffuse localization pattern before A-band components.
In order to determine whether thick filaments and M-bands remain intact throughout cell
division and how myofibril reassembly occurs, we compared the localization pattern of
different sarcomeric proteins in cultured cardiomyocytes in anaphase, telophase and late
cytokinesis. The different stages of the cell cycle were judged as above either by staining for
phosphorylated histone H3 (Figure 2.3 D-F) or for tubulin (Figure 2.3 J-L; P-R). While
cardiac alpha-actin shows a completely diffuse localization pattern in anaphase
cardiomyocytes (Figure 2.3 A), M-bands and A-bands only start to get disassembled at this
stage as indicated by the partially still cross-striated staining pattern obtained for myomesin
and sarcomeric myosin heavy chain (Figure 2.3 B and C, respectively). Complete myofibril
disassembly as indicated by diffuse staining for M-band, thin and thick filament proteins is
only seen in cardiomyocytes in telophase (Figure 2.3 G, H, I). Reassembly of myofibrils
occurs quite fast after cytokinesis since cross-striations for all investigated sarcomeric
proteins can be seen soon after the cells have started to segregate from each other (Figure 2.3
M, N, O). These results suggest that in dividing cardiomyocytes a biphasic disassembly of
myofibrils occurs, with Z-disc and thin filament associated components being disassembled
before A- and M-bands. Nevertheless, in order to be able to go through telophase and to
complete cytokinesis, disassembly of the entire myofibrils seems to occur in cardiomyocytes.
After cell division reassembly happens soon, leading to a cross-striated localization pattern
that is indistinguishable from neighbouring non-dividing cells.
47
Chapter 2
Myofibril disassembly in dividing cardiomyocytes
Figure 2.2: Single confocal sections showing the comparison of the disassembly level of various sarcomeric
proteins present in cultured cardiomyocytes during metaphase (B, D, F, H, J, L, N). While cardiac alpha-actin
(E) as well as Z-disc associated epitopes like alpha-actinin (A) and titin T12 (C) display a mostly diffuse staining
pattern throughout the entire cytoplasm already at this stage; sarcomeric myosin heavy chain (G), myosin
binding protein-C (I) and M-band associated epitopes like myomesin (K) and titin T51 (M) show still an intact
localization pattern. Bar represents 10 µm.
48
Chapter 2
Myofibril disassembly in dividing cardiomyocytes
Figure 2.3: Localization pattern of different sarcomeric proteins in cultured cardiomyocytes during later stages
of mitosis in single confocal sections. Late metaphase/early anaphase was visualized by staining for
phosphorylated histone H3 (D-F), the later stages of mitosis by staining for tubulin (J-L and P-R). The
sarcomeric protein stained for in the left column was cardiac alpha-actin, in the middle myomesin and right
sarcomeric myosin heavy chain as indicated above. While cardiac alpha-actin (A) is completely diffuse at late
metaphase, myomesin (B) and sarcomeric myosin heavy chain (C) only start to disassemble at this stage (small
arrowheads point at still intact myofibrils). Only when chromosomes are being segregated, cardiac alpha-actin
49
Chapter 2
Myofibril disassembly in dividing cardiomyocytes
(G), myomesin (H) as well as myosin heavy chain (I) show a diffuse pattern with some remaining aggregates,
especially in the case of the latter. During cytokinesis, as identified by tubulin concentration in the midbody (PR), all sarcomeric proteins start reassembly to myofibrils and display a cross-striated pattern again (M-O). The
arrows point at the dividing cells. Bar represents 10 µm.
To exclude that these differences in myofibril assembly of different parts of the sarcomere are
caused by slight temporal differences between the analyzed cardiomyocytes we investigated
the organization of the Z-disc protein alpha-actinin and MyBP-C as marker of the thick
filaments in the same cell (Figure 2.4). In interphase cells cross-striations can be seen for both
sarcomeric components (Figure 2.4 A-C). However, in metaphase a clear distinction in the
organization is apparent with alpha-actinin being mainly diffuse (Figure 2.4 E), while MyBPC displays still distinct double bands in the same cell (Figure 2.4 F). Only by anaphase both
proteins have attained a diffuse localization pattern with some aggregated material (Figure 2.4
H, I), which is retained in early telophase (Figure 2.4 K, L). These observations show clearly
that myofibril disassembly happens in at least two steps, with Z-disc material being
disassembled before the thick filaments. A compilation of the state of assembly for different
parts of the sarcomere at a given stage of cell division can be found in Table 2.1.
2.2.3 Myofibril disassembly also occurs in dividing cardiomyocytes in situ
Cultured cardiomyocytes display important differences compared to cardiomyocytes in situ.
There are obvious differences in cellular shape and in the surrounding of the cells, but there
are also differences in cellular processes like responsiveness to growth factors (Armstrong et
al., 2000) and in myofibrillogenesis (Ehler et al., 1999). Therefore we wanted to find out
whether the disassembly of myofibrils with the delay between different parts of the sarcomere
could also be observed in cardiomyocytes in situ. Whole mount preparations of embryonic
mouse hearts were stained for phosphorylated histone H3 and at the same time for the cellcell contact protein beta-catenin to be able to visualize the cell borders. In addition, different
components of the sarcomere were stained and the whole mount preparations were
subsequently analysed by confocal microscopy. Interestingly, also in situ myofibrils are
disassembled in dividing cardiomyocytes with a similar delay between different parts of the
sarcomere as in cultured cardiomyocytes. In metaphase cardiomyocytes, Z-disc associated
epitopes like alpha-actinin or titin T12 are already completely diffuse (Figure 2.5 B, E) while
50
Chapter 2
Myofibril disassembly in dividing cardiomyocytes
Figure 2.4: Sequential disassembly of different parts on the sarcomere analyzed in single confocal sections.
Embryonic rat cardiomyocytes were stained for alpha-actinin (panel B, E, H, K; red in overlay A, D, G, J) and
for MyBP-C (panel C, F, I, L; green in overlay) as indicated above the columns. The stage of cell division was
identified by staining for tubulin (blue in overlay). In the interphase cell, clear cross-striations can be seen for
both sarcomeric proteins. At metaphase the localization pattern of alpha-actinin is already diffuse (arrow), while
double bands are still visible for MyBP-C (arrowheads), which only redistributes by the anaphase and telophase
stage. Bar represents 10 µm.
51
Chapter 2
Myofibril disassembly in dividing cardiomyocytes
Mitotic stage
Prophase
Metaphase
Anaphase
Telophase
Cytokinesis
Mitotic stage
Metaphase
Mitotic
stage
Metaphase
State of
myofibril
assembly
+++
++
+
-
State of
myofibril
assembly
+++
++
+
+++
++
+
+++
++
+
+++
++
+
+++
++
+
-
alpha-actinin
myomesin
100% (30/30)
0% (0/30)
0% (0/30)
0% (0/30)
0%
(0/65)
0% (0/65)
8% (5/65)
92% (60/65)
0% (0/28)
0%
(0/28)
0% (0/28)
100% (28/28)
0% (0/32)
0%
(0/32)
9.37% (3/32)
90.62% (29/32)
0% (0/20)
45% (9/20)
55% (11/20)
0%
(0/20)
100% (25/25)
0% (0/25)
0% (0/25)
0% (0/25)
60% (18/30)
40% (12/30)
0% (0/30)
0% (0/30)
0% (0/20)
0% (0/20)
10% (2/20)
90% (18/20)
0% (0/28)
0% (0/28)
0% (0/28)
100% (28/28)
0% (0/20)
100% (20/20)
0% (0/20)
0% (0/20)
cardiac alphaactin
0%
0%
10%
90%
(0/30)
(0/30)
(3/30)
(27/30)
State of
myofibril
assembly
titin T12
(Z-disc)
+++
++
+
-
0% (0/26)
0% (0/26)
34.6% (9/26)
65.3%(17/26)
M-band
Z-disc
cardiac
troponin I
sarc MyHC
MyBP-C
0% (0/20)
0% (0/20)
15% (3/20)
85% (17/20)
25% (6/24)
62.5%(15/24)
12.5% (3/24)
0% (0/24)
53% (24/45)
47% (21/45)
0% (0/45)
0% (0/45)
titin 9D10
(I-band)
titin K58
(A-band)
titin T51
(M-band)
0%
0%
40%
60%
(0/20)
(0/20)
(8/20)
(12/20)
45%
55%
0%
0%
(9/20)
(11/20)
(0/20)
(0/20)
80% (20/25)
20% (5/25)
0% (0/25)
0% (0/25)
+++ intact myofibrils, ++ some disorganization, + partial disassembly, - complete disassembly
Table: 2.1 Quantification of the state of myofibril assembly in cardiomyocytes during mitosis based on number
of observed cases over total number of cells analyzed.
52
Chapter 2
Myofibril disassembly in dividing cardiomyocytes
Figure 2.5: Single confocal sections of whole mount preparations of embryonic mouse hearts labelled with
antibodies against different sarcomeric proteins (B, E, H, K; red in overlay A, D, G, J) and against
phosphorylated histone H3 together with antibodies against beta-catenin to delineate the cell-cell contacts (C, F,
I, L; green in overlay). Also in dividing cardiomyocytes in the heart in situ, alpha-actinin (A, B) and titin T12
(D, E), which are Z-disc associated proteins/epitopes, are diffuse at a time when the localization pattern of
myomesin (G, H) remains still quite intact (arrowheads delineate the cell borders, small arrows point at intact
myofibrils in neighboring cardiomyocytes (D, E; J, K) or in dividing cells (G, H). The staining for titin T51 (J,
K), which is an M-band associated titin epitope, becomes diffuse only by anaphase, similar to myomesin (data
not shown). Continuous staining for beta-catenin along the plasma membrane also in dividing cardiomyocytes
indicates that cardiomyocytes retain their contacts to the neighboring cells during division. Bar represents 10
µm.
53
Chapter 2
Myofibril disassembly in dividing cardiomyocytes
the M-band protein component myomesin still shows a partially cross-striated localization
pattern (Figure 2.5 H, small arrows). Only in late anaphase cardiomyocytes also M-band
epitopes like titin T51, myomesin and sarcomeric myosin heavy chain start to appear in a
diffuse localization as well (Figure 2.5 K, data not shown). Therefore myofibril disassembly
happens also during cytokinesis in the developing heart in situ and shows a similar biphasic
dynamics as in cultured cardiomyocytes.
In addition, dividing cardiomyocytes in situ stay tightly connected to their presumably
contracting neighboring cells and there is only little change in the overall cellular shape. The
staining for the adherens junction protein beta-catenin remains continuous in dividing cells,
similar to the localization pattern seen in the surrounding non-dividing cardiomyocytes. At
this stage of development, the segregation of intercalated disc proteins to the sites of terminal
myofibril insertion has not yet been achieved and adherens junctions as well as other types of
cell-cell contacts are still distributed all around the plasma membrane (Perriard et al., 2003).
2.2.4 How is myofibril disassembly regulated?
To assess whether the myofibrils in dividing cardiomyocytes are merely disassembled and
reassembled afterwards or whether protein degradation plays a role as well in this process we
stained cultured cardiomyocytes with antibodies against ubiquitin (Figure 2.6 B, E, H) in
combination with alpha-actinin to delineate the myofibrils (Figure 2.6 A, D, G) and with
tubulin to assess the stage of cell division (Figure 2.6 C, F, I). Ubiquitination is the first step
in non-lysosomal protein degradation (Hershko and Ciechanover, 1992) and we do indeed
find an upregulation of ubiquitin expression in dividing cardiomyocytes. While in interphase
cardiomyocytes the signal for the ubiquitin antibody is rather weak and mainly associated
with the nuclei as reported previously (Hilenski et al., 1992); Figure 2.6 B, cells in top left
corner of E); once cardiomyocytes enter mitosis, ubiquitin starts to be spread throughout the
cytoplasm (Figure 2.6 B, arrow). The ubiquitin fluorescence increases throughout telophase
and remains high during cytokinesis, suggesting that ubiquitination of proteins takes place
also in dividing cardiomyocytes (Figure 2.6 E, H). Due to the low frequency of cell division
in our cultures as well as in the developing heart in situ, we were unable to analyse by
biochemical means, whether myofibrillar proteins themselves are ubiquitinated. However, the
high intensity of the ubiquitin signal could mean that protein degradation also of components
54
Chapter 2
Myofibril disassembly in dividing cardiomyocytes
of the sarcomere or of proteins, which control sarcomere integrity, might happen during cell
division.
Figure 2.6: Expression of ubiquitin is upregulated in dividing cardiomyocytes. Cultured cardiomyocytes were
stained with antibodies to sarcomeric alpha-actinin (A, D, G) together with antibodies against ubiquitin (B, E, H)
and against tubulin (C, F, I) to identify the stage of cell division. Single confocal section reveal that while
interphase cardiomyocytes display only little signal for ubiquitin (panel B); a drastic increase in the signal for
ubiquitin can be detected in cardiomyocytes in metaphase (panel E) as well as in cytokinesis (panel H). There is
no clear-cut colocalization between alpha-actinin and ubiquitin. Bar represents 10 µm.
55
Chapter 2
Myofibril disassembly in dividing cardiomyocytes
Further evidence for the role of ubiquitin-mediated degradation for myofibril disassembly
comes from experiments, where inhibitors that interfere with the proteasome pathway were
used on dividing cardiomyocytes. Treatment with MG132 leads to metaphase cells that still
display cross-striations in the alpha-actinin staining, while control cells show a completely
diffuse localization of this protein (Figure 2.7).
We conclude that the myofibrils have to be disassembled to achieve successful cell division in
cardiomyocytes and that this disassembly process is probably regulated by factors, which are
part of ubiquitination pathways.
Figure 2.7: Disassembly of myofibrils is delayed in cardiomyocytes that were treated with MG132 to inhibit
proteasome degradation. Single confocal sections of cardiomyocytes stained with monoclonal antibodies to
sarcomeric alpha-actinin (B, D; red in A, C) and for tubulin (blue in A, C) as well as for phosphorylated histone
(green in A, C) to identify the stage of mitosis. While in control cells at metaphase all the alpha-actinin is
localized in a diffuse fashion throughout the entire cell (A, B), cross-striated myofibrils can still be seen in
MG132 treated cardiomyocytes (C, D). Bar represents 10 µm.
