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Pharmacology & Therapeutics 128 (2010) 191–227
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Pharmacology & Therapeutics
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p h a r m t h e r a
Associate Editor: P. Molenaar
Molecular distinction between physiological and pathological cardiac hypertrophy:
Experimental findings and therapeutic strategies
Bianca C. Bernardo a, Kate L. Weeks a,b, Lynette Pretorius a,c, Julie R. McMullen a,⁎
a
b
c
Cardiac Hypertrophy Laboratory, Baker IDI Heart & Diabetes Institute, Melbourne, Australia
Faculty of Medicine, Dentistry and Health Sciences, Department of Biochemistry and Molecular Biology, University of Melbourne, Melbourne, Australia
Faculty of Medicine, Nursing and Health Sciences, Department of Medicine (Alfred Hospital), Monash University, Melbourne, Australia
a r t i c l e
i n f o
Keywords:
Heart failure
Phosphoinositide 3-kinase
Insulin-like growth factor 1
Physiological cardiac hypertrophy
Pathological cardiac hypertrophy
Gender differences
Therapeutic applications
a b s t r a c t
Cardiac hypertrophy can be defined as an increase in heart mass. Pathological cardiac hypertrophy (heart
growth that occurs in settings of disease, e.g. hypertension) is a key risk factor for heart failure. Pathological
hypertrophy is associated with increased interstitial fibrosis, cell death and cardiac dysfunction. In contrast,
physiological cardiac hypertrophy (heart growth that occurs in response to chronic exercise training, i.e. the
‘athlete's heart’) is reversible and is characterized by normal cardiac morphology (i.e. no fibrosis or
apoptosis) and normal or enhanced cardiac function. Given that there are clear functional, structural,
metabolic and molecular differences between pathological and physiological hypertrophy, a key question in
cardiovascular medicine is whether mechanisms responsible for enhancing function of the athlete's heart
can be exploited to benefit patients with pathological hypertrophy and heart failure. This review summarizes
key experimental findings that have contributed to our understanding of pathological and physiological
heart growth. In particular, we focus on signaling pathways that play a causal role in the development of
pathological and physiological hypertrophy. We discuss molecular mechanisms associated with features of
cardiac hypertrophy, including protein synthesis, sarcomeric organization, fibrosis, cell death and energy
metabolism and provide a summary of profiling studies that have examined genes, microRNAs and proteins
that are differentially expressed in models of pathological and physiological hypertrophy. How gender and
sex hormones affect cardiac hypertrophy is also discussed. Finally, we explore how knowledge of molecular
mechanisms underlying pathological and physiological hypertrophy may influence therapeutic strategies for
the treatment of cardiovascular disease and heart failure.
© 2010 Elsevier Inc. All rights reserved.
Abbreviations: 4E-BP1, 4E binding protein 1; ACE, Angiotensin converting enzyme; ACEI, Angiotensin converting enzyme inhibitors; Adr, adrenaline; Ang II, Angiotensin II; ANP,
Atrial natriuretic peptide; ARB, Angiotensin receptor blockers; ARK, Adrenergic receptor kinase; ARs, Adrenergic receptors; AT1, Angiotensin type 1 receptor; BNP, B-type natriuretic
peptide; ca, constitutively active; CaM, calmodulin; CaMK, calcium/calmodulin-dependent protein kinases; c-fos, c-fos oncogene; c-jun, c-jun oncogene; c-myc, c-myc oncogene; Cn/
CN, calcineurin; CoA, coenzyme A; CREB, cAMP response element-binding protein; CT-1, cardiotrophin 1; cTNT, cardiac troponin T; DAG, diacylglycerol; DCM, dilated
cardiomyopathy; dn, dominant negative; ECM, extracellular matrix; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; eIF2, eukaryotic initiation factor 2; eIF4E,
eukaryotic initiation factor 4E; ER, estrogen receptor; ERK, extracellular signal-regulated kinases; ETA, endothelin type A receptor; ET-I, endothelin 1; FAK, focal adhesion kinase;
GATA, GATA protein binding; Gp, guanine nucleotide binding proteins; GPCR, G protein-coupled receptor; Grb2, growth factor receptor bound protein 2; GSK3, glycogen synthase
kinase 3; HDAC, histone deacetylase; HIF, hypoxia-inducible factor; HSF1, heat shock transcription factor 1; Hsp, heat shock protein; HW/BW, heart weight/body weight ratio; IGF1,
insulin-like growth factor 1; IGF1R, insulin-like growth factor 1 receptor; IP3, inositol 1,4,5-trisphosphate; JAK, Janus kinase; JNK, c-Jun amino-terminal kinase; JVS, juvenile visceral
steatosis; KO, knockout; LIF, leukemia inhibitory factor; LVPW, left ventricular posterior wall; MAPK, mitogen activated protein kinase; MCAD, medium chain acyl coenzyme A
dehydrogenase; MCIP, mitogen-enriched calcineurin-interacting protein; mCPT-1, muscle-type carnitine palmitoyltransferase 1; MEF2, myocyte enhancer factor 2; MEK, mitogen
activated protein kinase kinase; MEKK, mitogen activated protein kinase kinase kinase; MHC, myosin heavy chain; miRNAs, microRNAs; MLC, myosin light chain; MLCK, myosin light
chain kinase; mTOR, mammalian target of rapamycin; Nab1, NGF1A-binding protein; NADPH, nicotinamide adenine dinucleotide phosphate; NE, noradrenaline, norepinephrine;
NFAT, nuclear factor of activated T cells; Ntg, non-transgenic; PDE, phosphodiesterase; PE, phenylephrine; PI3K, phosphoinositide 3-kinase; PKA, protein kinase A; PKC, protein
kinase C; PKD, protein kinase D; PLB, phospholamban; PLC, phospholipase C; PPAR, peroxisome proliferation-activated receptors; pS6, phosphorylation of 40 S ribosomal S6 protein;
Rab, member of RAS oncogene family; Raf1, member of RAS oncogene family; Ran, member of RAS oncogene family; Ras, Ras oncogene; Rho, rhodopsin; ROS, reactive oxygen
species; RTK, receptor tyrosine kinase; S6K, ribosomal S6 kinase; SERCA, sarcoplasmic reticulum Ca2+-ATPase; Src, Rous sarcoma oncogene; STAT, signal transducer and activator of
transcription; TAK, TGF-β activated kinase; Tg, transgenic; TGF, transforming growth factor; TNF, tumor necrosis factor; TR, thyroid hormone receptor; Ub, ubiquitin; WT, wildtype.
⁎ Corresponding author. P.O. Box 6492 St Kilda Road Central, Melbourne Victoria 8008, Australia. Tel.: +61 385321194; fax: +61 385321100.
E-mail address: [email protected] (J.R. McMullen).
0163-7258/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.pharmthera.2010.04.005
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B.C. Bernardo et al. / Pharmacology & Therapeutics 128 (2010) 191–227
Contents
1.
2.
Cardiac hypertrophy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Experimental and genetic mouse models utilized in the identification of
signaling pathways that mediate cardiac hypertrophy . . . . . . . . . . . . . . . . . . . . . . .
3.
Overview of signaling cascades/proteins implicated
in mediating physiological and pathological cardiac growth . . . . . . . . . . . . . . . . . . . .
4.
Molecular mechanisms associated with structural features of pathological and physiological hypertrophy
5.
Molecular mechanisms associated with differences in
energy metabolism in pathological and physiological hypertrophy . . . . . . . . . . . . . . . . .
6.
Characteristic gene expression changes associated with pathological and physiological hypertrophy .
7.
Gender differences in cardiac hypertrophy . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.
Therapeutic strategies for the treatment of heart failure . . . . . . . . . . . . . . . . . . . . . .
9.
Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Cardiac hypertrophy
1.1. Introduction and overview
Cardiac hypertrophy can broadly be defined as an increase in heart
mass. Growth of the postnatal heart is closely matched to its
functional load (Zak, 1984). In response to an increase in load (e.g.
pressure overload in a setting of hypertension), the heart must work
harder than under normal conditions. To counterbalance the chronic
increase in wall stress the muscle cells within the heart enlarge
leading to an increase in size and mass (Cooper, 1987; Sugden & Clerk,
1998; Hunter & Chien, 1999). The increase in heart mass is largely due
to an increase in ventricular weight. In the subsequent sections we
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have described cardiac hypertrophy at the cellular level, different
types of cardiac hypertrophy (pathological and physiological), the
molecular mechanisms responsible for different forms of cardiac
hypertrophy, gender differences, and possible treatment strategies
based on the distinct molecular mechanisms associated with
physiological and pathological cardiac hypertrophy.
1.2. Cardiac hypertrophy at the cellular level
The heart is composed of cardiac myocytes (muscle cells), nonmyocytes (e.g. fibroblasts, endothelial cells, mast cells, vascular smooth
muscle cells), and the surrounding extracellular matrix (Nag, 1980; Zak,
1984). Ventricular cardiac myocytes make up only one-third of the total
Fig. 1. Cellular processes involved in the development of cardiac hypertrophy. ECM: extracellular matrix, FAO: fatty acid oxidation, GPCR: G protein-coupled receptor, MAPK:
mitogen-activated protein kinase, PI3K: phosphoinositide 3-kinase, ROS: reactive oxygen species, SERCA: sarcoplasmic reticulum Ca2+ ATPase.
B.C. Bernardo et al. / Pharmacology & Therapeutics 128 (2010) 191–227
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cell number, but account for 70–80% of the heart's mass (Nag, 1980; Zak,
1984; Popescu et al., 2006). In mammals, at birth or soon after, the
majority of cardiac myocytes lose the ability to proliferate, thus heart
growth occurs primarily via an increase in myocyte size (Soonpaa et al.,
1996). The inability of adult cardiac myocytes to divide has come under
some debate (Anversa & Nadal-Ginard, 2002; Anversa et al., 2002;
Pasumarthi & Field, 2002). However, estimates of DNA labeling indicate
that DNA synthesis is taking place in a very small fraction of the total
adult cardiac myocyte population (Nakagawa et al., 1988; Soonpaa et al.,
1996; MacLellan & Schneider, 2000; Anversa et al., 2002; Pasumarthi &
Field, 2002), indicating that the postnatal heart enlarges primarily by an
increase in myocyte size.
Myocytes are composed of bundles of myofibrils. Myofibrils contain
myofilaments which consist of sarcomeres, the basic contractile unit of
the heart. Myocytes are arranged in a circumferential and spiral
orientation around the left ventricle, and need to contract simultaneously to ensure the heart pumps with a normal rhythm. Intercalated
discs, located at the bipolar ends of cardiac myocytes, are responsible for
maintaining cell–cell adhesion while allowing contractile force to be
transmitted between adjacent cardiac myocytes (Estigoy et al., 2009).
Growth of cardiac myocytes is dependent on the initiation of several
events in response to an increase in functional load, including activation
of signaling pathways, changes in gene expression, increases in the rate
of protein synthesis, and the organization of contractile proteins into
sarcomeric units (Fig. 1). Cardiac myocytes appear to have an intrinsic
mechano-sensing mechanism. Stretch sensitive ion channels present in
the plasma membrane of cardiac myocytes and structural proteins (such
as integrins) play a role in linking the extracellular matrix, cytoskeleton,
sarcomere, calcium handling proteins and nucleus (Knoll et al., 2003;
Hoshijima, 2006). Thus, there is an interactive continuum from integrins
at the cell surface to the contractile apparatus and nucleus (Fig. 1).
known association between cardiac hypertrophy and nearly all forms of
heart failure (Levy et al., 1990). Cardiac hypertrophy is also an
independent risk factor for myocardial infarction, arrhythmia and
sudden death (Levy et al., 1990). In response to a chronic increase in
load, there is an initial increase in heart mass to normalize wall stress
and permit normal cardiovascular function at rest i.e. compensated
growth. However, if the chronic increase in wall stress is not relieved,
the hypertrophied heart can dilate, contractile function falls and the
heart can fail.
Heart failure affects approximately 1–3% of people in Western
society. The incidence of heart failure increases with age, affecting 3–4%
of those over 45 years old, 5% of those aged between 60 and 69 years of
age, and 10% of people over the age of 70 (Davies et al., 2001; AIHW,
2004; Thom et al., 2006; Lloyd-Jones et al., 2009). Symptoms of heart
failure patients include fatigue, insomnia, anxiety, depression, shortness
of breath, edema, dizziness, and nausea, all of which contribute to a
reduced quality of life for these patients (Blinderman et al., 2008). With
an aging population, rising rates of obesity and diabetes, as well as the
availability of interventions that prolong survival following cardiac
insults, the incidence of heart failure is likely to rise over the coming
decades. The costs associated with an expanding number of patients and
specialized treatment strategies are expected to contribute significantly
to the economic burden caused by heart failure (Blinderman, et al.,
2008). Currently there is no cure for heart failure, and long term survival
following heart failure remains poor, with one third of patients dying
within a year of diagnosis (Zannad et al., 1999; Cowie et al., 2000;
Bleumink et al., 2004; McMurray & Pfeffer, 2005). Thus, a number of
studies have focused on identifying the molecular mechanisms
associated with cardiac hypertrophy and the transition to heart failure,
to identify new therapeutic targets to prevent or reverse cardiac
hypertrophy and heart failure.
1.3. Association between cardiac hypertrophy and heart failure
1.4. Cardiac hypertrophy and the athlete's heart
Understanding the molecular mechanisms responsible for the
induction of cardiac hypertrophy has been of great interest due to the
The athlete's heart has generally been defined as a benign increase in
heart mass, associated with morphological alterations, that represents a
Fig. 2. Cardiac hypertrophy can be classified as physiological, which occurs during pregnancy or in response to chronic exercise training, is reversible and characterized by normal
cardiac morphology and function. In contrast, hypertrophy that occurs in settings of disease is detrimental for cardiac structure and function and can lead to heart failure.
Developmental hypertrophy is associated with the normal growth of the heart after birth until adulthood. RV: right ventricle, LV: left ventricle. Normal/ physiological heart growth is
shown in green, pathological heart growth is shown in red.
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physiological adaptation to chronic training. Though, with attention
from media reports of sudden death in young athletes, it has been
questioned whether highly trained athletes develop pathological
conditions. Notably, there is currently no evidence in the healthy
population (excluding persons with underlying cardiovascular disease
or genetic disorders) showing that remodeling due to exercise training
leads to long-term cardiac disease progression, cardiovascular disability,
or sudden cardiac death (Maron & Pelliccia, 2006). The overall risk of
sudden death in athletes is not well defined but considered low. A 12year survey in high-school athletes participating in organized sports in a
US state (Maron et al., 1998) reported a frequency of sudden death of
1:200,000 per year (based on only 3 deaths among 1.4 million students;
including 27 sports). Sudden death in young trained athletes in response
to physical exertion has largely been causally linked to congenital but
clinically unsuspected cardiovascular disease (see Maron, 2003). In
large autopsy-based surveys of athletes in the US, hypertrophic
cardiomyopathy is the most common cause of sudden death (accounting for about one-third of events), followed by congenital coronaryartery anomalies. A wide range of other, largely congenital malformations account for the remaining sudden deaths from cardiovascular
disease among athletes (Maron, 2003). Thus, it is generally accepted
that cardiac hypertrophy in response to exercise is protective, in some
instances improves cardiac function, and does not progress to heart
failure. A comprehensive understanding of why cardiac hypertrophy
progresses to heart failure in a setting of disease, but does not in
response to exercise, is considered important for identifying and
targeting the critical molecular mechanisms responsible for the
transition from hypertrophy to heart failure.