56
Chapter 2
Myofibril disassembly in dividing cardiomyocytes
2.3 DISCUSSION
By comparing the distribution of different components of the sarcomere in triple-stained
dividing cardiomyocytes in culture and in the developing heart at different stages of mitosis
we were able to find out that myofibril disassembly occurs prior to cytokinesis in at least two
steps. Disassembly of individual components of the myofibrillar apparatus like sarcomeric
alpha-actinin or sarcomeric myosin heavy chain in cultured cardiomyocytes was described
previously by others (Conrad et al., 1991; Li et al., 1996; Li et al., 1997b; Du et al., 2003);
however, so far no comparative analysis was performed. Our novel finding is that
disassembly of the myofibrils shows a biphasic dynamics. Proteins or epitopes of proteins that
are associated with the Z-disc or the thin filaments display a diffuse localization pattern at a
time when the thick filaments are still comparatively intact as indicated by a cross-striated
pattern for proteins like myosin heavy chain, MyBP-C or myomesin. A schematic
representation of myofibril disassembly during cell division at the level of the individual
sarcomere is depicted in Figure 2.8.
Figure 2.8: Schematic representation of sarcomere disassembly during cell division. Thick (myosin and
associated proteins) filaments are represented in dark blue, thin (actin and associated proteins) filaments in
yellow and titin filaments in red. The Z-disc is shown in green, the M-band in purple. In metaphase, Z-disc
associated proteins are already disassembled (indicated by font in italics), while the thick filaments and the Mband still remain intact, only to be disassembled in late anaphase.
57
Chapter 2
Myofibril disassembly in dividing cardiomyocytes
This process is exactly reversed to the time-course of myofibril assembly, where the first
organized complexes consist of alpha-actinin, actin and Z-disc epitopes of titin at a time when
all the thick filament components are still completely diffuse throughout the sarcomere (Ehler
et al., 1999). On the other hand, myofibril disassembly as it occurs during myopathies or
muscle wasting is also characterized by degradation of Z-disc and I-band material before the
rest of the sarcomere is affected (Taylor et al., 1995). During this process, degradation of
myofibrils seems to be mainly regulated by the activity of muscle-specific calpain, which can
associate with the I-band region of titin (Kinbara et al., 1998) and thereafter by the ubiquitinproteasome pathway (Hasselgren and Fischer, 2001). A possible explanation for the
prolonged existence of intact thick filaments comes from their structure and way of assembly.
Myosin molecules are characterized by the ability to associate to bipolar filaments on their
own in the test tube (Margossian et al., 1987). In addition, the putative M-band cross-linking
molecule, myomesin, which seems to be important to provide a link between titin and myosin,
binds so strongly to titin, that it can even be detected on isolated titin molecules (Fürst et al.,
1989). It appears that once the myosin molecules have been integrated by myomesin, they
represent a rather stable structure. Also during myofibrillogenesis in cultured cardiomyocytes
so-called floating A-bands have been observed, which were then integrated into nascent
myofibrils (Schultheiss et al., 1990); suggesting first, that the assembly of thick and thin
filaments is an independent process and that also their disassembly might be regulated
autonomously.
How can M-bands still stay in register at a time when Z-disc epitopes of titin are already
localized in a completely diffuse fashion? It has been thought that titin provides a basic
cytoskeletal framework together with alpha-actinin and myomesin for myofibril assembly
(Ehler et al., 1999). While this seems indeed to be the case for myofibrillogenesis, as
indicated by the absence of properly formed myofibrils in cells that lack titin or express only
truncated forms of it (van der Ven et al., 2000; Xu et al., 2002), there must be another means
to hold the M-bands at least temporarily in place in dividing cardiomyocytes. One possibility
is the intermediate filament network, consisting of desmin in cardiomyocytes. However,
desmin filaments are mainly concentrated around the Z-disc and evidence for their
continuation along the myofibrils to the M-band is still conflicting (Small et al., 1992). In
addition, the cross-striated pattern that desmin filaments show in cardiomyocytes in situ gets
completely lost in cultured cardiomyocytes during the first days and reorganization only takes
place after prolonged culture periods (Ehler and Perriard, 2000). Another possibility for
registering is a link from the myofibrils to the plasma membrane. The costameres, the major
58
Chapter 2
Myofibril disassembly in dividing cardiomyocytes
integrating complex between the myofibrils and the membrane do show a cross-striated
pattern in situ as well as in cultured cells, however, they are situated at the Z-disc region
(Pardo et al., 1983). It is still not quite clear whether and how myofibrils are connected to the
lateral membrane at the M-band level. Previously it was thought that skelemin would serve as
a linker between M-bands and the membrane (Price, 1987) but since skelemin has been
identified as a splice variant of the integral M-band protein myomesin and it could be shown
that embryonic M-bands consist exclusively of this isoform, this possibility is highly unlikely
(Steiner et al., 1999; Agarkova et al., 2000). Another candidate for the linkage of M-bands to
the membrane is spectrin, based on its localization pattern (Williams and Bloch, 1999; Flick
and Konieczny, 2000). Recent evidence has suggested an interaction between ankyrin, a
binding partner of spectrin in erythrocytes and the giant M-band protein obscurin (Bagnato et
al., 2003). It remains to be determined whether this multiprotein complex is indeed the
filamentous material that provides a link between the M-band and the plasma membrane, as
proposed by electron microscopy results (Pierobon-Bormioli et al., 1981; Nakamura et al.,
1983).
In contrast to most cell lines, dividing cardiomyocytes in vitro and even more so in situ do not
round up completely during mitosis and stay in relatively close association with their
neighboring cells and the extracellular matrix. This was especially apparent when we studied
dividing cells in whole mount preparations of embryonic hearts, where the cell-cell contact
sites stayed absolutely intact in cells that were positive for phosphorylated histone H3. This is
in good agreement with pioneering ultrastructural studies on dividing cardiomyocytes, where
it could also be shown that no loosening of cell-cell contacts occurs (Manasek, 1968). The
fact that the contractile work of the cardiac tissue has to go on while individual
cardiomyocytes divide, probably does not allow to interfere too much with the integrity of the
tissue.
Currently it is not quite clear whether the myofibrils are only disassembled and the sarcomeric
proteins are recycled again after cytokinesis or whether protein degradation of myofibrillar
components takes place as well. The drastic upregulation of ubiquitin expression in dividing
cardiomyocytes suggests that protein degradation is important; however, possibly this is
mainly the case for proteins that are immediately involved in cell cycle regulation like the
cyclins and the cdks (Peters, 2002). On the other hand, the increased expression levels of
ubiquitin could also indicate a proteasome-independent function, as described recently (see
review by (Schnell and Hicke, 2003). However, the results from the MG132 experiment point
towards a role of protein degradation in regulating myofibril disassembly, although not
59
Chapter 2
Myofibril disassembly in dividing cardiomyocytes
necessarily degradation of sarcomeric proteins. The fact that although no longer assembled
into clear cross-striations, structural components like alpha-actinin, myomesin and titin can
still be stained by antibodies and show a subcellular localization that is distinct from the
signal for ubiquitin rather argues for a recycling of these sarcomeric components. Similar
recycling processes seem to occur when adult rod-shaped cardiomyocytes adapt to culture
conditions. While they flatten and attach to the culture dish in the presence of serum, their
myofibrillar material is aggregated to a clump; once the cells have spread, myofibrils are
reassembled and beating activity is resumed (Messerli et al., 1993). In addition, it has been
shown that myofibril assembly in cultured myocytes was not blocked by addition of
cycloheximide, suggesting that protein synthesis is not essential (Rumyantsev, 1977).
At present the factors that regulate myofibril disassembly during mitosis have not been
identified yet. The existence of a putative Z-disc degradation factor was already postulated
thirty years ago (Rumyantsev, 1977), however, its molecular nature is still unclear. Numerous
proteins that are either integral components or transiently associated with the Z-disc have
been identified in the recent years (Faulkner et al., 2001), but for none of them a direct
involvement in the organization of cardiomyocyte proliferation was shown so far. Also at the
M-band numerous proteins that are potentially involved in signalling pathways have been
shown in addition to bona fide structural sarcomeric proteins. Among these are e.g. the
MURFs, muscle-specific ring finger proteins that are associated with the ubiquitin pathway
(Bodine et al., 2001; McElhinny et al., 2002; Pizon et al., 2002) and have been shown to be
involved in the regulation of muscle atrophy. Obscurin, is a giant M-band protein that
possesses several domains that have been associated with different signalling pathways and
also contains a Rho guanine exchange factor domain, suggesting that it could be involved in
the activation of the small GTPase Rho (Young et al., 2001). Activation of the Rho signalling
pathway seems to be important for cardiomyocyte proliferation during development, as
demonstrated by the embryonic lethality of the conditional expression of the Rho inhibitor
GDIα in the heart (Wei et al., 2002). In conclusion, these proteins might represent a sensory
machine that is associated with the sarcomere under normal conditions but which might adopt
a second function as signalling molecules under conditions of additional work load, stress and
myofibril disassembly during cytokinesis or in disease. Unfortunately, the low rate of division
in culture as well as in situ does not permit a thorough biochemical analysis of the identity of
the ubiquitinated proteins at present; however based on results published on proteins for other
cell types an ubiquitination of e.g. the MURFs is rather likely to occur. Preliminary
experiments in our lab have shown that the signals for MURF-2 and for ubiquitin show a
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Myofibril disassembly in dividing cardiomyocytes
similar subcellular localization in dividing cultured cardiomyocytes (Ahuja, Ehler, Gautel and
Perriard, unpublished observations).
It is astonishing that the cells should break down such an elaborate structure as the myofibrils
to undergo mitosis and it will be exciting to find out about the signalling pathways that lead to
this disassembly and possibly degradation process. The observation that the myofibrils have
to be disassembled for cytokinesis to occur might provide also a simple mechanistic
explanation, why cardiomyocytes cease to divide after birth. With the hypertrophic growth
that is caused by the increased workload on the heart after birth, this disassembly - reassembly
process might be simply too costly from an energetic point of view. In addition, too many
cytoskeletal elements in the form of myofibrils might physically impede cell division as well.
This together with the alterations in the expression levels of proteins that regulate the cell
cycle and cytokinesis, respectively might contribute to the uncoupling of karyokinetic and
cytokinetic events as seen in postnatal rodent cardiomyocytes (Li et al., 1996; Georgescu et
al., 1997; Poolman and Brooks, 1998).
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CHAPTER 3
Probing the role of septins in cardiomyocytes
Preeti Ahuja1, Evelyne Perriard1, William Trimble2, Jean-Claude Perriard1 and
Elisabeth Ehler3,4
1
2
3
Institute of Cell Biology, ETH Zurich-Honggerberg, CH-8093 Zurich, Switzerland
Hospital for Sick Children, University of Toronto, Toronto, Ontario, M5G1X8, Canada
The Randall Division of Cell and Molecular Biophysics, King’s College London, London
SE1 1UL, UK
4
The Cardiovascular Division, King’s College London
Accepted in Experimental Cell Research (2006)
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3.1 SUMMARY
Heart growth in the embryo is achieved by division of differentiated cardiomyocytes. Around
birth cardiomyocytes stop dividing and heart growth occurs only by volume increase of the
individual cells. Cardiomyocytes seem to lose their capacity for cytokinesis at this
developmental stage. Septins are GTP-binding proteins that have been shown to be involved
in cytokinesis from yeast to vertebrates. We wanted to determine whether septin expression
patterns can be correlated to the cessation of cytokinesis during heart development. We found
high expression of only SEPT2, SEPT6, SEPT7 and SEPT9 in heart, in a developmentally
regulated fashion, with high levels in the embryonic heart, downregulation around birth and
no detectable expression in the adult. In dividing embryonic cardiomyocytes all septins
localize to the cleavage furrow. We used drugs to probe for the functional interactions of
SEPT2 in dividing embryonic cardiomyocytes. Differences in the effects on subcellular septin
localization in cardiomyocytes were observed, depending whether a Rho kinase (ROCK)
inhibitor was used or whether actin and myosin were targeted directly. Our data show a tight
correlation of high levels of septin expression and the ability to undergo cytokinesis in
cardiomyocytes. In addition, we were able to dissect the different contributions of ROCK
signaling and the acto-myosin cytoskeleton to septin localization to the contractile ring using
cardiomyocytes as an experimental system.
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3.2 RESULTS
3.2.1 Expression of septins in heart during development
In order to determine which septins are expressed in mouse heart and whether there is any
differential regulation during development we performed immunoblotting with antisera raised
against different septins. The expression of ten different septins was investigated:SEPT1
(Diff6), SEPT2 (Nedd5), SEPT3 (G septin), SEPT4 (Bh5), SEPT5 (CDCrel-1), SEPT6
(KIAA0128), SEPT7 (Cdc10), SEPT8 (KIAA0202), SEPT9 (MSF) and SEPT11 (FLJ10849).
The sequences chosen for each peptide, as indicated in the methods, were isoform-specific
and did not significantly overlap with any other proteins in the GenBank database. Only four
septins, SEPT2, SEPT6, SEPT7 and SEPT9 showed considerable expression levels in heart
tissue (Figure 3.1, lanes 2-5), while the other septin antibodies only gave a signal in the
positive control brain tissue (Figure 3.1, lane 1). It should be noted that many septin genes
undergo alternative splicing, only some of which has been characterized, and it remains
possible that the peptide-specific antisera could fail to detect all of the protein products from
each gene. However, of the isoforms detected by the antibodies, SEPT2 and SEPT6 are
expressed at higher levels in the samples from embryonic heart (Figure 3.1, lane 3), get
downregulated by the first postnatal week and are absent in adult heart (Figure 3.1, lane 2).
Equal loading of cardiac tissue at different developmental stages is demonstrated by
comparable intensities for cardiac actin expression. In the case of SEPT7, two isoforms are
detected with the larger form being less abundant in brain and more abundant in heart. Again,
both isoforms appear to be downregulated as development proceeds. In contrast, SEPT9
levels appear to rise during these initial developmental stages before declining to low levels in
the adult heart. This suggests that cardiomyocytes express only a subset of septins at
detectable levels. In addition, septins are only highly expressed in cardiomyocytes that are
still able to divide and do not seem to play a major role in the adult heart.
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Figure 3.1: Expression of different septin family members in the heart during development. The expression of
SEPT1 to 9 and SEPT11 was analysed by immunoblotting of cell lysates from embryonic (E14) heart (lane 3),
day of birth (P0; lane 4), day eight (P8; lane 5), and adult ventricular muscle (lane 2). Only four septins, SEPT2
(Nedd5), SEPT6 (KIAA0128), SEPT7 (Cdc10) and SEPT9 (MSF) were detected in heart, while all antibodies
showed a signal in the control sample of brain tissue (lane 1). They were expressed in a developmental-stage
specific fashion. The expression is high in samples from embryonic heart, starts to get down regulated after birth
and is completely absent or marginally present in the adult heart (lane 2). Equal loading of heart samples is
demonstrated by immunostaining for cardiac actin.
65
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Septins in heart
3.2.2 Localization pattern of the different septins in cultured embryonic rat
cardiomyocytes during cytokinesis
To find out where the septins are localized in the cell, we determined the subcellular
distribution of SEPT2, SEPT6 and SEPT9 in cultured rat embryonic cardiomyocytes by triple
immunofluorescence (Figure 3.2a). We were unable to investigate the subcellular location of
SEPT7, since none of the several antibodies that we tested worked for immunofluorescence.
Cardiomyocytes undergoing cytokinesis were identified by staining with an antibody specific
for tubulin, which finally amasses in the midzone region before the cells pinch off each other
(Figure 3.2(a) J, K, L; blue signal in A, B, C). Sarcomeric alpha-actinin, which is a
component of the sarcomeric Z-disc, served as a myofibrillar marker (Figure 3.2(a) D, E, F;
red in A, B, C) and is localized in a cross striated pattern in interphase and prophase
cardiomyocytes. In metaphase, anaphase and telophase however, the localization of alphaactinin becomes completely diffuse and only in late telophase the cross-striated localization
pattern begins to re-establish (Ahuja et al., 2004). SEPT2, SEPT6 and SEPT9 could only be
detected during late telophase of cardiomyocytes and showed midbody localization (Figure
3.2(a) G, H, I, green in A, B, C; indicated by arrows). SEPT2 and SEPT6 are expressed at
higher levels than SEPT9 and also display localization throughout the entire midbody region,
while SEPT9 seems to be confined to the edges. In addition to the signal at the cleavage
furrow, SEPT2 showed a cytoplasmic signal that was at times associated with the actin
filaments (compare with the localization of alpha-actinin). Expression of septins during late
telophase and their localization at the midbody during this time, suggests that they are
involved in cytokinesis of dividing cardiomyocytes, similar to observations made in yeast and
other mammalian cell types (Kinoshita et al., 1997; Surka et al., 2002). The sequential
condensation of the septin signal during cleavage furrow formation is demonstrated in Figure
3.2(b), which shows the distribution of SEPT2 at late anaphase, telophase and cytokinesis in
dividing cardiomyocytes.
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Septins in heart
Figure 3.2(a): Localization pattern of the three different septins in cultured embryonic rat (E14) cardiomyocytes
(ERC) during cytokinesis. Confocal micrographs of ERC stained for the individual septin (panels G, H, I, green
in overlay), for the myofibrillar protein sarcomeric alpha-actinin (panels D, E, F, red in overlay) and for tubulin
to assess the stage of mitosis (panels J, K, L, blue in overlay). While SEPT2 and SEPT6 show midbody
localization and some actin association in the case of SEPT2, SEPT9 shows in addition colocalization with the
microtubules. Bar represents 10 µm.
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Chapter 3
Septins in heart
Figure 3.2(b): Distribution of SEPT2 at different stages of cell division in cardiomyocytes. Confocal
micrographs of cultured ERCs stained for SEPT2 (panel G, H, I; green in overlay), sarcomeric alpha-actinin (D,
E, F; red in overlay) and tubulin (panel J, K, L; blue in overlay) show the gradual condensation of the SEPT2
signal to the constricting cleavage furrow during cytokinesis. Bar represents 10 µm.
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Chapter 3
Septins in heart
3.2.3 Effect of a cytokinetic inhibitor on septin localization in embryonic rat
cardiomyocytes
To identify the specific role of septins in cardiomyocytes, we next investigated the effect of a
cytokinetic inhibitor on septin localization in dividing cardiomyocytes. Cultured rat
embryonic cardiomyocytes were treated with the ROCK Kinase inhibitor, Y-27632 (Figure
3.3), which is known to inhibit cytokinesis by preventing the formation of the cleavage furrow
(Ishizaki et al., 2000; Kosako et al., 2000). In untreated cells, SEPT2, (Figure 3.3 C, single
arrow) and SEPT6, (Figure 3.3 K, single arrow) were mainly localized at the cleavage furrow,
while in cells treated with Y-27632, SEPT2, (Figure 3.3 G, double arrow) and SEPT6, (Figure
3.3 O, double arrow), got redistributed. Interestingly, we saw a differential effect on the
distribution of these two septins. While SEPT2 relocalizes to areas in the periphery of the cell,
SEPT6 gets associated with the spindle in Y-27632 treated cardiomyocytes. SEPT2 and
SEPT6 have been suggested to interact closely in other cell types and to our knowledge this is
the first time that a distinct effect can be seen on the distribution of these two septins in a cell
type (Macara et al., 2002). The apparent elevation of septin expression levels during the phase
of cytokinesis seems to be independent of ROCK signaling for both SEPT2 and SEPT6.
However, their assembly in the future midbody region of dividing cardiomyocytes does
require active ROCK and cleavage furrow formation.
3.2.4 Effect of Cytochalasin D on SEPT2 localization in embryonic rat cardiomyocytes
In interphase and postmitotic cells, SEPT2 is known to co-localize with actin stress fibers
(Kinoshita et al., 1997). It has been suggested that intact actin fibers are necessary for the
maintenance of SEPT2-containing fibers (Kinoshita et al., 1997). In order to find out whether
septin distribution is also affected in cardiomyocytes upon actin depolymerization, we
incubated the cells with an inhibitor of actin polymerization, Cytochalasin D (Figure 3.4). In
untreated cells, SEPT2, (Figure 3.4 E, single arrow) mainly localizes at the midbody of the
dividing cell while in the cells treated with Cytochalasin D, SEPT2, (Figure 3.4 F, double
arrows) appears to be disrupted and accumulates in puncta and small circles near the
membrane. These SEPT2 aggregates and rings in treated cells are reminiscent of septin
localization in Cytochalasin D treated fibroblasts (Kinoshita et al., 2002). No truly separated
cells could be observed in cytochalasin-treated cultures, despite the fact that furrow ingression
looks initially normal. The actin in the myofibrils of the cardiomyocytes remains unaffected
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Chapter 3
Septins in heart
by Cytochalasin D treatment, as shown previously for mature myofibrils (Rothen-Rutishauser
et al., 1998). These results suggest that in cardiomyocytes a distinct population of actin fibers,
which is affected by Cytochalasin D, is necessary for the redistribution of septins to the
midbody region and thus cytokinesis.
Figure 3.3: Prevention of cytokinesis affects septin localization in embryonic rat cardiomyocytes.
Immunofluorescence analysis by confocal microscopy of control ERC (panels A-D; I-L) and ERC treated with
Y-27632, an inhibitor of cytokinesis (panels E-H; M-P), stained for the individual septins, SEPT2, (panels C, G,
green in overlay A, E) and SEPT6, (panels K, O, green in overlay I, M) respectively. Sarcomeric alpha-actinin
was stained as a myofibrillar marker (panels B, F, J, N, red in overlay) and tubulin to assess the stage of mitosis
(panels D, H, L, P, blue in overlay). Localization of SEPT2 was mainly found at the cell periphery (panel G,
indicated by double arrow) after the treatment with the cytokinetic inhibitor Y-27632 at the concentration of 100
µM for 4 hours, while SEPT6 got associated with the microtubular spindle (panel O; double arrows). In control
cells for both the septins (panel C, K indicated by single arrow) mainly contractile ring localization could be
found. Bar represents 10 µm.
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Chapter 3
Septins in heart
Figure 3.4: Inhibition of actin polymerization affects SEPT2 localization in embryonic rat cardiomyocytes.
Immunofluorescence analysis by confocal microscopy of control ERC (panels A, C, E, G) and ERC treated with
Cytochalasin D, an inhibitor of actin polymerization (panels B, D, F, H), stained for SEPT2, (panels E, F, green
in overlay). Cardiac actin was stained (panels C, D, red in overlay) as a myofibrillar marker and tubulin to assess
the stage of mitosis (panels G, H, blue in overlay). SEPT2 in control cells was mainly found at the midbody of
the dividing cells (single arrow in E). After the treatment with Cytochalasin D at the concentration of 100µg/ml
for 1hour, SEPT2 localization appears to be disrupted and it accumulates in dots and circles (double arrow in F;
circles pointed out by arrowheads). Bar represents 10 µm.
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3.2.5 Interference with different parts of the cytoskeleton has distinct effects on SEPT2
and non muscle myosin IIB distribution in embryonic rat cardiomyocytes
The localization pattern that SEPT2 adopted following treatment with Y-27632 is very
reminiscent of the distribution of non muscle (nm) myosin IIB in cultured cardiomyocytes,
namely in peripheral areas of the cell. Since nm myosin IIB is also involved in the contractile
ring we wanted to analyze its distribution in cultured cardiomyocytes following drug
treatment and compare it with the one of SEPT2 (Figure 3.5). Determination of the stage of
mitosis was performed by DAPI staining of the chromosomes (data not shown). In control
cardiomyocytes that are dividing nm myosin IIB can be detected at the cleavage furrow
(indicated by an asterisk in all overlay panels), where it colocalizes with SEPT2. Nm myosin
IIB is excluded from areas that contain myofibrils, but there is an additional signal in the
periphery, where the cells spread (Figure 3.5 A-D; arrowheads in C). Inhibition of ROCK
signaling with Y-27632 leads to a redistribution of SEPT2 to the same peripheral areas, while
the organization of nm myosin IIB is similar to control cells (Figure 3.5 E-H). Upon
Cytochalasin D treatment SEPT2 accumulates to aggregates and rings, where it colocalises
with nm myosin IIB (Figure 3.5 I-L). Interestingly, inhibition of myosin ATPase activity by
incubation with Blebbistatin results in a segregation of the SEPT2 and the nm myosin IIB
signal. While the former is now found preferentially associated with the plasma membrane
(arrowheads in 5N), nm myosin IIB localization seems to be little affected (Figure 3.5 M-P).
Long-term incubation (>2h) of the cardiomyocytes with Blebbistatin leads to a shrunken
phenotype with many blebbings at the periphery, indicating that this cell type is extremely
sensitive to this drug. Taken together these results suggest that 1) SEPT2 has a tendency to
cosegregate with nm myosin IIB but only if the activity of the myosin ATPase is maintained,
2) interaction with additional proteins that are subjects to ROCK regulation keep SEPT2 in
the cleavage furrow and prevent it from associating with nm myosin IIB in the cellular
periphery and 3) interference with the actin cytoskeleton leads to disruption of the distribution
of nm myosin IIB and its relocation in aggregates together with SEPT2 (see also discussion
and Figure 3.6).
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Septins in heart
Figure 3.5: Comparative analysis of the localization of SEPT2 and nm myosin IIB after treatment with
differentdrugs. Confocal micrographs of cultured ERC treated with Y-27632 (1mM) for 4h (panels E-H),
Cytochalasin D (100 µg/ml) for 1h (panels I-L) or Blebbistatin (100µM) for 2h (panels M-P) just before fixation
or control cells (A-D). Cells were triple-stained for SEPT2 (B, F, J, N; red in overlay), nm myosin IIB (C, G, K,
O; green in overlay) and sarcomeric alpha-actinin to identify cardiomyocytes (D, H, L, P). Determination of the
stage of mitosis was performed by DAPI staining (not shown). In control cells SEPT2 is restricted to the
cleavage furrow, but redistributes to the cellular periphery after treatment with Y-27632. There it colocalizes
with nm myosin IIB, which is generally present in the peripheral areas of cell spreading and attachment
(arrowheads in C). Depolymerization of the actin filaments leads to SEPT2 being localized in small aggregates
throughout the cytoplasm, while interference with myosin activity results in SEPT2 association with the plasma
membrane (arrowheads in N). With the exception of the latter case, SEPT2 is usually found in close association
with nm myosin IIB. Dividing cells are indicated by two arrows in the overlay, the region of the cleavage furrow
is indicated by an asterisk. Bar represents 10 µm.
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Chapter 3
Septins in heart
3.3 DISCUSSION
Analysis of septin expression in the heart during development revealed that from the 12
mammalian septin genes that were characterized so far (Martinez and Ware, 2004) only a
subset of four, namely SEPT2, SEPT6, SEPT7 and SEPT9 are expressed at the protein level
and that they display highest expression at developmental stages when cardiomyocytes still
possess the ability to undergo cytokinesis. Unfortunately, many septin genes undergo
alternative splicing and we cannot rule out the possibility that splice variants of some of the
septins may not be recognized by our antibodies. However, they do recognize abundant forms
in many tissues and all recognize a band in the brain. Consistent with this different expression
levels of distinct septins have been described in other tissues, for example while SEPT2
seems to be fairly ubiquitously expressed, SEPT4 is mainly found in spleen, brain, kidney
lung and testis (Xie et al., 1999), tissue types which also show high expression levels of
SEPT9 in addition to its expression in heart (Surka et al., 2002). Expression profiling by
electronic northern blots of the entire septin gene family also revealed ubiquitous expression
of some septins and tissue specific expression of others, mainly in lymphoid or brain tissues
(Hall et al., 2005).
Recently another septin, SEPT13, has been identified that shows
expression in heart at least at the mRNA level (Hall et al., 2005); due to the lack of antibody
we were not able to characterize this septin in cardiomyocytes. The four septins that we
identified in heart are representatives of four evolutionary distinct classes of septins
(Kinoshita, 2003), suggesting that cardiomyocytes may only express the minimal required set
of septins to form a complex (Versele and Thorner, 2005). In addition, cardiomyocytes show
a developmental stage specific expression of septins with high levels being only expressed at
stages when cytokinesis still occurs (i.e. embryonic and perinatal). Septins have been
suggested to play a role for vesicle trafficking, cytoskeletal remodeling and apoptosis, but do
not seem to be required at high amounts in adult cardiomyocytes. We suggest that at
embryonic stages similar levels of septin expression occur in dividing and interphase
cardiomyocytes. Our explanation for the lack of signal that we observe in surrounding
interphase cells is that the signal intensity in the cleavage furrow is too high to allow for
visualization of diffuse septins in the neighbouring cells without saturation. Drugs that lead to
septin aggregation either in the cytoplasm like Cytochalasin D or at the plasma membrane like
Blebbistatin do reveal the presence of septin expression also in interphase cells.
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Septins in heart
Upon treatment with Y-27632 we observed a differential effect on the localization of SEPT2
compared to SEPT6. While SEPT2 cosegregated with nm myosin IIB to the periphery of the
cell, SEPT6 started to show association with the spindle. Mitotic spindle association of septin
family members that are known to target to the actin cytoskeleton has been observed under
other experimental conditions as well (Spiliotis et al., 2005) and its significance remains to be
determined.