1.5. Distinct forms of cardiac growth and hypertrophy
1.5.1. Pathological and physiological cardiac growth and hypertrophy
Cardiac growth or hypertrophy can broadly be classified as either
physiological (“normal”) or pathological (“detrimental”). Physiological heart growth includes normal postnatal growth, pregnancyinduced growth, and exercise-induced cardiac hypertrophy. In
contrast, pathological growth occurs in response to chronic pressure
or volume overload in a disease setting (e.g. hypertension, valvular
heart disease), myocardial infarction or ischemia associated with
coronary artery disease, or abnormalities/conditions that lead to
cardiomyopathy (e.g. inherited genetic mutations, diabetes) (Fig. 2).
Both physiological and pathological heart growth are associated with
an increase in heart size, however pathological hypertrophy is also
typically associated with loss of myocytes and fibrotic replacement,
cardiac dysfunction, and increased risk of heart failure and sudden
death (Levy et al., 1990; Weber et al., 1993; Cohn et al., 1997). In
contrast, physiological growth is associated with normal cardiac
structure, normal or improved cardiac function, and is reversible in
the instance of exercise- or pregnancy-induced hypertrophy (Ferrans,
1984; Schaible & Scheuer, 1984; Fagard, 1997) (Fig. 2).
1.5.2. Concentric and eccentric hypertrophy
Pathological and physiological hypertrophy has classically been
subdivided as concentric or eccentric. These classifications are based on
changes in shape, which is dependent on the initiating stimulus
(Grossman et al., 1975; Pluim et al., 2000) (Fig. 3). Concentric
hypertrophy refers to an increase in relative wall thickness and
cardiac mass, with a small reduction or no change in chamber volume.
Concentric hypertrophy is characterized by a parallel pattern of
sarcomere addition leading to an increase in myocyte cell width
(Fig. 3). Eccentric hypertrophy refers to an increase in cardiac mass
with increased chamber volume, i.e. dilated chambers. Relative wall
thickness may be normal, decreased, or increased. In eccentric
hypertrophy, addition of sarcomeres in series leads to an increase in
myocyte cell length (Fig. 3) (Grossman et al., 1975).
A pathological stimulus causing pressure overload (e.g. hypertension, aortic stenosis) produces an increase in systolic wall stress which
results in concentric hypertrophy (Grossman et al., 1975). In contrast,
a stimulus causing volume overload (e.g. aortic regurgitation,
arteriovenous fistulas) produces an increase in diastolic wall stress
and results in eccentric hypertrophy (Grossman et al., 1975; Pluim et
al., 2000). Clinical studies suggest that eccentric cardiac hypertrophy
induced by pathological stimuli poses a greater risk than concentric
cardiac hypertrophy (Berenji et al., 2005).
Physiological stimuli can also produce concentric or eccentric
hypertrophy. Aerobic exercise (also referred to as endurance training,
Fig. 3. Different stimuli induce different forms of cardiac hypertrophy. Pressure overload causes thickening of the left ventricle wall due to the addition of sarcomeres in parallel and
results in concentric hypertrophy. Volume overload induces an increase in muscle mass via the addition of sarcomeres in series and results in eccentric hypertrophy.
B.C. Bernardo et al. / Pharmacology & Therapeutics 128 (2010) 191–227
195
Fig. 4. Four distinct features of cardiac hypertrophy include heart size, cardiac function, cardiac fibrosis and gene expression. The upper left quadrant shows representative images of
mouse hearts that have been subjected to a pathological (aortic banding; band) stimulus for one week, compared with sham (sham) operated controls, or mice subjected to a
physiological stimulus for four weeks (chronic swim training; exercise) compared with sedentary controls. An increase in heart size is observed in mice that have undergone aortic
banding (band) or chronic swim training (exercise) compared to sham and sedentary controls. The upper right quadrant depicts cardiac function as shown by M-mode
echocardiography. Cardiac function is depressed in a mouse model of pathological (decompensated) hypertrophy but is conserved in a mouse model of physiological hypertrophy
(exercise). The lower left quadrant depicts histological analysis of heart sections stained with Masson's trichrome; increased fibrosis is shown in blue and is only present in
pathological hypertrophy. Representative sections from the left ventricular wall of untrained mice (control), aortic banded mice (pressure overload, band) and swim trained mice
(exercise). Bar = 10 µm. Sections from sham operated mice were similar to those of untrained (control). The lower right quadrant shows gene expression changes associated with
cardiac hypertrophy. Representative Northern blot showing total RNA from ventricles of sham, aortic banded (band), untrained (sedentary) and trained (exercise) mice. Expression
of GAPDH was determined to verify equal loading of RNA. There is increased expression of atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), β-myosin heavy chain
(β-MHC) and α-skeletal actin, and decreased expression of sarcoplasmic reticulum Ca2+ ATPase (SERCA) and α-MHC in mice subjected to aortic banding (band) compared to
controls, while gene expression remains relatively unchanged in exercised trained mice compared to sedentary controls.
isotonic or dynamic exercise e.g. long-distance running, swimming)
and pregnancy increase venous return to the heart resulting in
volume overload and eccentric hypertrophy (Zak, 1984; Pluim et al.,
2000; Eghbali et al., 2005). This type of eccentric hypertrophy is
usually characterized by chamber enlargement and a proportional
change in wall thickness, whereas eccentric hypertrophy in settings of
disease is generally associated with thinning of the ventricular walls.
Strength training (also referred to as isometric or static exercise, e.g.
weight lifting, wrestling, throwing heavy objects) results in a pressure
load on the heart rather than volume load and concentric hypertrophy
(Zak 1984; Pluim et al., 2000) (Fig. 3).
1.6. Distinct features of pathological and physiological hypertrophy
Despite comparable increases in heart size, pathological and
physiological hypertrophy are associated with distinct 1) structural
and functional, 2) metabolic, and 3) biochemical and molecular
features (Fig. 4, Table 1).
Table 1
Features of pathological and physiological hypertrophy.
Feature
Pathological cardiac hypertrophy
Physiological cardiac hypertrophy
Stimuli
Disease
Pressure or volume overload
Cardiomyopathy (familial, viral, diabetes, metabolic, alcoholic/toxic)
Increased myocyte volume
Formation of new sarcomeres
Increase in heart size
Yes
Yes
Upregulation of ANP, BNP, β-MHC, and α-skeletal actin
Downregulation of SERCA2a, α-MHC
Depressed
Decreased fatty acid oxidation
Increased glucose utilization
No
Yes
Aerobic exercise training
Postnatal growth
Pregnancy
Increased myocyte volume
Formation of new sarcomeres
Increase in heart size
No
No
Relatively unchanged
Normal or increased
Normal or enhanced
Enhanced fatty acid oxidation
Enhanced glucose utilization
Yes
No
Cardiac morphology
Cardiac fibrosis
Apoptosis
Fetal gene expression
Expression of genes associated with contractile function
Cardiac function
Metabolism
Reversible
Association with heart failure and mortality
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1.6.1. Structural and functional features
Cardiac hypertrophy is associated with structural remodeling of
components of the ventricular walls to accommodate increases in
myocyte size, including changes in the fibrillar collagen network
and angiogenesis. Under basal conditions or a setting of physiological hypertrophy, the fibrillar collagen network provides structural
integrity of adjoining myocytes, facilitating myocyte shortening
which translates into efficient cardiac pump function (Gunasinghe
& Spinale, 2004). Pathological hypertrophy is associated with cell
death (apoptosis, necrosis) and the loss of myocytes is replaced
with excessive collagen (known as fibrosis). The main fibrillar
collagen present in cardiac fibrosis is type 1 collagen. Excessive
accumulation of collagen stiffens the ventricles, which impairs
contraction and relaxation, impairs the electrical coupling of cardiac
myocytes with extracellular matrix proteins, and reduces capillary
density. Fibrosis and reduced capillary density increases oxygen
diffusion distances, leading to myocardial ischemia, and is likely to
contribute to the transition from hypertrophy to failure (Gunasinghe & Spinale, 2004).