The minimal set of septins that is expressed in cardiomyocytes comprises all the components
that are required for septin complex formation (Versele and Thorner, 2005). At the moment it
is not quite clear whether these kind of complexes are necessarily formed at all times in the
cellular environment. For example SEPT2 and SEPT9 associate with distinct cytoskeletal
filament systems, the actin filaments and the microtubuli, respectively (Kinoshita et al., 1997;
Surka et al., 2002). In addition, differential upregulation of distinct septins has been shown in
disease (Hall et al., 2005). Further indications for the existence of a crosstalk between the
expression of different septin isoforms but not necessarily in the stoichiometric amounts to
form complexes come from the analysis of the SEPT5 knockout mouse, which shows a
concomitant upregulation of SEPT2 and a downregulation of SEPT7 (Peng et al., 2002).
The most important observations that we have made in our studies concern the distinct
behavior of SEPT2 following the application of drugs that are known to interfere with
different aspects of cytokinesis. Inhibition of Rho signaling resulted in SEPT2 redistributing
to the cellular periphery to colocalize with non-sarcomeric nm myosin, interference with actin
filaments other than the myofibrils resulted in aggregates of SEPT2 and nm myosin
throughout the cell, while blocking of myosin ATPase activity lead to dissociation of the
septin and nm myosin signals and to an aggregation of SEPT2 at the plasma membrane (see
Figure 3.6). The membrane association of septins is probably mediated by a conserved
polybasic region that can bind phosphatidylinositol 4,5 biphosphate (PtdIns(4,5)P2) (Zhang et
al., 1999). Some of the effects that we observe are probably due to side effects of the drugs
that were employed. For example Y-27632 inhibits ROCK, but also downstream
phosphorylation of MLC, which in turn is required for nm myosin IIB assembly at the
contractile ring; in addition the concentrations that we used will also inhibit citron kinase,
another player in the establishment of the contractile ring. Blebbistatin is claimed to be one of
the most specific inhibitors of myosin ATPase and only works on non muscle and skeletal
muscle myosins but not on smooth muscle myosins or other members of the myosin family
(Straight et al., 2003). This specificity was explained on a structural basis recently by the
identification of the Blebbistatin contact residues in the myosin molecule (Allingham et al.,
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Septins in heart
2005). While there are similarities between the three-dimensional structure of the active sites
of myosin and P-loop proteins such as the septins (Smith and Rayment, 1996), no threedimensional structure of a septin is available at the moment and it is therefore not quite clear
whether Blebbistatin might also have a side effect on septins, which might result in this
membrane association of SEPT2.
Our high resolution confocal microscopy studies allowed us also to delineate subtle
differences between septin and nm myosin localization at the cleavage furrow, with septin
being localized always more centrally than nm myosin IIB (see Figure 3.6 inset) while actin,
as deduced from the signal of alpha-actinin, is localized more distally. This distribution is
even maintained in Y-27632 treated cultured cardiomyocytes, where again the residual septin
and nm myosin IIB in the region of the prospective cleavage furrow are localized more
centrally than actin. These observations support the hypothesis that one of the functions of
septins is to act as a scaffold for the assembly of a contractile ring; presumably via the
interaction with anillin (Straight et al., 2005).
Figure 3.6: Model of the distribution of SEPT2, nm myosin IIB and actin in dividing ERCs under different
culture conditions. The proposed location of the actin is based on staining for alpha-actinin. In control
cardiomyocytes nm myosin heavy chain is localized at the areas of cell spreading in the periphery and in
addition also at the cleavage furrow together with SEPT2 and actin. Careful analysis of the confocal images
revealed that septin is always localized further towards the middle of the cleavage furrow than nm myosin IIB
and actin. Incubation with Y-27632 leads to an inhibition of ROCK, its downstream target MLCK and at the
concentration used also of citron kinase. Furrowing is reduced although some residual assembly of contractile
ring proteins can still be seen; again septin and nm myosin IIB are localized more centrally than actin. The rest
of the septin gets redistributed to the peripheral areas of the cells, where it colcalizes with nm myosin IIB.
Cytochalasin D leads to a depolymerization of all actin filaments except the myofibrils. Furrowing is reduced
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Septins in heart
and septin, nm myosin IIB and actin colocalize in aggregates throughout the cytoplasm. Blebbistatin inhibits the
myosin ATPase activity and leads to a complete diffuse distribution of nm myosin IIB throughout the cytoplasm.
While actin is still present in fibers that span the cytoplasm, septin relocalizes and shows strong association with
the plasma membrane.
In conclusion, our results on septin expression during heart development have provided a
possible explanation why karyokinesis and cytokinesis become uncoupled in cardiomyocytes
after birth, namely the reduced levels of septin expression. This reduction cannot be explained
alone by hypertrophic growth and thus dilution of the septin signal in the protein samples by
an excess of myofibrillar material, since dramatic increases in cardiomyocyte size and
myofibril content are only seen later (Leu et al., 2001). In addition, the drug treatment
experiments using a highly differentiated cell system with an elaborated cytoskeleton such as
the cardiomyocyte have enabled us to have a closer look at the function of SEPT2 at the
cellular level.
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CHAPTER 4
Analysis of the cytokinetic potential of
cardiomyocytes during development and disease
Preeti Ahuja1, Evelyne Perriard1, Thierry Pedrazzini2, Shinji Satoh3, Jean-Claude
Perriard1 and Elisabeth Ehler4,5
1
2
3
Institute of Cell Biology, ETH Zurich-Honggerberg, CH-8093 Zurich, Switzerland
Division of Hypertension and Vascular Medicine, University of Lausanne Medical School,
CH-1011 Lausanne, Switzerland
Medical Institute of Bioregulation, Kyushu University, 4546 Tsurumihara, 874-0838, Beppu
Japan
4
The Randall Division of Cell and Molecular Biophysics, King’s College London, London
SE1 1UL, UK
5
The Cardiovascular Division, King’s College London
To be submitted (2006)
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4.1 SUMMARY
The heart is the first functional organ in the embryo. In the early postnatal period the
cardiomyocytes stop dividing and growth is mainly achieved by increase in cell volume.
However, in some species cardiomyocytes can undergo an additional incomplete mitosis
where karyokinesis takes place in the absence of cytokinesis leading to binucleation. There
are several key players known that are important for cytokinesis such as small GTPases like
RhoA, Rac1, Cdc42, septins along with their downstream effectors like ROCK I, ROCK II,
Citron Kinase. We demonstrate here for the first time that cardiomyocytes show a
developmental stage specific expression of all the proteins associated with the formation of
the actomyosin ring with high levels being only expressed at stages when cytokinesis still
occurs (i.e. embryonic and perinatal). This suggests that downregulation of expression of
many regulatory and cytoskeletal components involved in cytokinesis may be responsible for
uncoupling of cytokinesis from karyokinesis in cardiomyocytes after birth. Also we found out
that while stressed myocardium during pathological hypertrophy tries to adapt to the
increased work load there is indeed an upregulation of karyokinetic and cytokinetic proteins,
despite the fact that the mitotic cycle is usually not resumed under these conditions. The
failure to undergo complete division could be due to the presence of stable, highly ordered
and functional sarcomeres in the adult myocardium or could be because of the absence of
degradation mechanisms (ubiquitin or calcium dependent) which facilitate division of
embryonic cardiomyocytes by disintegrating myofibrils.
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4.2 RESULTS
4.2.1 Expression and localization of various actomyosin ring associated proteins in the
heart during development
To ascertain whether neonatal rodent cardiomyocytes stop dividing due to the lack of their
entire cytokinesis machinery or by downregulation of an individual regulatory component, the
expression pattern of different Rho GTPases and their downstream effectors were investigated
at different stages of mouse heart development by immunoblotting with specific antibodies.
We analyzed different cytokinetic markers; RhoA (panel A), Rac1 (panel B), Cdc42 (panel
C), p-Cofilin (panel D), ROCK II (panel E), ROCK I (panel F) and a karyokinetic marker
PCNA (panel G) in embryonic day 14 (lane 1), day of birth P0 (lane 2), P8 (lane 3) and adult
mouse heart (lane 4) along with adult mouse kidney tissue lysate (lane 5) as a positive control
for all the cytokinetic markers (Figure 4.1). In the embryonic heart (Figure 4.1, lane 1), high
levels of expression of all the cytokinetic as well as karyokinetic markers could be found,
which get downregulated by the first postnatal week and were almost absent in adult heart
(Figure 4.1, lane 4). This suggests that the Rho GTPases and their modulators tested here are
highly expressed in cardiomyocytes that are still able to divide and do not seem to play a
major role in the adult heart.
Subcellular distributions of the Rho GTPases RhoA, Cdc42 and Rac1 in cultured rat
embryonic cardiomyocytes (E14) were determined by triple immunofluorescence (Figure
4.2). Cardiomyocytes undergoing cytokinesis were identified by staining with an antibody
specific for tubulin (Figure 4.2 J, K, L; blue in overlay), which finally concentrates in the
midzone region before the cells pinch off to form daughter cells (indicated by double arrows
in the Figure 4.2 A, B, C). Myosin binding protein C (MyBP-C), which is a component of the
sarcomeric A-band, served as a myofibrillar marker (Figure 4.2 D, E, F; red in overlay).
MyBP-C is localized in a cross-striated pattern in interphase, prophase and metaphase
cardiomyocytes, however, the localization of MyBP-C becomes completely diffuse during
anaphase and only after cytokinesis the cross-striated localization pattern begins to reestablish (Ahuja et al., 2004). We found that only during late telophase of cardiomyocytes,
RhoA, Cdc42 and Rac1 were expressed at high levels and restricted at the midbody region of
dividing cardiomyocytes (Figure 4.2 G, H, I, indicated by arrow).
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Expression of Rho GTPases and their downstream effectors during late telophase and their
localization at the midbody during embryonic stage implies that they are involved in
cytokinesis of dividing cardiomyocytes, similar to what has been observed in most
mammalian cells (Guertin et al., 2002). Thus, downregulation of many regulatory and
cytoskeletal components involved in cytokinesis may be responsible for uncoupling
cytokinesis from karyokinesis in cardiomyocytes after birth.
Figure 4.1: Expression pattern of different cytokinesis associated proteins during developmental stages of the
heart. Cell lysates from embryonic heart (E14, lane 1), day of birth (P0, lane 2), day eight (P8, lane 3), adult
mouse ventricular muscle (lane 4), and adult mouse kidney tissue lysate (positive control, lane 5) were analysed
by immunoblotting for expression of several actomyosin associated proteins; RhoA (panel A), Rac1 (panel B),
Cdc42 (panel C), p-Cofilin (panel D), ROCK II (panel E), ROCK I (panel F) and PCNA (panel G) as a
karyokinetic protein. Equal amounts of heart tissue were loaded; α-cardiac actin was used as loading control
(bottom panel). All markers coupled with karyokinesis and cytokinesis are expressed high in samples from
embryonic heart, start to get downregulated after birth and are completely or almost absent in the adult heart.
Equal amounts of total proteins were loaded in each lane, but the intensity of the bands obtained for several
antibodies does not reflect the relative amounts of these proteins compared to each other.
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Figure 4.2: Localization of three different actomyosin ring associated proteins in cultured embryonic rat (E14)
cardiomyocytes (ERC) during division. Confocal micrographs of ERC stained for RhoA, Rac1, Cdc42 (panels
G, H, I respectively and green in overlay), for the myofibrillar protein Myosin Binding Protein-C (MyBP-C),
(panels D, E, F and red in overlay) and for tubulin to assess the stage of mitosis (panels J, K, L and blue in
overlay). Double arrows in panels A, B and C point to the cells undergoing cytokinesis. All the three GTPases
indicated by arrows in panels (G, H and I) show strong midbody localization in embryonic cardiomyocytes
undergoing cytokinesis. Bar represents 10 µm.
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4.2.2 Re-expression of cytokinesis associated proteins in heart during hypertrophy
After having shown that karyokinetic and cytokinetic proteins are downregulated after birth
and are absent in adult heart, we now wanted to explore the possibility of cardiomyocytes
making an attempt to undergo complete cell division during the diseased state of the heart. It
is believed that cell cycle re-entry is an innate response to hypertrophy and that there is a
partial progress through the cell cycle under these conditions, which signifies an important
adaptive aspect of the myocardium to stress (Ferrans and Rodriguez, 1987; Li et al., 1998;
Capasso et al., 1992). To test, whether cytokinetic associated markers also reappear in heart
during myocardial response to hypertrophy, well-established in vitro and in vivo mouse and
rat models for hypertrophy were investigated.
In vivo models of pathological hypertrophy
4.2.2.1 β-adrenergic stimulated and hypertension induced one-kidney, one clip (1K1C)
mouse heart
In order to determine whether there is re-appearance of cytokinetic markers during
myocardial response to hypertrophy, immunoblots were performed on β-adrenergic receptor
agonist isoproterenol stimulated mouse heart and on hypertension induced one-kidney, one
clip (1K1C) mouse heart (Wiesel et al., 1997) cell lysates (Figure 4.3) with antibodies against
different cytokinesis coupled proteins; RhoA (panel A), Cdc42 (panel B), Rac1 (panel C),
ROCKII (panel D), and ROCKI (panel E) and the karyokinetic protein PCNA (panel F). A
marked increase in the expression of all the cytokinesis associated proteins tested here along
with PCNA could be observed in isoproterenol treated for one week (lane 2) and two weeks
(lane 3) compared to the control (lane 1) and also in 1K1C mouse (lane 5) in contrast to
control, sham operated mouse heart (lane 4). These results suggest that under a stressed state,
cardiomyocytes do in fact attempt to re-enter the cell cycle, as all karyokinetic and cytokinetic
associated proteins investigated here get upregulated; however, somehow cells cannot
complete the cell cycle consequentially leading to even an increased binucleation or
polyploidy level.
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Figure 4.3: Re-expression of cytokinesis coupled proteins in β-adrenergic stimulated and in hypertension
induced one-kidney, one clip (1K1C) mouse heart. Immunoblot with different cytokinetic proteins on SDS
samples of isoproterenol treated and 1K1C mouse heart together with their respective controls. There is a marked
increase in the expression of all the cytokinesis coupled proteins; RhoA (panel A), Cdc42 (panel B), Rac1 (panel
C), ROCKII (panel D), and ROCKI (panel E), including PCNA (panel F) in hypertrophic samples of
isoproterenol treated mice after one week (lane 2) and two weeks (lane 3) compared to the control (lane 1) and in
the 1K1C mouse (lane 5) in contrast to its control (lane 4), sham operated mouse heart. Equal amounts of the
heart tissue were loaded; α-cardiac actin was used as loading control (bottom panel).
4.2.2.2 Hypertension-induced hypertrophy of Dahl salt sensitive rats
To further test our hypothesis of cardiomyocytes making an attempt to undergo complete cell
division during hypertrophy by expressing karyokinetic and cytokinetic markers,
hypertension-induced hypertrophic hearts of Dahl salt-sensitive (DS) rats (Satoh et al., 2003)
were examined (Figure 4.4). This animal model develops hypertension, thus promoting
myocardial hypertrophy, left ventricular (LV) dilation, and heart failure when fed on a highsalt diet (Inoko et al., 1994; Doi et al., 2000). Immunoblot analysis was performed on left
ventricular tissue lysates obtained from three groups of Dahl salt rats which were on a high
salt diet: (1) Dahl salt resistant (DR) rats served as a normotensive control, (Figure 4.4 (A),
group 1, lane SR-14 and FW-2). (2) Dahl salt sensitive (DS) rats, (Figure 4.4 (A), group 2,
lane SS-62, SS-64 and SS-66) and (3) DS rats treated with Y-27632, a selective ROCK
inhibitor (Figure 4.4 (A), group3, lane SS-61, SS-67 and SS-68) were probed with different
cytokinesis associated proteins; RhoA, Cdc42, Rac1, ROCK II, and ROCK I.
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Figure 4.4: (A): Re-expression of cytokinesis coupled proteins in hypertension-induced hypertrophy of Dahl salt
sensitive rats. Immunoblot on Dahl salt sensitive (DS) and resistant rats (DR) fed on high salt diet with different
cytokinesis related protein markers. SDS samples of left ventricular tissue of three groups: (1) DR rats as a
normotensive control, (lane SR-14 and FW-2). (2) DS rats with no treatment, (lane SS-62, SS-64 and SS-66) (3)
DS rats treated with Y-27632, a selective ROCK inhibitor (lane SS-61, SS-67 and SS-68) were probed with
different cytokinesis associated proteins; RhoA, Cdc42, Rac1, ROCKII, and ROCKI, including PCNA as a
karyokinetic marker. DS rats, which did not undergo any treatment (group 2) and developed hypertrophy showed
high expression of all the cytokinetic markers including PCNA in comparison to DR rats (group1), which served
as normotensive controls. DS rats treated with Y-27632 (group 3) showed a slightly blunted response in their
expression of cytokinesis markers in contrast to DS rats with no treatment (group2). Equal amounts of heart
tissue were loaded; α-cardiac actin was used as loading control (bottom panel). (B) Quantification of expression
of cytokinetic proteins in Dahl salt induced hypertrophy model. Data are presented as mean ± standard error of
calculated ratios of arbitrary units from three sets of each experiment.
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DS rats, which did not undergo any treatment (Figure 4.4 (A), group 2) and had developed
hypertrophy displayed high expression of all the cytokinetic markers in contrast to control DR
rats (Figure 4.4 (A), group1). It has been previously shown that ROCK pathway inhibition in
DS sensitive rats blunts the development of left ventricular hypertrophy (LVH), and thereby,
preserves myocardial contractile function (Satoh et al., 2003). Hence as anticipated, DS rats
treated with Y-27632, an inhibitor of ROCK kinase (Figure 4.4 (A), group 3) showed a
slightly blunted response in expression of all the cytokinetic markers checked, in contrast to
DS rats with no treatment (Figure 4.4 (A), group2).
Quantification of expression of cytokinetic proteins in the Dahl salt induced hypertrophy
model (Figure 4.4 (B)) clearly shows that cytokinesis associated proteins are elevated in Dahl
salt sensitive rats compared to Dahl salt resistant rats, when fed on a high salt diet. Thus,
implying that cardiomyocytes do make an attempt to complete division by upregulating both
karyokinetic and cytokinetic machinery during hypertrophy.
4.2.2.3 Angiotensin over-expressing mice characterized by hypertrophy
Ventricular extracts of transgenic mice over-expressing angiotensinogen in cardiomyocytes
(Mazzolai et al., 1998), (Figure 4.5 (A)) and transgenic mice over-expressing angiotensinogen
treated with the β adrenergic receptor agonist, isoproterenol for four weeks (Figure 4.5 (B)),
were probed for cytokinesis associated proteins; RhoA, Rac1, Cdc42, ROCKII, and ROCKI.
As expected, there was a marked increase in the expression of all the cytokinesis associated
proteins in cell lysates of mouse hearts over-expressing angiotensin (Figure 4.5, group 2 of A)
and in angiotensinogen mice treated with isoproterenol (Figure 4.5, group 2 of B) when
compared with their respective controls (Figure 4.5, group 1 of A) and (Figure 4.5, group 1
(angiotensinogen over-expression alone) and group 3 (wild type) of B).
All these results imply that indeed there is division potential in the diseased hearts as evident
from upregulated karyokinetic and cytokinetic protein markers in all the in vivo models tested
here, to form functional cardiomyocytes, however; there is an obstruction which impedes the
cells to do that and thus consequently repair the damaged myocardium.
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Figure 4.5: Re-expression of cytokinesis associated proteins in angiotensin over-expressing mice characterized
by hypertrophy. Immunoblot on SDS samples of (A) transgenic mice overexpressing angiotensinogen in
cardiomyocytes and in (B) transgenic mice overexpressing angiotensinogen treated with β adrenergic receptor
agonist, isoproterenol for four weeks, probed with cytokinesis associated proteins; RhoA, Rac1, Cdc42,
ROCKII, and ROCKI. There is a marked increase in the expression of all the cytokinesis associated proteins in
cell lysates of mouse hearts overexpressing angiotensin (group 2; of A) and in angiotensinogen mice treated with
isoproterenol (group 2; of B) compared with controls (group 1; of A) and (group 1(angiotensinogen
overexpressing mice alone) and 3 (wild type); of B) respectively. Equal amounts of heart tissue were loaded; αcardiac actin was used as loading control (bottom panel).
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4.2.3 Localization of upregulated cytokinetic protein markers in diseased heart
We next investigated the subcellular distribution of upregulated cytokinetic proteins in the
diseased hearts by immunofluorescence. Cryosections of wild type (Figure 4.6 A-D, I-L) and
1K1C mouse hearts (Figure 4.6 E-H, M-P)) were triple stained with antibodies to M protein
(Fig. 4.6 B, F, J, N; red in overlay) to identify the cardiomyocytes, RhoA (Figure 4.6 C, G;
green in overlay A,E) and Cdc42 (Figure 4.6 K, O; green in overlay I, M), were used as
cytokinesis associated protein markers while non-muscle myosin heavy chain IIB (Figure 4.6
D, H, L, P; blue in overlay) was used to visualize cardiac fibroblasts. Single confocal sections
revealed an absence of expression of cytokinesis-associated proteins in wild type sections
(Figure 4.6 C, K) while at the same time 1K1C sections (Figure 4.6 G, O) displayed a drastic
increase in cytoplasmic and myofibrillar distribution for both the cytokinetic proteins RhoA
(Figure 4.6 G) and Cdc42 in the cardiomyocytes (Figure 4.6 O). Additionally there was an
increase in fibrosis in sections from 1K1C hearts, as determined by staining for non-muscle
myosin heavy chain IIB (Figure 4.6 H, P) in contrast to sections from sham-operated animals
(Figure 4.6 D, L).
4.2.4 Re-expression of cytokinesis associated proteins in an in-vitro model of
hypertrophy
To verify the results obtained from in vivo models of hypertrophy, a well-documented in vitro
model of hypertrophy in cardiomyocytes (Deng et al., 2000) was next investigated. Primary
cultures of neonatal rat cardiomyocytes (NRC) were grown in serum free medium containing
hypertrophy inducing agents; phenylephrine (α-adrenergic receptor agonist with weak ßadrenergic receptor agonist activity; 100µM), isoproterenol (ß- adrenergic receptor agonist;
10µM) and norepinephrine (α and ß- adrenergic receptor agonist; 10µM) or vehicle for 72
hours. Triplicate samples of the respective condition were probed on immunoblots for
cytokinesis associated proteins with antibodies (Figure 4.7); RhoA, Rac1, ROCK II, p-cofilin,
polo like kinase and as karyokinetic markers PCNA, p-His H3 were chosen. All the triplicate
samples of primary cultures of neonatal rat cardiomyocytes treated with phenylephrine,
isoproterenol and norepinephrine showed a marked increase in the expression of all
karyokinetic and cytokinetic coupled proteins in contrast to the control cardiomyocytes.
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Quantification of expression of cytokinetic proteins in an in-vitro model of induced
hypertrophy (Figure 4.7 (B)) shows that cytokinesis associated proteins are elevated during
hypertrophic conditions induced by the agonists in comparison to the control neonatal rat
cardiomyocytes.
Figure 4.6: Confocal micrographs illustrating the localization of upregulated cytokinetic protein markers in
diseased heart. Cryosections of wild type (panels A, B, C, D and I, J, K, L) and 1K1C mouse heart (panels E, F,
G, H and M, N, O, P) triple stained with antibodies to M protein (panels B, F, J, N and red in overlay) to
visualize cross striations in the cardiomyocytes, RhoA in (panels C, G and green in overlay A, E), Cdc42 in
(panels K, O and green in overlay I, M), as cytokinesis associated protein marker and non-muscle myosin heavy
chain IIB (panels D, H, L, P and blue in overlay) to visualize cardiac fibroblasts. Single confocal sections reveal
that wild type sections (panels C and K) shows no cytokinetic proteins while at the same time 1K1C sections
(panels G and O) displays a drastic increase in signal for both the cytokinetic proteins RhoA (panel G) and
Cdc42 (panel O) in cardiomyocytes along with increased fibrosis (panels H and P) in contrast to their respective
control (panel D and L). Bar represents 10 µm.
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Figure 4.7: Re-expression of cytokinesis associated proteins in an in-vitro model of hypertrophy. (A)
Immunoblot on cell lysates of primary cultures of neonatal rat cardiomyocytes (NRC) grown in serum free
medium containing hypertrophy inducing agents; phenylephrine (α-AR agonist with weak ß-AR agonist activity;
100µM), isoproterenol (ß-AR agonist; 10µM) and norepinephrine (α and ß-AR agonist; 10µM) or vehicle for 72
hours. Triplicate samples of each condition were probed with cytokinesis associated proteins; RhoA, Rac1,
ROCK II, p-cofilin, polo like kinase and for karyokinetic markers; PCNA, p-His H3. There is marked increase in
the expression of all karyokinetic and cytokinetic proteins tested here, in phenylephrine, isoproterenol and
norepinephrine induced hypertrophic cardiomyocytes in contrast to control neonatal rat cardiomyocytes.
(B) Quantification of expression of cytokinetic proteins in hypertrophic neonatal rat cardiomyocytes. Data are
presented as mean ± standard error of calculated ratios of arbitrary units from three sets of each experiment.
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4.2.5 Subcellular distribution of RhoA in primary cultures of neonatal rat
cardiomyocytes (NRC) following the treatment with hypertrophy inducing agents
Immunostaining of neonatal rat cardiomyocytes (NRC) following the treatment with
hypertrophy inducing agents was subsequently done to determine the localization of one of
the upregulated cytokinetic proteins, RhoA. Cardiomyocytes were stained for the cytokinetic
marker, RhoA; (Figure 4.8 I-L; green in overlay), for the myofibrillar protein, cardiac actin,
(Figure 4.8 E-H; red in overlay) and DAPI to detect any binucleated cell resulting from
incomplete cell division (Figure 4.8 M-P; blue in overlay). Single confocal sections revealed
that while control cardiomyocytes displayed no signal for RhoA (Figure 4.8 I); a drastic
increase in the signal for RhoA (Figure 4.8 J, K, L) could be observed, spread throughout the
cytoplasm of the cardiomyocytes following the treatment with the hypertrophy inducing
agents; phenylephrine (100µM), isoproterenol (10µM) and norepinephrine (10µM)
respectively for 72 hours. Arrows in panels (Figure 4.8 N, O, P) point to the cardiomyocytes
with two nuclei and the asterisk in panel (Figure 4.8 N and B in overlay) points to a
tetranucleated cardiomyocyte which resulted due to the absence of cytoplasmic division or
cytokinesis in these cells.
4.2.6 Binucleation in hypertrophic neonatal rat cardiomyocytes
The consequential binucleation due to the lack of cytokinesis in primary cultures of neonatal
rat cardiomyocytes following the treatment with hypertrophy inducing agents was quantified
by counting the number of mononucleated and binucleated cells from 200 isolated
cardiomyocytes from two individual data sets obtained by confocal microscopy and is
expressed in percentages (Figure 4.8). The results clearly show that the percentage of
binucleation in cardiomyocytes treated with phenylephrine (100µM), isoproterenol (10µM)
and norepinephrine (10µM) goes up by almost a factor of two.
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Figure 4.8: Expression of RhoA is upregulated in primary cultures of neonatal rat cardiomyocytes (NRC)
following the treatment with hypertrophy inducing agents. (a) Confocal micrographs of neonatal rat
cardiomyocytes stained for cytokinetic marker, RhoA (panels I, J, K, L and green in overlay), for the
myofibrillar protein cardiac actin, (panels E, F, G, H and red in overlay) and for DAPI to observe binucleation
(panels M, N, O, P and blue in overlay). Single confocal sections reveal that while control cardiomyocytes
display no signal for RhoA (panel I); a drastic increase in the signal for RhoA (panel J, K and L) distributed
through out the cytoplasm can be seen following the treatment with the hypertrophy inducing agents;
phenylephrine (100µM), isoproterenol (10µM) and norepinephrine (10µM) respectively for 72 hours. Arrows in
panels (N, O and P) point to the binucleated cells and the asterisk in panel (N and B in overlay) points to a
cardiomyocyte with four nuclei. Bar represents 10 µm. (b) Quantification of binucleation in hypertrophic
neonatal rat cardiomyocytes. Data are presented as mean ± standard error of arbitrary ratios from the percentage
of mononucleated and binucleated cells counted from 200 isolated cardiomyocytes from 2 individual sets using
confocal microscope.
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We conclude from our results that downregulation of many regulatory and cytoskeletal
components involved in cytokinesis may be responsible for uncoupling of cytokinesis from
karyokinesis in cardiomyocytes after birth. Furthermore, the potential to undergo complete
division does exist in the cardiomyocytes as determined by the re-expression of karyokinetic
and cytokinetic proteins during hypertrophy. The failure to undergo complete division could
be due to the presence of stable, highly ordered and functional sarcomeres in the adult
myocardium or could be due to the absence of degradation mechanisms (ubiquitin or calcium
dependent) which facilitate division of embryonic cardiomyocytes by disintegrating the
myofibrils (Ahuja et al., 2004).