1.6.2. Cardiac metabolism
In the normal healthy heart, fatty acid oxidation is the main
metabolic pathway responsible for generating energy, accounting for
60–70% of ATP production (van der Vusse et al., 1992); glucose and
lactate metabolism account for approximately 30% of ATP synthesis.
The heart is capable of switching energy substrates depending on
workload and the relative concentrations of fuel molecules in the
bloodstream (see van der Vusse et al., 1992). This is considered an
adaptive mechanism which allows the heart to produce a continuous
supply of ATP under various physiological conditions (e.g. fasting,
during exercise, etc.).
Pathological cardiac hypertrophy is associated with decreases in
fatty acid oxidation and increases in glucose metabolism (Allard et al.,
1994; Christe & Rodgers 1994; Davila-Roman et al., 2002). This switch
in substrate utilization may be a protective mechanism, allowing the
heart to produce more ATP per molecule of oxygen consumed (see
van Bilsen et al., 2009). This is reminiscent of what occurs during fetal
cardiac development, when oxygen supply is limited and fatty acid
transport and metabolism are impaired (due to carnitine deficiency
and delayed maturation of enzymes involved in fatty acid oxidation).
Thus, glucose is the primary substrate used by the fetal heart to
generate ATP (Ostadal, et al., 1999). In contrast, physiological cardiac
hypertrophy induced by exercise training is characterized by
enhanced fatty acid and glucose oxidation (Gertz et al., 1988). Of
note, in advanced pathological hypertrophy and failure, glucose
metabolism decreases as the heart becomes resistant to insulin,
reducing the overall ability of the heart to generate sufficient ATP (see
Neubauer, 2007).
1.6.3. Biochemical and molecular features
In the 1970s and early 1980s it was recognized that physiological
hypertrophy (induced by exercise training/ thyroid hormone) was
associated with elevations in myosin ATPase activity and enhancement of contractility, whereas pathological hypertrophy (induced by
renal hypertension, aortic banding) was associated with decreased
myosin ATPase activity and depressed contractile function (WikmanCoffelt et al., 1979; Rupp, 1981). Since then, there have been a number
of studies demonstrating that physiological and pathological cardiac
hypertrophy are associated with some distinct biochemical and
molecular signatures. Iemitsu et al. (2001) compared mRNA expression in a rat model of pathological cardiac hypertrophy (spontaneously hypertensive rat) and physiological hypertrophy (chronic swim
training). The investigators reported a distinct pattern of gene
expression in the two models (Iemitsu et al., 2001). It is now well
recognized that pathological cardiac hypertrophy is associated with
distinct alterations in cardiac contractile proteins (α- and β-myosin
heavy chain (MHC)), increased expression of fetal genes (e.g. atrial
natriuretic peptide (ANP), B-type natriuretic peptide (BNP), αskeletal actin) but down-regulation of calcium-handling proteins
(e.g. sarcoplasmic reticulum Ca2+-ATPase 2a (SERCA2a)). Since this
time, generation of numerous transgenic and knockout mouse models
in combination with models of physiological and pathological
hypertrophy have allowed investigators to delineate signaling
proteins that appear to play distinct roles in regulating physiological
and pathological cardiac hypertrophy (discussed in detail in
Section 3).
2. Experimental and genetic mouse
models utilized in the identification of
signaling pathways that mediate cardiac hypertrophy
The definition of cardiac hypertrophy as either physiological or
pathological has not been without contention (Dorn et al., 2003).
However, there is now substantial evidence from animal studies that
the different phenotypes associated with pathological and physiological hypertrophy can be due to distinct stimuli and activation of some
distinct signaling pathways, at least under certain conditions.
Studies utilizing genetic mouse models (transgenic and knockout)
alone or in combination with morphologically distinct models of
hypertrophy (e.g. pathological, physiological, concentric and eccentric) have become powerful tools for understanding molecular
pathways responsible for different forms of heart growth in vivo.
Genetic mouse models have typically utilized the α-MHC promoter to
achieve cardiac myocyte specific expression (Subramaniam et al.,
1991), or the Cre-loxP system to generate cardiac-specific or inducible
knockout mice (Chien, 2001). Inducible transgenic mouse models
have also been valuable as they allow investigators to switch on the
activity of the protein of interest in myocytes by injection of a drug
(e.g. tamoxifen) at a specific time point, i.e. after completion of
developmental growth (Fan et al., 2005b; Hoesl et al., 2008; Lu et al.,
2009; Ruan et al., 2009). Commonly used experimental mouse models
of pathological hypertrophy include pressure overload (constriction/
banding of the renal, abdominal, ascending or transverse aorta),
volume overload (aortocaval shunt), and minipump infusions of
vasoactive substances (e.g. isoproterenol, angiotensin II (Ang II)).
Physiological models include treadmill running, freewheel running
and chronic swim training. Below, we have described hypertrophic
triggers/stimuli, signaling proteins and cascades which appear to play
an important role for the development of pathological or physiological
hypertrophy.
2.1. Hypertrophic triggers/stimuli
In response to hemodynamic overload, cardiac myocytes are
subjected to mechanical stretch, and autocrine and paracrine humoral
factors including Ang II, endothelin 1 (ET-1), insulin-like growth
factor 1 (IGF1), transforming growth factor-β (TGF-β) and cardiotrophin 1 (CT-1) are released. These factors bind to receptors on
cardiac cells, in turn activating intracellular signaling pathways that
leads to cell growth. Signaling cascades and proteins responsible for
cardiac growth and hypertrophy are complex and extensive crosstalk
has been identified (Fig. 5). The subsequent sections of this review
focus on signaling cascades and proteins that have been reported to
play distinct roles in regulating pathological and physiological
hypertrophy.
2.1.1. Physiological and pathological triggers/stimuli
Human and animal studies have demonstrated that certain factors
are preferentially released in response to pathological and physiological stimuli. It is well recognized that IGF1 is released during postnatal
development and in response to exercise training (Yeh et al., 1994;
Conlon and Raff 1999; Koziris et al., 1999; Neri Serneri et al., 2001b;
B.C. Bernardo et al. / Pharmacology & Therapeutics 128 (2010) 191–227
197
Fig. 5. A schematic of the major signaling pathways involved in cardiac hypertrophy, showing cross-talk and integration of various pathways. N.B. due to the complex nature of
signaling cascades and on-going discoveries it was not possible to illustrate all interactions.
Perrino et al., 2006), and IGF1 levels in the heart were increased in
swim-trained rats (Scheinowitz et al., 2003). Furthermore, production
of cardiac IGF1 (but not Ang II or ET-1) was increased in professional
athletes compared with control subjects (Neri Serneri et al., 2001b). In
contrast, pressure overload is associated with elevated levels of Ang II,
catecholamines and ET-1 (Schunkert et al., 1990; Arai et al., 1995;
Yamazaki et al., 1999; Rapacciuolo et al., 2001; Yayama et al., 2004),
and cardiac formation of Ang II was increased in heart failure patients
with hypertrophied hearts (Neri Serneri et al., 2001a).
3. Overview of signaling cascades/proteins implicated
in mediating physiological and pathological cardiac growth
The best characterized signaling cascades responsible for mediating physiological and pathological cardiac hypertrophy are the IGF1phosphoinositide 3-kinase [PI3K, (p110α)]-Akt pathway and Gαq
signaling (downstream of G protein-coupled receptors (GPCR)
activated by Ang II, ET-1 and catecholamines), respectively (Fig. 6).
Other signaling pathways associated with physiological cardiac
hypertrophy and/or protection include the gp130/JAK/STAT pathway,
thyroid hormone signaling, and heat shock transcription factor 1
(HSF1). In contrast, pathological hypertrophy has also been associated
with abnormalities leading to enhanced PI3K(p110γ), mitogen
activated protein kinases (MAPKs), protein kinase C (PKC) and D
(PKD), and calcineurin.
3.1. Signaling proteins/pathways
implicated in mediating physiological hypertrophy
3.1.1. IGF1-PI3K(p110α)-Akt pathway
Substantial evidence from genetic mouse models has demonstrated the critical role of the IGF1-PI3K(p110α)-Akt pathway in
regulating physiological cardiac growth.
3.1.1.1. Insulin-like growth factor (IGF1) receptor signaling. IGF1 is best
known for being produced by the liver in response to growth
hormone stimulation and is essential for normal fetal and postnatal
growth and development (Adams et al., 2000). IGF1 is also produced
by the heart (see reviews: Ren et al., 1999; McMullen, 2008) and binds
to a cell surface receptor, insulin-like growth factor 1 receptor
(IGF1R), a receptor tyrosine kinase that activates downstream
signaling proteins. A number of studies have examined the role of
IGF1 in the heart using gene targeted mice.
a) Mice with increased cardiac myocyte specific expression of IGF1
(human IGF1B transgene expression driven by the α-MHC
promoter) had enlarged hearts with normal cardiac function
(Reiss et al., 1996). A confounding factor of this study was that
transgene expression increased IGF1 secretion from cardiac
myocytes which resulted in a significant rise in systemic plasma
levels of IGF1 (approximately 80%) and an increase in other organ
weights. The increase in heart size was attributed to an increase in
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cardiac myocyte number rather than myocyte size. This result was
unexpected given that the majority of mammalian cardiac
myocytes are thought to lose their ability to proliferate at birth
or within the first few weeks of postnatal life (Soonpaa et al.,
1996).
b) In a second study, IGF1 transgenic mice were generated using the
α-skeletal actin promoter (transgene expression in heart and
skeletal muscle) (Delaughter et al., 1999). Serum IGF1 levels were
not elevated but persistent transgenic expression was associated
with increases in gut, liver, and spleen weight (Fiorotto et al.,
2003). Up to 10 weeks of age, IGF1 transgenic mice displayed
cardiac hypertrophy, which was associated with enhanced cardiac
systolic function i.e. physiological hypertrophy (Delaughter et al.,
1999). However, the investigators concluded that the “physiological” cardiac phenotype ultimately progressed to pathological
hypertrophy, because mice had depressed cardiac function by
12 months of age (Delaughter et al., 1999).
c) We examined the role of IGF1 specifically in cardiac myocytes by
over-expressing the IGF1R rather than IGF1 (transgenic expression
of human IGF1R using the α-MHC promoter) (McMullen et al.,
2004b). Expression of the IGF1R was considered an advantage
because it allowed examination of IGF1 signaling in the absence of
effects of secreted IGF1 on other tissues or non-myocytes. At
3 months of age, IGF1R transgenic mice had enlarged hearts
(approximately 40% increase in weight, with a proportional
increase of all chambers and ventricular wall thickness), increased
myocyte size, no evidence of histopathology (e.g. necrosis, fibrosis,
myocyte disarray) and enhanced systolic function (McMullen et
al., 2004b). These characteristic features of physiological hypertrophy were maintained at 12–16 months of age (IGF1R transgenic
mice had enhanced systolic function in comparison to nontransgenic mice) (McMullen et al., 2004b). Thus, IGF1R transgenic
mice did not progress to pathological hypertrophy with aging,
which was observed in the earlier study (model b) (Delaughter et
al., 1999). Consistent with the hypothesis that IGF1 activates PI3K
(p110α) and Akt (a known downstream target of PI3K) to induce
physiological cardiac hypertrophy (Fig. 6), activation of PI3K and
phosphorylation of Akt were elevated in hearts of IGF1R transgenic
mice (McMullen et al., 2004b). In contrast, signaling proteins
downstream of Gαq (implicated in pathological hypertrophy)
including MAPKs and calcineurin were not activated in hearts of
IGF1R transgenic mice (McMullen et al., 2004b).
d) In corroboration with the idea that IGF1 signaling is critical for
physiological heart growth, cardiac myocyte-specific ablation of
the IGF1R gene in mice attenuated the hypertrophic response to
swim exercise training compared to non-transgenic mice (Kim et
al., 2008b). A basal cardiac phenotype was not observed.
3.1.1.2. Phosphoinositide 3-kinase (PI3K, p110α) signaling. PI3Ks are a
family of enzymes and have been linked to a diverse group of cellular
functions, particularly cell growth, survival, differentiation, and
proliferation (Cantley, 2002). PI3K is a lipid kinase that releases
inositol lipid products from the plasma membrane which in turn
mediate intracellular signaling (Toker & Cantley, 1997; Vanhaesebroeck et al., 1997). Activation of PI3Ks is coupled to both receptor
tyrosine kinases (e.g. insulin receptor and IGF1R) and GPCRs. There
are three major classes of PI3Ks (classes I, II and III), which are
classified based on sequence homology in the catalytic domain,
structure and substrate specificity (Vanhaesebroeck et al., 2001; Kok
et al., 2009). Class I PI3Ks are heterodimers and further divided into
Fig. 6. A schematic overview of pathological and physiological hypertrophy outlining key differences in initiating stimuli, signaling pathways, cellular responses and cardiac function.
For simplicity we have focussed on the best characterized signaling pathways implicated in mediating pathological (shaded red) and physiological (shaded green) cardiac
hypertrophy. Other important mediators are described in detail in Section 3. Ang II: angiotensin II, ET-1: endothelin-1, GPCR: G protein-coupled receptor, IGF-1: insulin-like growth
factor 1, MAPK: mitogen-activated protein kinase, NE: norepinephrine, PI3K(p110α): phosphoinositide 3-kinase p110α, RTK: receptor tyrosine kinase.
B.C. Bernardo et al. / Pharmacology & Therapeutics 128 (2010) 191–227
the subclasses IA and IB. Class IA PI3Ks consist of a p110 catalytic
subunit (α, β or δ) and a p85 or p55 regulatory subunit. The only Class
Iβ PI3K is p110γ, which is regulated by p101 (Vanhaesebroeck et al.,
1997). Of the Class I PI3Ks, p110α and p110γ are abundantly
expressed in the heart. p110β is also expressed in the heart but at a
lower level (Crackower et al., 2002). The p110δ isoform of PI3K is
exclusively expressed in leukocytes (Vanhaesebroeck & Waterfield,
1999).
PI3Ks were first shown to regulate organ size in Drosophila. Overexpression of the Drosophila PI3K homolog, Dp110, resulted in
formation of larger wings and eyes (Leevers et al., 1996). In contrast,
expression of a catalytically inactive Dp110 caused the opposite
phenotype, i.e. smaller wings and eyes (Leevers et al., 1996). Null
homozygous mice for p110α were embryonically lethal due to
proliferation defects in the embryo (Bi et al., 1999). Evidence that
PI3K activity regulates heart size was obtained from studies that
expressed a cardiac-specific constitutively active (ca) form of PI3K
(p110α) or dominant negative (dn) form of PI3K(p110α) in
transgenic mice utilizing the α-MHC promoter (Shioi et al., 2000;
McMullen et al., 2003). PI3K activity was increased by 6.5-fold in
hearts of caPI3K transgenic mice, and hearts were 20% larger than
non-transgenic mice. The increase in heart size of the caPI3K mice was
proportional, resembling physiological hypertrophy. In contrast, PI3K
activity was 77% lower in hearts of dnPI3K transgenic mice, which
resulted in a 17% decrease in heart size compared to non-transgenic
mice (Shioi et al., 2000). Changes in heart size were due to changes in
myocyte size rather than number (Shioi et al., 2000). Cardiac function,
structure and life span were normal in caPI3K and dnPI3K transgenic
mice under basal conditions (Shioi et al., 2000; McMullen et al., 2003).
These studies demonstrated that PI3K(p110α) is critical for physiological postnatal growth of the heart.