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4.3 DISCUSSION
Cardiomyocytes loose their ability to proliferate soon after birth (Zak, 1974; Clubb and
Bishop, 1984; Oparil et al., 1984; Tam et al., 1995; Li et al., 1996). Before terminal
withdrawal from the cell cycle, rodent cardiomyocytes undergo a final round of incomplete
cell division, during which karyokinesis gets uncoupled from cytokinesis, resulting in
binucleated cardiomyocytes. We wanted to investigate the hypothesis of cytokinesis being
controlled at the signal transmission level i.e. the cells do not produce the necessary signal for
promotion to cytokinesis and thus remain undivided. We checked the expression and
localization of various signaling proteins associated with the formation of the contractile ring
in the heart during development. The contractile ring has been investigated in postnatal
cardiomyocytes from rat (Li et al., 1997a; Li et al., 1997b). However, these localization
studies were restricted only to F-actin and non-muscle myosin and none of the other proteins
involved in cytokinesis were analyzed. We demonstrate here for the first time that
cardiomyocytes show a developmental stage specific expression of all the proteins like RhoA,
Cdc42, Rac1, ROCK-I, ROCK-II, p-cofilin associated with the formation of the actomyosin
ring with high levels being only expressed at stages when cytokinesis still occurs. The results
were surprising, as all of these proteins have been implicated to play a role in regulating other
actomyosin dynamics of the cell like formation of stress fibres, focal adhesions, migration,
maintaining morphology (Ridley, 1995; Narumiya, 1996; Etienne-Manneville and Hall, 2002;
Zhao and Rivkees, 2003), but it appears that they are only expressed in cardiomyocytes that
are still able to divide and do not seem to play a major role in the adult heart.
Vertebrates respond to injury through activation of committed progenitor cells or stem cells
(e.g. bone marrow) or through proliferation of differentiated cells (liver or endothelial cells),
(for review see Michalopoulos and DeFrances, 1997; Gage, 1998). In skeletal muscle,
committed progenitor cells (satellite cells) located below the basal lamina are induced to
proliferate in response to injury (Mauro, 1961). In contrast to skeletal muscles, a population
of myosatellite-type cells, a potential source of regeneration, is not formed during
cardiomyogenesis. The failure of the adult mammalian heart to reactivate the cell cycle
accounts for a major difficulty in restoration of function to the damaged heart (Armstrong et
al., 2000; Olson and Schneider, 2003). Nevertheless, mammalian cardiomyocytes retain the
ability to undergo partial cell cycle reactivation following hypertrophic stimulation. The
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increase in the activities of the G1/S phase cyclin-Cdk complexes has been described during
development of pressure overload-induced LVH in rats (Li et al., 1998). Capasso et al showed
that ventricular loading is coupled with DNA synthesis in adult cardiomyocytes after acute
and chronic myocardial infarction in rats (Capasso et al., 1992). Similarly, it was found that
ventricular failure occurring after acute myocardial infarction (Reiss et al., 1996) or
conditions of global ischemia (Reiss et al., 1993), upregulates the mRNA levels of PCNA and
histone-H3. In humans, it has been repeatedly identified that after myocardial injury the
ploidy level and number of nuclei per myocyte increases (Beltrami et al., 1997; Herget et al.,
1997). Also, the percentage of cardiomyocytes expressing PCNA increases in diseased human
hearts particularly in response to hypertrophy (Arbustini et al., 1993). Nevertheless, none of
these studies documented an increase in the number of cardiomyocytes subsequent to
hypertrophic stimulation. Thus, it seems that the heart is endowed with a programme that
protects against uncontrolled proliferation of contracting cardiomyocytes. However, recently
it was shown that p38 MAP kinase inhibition along with growth factor stimulation in vitro
can induce proliferation in adult mammalian cardiomyocytes (Engel et al., 2005).
In order to determine whether cytokinetic potential exists in heart during myocardial response
to hypertrophy, well-established in vitro and in vivo mouse and rat models for hypertrophy
were investigated. All our in vitro and in vivo model results reveal that while stressed
myocardium tries to adapt to the increased work load there is indeed an upregulation of
contractile ring associated proteins, representing the fact that cardiomyocytes do make an
attempt to undergo complete division. These results demonstrate that molecules and
mechanisms involved in maintaining embryonic growth are redeployed in response to
pathological cardiac hypertrophy in the adult heart. However, it seems that there is a
mechanism of hindrance which impedes the cleavage of dividing myocytes resulting in
increase in the level of binucleation or ploidy. We believe that this mechanical hindrance
could be posed by the presence of stable, highly ordered and functional sarcomeres in the
adult myocardium. It is known that during the progressive differentiation from embryonic to a
postnatal stage of cardiomyocytes, there is a gradual increase in the size, number and
complexity of organization of myofibrils (Zak, 1974; Rumyantsev, 1977; Ehler et al., 1999;
Perriard et al., 2003; Hirschy et al., 2006). This, together with the downregulation in the
expression levels of proteins that regulate cell cycle and cytokinesis respectively, might
contribute to uncoupling of karyokinetic and cytokinetic events as seen in the postnatal
cardiomyocytes. In addition, embryonic cardiomyocytes have adapted an effective mechanism
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of disassembly of myofibrils to undergo cell division (Kaneko et al., 1984; Ahuja et al.,
2004). To go through cell division, disassembly of the contractile elements, the myofibrils,
has to take place. This disassembly occurs in two steps with Z-disc and thin (actin)-filamentassociated proteins getting disassembled before disassembly of the M-bands and the thick
(myosin) filaments (Ahuja et al., 2004). A possible role of ubiquitin dependent degradation
during this process has been suggested, which consequently facilitates the disassembly of
stable myofibrils in embryonic heart hence making the division possible (Ahuja et al., 2004).
This might explain why embryonic cardiomyocytes retain the potential to proliferate whereas
adult cardiomyocytes do not. Another reason for the inability of cardiomyocytes to complete
cell division during hypertrophy could be the absence of these degradation mechanisms which
could disassemble the stable myofibrils despite the fact that the cytokinetic machinery is
primed up during this period. Activation of ubiquitin dependent degradation mechanism has
been proposed during the progression of compensated hypertrophy to heart failure (Hein et
al., 2003). It was suggested that ubiquitin binds to contractile or membrane proteins destined
for degradation but because of proteasomal insufficiency, the complexes are accumulated
which might cause nuclear fragmentation. However, the role of ubiquitin dependent
degradation machinery in response to hypertrophic stimulation needs to be established in
detail.
In conclusion, our results suggest that after birth cardiomyocytes downregulate many
regulatory and cytoskeletal components involved in cytokinesis which may be responsible for
uncoupling cytokinesis from karyokinesis in cardiomyocytes. In addition, under stressed or
hypertrophic conditions adult cardiomyocytes retain the potential to divide again as
determined by the redeployment of the division machinery during this time. The mechanisms
which do not allow complete division to occur at this time needs to be understood in detail
and will determine the future of research into myocardial regeneration. The developmentally
regulated cessation of cytokinesis makes cardiomyocytes an interesting model system to
investigate the factors involved in regulation of cytokinesis.
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Additional results
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Additional results
5. Additional results
Apart from all the results documented in previous chapters there are some other interesting
observations made which are significant and merit to be mentioned here.
5.1 Role of calcium dependent calpain mediated degradation in dividing cardiomyocytes
During skeletal muscle wasting it has been shown that increased calcium dependent calpain
mediated activity provides an early and perhaps a rate limiting step, accounting for
degradation of Z-disc associated proteins and release of actin and myosin from the myofibrils
which are then subsequently ubiquitinated and degraded by the proteasome (Hasselgren et al.,
2005). As shown earlier in chapter 2, ubiquitin expression gets upregulated in dividing
embryonic cardiomyocytes (Figure 2.6) suggesting that protein degradation also of
components of the sarcomere or of proteins, which control sarcomere integrity, might happen
during cell division. To assess the role of calpain mediated degradation also in dividing
cardiomyocytes, we stained cultured cardiomyocytes with antibodies against heart specific
calpain-1 (Figure 5.1 C, G, K; green in overlay) in combination with MyBP-C to delineate the
myofibrils (Figure 5.1 B, F, J; red in overlay) and with tubulin to assess the stage of cell
division (Figure 5.1 D, H, L; blue in overlay). While in interphase cardiomyocytes the signal
for the calpain antibody is rather weak; (Figure 5.1 C, cell in top left corner); once
cardiomyocytes enter mitosis, calpain just like ubiquitin starts to be spread throughout the
cytoplasm (Figure 5.1 C, G, K). Due to the low frequency of cell division in our cultures as
well as in the developing heart in situ, we were unable to analyse by biochemical means,
whether myofibrillar proteins themselves were degraded by ubiquitin or calcium dependent
pathways. However, the high intensity of the ubiquitin and calpain signal could mean that
protein degradation also of components of the sarcomere, takes place during cell division.
5.2 Expression of calpain-1 in heart during development
To determine the activity of calpain in heart during development, we performed
immunoblotting with antibodies against heart specific calpain-1 at different stages of mouse
heart development (Figure 5.2). In the embryonic heart (Figure 5.2, lane 1), high levels of
expression of calpain could be found, which get downregulated by the first postnatal week
and are almost absent in adult heart (Figure 5.2, lane 4). This suggests that calpain is highly
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expressed in cardiomyocytes that are still able to divide and does not seem to play a major
role in the adult heart.
Figure 5.1: Expression of calpain-1 is upregulated in dividing embryonic cardiomyocytes. Cultured
cardiomyocytes were stained with antibodies to MyBP-C (B, F, J; red in overlay) together with antibodies
against calpain-1 (C, G, K; green in overlay) and against tubulin (D, H, L; blue in overlay) to identify the stage
of cell division. Single confocal sections reveal that while interphase cardiomyocytes display only little signal
for calpain-1 (panel C, top left); a drastic increase in the signal for calpain can be detected in cardiomyocytes in
metaphase (panel C, G) as well as in late anaphase (panel K). Bar represents 10 µm.
Figure 5.2: Expression pattern of calpain-1 during different developmental stages of the heart. Cell lysates from
embryonic heart (E14, lane 1), day of birth (P0, lane 2), day eight (P8, lane 3), adult mouse ventricular muscle
(lane 4), were analysed by immunoblotting for expression of calpain-1. Equal amounts of heart tissue were
loaded; cardiac α-actin was used as loading control (bottom panel).
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5.3 Disassembly of myofibrils delayed after treatment with NCO-700
To further address the role of calpain-mediated degradation for myofibril disassembly, an
inhibitor that interferes with the calcium dependent calpain pathway was used on cultured
embryonic cardiomyocytes. Treatment with NCO-700 leads to metaphase cells that still
display cross-striations in the alpha-actinin staining, while control cells show diffuse
localization of these proteins (Figure 5.3). Another myofibrillar protein tested was the M-band
protein myomesin which displays a comparatively intact localization pattern compared to the
Z-disc protein alpha-actinin during metaphase (Ahuja et al., 2004). After NCO-700 treatment
myomesin staining revealed cells with comparatively even more intact striations in contrast to
untreated ones.
Figure 5.3: Disassembly of myofibrils is delayed in cardiomyocytes that were treated with NCO-700 to inhibit
calcium dependent-calpain degradation. Single confocal sections of cardiomyocytes stained with monoclonal
antibodies to myomesin (B; red in A, both panels), sarcomeric alpha-actinin (D; red in C, both panels) and for
tubulin (blue in A, C; both panels) as well as for phosphorylated histone (green in A, C; both panels) to identify
the stage of mitosis. While in control cells at metaphase alpha-actinin is localized in a diffuse fashion (D; first
panel), cross-striated myofibrils can still be seen in NCO-700 treated cardiomyocytes (D; second panel). For
myomesin, control cells display comparatively intact striation than alpha-actinin (B; first panel), however after
the treatment displays even better cross-striated pattern than the control (B; second panel). Bar represents 10 µm.
This experiment further confirms our observation of myofibrils being disassembled to achieve
successful cell division in cardiomyocytes and that this disassembly process is probably
regulated by factors, which are part of calpain and ubiquitin dependent pathways.
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5.4 Role of Cullin3 in dividing cardiomyocytes
To further characterize the role of ubiquitin dependent proteolysis in embryonic heart during
division, we next investigated the role of cullin3, an ubiquitin ligase (E3). Cullin family
proteins form a part of SCF (Skp1-Cullin-F-box) complexes, which are a major class of E3
ligases that are required to selectively target substrates for ubiquitin-dependent degradation by
the 26S proteasome (for review see Deshaies, 1999). Among the characterized cullins, cullin1
(CUL-1)-containing SCF (Skp1-Cul1/Cdc53-F-box protein), cullin3 (CUL-3)-containing
BCR (BTB domain-Cul3-RING), and cullin4 (CUL-4)-containing VDC (V-DDB1-Cul4A)
complexes promote specific protein ubiquitination and degradation that are critical for cellcycle progression, cell differentiation and signal transduction (Willems et al., 2004; Petroski
and Deshaies, 2005). Conjugation of the ubiquitin-like protein Nedd8 to the cullin subunit
(neddylation) positively regulates activity of SCF complexes, most likely by increasing their
affinity for the E2 conjugated to ubiquitin (Kawakami et al., 2001). Neddylation is essential
for cullin-organized ligase activities in vivo, as observed for Drosophila melanogaster CUL-1
in regulating the protein stability of cyclin E (Ou et al., 2002), Caenorhabditis elegans CUL-3
in MEI-1-katanin (Pintard et al., 2003) and fission yeast CUL3 in Btb3p (Geyer et al., 2003).
Recent evidence suggests that CUL-3 inactivation by RNAi results in cytokinetic defects in
HeLa cells, consequently leading to multinucleation (I. Sumara and M. Peter personal
communication). Currently the downstream targets of CUL-3 involved during the cytokinesis
process are being investigated in M. Peter’s laboratory, at ETH.
To check the expression of CUL-3, an E3 ligase specifically implicated during cytokinesis,
we performed western blotting with antibodies against CUL-3 at different stages of mouse
heart development (Figure 5.4). In the embryonic heart (Figure 5.4, lane 1), active or
neddylated form of cullin3 expression could be found, which is essential for cullin-organized
ligase activity in vivo. This gets downregulated by the first postnatal week and is almost
absent in adult heart, judged by the disappearance of the neddylated band (Figure 5.4, lane 4).
However, low levels of inactive cullin3 are still expressed in the adult heart. It thus appears
that the cullin3 pathway, part of ubiquitin dependent proteolysis, is active in cardiomyocytes
that are still able to divide and does not seem to play a major role in the adult heart.
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Figure 5.4: Expression pattern of CUL-3 during developmental stages of the heart. Cell lysates from embryonic
heart (E14, lane 1), day of birth (P0, lane 2), day eight (P8, lane 3), adult mouse ventricular muscle (lane 4),
were analysed by immunoblotting for expression of CUL-3. The neddylated form of CUL-3 can be detected in
samples from embryonic heart, starts to get downregulated after birth and is completely absent in the adult heart.