It was later demonstrated that PI3K(p110α) is also critical for
physiological exercise-induced growth of the heart but not pathological hypertrophy. Adult dnPI3K transgenic mice were subjected to a
physiological stimulus (exercise; chronic swim training) and a
pathological stimulus (pressure overload; ascending aortic banding).
dnPI3K mice showed significant hypertrophy in response to pressure
overload, but an attenuated hypertrophic response to swim training,
compared with non-transgenic mice (McMullen et al., 2003; McMullen et al., 2007). These studies were also confirmed using a musclespecific knockout approach of the p85α/p55α/p50α and p85β
(global) regulatory subunits in mice (Luo et al., 2005), as well as
cardiac-specific ablation of p110α (Lu et al., 2009). These mice showed
a decrease in heart weight to body weight ratio of approximately 20%
and 16%, respectively (Luo et al., 2005; Lu et al., 2009), similar to that
reported in dnPI3K mice (Shioi et al., 2000; McMullen et al., 2003).
The small heart phenotype of p85α/p85β knockout mice was
accompanied by a reduction in mean myocyte cell area, and mice
exhibited an attenuated hypertrophic response to exercise training
(Luo et al., 2005). Together, these studies support the critical role of
class IA PI3Ks in the regulation of physiological cardiac hypertrophy.
Finally, to assess whether PI3K(p110α) was the critical mediator
responsible for physiological growth in the IGF1R transgenic mice, we
crossed transgenic mice over-expressing IGF1R with dnPI3K transgenic mice and examined heart size. Hearts of double transgenic mice
(i.e. expressing both the IGF1R and dnPI3K transgenes) were not
significantly different in size to that in dnPI3K mice alone (McMullen
et al., 2004b). This result demonstrated that the physiological heart
growth in the IGF1R transgenic mice was dependent on PI3K(p110α)
signaling (McMullen et al., 2004b).
3.1.1.3. Akt. Akt, a serine/threonine kinase (also known as protein
kinase B), is a well characterized target of PI3K. The Akt family is
involved in a number of cellular processes, including cell survival, cell
cycle, metabolism, and protein synthesis. There are three isoforms of
Akt (Akt1, Akt2 and Akt3), each is encoded by distinct genetic loci, in
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which the genes code for enzymes that are members of the serine/
threonine-specific protein kinase family (Matsui & Rosenzweig,
2005). The Akt homologue Dakt regulates cell and organ growth in
Drosophila, in the same manner as PI3K; activation of Dakt increased
cell size whereas loss-of-function caused a reduction in cell size, but
had no impact on cell proliferation (Verdu et al., 1999; Rintelen et al.,
2001). In mammals, Akt1 null mice had a 20% reduction in body
weight (Cho et al., 2001). Although all three isoforms are broadly
expressed, only Akt1 and Akt2 are highly expressed in the heart
(Matsui & Rosenzweig 2005).
Initial characterization of the cardiac phenotypes of Akt transgenic
mice led to confounding results. Phenotypes ranged from absence of
hypertrophy associated with protection from ischemia–reperfusion
injury to substantial hypertrophy associated with a pathological
phenotype and premature death (Condorelli et al., 2002; Matsui et al.,
2002; Shioi et al., 2002; Shiraishi et al., 2004; Shiojima et al., 2005). The
varying phenotypes have been attributed to different degrees of Akt
activation, angiogenesis, and subcellular localization. Of note, Akt can be
activated by both receptor tyrosine kinases (e.g. IGF1R) and GPCR
(Fig. 5), and appears to be differently regulated depending on the
initiating stimulus. Myostatin, an inhibitor of cardiac growth, reduced
GPCR-induced Akt phosphorylation but not receptor tyrosine kinase
(IGF1R)-induced phosphorylation in neonatal cardiac myocytes (Morissette et al., 2006). The biological significance of this differential
activation is currently unclear.
More recent studies in Akt knockout mice suggest Akt1 is required
for physiological rather than pathological heart growth. Akt1
knockout mice (normal cardiac phenotype under basal conditions)
showed a blunted hypertrophic response to swim training but not to
pressure overload (DeBosch et al., 2006b). These findings are
reminiscent of those in mice with reduced PI3K activity (McMullen
et al., 2003; Luo et al., 2005). It is now generally accepted that Akt1
mediates cardiac cell growth whereas Akt2 is important for cardiac
metabolism (DeBosch et al., 2006a,b).
Glycogen synthase kinase 3 (GSK3), a cellular substrate of Akt, is
an important regulatory kinase with a number of cellular targets,
including cytoskeletal proteins and transcription factors. Both GSK3
isoforms (GSK3α and GSK3β) are expressed in the heart (Ferkey &
Kimelman, 2000; Harwood, 2001; Hardt & Sadoshima, 2002). Initial
reports demonstrated that GSK3β negatively regulated heart growth
and that inhibition of GSK3β by hypertrophic stimuli was an
important mechanism for stimulating growth (Haq et al., 2000;
Morisco et al., 2000; Morisco et al., 2001; Antos et al., 2002; Badorff et
al., 2002). More recent studies have shown that GSK3α inhibits
postnatal cardiac growth and reduces pressure overload-induced
hypertrophy (Zhai et al., 2007). However, transgene expression was
associated with increased fibrosis and apoptosis both under basal
conditions and during pressure overload. Furthermore, the reduced
hypertrophic phenotype in response to pressure overload was
associated with severe cardiac dysfunction and heart failure (Zhai et
al., 2007). Interestingly, the GSK3α and GSK3β isoforms appear to
have distinct roles in a setting of pressure overload. Phosphorylation
of GSK3β was essential for the development of pathological
hypertrophy whereas phosphorylation of GSK3α played a compensatory role (Matsuda et al., 2008). Thus, selective modulation of the
phosphorylation status of the two isoforms may be required to
maximize the therapeutic potential of modulating this kinase.
3.1.2. Gp130/JAK/STAT pathway
Leukemia inhibitory factor (LIF), CT-1, and other members of the
interleukin-6 cytokine family activate the gp130 receptor associated
with the LIF receptor (Fig 5). Once activated, this cytokine receptor
interacts with janus kinase 1 (JAK1), leading to phosphorylation of the
signal transducer and activator of transcription (STAT) class of
transcription factors (Kodama et al., 1997; Pellegrini & DusanterFourt 1997; Aoki & Izumo, 2001; Molkentin & Dorn 2001). Cardiac-
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specific transgenic mice over-expressing STAT3 displayed cardiac
hypertrophy that was protective against doxorubicin-induced cardiomyopathy (Kunisada et al., 2000). In contrast, mice with
ventricular deletion of gp130 had normal cardiac structure and
function under basal conditions, but displayed a rapid onset of dilated
cardiomyopathy in response to pressure overload (Hirota et al., 1999).
However, expression of a dominant negative mutant of gp130 (to
decrease activation of this pathway) appeared to protect transgenic
mice against pressure overload-induced hypertrophy (Uozumi et al.,
2001). Despite this discrepancy, the majority of data in the literature
suggests that the gp130/JAK/STAT pathway has a protective role in the
heart (see Fischer & Hilfiker-Kleiner, 2007; Boengler et al., 2008;
Fischer & Hilfiker-Kleiner, 2008).
3.1.3. Thyroid hormone receptor signaling
Thyroid hormone is a classic hormonal mediator of normal
postnatal heart growth. The thyroid gland secretes two biologically
active hormones: thyroxine (T4, prohormone) and triiodothyronine
(T3). T4 and T3 diffuse across the plasma membrane and T4 is
converted to T3 (Danzi & Klein 2002; Dillmann, 2002). Postnatal heart
growth was reduced in a setting of depressed thyroid gland activity,
whereas administration of excess thyroid hormone to animals led to
an increase in heart weight (Bedotto et al., 1989; Hudlicka & Brown
1996). Furthermore, administration of T4 in humans was associated
with increased heart mass but no fall in systolic function (Ching et al.,
1996), and patients with chronic hyperthyroidism have increased
cardiac contractility which is often associated with cardiac hypertrophy (Forfar et al., 1982; Feldman et al., 1986). Thus, it has been
suggested that thyroid hormone induces physiological heart growth.