However, low levels of inactive cullin3 are still expressed in the adult heart. Equal amounts of heart tissue were
loaded; α-cardiac actin was used as loading control (bottom panel).
5.5 Subcellular distribution of Cullin3 in dividing cardiomyocytes
To find out where cullin3 is localized in the cells, we determined the subcellular distribution
of CUL-3 in cultured rat embryonic cardiomyocytes by triple immunofluorescence (Figure
5.5). Cardiomyocytes were stained for CUL-3; (Figure 5.5 G, H, I; green in overlay), for the
myofibrillar protein sarcomeric alpha-actinin, (Figure 5.5 D, E, F; red in overlay) and tubulin
to detect the mitotic stages (Figure 5.5 J, K, L; blue in overlay). Single confocal sections
revealed that while control cardiomyocytes displayed no signal for CUL-3; a drastic increase
in the signal (Figure 5.5 G, H I) could be observed, which is mainly associated with the
spindle during late anaphase and telophase and the midbody of the cells in cytokinesis. The
localization pattern of CUL-3 was consistent with an active role of CUL-3 in final stages of
division, cytokinesis in particular. At present the target substrates for ubiquitin-dependent
proteolysis are not known during division of embryonic cardiomyocytes and further work is
required to determine the role of protein degradation in regulating myofibrillar disassembly
during division in embryonic heart.
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Figure 5.5: Expression of CUL-3 is upregulated in dividing cardiomyocytes. Cultured cardiomyocytes were
stained with antibodies to sarcomeric alpha-actinin (D, E, F and red in overlay) together with antibodies against
CUL-3 (G, H, I and green in overlay) and against tubulin (J, K, L and blue in overlay) to identify the stage of cell
division. Single confocal sections reveal that while interphase cardiomyocytes display only little signal for CUL3 (panel I, bottom); a drastic increase in the signal for CUL-3 can be detected in cardiomyocytes in anaphase
(panel G), telophase (panel H) and cytokinesis (panel I). Bar represents 10 µm.
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5.6 Re-expression of Cullin3 in heart during hypertrophy
As determined and stated in chapter 4 our results revealed that while stressed myocardium
tries to adapt to the increased work load during hypertrophy there is indeed an upregulation of
cytokinetic proteins associated with the contractile ring, representing the fact that
cardiomyocytes do make an attempt to undergo complete division during this time. However,
it seems that there is some hindrance which impedes the cleavage of dividing myocytes
resulting in incomplete division. One reason which we hypothesize for the inability of
cardiomyocytes to complete cell division during hypertrophy is the absence or insufficiency
of these degradation mechanisms which disassemble the stable myofibrils in embryonic
cardiomyocytes, thereby facilitating division. Activation of ubiquitin dependent degradation
mechanisms has been proposed during the progression of compensated hypertrophy to heart
failure (Hein et al., 2003). It was suggested that ubiquitin binds to contractile or membrane
proteins destined for degradation but because of proteasomal insufficiency, the complexes are
accumulated which might cause nuclear fragmentation. However, the role of the ubiquitin
dependent degradation machinery in response to hypertrophic stimulation needs to be
established in detail.
In an attempt to study the role of ubiquitin dependent degradation during hypertrophy, we
determined the expression of CUL-3, an E3 ligase, during hypertrophy in ventricular extracts
of transgenic mice over-expressing angiotensinogen in cardiomyocytes (Mazzolai et al.,
1998), and the same treated with β adrenergic receptor agonist, isoproterenol for four weeks
(Figure 5.6). Even though the neddylated form of CUL-3 does not accumulate in samples
from hypertrophic hearts, the intensity of the band observed is stronger compared to the wild
type heart samples. However, much more work is required to evaluate the significance of this
finding and to determine the functional role and expression profile of different ubiquitin
ligases like CUL-3 or for proteasome during cardiac development and disease.
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Figure 5.6: Re-expression of CUL-3 in angiotensin over-expressing mice characterized by the hypertrophy.
Immunoblot on SDS samples of transgenic mice overexpressing angiotensinogen and treated with β adrenergic
receptor agonist, isoproterenol for four weeks, probed with antibodies against cullin3. Even though the
neddylated form of CUL-3 does not accumulate in samples from hypertrophic hearts, the intensity of the band
observed is stronger compared to the wild type heart. Equal amounts of heart tissue were loaded; α-cardiac actin
was used as loading control (bottom panel).
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General discussion and outlook
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General discussion and outlook
6. General discussion and outlook
The differentiative and proliferative capacity of cardiac muscle cells during development and
their potential to regenerate during disease has in recent years become a topic of great interest
and scrutiny (Leri et al., 2002; Keating, 2004; Busk et al., 2005; Engel et al., 2005; Laflamme
and Murry, 2005). The work presented here aims to address several key issues of
cardiomyocyte cell division during development and disease. One of the novel findings of my
study was to demonstrate that during embryonic cardiomyocyte division, myofibril
disassembly occurs in a sequential manner. Myofibrillar proteins that are associated with the
Z-disc or the thin filaments display a diffuse localization pattern at a time when the thick
filaments are still comparatively intact. It is likely that ubiquitin dependent degradation is
involved during this process, which consequently facilitates the disassembly of stable
myofibrils in the embryonic heart hence making cell division possible (Ahuja et al., 2004).
Unfortunately, the low rate of division in culture and in situ did not permit a thorough
biochemical analysis of the ubiquitinated proteins. One approach to identify the ubiquitinated
substrates would be to first synchronize the cultures of dividing embryonic cardiomyocytes
(for e.g. with nocodazole) and then to co-immunoprecipitate ubiquitin with probable
sarcomeric protein targets. The co-precipitates can then be analyzed by mass spectrometry.
Further interaction between ubiquitin and the target protein can be confirmed by pair wise coimmunoprecipitation or alternatively by yeast two hybrid techniques. Preliminary experiments
in our laboratory showed that the subcellular signals for ubiquitin and MURF-2, a ubiquitin
ligase are upregulated in cultured dividing embryonic cardiomyocytes (Ahuja, Ehler, Gautel
and Perriard, unpublished observations). In addition, we observed high expression of another
ubiquitin ligase, CUL-3 in dividing embryonic cardiomyocytes. It was recently shown, that
MURF-1 degrades troponin I in heart (Kedar et al., 2004). The interaction of MURF-1 with
the myofibrillar giant protein titin at the M-band during sarcomere assembly has also been
seen (Pizon et al., 2002), although the functional consequences of this interaction remains to
be explored. The examination of the functional role and natural substrates for ubiquitin ligases
like MURFs and CUL-3 during division would provide deeper insight into the process of
ubiquitin mediated proteolysis in heart during division.
Cytokinesis is uncoupled from karyokinesis in cardiomyocytes during early postnatal
development. An understanding of this process is of fundamental interest as it may hold the
key to our understanding of (i) termination of cell division after birth in cardiomyocytes and
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(ii) lack of replacement of damaged cardiac tissue in adult vertebrates. We suspect that this
uncoupling phenomenon occurs because cardiomyocytes show a developmental stage specific
expression of all the proteins that are coupled with the formation of the contractile ring such
as Rho GTPases and their downstream effectors with high levels being only expressed at
stages when cytokinesis still occurs (Ahuja et al., In preparation). This suggests that the
downregulation of many regulatory and cytoskeletal components involved in cytokinesis may
be responsible for uncoupling cytokinesis from karyokinesis in cardiomyocytes after birth. If
this is true, then inhibition of these signalling pathways implicated in the induction of
cytokinesis with an antagonist should lead to multinucleated cells in the embryonic heart. On
the contrary, over-expression with an agonist of these signalling pathways in neonatal
cardiomyocytes should overcome the block and drive them through cytokinesis. Further work,
combining different approaches like time-lapse analysis of cardiomyocytes with a cytokinetic
marker and a nuclear envelope protein marked with different fluorescent tags would yield
more information regarding the exact timings of localization of these markers to the cleavage
furrow. A major drawback of the currently used transient transfection method for expression
of GFP/RFP-tagged myofibrillar components is the low transfection rate of primary
cardiomyocyte cultures. This, combined with low proliferation activity, is likely to render any
interpretation difficult. In order to increase the number of transgenic cells, alternative
strategies like employing recombinant adenovirus for transfection can be envisaged. It would
be also of interest to examine the phenotypes of embryonic cardiomyocytes when expression
of these cytokinetic markers is abrogated by RNAi or when their respective gene(s) are
knocked out. This may help to establish a functional role of these proteins in the cessation of
cytokinesis in cardiomyocytes during heart development.
Although cell cycle exit may be necessary to facilitate myocardial function under normal
conditions (Ahuja et al., 2004), many cardiovascular diseases are associated with a significant
loss of working myocytes. Therefore, development of means to reactivate the cardiomyocyte
cell cycle in a controlled manner can be extremely beneficial for myocardial repair. Although
several groups have been able to show re-entry of cardiomyocytes into the cell cycle by
genetically manipulating cell cycle regulatory factors, thus far true evidence of cytokinesis in
adult cardiomyocytes has yet to be demonstrated (for review see Field, 2004; Regula et al.,
2004). In our study we found that while the pathologically stressed myocardium tries to adapt
to the increased work load during hypertrophy, this response is accompanied by an
upregulation of both karyokinetic and cytokinetic proteins. These results demonstrate that
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molecules and mechanisms involved in maintaining embryonic growth are redeployed in
response to pathological cardiac hypertrophy in the adult heart.
We hypothesize that failure to undergo complete division could be due to (i) the presence of
highly stable, ordered and functional sarcomeres in the adult myocardium or (ii) the absence
of degradation mechanisms (ubiquitin or calcium dependent) which allows division in
embryonic cardiomyocytes by disintegrating myofibrils. One approach to prove the first
hypothesis could be by loosening up the stable myofibrillar cytoskeleton and thus promoting
cytokinesis. Myofibrillar breakdown can be induced by the over-expression of cytoplasmic
actin isoforms (Von Arx et al., 1995), thin filament associated proteins like tropomodulin or
mutant contractile proteins that are known to induce weakening of myofibrillar structures
(Sussman et al., 1998). Alternatively, myofibrils can be destabilised using inhibitors of
collagen synthesis like cis-hydroxyl-L-proline and ethyl-3,4-dihydroxybenzoate, that were
previously shown to interfere with the myofibril formation in cardiomyocytes (Fisher and
Periasamy, 1994). Alternatively, one could use different stem cell types that differentiate into
cardiac muscle cells in vitro like mouse embryonic stem (ES) cells or P19 embryonal
carcinoma (EC) cells. These cells have been previously used as model systems to study
cardiomyocyte differentiation that led to detailed insights into the functional role of cardiac
specific transcription factors, signaling pathways and cardiac commitment and repair (for
review see Pasumarthi and Field, 2002; van der Heyden and Defize, 2003). Cardiomyocytes
derived from both ES and P19Cl6 cell lines can also be used as controls for cultured adult
cardiomyocytes in order to show that with less mature and fewer myofibrils there is no
physical barrier for cardiomyocytes to complete division. Owing to their high proliferation
rates these model systems can in addition be used to identify the sarcomeric substrates for
ubiquitin dependent proteolysis during division by biochemical assays. However, in our
laboratory we were unable to derive an adequate amount of cardiomyocytes from these cells
for performing biochemical experiments.
The second hypothesis, of the absence of degradation mechanisms which might maintain the
stability of myofibrils during hypertrophy, also needs to be looked at. It seems from our
preliminary results that activation of CUL-3, an E3 ligase, takes place during hypertrophy. In
addition, another group proposed the activation of ubiquitin dependent degradation
mechanisms during the progression of compensated hypertrophy to heart failure (Hein et al.,
2003). It was suggested that ubiquitin binds to contractile or membrane proteins destined for
degradation but because of proteasomal insufficiency, the complexes are accumulated which
causes nuclear fragmentation. Thus, proteasomal insufficiency could be another limiting
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factor which does not allow the disintegration of myofibrils during hypertrophy. Thus far, not
much information about the functional role and expression profile of different ubiquitin
ligases or for the proteasome is known during cardiac development and disease and the
subject surely needs to be investigated further in detail.
Research into myocardial regeneration has an exciting future, especially if the factors that
regulate the withdrawal of cardiomyocytes from the cell cycle are understood. The gradual
increase in the size, number and complexity of organization of myofibrils during the
progressive differentiation from embryonic to a postnatal stage might be an important factor
regulating the withdrawal from cell cycle. With the hypertrophic growth drawing in during
development and after birth caused by the increased workload on the heart, division might be
simply too costly from an energetic point of view. This together with the downregulation in
the expression levels of proteins that regulate cell cycle and cytokinesis respectively might
contribute to cessation of cell cycle in cardiomyocytes after birth. Investigation of model
organisms that exhibit an inherent capacity for myocardial repair like newt and zebrafish may
also shed light on additional regulatory mechanisms that can be exploited to stimulate a
similar regenerative response in the mammalian heart. In summary, the field is still at its
infancy with some progress having been made through my work. There are certainly new and
exciting discoveries yet to be made and new insights to be gained.
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Material and methods
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Material and methods
7 Material and methods
7.1 Isolation and culture of embryonic and neonatal rat cardiomyocytes (ERC and
NRC)
Embryonic (day 14) and neonatal rat ventricular hearts were dissected, digested with
collagenase (Worthington Biochemical Corp., Free-hold, NJ, USA) and pancreatin (Gibco
Laboratories, Grand Island, NY, USA) and cultured in Maintenance medium which contained
20% medium M199, 75% DBSS-K, 4% horse serum, 4 mM glutamine, 1%
penicillin/streptomycin, 0.1 mM phenylephrine; (DBSS-K: 6.8 g/l NaCl, 0.14 mM NaH2PO4,
0.2 mM CaCl2, 0.2 mM MgSO4, 1 mM dextrose, 2.7 mM NaHCO3) in fibronectin-coated (10
µg/ml; Sigma) plastic dishes (Nunc; (Ahuja et al., 2004)). The maintenance medium for
neonatal rat cardiomyocyte maintenance was alike but lacked serum and phenylephrine.
7.2 Fixation and staining of cultured cardiomyocytes
The cells were rinsed briefly in MP buffer (Ahuja et al., 2004), were either fixed for 10
minutes with 4% paraformaldehyde in MP buffer, then washed with MP buffer again and
permeabilized with 0.2% Triton X-100 in MP for 5 min or alternatively fixed in methanol for
5 minutes at –20˚C. Primary and secondary antibodies were diluted using 1% BSA in PBS
and incubations were carried out at room temperature for 1 h. After final washing three times
with PBS, the cells were mounted in 0.1 M Tris-HCl (pH 9.5)-glycerol (3:7) including 50
mg/ml n-propyl gallate as anti-fading reagent (Ahuja et al., 2004) and the coverslips were
sealed with nail polish.