The biological effects of thyroid hormone have largely been
attributed to nuclear transcriptional mechanisms. T3 passes through
the nuclear membrane to bind to nuclear thyroid hormone receptors
(TRs), which act as transcription factors to directly repress or activate
cardiac genes (Lazar & Chin 1990; Lazar 1993; Mangelsdorf et al.,
1995; Danzi & Klein, 2002; Dillmann, 2002; Harvey & Williams, 2002).
Thyroid hormone has been shown to regulate α- and β-MHC, cardiac
troponin, SERCA2a, and voltage gated potassium channels (Izumo et
al., 1986; Rohrer & Dillmann, 1988; Nishiyama et al., 1998; Danzi &
Klein, 2002). In mammals TRs are encoded by two genes: TRα and TRβ
(Lazar, 1993; Harvey & Williams, 2002). Thyroid hormone treatment
increased heart mass by approximately 64% in wildtype mice, 44% in
TRα knockout mice but only 6% in TRβ knockout mice, suggesting that
TRβ plays a predominant role in regulating heart growth (Weiss et al.,
2002).
More recently, cytosolic and membrane-initiated effects of thyroid
hormones have been reported (Bassett et al., 2003; Farach-Carson &
Davis, 2003). Kenessey and Ojamaa demonstrated a direct interaction of
cytosol-localized TRα1 with the p85α regulatory subunit of PI3K in
neonatal rat ventricular myocytes (Kenessey & Ojamaa, 2006). This
interaction was shown to be critical for T3-induced protein synthesis.
The authors concluded that rapid T3 mediated activation of PI3K by
TRα1 may underlie the mechanisms via which thyroid hormone
induces physiological heart growth (Kenessey & Ojamaa, 2006) (Fig 5).
3.1.4. Heat shock transcription factor 1
Sakamoto and colleagues identified HSF1 as a promising critical
mediator of physiological cardiac hypertrophy from a genetic profiling
study that compared gene expression in hearts from rats subjected to
pressure overload with exercise trained rats (voluntary running
wheel). Gene expression of HSF1, which regulates heat shock proteins
(Hsp) including Hsp70 and Hsp27, was upregulated in hearts from
exercise trained rats but not in hearts subjected to pressure overload
(Sakamoto et al., 2006). To examine whether HSF1 had a protective
role in exercise-induced cardiac hypertrophy, HSF1-deficient heterozygote mice (HSF1+/−) were subjected to voluntary wheel running
for 4 weeks. Interestingly, exercise-induced hypertrophy was not
blunted in HSF1+/− but cardiac function was significantly reduced
(Sakamoto et al., 2006).
3.2. Signaling proteins/pathways
implicated in mediating pathological hypertrophy
3.2.1. G proteins
G proteins can be divided into two main subgroups: heterotrimeric
G proteins and small-molecular-weight monomeric G proteins (small
G proteins).
3.2.1.1. Heterotrimeric G proteins. Heterotrimeric G proteins consist of
three subunits (α, β and γ) and couple to GPCR. Binding of an agonist
to the GPCR leads to dissociation of the Gα and Gβγ subunits,
followed by activation of downstream signaling pathways (Gutkind,
1998a,b; Rockman et al., 2002). Isoforms of the heterotrimeric G
proteins are largely determined by the isoform of the α subunits,
which fall into four subfamilies: Gs, Gi, G12, and Gq (e.g. Gαq, Gα11)
(Simon et al., 1991; Neer, 1995).
In response to a pathological stimulus (e.g. pressure overload),
hormones/vasoactive factors such as Ang II, ET-1 and noradrenaline
(norepinephrine, NE) are released and induce cardiac growth
(Schunkert et al., 1990; Arai et al., 1995; Yamazaki et al., 1999;
Rapacciuolo et al., 2001; Yayama et al., 2004). These ligands bind to
GPCR: Ang II receptor type 1 (AT1 receptor), endothelin receptors (ETA
and ETB) and α1-adrenergic receptors (ARs), respectively. This causes
activation of Gαq/11 and downstream signaling proteins, including
phospholipase C (PLC), MAPKs, PKC and protein kinase A (PKA).
Transgenic mouse studies have highlighted the critical role of Gαq/11
in mediating pathological cardiac hypertrophy (Fig. 6). Cardiacspecific transgenic mice over-expressing Gαq developed cardiac
hypertrophy that was associated with cardiac dysfunction and
premature death (D'Angelo et al., 1997; Mende et al., 1998). In
contrast, mice lacking G proteins (Gαq/11) in cardiac myocytes and
cardiac-specific transgenic mice expressing a peptide specific for
inhibiting Gq-coupled receptor signaling displayed no hypertrophy or
a significantly blunted response to pressure overload (Akhter et al.,
1998; Wettschureck et al., 2001). Taken together, these studies
suggest that the Gαq/11 pathway is important for the induction of
pathological hypertrophy.
3.2.1.1.1. Angiotensin II receptors. Ang II is the principal vasoactive
substance of the renin–angiotensin system with a variety of
pathophysiological actions in the cardiovascular system via systemic
and local effects including vasoconstriction, aldosterone release, and
cell growth (Zimmerman & Dunham, 1997; de Gasparo et al., 2000).
Two pharmacologically distinct Ang II receptors have been cloned
(AT1 and AT2). Rodents have two AT1 receptor isoforms (AT1A and
AT1B) (de Gasparo et al., 1995; Lorell 1999). The heart contains a local
renin–angiotensin system which is activated in response to hemodynamic stress (e.g. pressure overload) (Yamazaki & Yazaki, 1997;
Lijnen & Petrov, 1999). It is well established that blocking Ang II
formation with angiotensin converting enzyme inhibitors (ACEI)
attenuates pressure overload-induced hypertrophy in animal models
and humans (Sadoshima et al., 1996; Zhu et al., 1997; Lijnen and
Petrov 1999; Yamazaki et al., 1999; Devereux 2000; Modesti et al.,
2000).
Hypertrophy due to activation of the AT1 receptor in rodent cardiac
neonatal myocytes or mouse models has been associated with
activation of MAPKs, increased intracellular calcium, PKC, and
transactivation of the epidermal growth factor receptor (EGFR)
(Sadoshima & Izumo, 1993; Miyata & Haneda, 1994; Sadoshima et
al., 1995; Kagiyama et al., 2002; Thomas et al., 2002; Chan et al., 2006).
Under basal conditions, single global Ang II receptor knockout mice
(AT1A, AT1B, AT2) have been reported to have no cardiac phenotype or
a small decrease in heart mass (Hein et al., 1995; Ichiki et al., 1995;
Hamawaki et al., 1998; Harada et al., 1998; Oliverio et al., 1998;
B.C. Bernardo et al. / Pharmacology & Therapeutics 128 (2010) 191–227
Gembardt et al., 2008). Interestingly, double knockouts lacking AT1A
and AT1B had smaller hearts than AT1A alone, whereas the reduction
was less pronounced in triple knockout mice lacking AT1A, AT1B and
AT2 (Gembardt et al., 2008). Cardiac-specific AT1 transgenic mice
developed pathological hypertrophy and died prematurely of heart
failure (Paradis et al., 2000). Unexpectedly, pressure overloadinduced hypertrophy was not blunted in AT1A knockout mice
(Hamawaki et al., 1998; Harada et al., 1998), possibly suggesting
that AT1B is able to compensate for loss of AT1A. Mice with a gain of
function mutated AT1A receptor did not display cardiac hypertrophy
but developed progressive cardiac fibrosis and diastolic function
abnormalities (Billet et al., 2007). Though, this study is confounded by
a modest increase in blood pressure in the mutant mice. Cardiacspecific AT2 transgenic mice had no cardiac phenotype (Masaki et al.,
1998), but pressure overload-induced hypertrophy was inhibited in
AT2 knockout mice (Senbonmatsu et al., 2000). While it is reasonably
clear that Ang II is critical for mediating cardiac hypertrophy, the
precise role of Ang II receptor subtypes requires further examination
(see Billet et al., 2008)). In addition, recent studies have demonstrated
the cardiovascular effects of a number of breakdown products of Ang
II including Ang 1–7, Ang III and Ang IV; as well as new mechanisms
concerning the functional regulation of Ang II receptors such as
receptor dimerization, ligand-independent activation, receptor-interacting proteins, and the existence of an agonistic antibody against the
AT1 receptor (Jones et al., 2008; Mogi et al., 2009). The role of these
peptides and new regulation of Ang II receptors in relation to cardiac
hypertrophy also requires further investigation.