7.3 PFA-fixed heart whole mount preparations
The hearts of E14.5 mouse embryos were dissected in PBS and rinsed briefly with MP buffer
followed by fixation in 4% PFA in MP buffer for 90 minutes. After several washes in PBS,
the hearts were treated with hyaluronidase (1 mg/ml; Sigma) in PBS for 45 minutes at RT
(room temperature) in order to remove the cardiac jelly and to ensure access for the antibodies
to the inner myocardial wall (Tokuyasu and Maher, 1987). This was followed by
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Material and methods
permeabilization with 0.2% Triton X-100 in PBS for 45 minutes. After further washes in PBS
and blocking with 5% NGS (normal pre-immune goat serum), 1% BSA (bovine serum
albumin) in PBS for 45 minutes, the hearts were incubated with the primary antibody
mixtures, diluted in blocking solution, shaking overnight at 4°C. After 4x2 hours washing in
PBS with 0.002% Triton X-100 (PBT), the secondary antibodies were applied for overnight at
4°C. The hearts were washed with PBT for 6x1 hour and mounted on slides as described
above (Ehler et al., 1999).
7.4 Antibodies used for immunofluorescence
Table 7.1 Primary antibodies used for immunofluorescence
Type and specificity
Dilution
Source
mM a sarc. α-actinin, clone EA53
1:500
Sigma
mM a myomesin, clone B4
1:50
Obtained from E. Perriard, ETH, Zürich, Switzerland
pR a MyBP-C
1:100
mRt a tubulin, clone YOL1/34
1:100
Abcam, UK
pR a β-catenin
1:200
Sigma
pR a ubiquitin
1:25
Sigma
pR a phos.Histone H3
1:500
Upstate Biotechnology, Luzern, Switzerland
1:5
Developmental Studies Hybridoma Bank, Iowa, USA
mM a titin clone T51
1:2
Obtained from Prof. Fürst, Postdam, Germany
mM a titin clone T12
1:2
Obtained from Prof. Fürst, Postdam, Germany
pRt a cardiac MyBP-C
1:100
mM a α-cardiac actin
1:50
Progen, Heidelberg, Germany
mM a non-muscle myosin IIB
1:20
Chemicon, Temecula, CA, USA
mM a RhoA
1:50
Santa Cruz, CA, USA
mM a sarc. myosin heavy chain,
clone A4.1025
Obtained from Prof. M.Gautel, King`s College,
London, UK
Obtained from Prof. M.Gautel, King`s College,
London, UK
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Material and methods
mM a Cdc42
1:100
Santa Cruz, CA, USA
mM a Rac1
1:100
BD Biosceinces
mM a M-protein, clone AA259
1:3
Obtained from Prof. Fürst, Postdam, Germany
mM a calpain-1, domain-II
1:100
Calbiochem, Switzerland
pR a MURF-2
1:20
pR a CUL-3
1:50
Obtained from Prof. M.Gautel, King`s College,
London, UK
Obtained from I.Sumara, ETH, Zürich, Switzerland
Polyclonal rabbit antibodies against different septins were characterized in the lab of Prof.
William Trimble (Beites et al., 1999; Xie et al., 1999; Surka et al., 2002; Xue et al., 2004).
With the exception of SEPT5, all anti-septin antibodies were raised in rabbits against the
following
human
sequence
peptides
unless
otherwise
indicated:
SEPT1
–
EDRQVPDASARTAQTLC (mouse); SEPT2 – MSKQQPTQFINPETGC (mouse); SEPT3 MSELPEPRPKPAVPC; SEPT4 - CDFPIPAVPPGTDPE; SEPT6 - MAATDIARQVGEGC;
SEPT7 - RILEQQNSSRTLEKNKKKGKIFC; SEPT8 - TLDERFSNAEPEPRC; SEPT9 TDAAPKRVEIQVPKPC; SEPT11 - MAVAVGRPSNEELRNC. The peptides were chosen
because they were unique to each septin and not significantly similar to any other proteins in
the GenBank database.
Crude serum was affinity purified and eluted from columns of
covalently bound peptide. Mouse monoclonal anti-SEPT5 was obtained from Chemicon
International (Temecula, CA). For immunofluorescence, SEPT 2, 6 and 9 were used at a
dilution of 1:500.
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Table 7.2 Secondary antibodies used for immunofluorescence
Type and specificity
Dilution
Source
G a R Cy2
1:100
Jackson Laboratory (distributed by Milan)
Do a Rt Cy3 (no cross-reaction with mouse Ig)
1:100
Jackson Laboratory (distributed by Milan)
Do a M Cy5 (no cross-reaction with rat Ig)
1:100
Jackson Laboratory (distributed by Milan)
G a M Cy3
1:1000
Jackson Laboratory (distributed by Milan)
G a M IgM (µ-chain specific) FITC
1:100
Sigma
H a M FITC (no cross-reaction with rat Ig)
1:50
Vector
G a R Cy5
1:100
Jackson Laboratory (distributed by Milan)
Do a M IgM (µ-chain specific) Cy5
1:100
Jackson Laboratory (distributed by Milan)
G a M IgA (a-chain specific) FITC
1:20
Sigma
Type and specificity
Dilution
Source
DAPI
1:100
Molecular Probes
Alexa 546-phalloidin
1:100
Molecular Probes
Table 7.3 Non-immune dyes
7.5 Confocal microscopy
The specimen were analysed using confocal microscopy on an inverted microscope DM
IRB/E equipped with a true confocal scanner SP2 AOBS, a PL APO 63x/1.32 oil and a PL
APO 100x/1.40 oil immersion objective (Leica) as well as argon, helium-neon and UV lasers.
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Material and methods
Image processing was done on a Silicon Graphics workstation using Imaris® (Bitplane AG,
Zürich), a 3D multichannel image processing software specialized for confocal microscopy
data sets.
7.6 SDS-PAGE and immunoblotting
Dissected ventricular hearts from E (embryonic day) 14 embryos, P (postnatal day) 0, P8 and
adult mice were freeze slammed in liquid nitrogen, resuspended in a modified version of
SDS-sample buffer (3.7 M urea, 134.6 mM Tris, pH 6.8, 5.4% SDS, 2.3% NP-40, 4.45% βmercaptoethanol, 4% glycerol and 6 mg/100 ml Bromophenol and boiled for 2 minutes (Ehler
et al., 2001). For cultured neonatal cardiomyocytes, the same SDS-sample buffer was used to
scrape the cells off the dish after their respective treatment.
The SDS-samples were run on 7.5% or 15% polyacrylamide minigels (Bio-Rad, Glattbrugg,
Switzerland). The proteins were blotted overnight onto nitrocellulose (Hybond-C extra,
Amersham, Zurich, Switzerland (Ehler et al., 2001). After the transferred proteins had been
visualized with Ponceau Red (Serva, Heidelberg, Germany), unspecific binding regions were
blocked with 5% non-fat dry milk (or 3% BSA for phosph. Histone H3/Cofilin) in washing
buffer (0.9% NaCl, 9mM Tris, pH 7.4, 0.1% Tween-20) for 1 hour at RT. Primary and
secondary horseradish peroxidase (HRP)-conjugated antibodies were diluted in washing
buffer with 0.1% milk and incubated for 1 hour, respectively, with intermittent washing in
washing buffer. After a final wash in washing buffer a chemiluminescence reaction was
performed according to the manufacturer (Amersham and Pierce, Socochim, Lausanne,
Switzerland, respectively) and results were visualized on Fuji Medical X-Ray films. Bands of
the expected molecular mass, as judged by Kaleidoscope Prestained Standards (Bio-Rad)
were present in the positive control with all the antibodies used.
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Material and methods
7.7 Antibodies used for immunoblotting
Table 7.4 Primary antibodies used for immunoblotting
Type and specificity
Dilution
Source
pR a phosph. Histone H3
1:1000
Upstate Biotechnology, Luzern, Switzerland
mM a α-cardiac actin
1:1000
Progen, Heidelberg, Germany
mM a RhoA
1:500
Santa Cruz, CA, USA
mM a Cdc42
1:1000
Santa Cruz, CA, USA
mM a Rac1
1:1000
BD Biosceinces
pR a phosph. cofilin
1:1000
pR a ROCK I
1:1000
mM a ROCK II
1:1000
BD Biosceinces
mM a PCNA
1:500
Santa Cruz, CA, USA
pR a Polo like kinase 1
1:1000
Upstate Biotechnology, Luzern, Switzerland
mM a calpain-1, domain-II
1:1000
Calbiochem, Switzerland
pR a CUL-3
1:500
Obtained from I.Sumara, ETH, Zürich, Switzerland
Obtained from James Bamburg, Colourado State
University, Colourado
Obtained from Didier Job, Institut National de la Santé
et de la Recherche Medicale, France
Table 7.5 Secondary antibodies used for immunoblotting
Type and specificity
Dilution
Source
HRPO G a M IgG
1:3000
DAKO Diagnostics AG, Zug, Switzerland
HRPO G a R IgG
1:3000
Calbiochem, Juro supply, Luzern, Switzerland
117
Chapter 7
Material and methods
7.8 Drug Treatment
Cultured embryonic rat cardiomyocytes were treated with ROCK inhibitor-Y-27632 (Alexis)
at a concentration of 1mM for 4 h, with Cytochalasin D (Sigma) at 100µg/ml for 1h or with
Blebbistatin (Calbiochem) at a concentration of 100µM for 2h just before fixation. Cultured
neonatal rat cardiomyocytes were treated with Phenylephrine (Sigma) at a concentration of
100µM, with Isoproterenol (Sigma) at 10µM and Norepinephrine (Sigma) at 10µM for 72 h
just before fixation or before preparing SDS-samples.
The proteasome inhibitor MG132 (Sigma) and calpain-1 inhibitor NCO-700 (Sigma) were
used at a concentration of 20 µM and 1mM for 3 and 1 hour respectively just before fixation.
7.9 Cryosections
Hearts rinsed in ice cooled PBS were frozen in liquid nitrogen using Isopentane as
cryoprotectant. The heart was mounted in Tissue tek OCT medium (Plano W.Plannet AG,
Wetzlar, Germany) and cryosections (20 µm) were cut in an HM560 MV cryostat (Microm,
Walldorf, Germany) at –20°C, then retrieved on gelatinized microscope slides for
immunostaining.
7.10 Immunofluorescence of cryosections
The protocol for staining cryosections was the same as that for isolated cells except that the
sections were blocked with 5% normal goat serum before the incubation with antibodies and
incubations with antibodies were made overnight at 4°C.
7.11 Densitometric analysis
X-Ray films were scanned in gray scale at 300dpi with a Hpscanjet Iicx hardware (Hawlett
Packard Company, Palo Alto, USA) and the digital image was submitted to densitometric
analysis using Image J software (National Institute of Health, Washington, USA). The
densitometric values represent the mean of the gray intensity of all the pixels present in a
selected area. For normalization, a densitometric analysis is performed in a selected reference,
e.g. cardiac actin content for standardization.
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Material and methods
7.12 Binucleation
Nuclei were stained with 4, 6-diamidino-2 phenylindole-2 HCl (DAPI). The percentage of
mononucleated and binucleated cells was counted from a sample of 200 isolated myocytes
from 2 individual sets using confocal microscopy images.
7.13 Statistical analysis
All statistical analyses and tests were carried out using Excel software (Microsoft, Redmond,
USA). Data are given as mean +/- standard deviation. Bars in the graphs represent standard
errors and differences analysed with a two-tailed T test with a P value below 0.05 were
considered significant.
7.14 Hypertrophic samples
Hypertension induced one-kidney, one clip (1K1C) mouse heart and sham operated mouse
heart (Wiesel et al., 1997), isoproterenol treated and its control mouse heart, angiotensin II
transgenic mouse heart alone and treated with isoproterenol (Mazzolai et al., 1998) were
provided by Prof. Thierry Pedrazzini (Division of Hypertension and Vascular Medicine,
University of Lausanne Medical School, CH-1011 Lausanne, Switzerland).
Dahl salt resistant rats (DR), sensitive (DS) and DS rats treated with Y-27632 heart extracts
(Satoh et al., 2003) were a kind gift from Prof. Shinji Satoh (Medical Institute of
Bioregulation, Kyushu University, Japan). Dissected ventricular hearts from all these animals
were freeze slammed in liquid nitrogen and afterwards processed as described in the SDSPAGE and immunoblotting protocol.
119
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PUBLICATIONS
Maitra, A., Aghi, P. and Prasad, Yogesh (2001). “SNaPshot technique for interrogation of
target SNPs for understanding disease association”Proceedings in Genetic Research,
Sultan Qaboos University. Abstract.
Ahuja, P., Perriard, E., Perriard, J.C., Ehler, E (2004). “Sequential myofibrillar breakdown
accompanies mitotic division of mammalian cardiomyocytes”. Journal of Cell
Science, 117(15): 3295-3306.
Feng, J., Lucchinetti, E., Ahuja, P., Pasch, T., Perriard, J.C., Zaugg, M., (2005). “Isoflurane
postconditioning prevents opening of the mitochondrial permeability transtion pore
through inhibition of glycogen synthase kinase-3β. Anesthesiology, 103(5): 987-95.
Ahuja, P., Perriard, E., Trimble, W., Perriard, J.C., Ehler, E (2006). “Probing the role of
septins in cardiomyocytes”. Experimental Cell Research.
Ahuja, P., Perriard, E., Perriard, J.C., Ehler, E. “Analysis of the cytokinetic potential of
cardiomyocytes during development and disease ”. In Preparation.
Krishnan, J., Ahuja, P., Bodenmann, S., Knapik, D., Turnbull, D.H., Gassmann, M., Perriard,
J.C. “Cardiac Development is driven by Hypoxia Inducible Factor 1a (HIF-1α)”.
In Preparation.
144
CURRICULUM VITAE
Preeti Ahuja
Born on March 11th, 1975 in New Delhi
Citizen of India
2002-2006
Ph.D thesis at the Institute of Cell Biology, Swiss Federal Institute of
Technology, Zürich, Switzerland
2001-2002
Research associate, Genetic Analysis Laboratory, Applied Biosystems
(ABI), LABINDIA, New Delhi
1999-2001
Microbiologist, Research and Development, J. Mitra and Company,
New Delhi, India
1996-1998
Masters degree with honors in Microbiology from the Punjab
University, Chandigarh, India
1993-1996
Bachelors degree with honors in Microbiology from the University of
Delhi, Delhi, India
1991-1993
High school in Delhi, India
1990-1991
Secondary school in Delhi, India
1981-1990
Primary school in Delhi, India
145