3.2.1.1.2. Endothelin-1 receptors. ET-1 is the predominant endothelin in the heart and is a potent hypertrophic stimulus in neonatal
cardiac myocytes (Shubeita et al., 1990). ET-1 binds to two GPCRs: ETA
and ETB. ETA receptors account for 90% of endothelin receptors on
cardiac myocytes (Kedzierski & Yanagisawa, 2001). Ang II increased
ET-1 levels in rat cultured cardiac myocytes, and an ETA antagonist
(BQ123) reduced Ang II-induced myocyte hypertrophy (Ito et al.,
1993). ETA receptor antagonists (alone or in combination with ETB
receptor antagonists) have been shown to attenuate pathological
hypertrophic responses in animal models (see Brunner et al., 2006).
Though, cardiac myocyte specific ETA knockout mice displayed a
normal hypertrophic response to Ang II (Kedzierski et al., 2003).
A number of clinical trials have assessed the potential of ET-1
antagonists to treat heart failure patients; results have largely been
disappointing, and/or remain unpublished (reviewed by (Seed et al.,
2001; Rich & McLaughlin, 2003; Kelland & Webb, 2006; Kirkby et al.,
2008)). In the majority of clinical trials using ET-1 antagonists [either
non-selective for ETA or ETB (e.g. bosentan, tezosentan) or selective for
ETA (e.g. darusentan)] heart failure patients developed adverse side
effects without improvement in cardiac remodeling or clinical
symptoms (Louis et al., 2001; Kalra et al., 2002; Luscher et al., 2002;
Rich & McLaughlin, 2003; Anand et al., 2004; Packer et al., 2005;
Kaluski et al., 2008).
3.2.1.1.3. Adrenergic receptors (ARs). Catecholamines activate ARs
(members of the GPCR superfamily) (Scheuer 1999; Lomasney &
Allen, 2001; Rockman et al., 2002). There are three major AR
subfamilies: α1-AR, α2-AR, and β-AR. At least 6 types of ARs are
present in the mammalian heart (three α1-ARs: α1A, α1B, α1D and
three β-ARs: β1, β2, β3), with β1-ARs predominating, accounting for
approximately 80% of the β-ARs in the healthy heart (Xiang & Kobilka,
2003; Barki-Harrington et al., 2004; Salazar et al., 2007; Woodcock et
al., 2008). ARs are coupled to Gαq, Gαs, and Gαi, leading to
modulation of adenylate cyclase, PLC and ion channels (Rockman et
al., 2002). Specifically, α1A, α1B, and α1D-ARs activate Gαq signaling,
β1-ARs couple to Gαs, and β2-ARs couple to Gαs and Gαi (Exton, 1985;
Garcia-Sainz et al., 1999; Rockman et al., 2002). Species-dependent
differences exist between the functions of β1- and β2-AR subtypes in
the heart which may be attributed to differential coupling to Gαs and
Gαi (see Kaumann et al., 1999). In the human heart, β2-ARs appear to
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largely couple to the Gαs/cAMP pathway (Kaumann et al., 1999). The
role of β3-ARs in the heart remains unclear (see Barki-Harrington et
al., 2004; Kaumann & Molenaar, 2008).
Heart failure patients have elevated circulating catecholamines
and increased adrenergic drive, which initially increases contractility
and may be beneficial. However, prolonged adrenergic drive is
detrimental and associated with desensitization and downregulation
of β-ARs (Bristow, 2000). This is consistent with findings from
cardiac-specific transgenic mice over-expressing β1-ARs. Before
15 weeks of age, transgenic mice displayed increased cardiac
contractility compared to controls. However, cardiac function progressively fell in β1-AR transgenic mice after 16 weeks and the mice
rapidly developed cardiac dysfunction and heart failure (Engelhardt
et al., 1999). Progressive deterioration in cardiac function with
chronic transgenic expression of β1-AR was later confirmed by
another report (Bisognano et al., 2000).
Cardiac-specific transgenic mice expressing a dominant negative
β-AR receptor kinase 1 (β-ARK1/GRK2; dominant negative mutant
restores β-AR signaling; β-ARK1 phosphorylates β-ARs leading to their
desensitization) were protected against pathological hypertrophy and
heart failure (Koch et al., 1995), and targeted deletion of GRK2 in
cardiac myocytes of mice prevented and rescued heart failure induced
by myocardial infarction (Raake et al., 2008). In this respect, it has been
difficult to explain why β-AR agonists are poorly tolerated in heart
failure patients but β-blockers have a protective role (Bristow, 2003;
Molenaar & Parsonage, 2005, discussed in further detail in Section 8.1).
Though it has been suggested that at the molecular level, inhibition of
β-ARK1/GRK2 shares a number of properties with β-blockade as
opposed to β-AR agonism (Rockman et al., 2002). For instance, β-AR
agonists promote desensitization and receptor downregulation due to
constant activation of the β-AR system. In contrast, inhibition of βARK1 may allow β-ARs to return to a more normal state of signaling
because desensitization will be inhibited (Rockman et al., 2002).
While it is generally accepted that chronic stimulation of the β-AR
system has an adverse effect on the heart that contributes to the
pathogenesis of heart failure, preservation of normal β-AR-G protein
coupling is critical during times of need, such as periods of stress and
during exercise (Christensen & Galbo, 1983; Lefkowitz et al., 2000;
Rockman et al., 2002). In this instance, acute versus chronic activation
of β-ARs may explain differences in phenotype observed with
pathological and physiological hypertrophy. Plasma resting levels of
catecholamines were significantly higher in a mouse model of
pathological hypertrophy induced by chronic pressure overload in
comparison to a mouse model of physiological hypertrophy induced
by swim training (Perrino et al., 2006).
The role of AR subtypes in mediating cardiac hypertrophy based on
genetically modified mouse models has been described in detail (see
Du, 2008). Global knockout mice deficient of β1 and β2-ARs displayed
an attenuated hypertrophic response to pressure overload, with
reduced fibrosis (Kiriazis et al., 2008). Based on various transgenic and
knockout models, α1B-AR and β2-AR appear to contribute to
maladaptation and the onset of heart failure in a setting of pressure
overload, whereas activation of α1A-AR may be beneficial (Du, 2008).
Of note, in rat neonatal cardiac myocytes, it was shown that β2-ARs
that couple with Gi proteins mediate cardiac protection due to
activation of the PI3K-Akt pathway (Chesley et al., 2000). Furthermore, α1-ARs were critical for normal postnatal heart growth in male
but not female mice (O'Connell et al., 2003), and protected the heart
against pressure overload-induced maladaptive hypertrophy (O'Connell et al., 2006).
3.2.1.2. Small G proteins (also called GTPases). The family of small G
proteins can be divided into 5 subfamilies (Ras, Rho, ADP ribosylation
factors, Rab, and Ran). Small G proteins act as molecular switches,
which link receptors to downstream signaling cascades. Ras and Rho
can be activated in myocytes in response to Ang II, ET-1,
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phenylephrine (PE) and mechanical stress and have been implicated
in the development of cardiac hypertrophy (Ramirez et al., 1997a;
Aoki et al., 1998; Aikawa et al., 1999; Chiloeches et al., 1999; Clerk and
Sugden, 2000; Clerk et al., 2001). Cardiac-specific transgenic mice
expressing a constitutively active form of Ras or over-expressing
Rab1a developed pathological cardiac hypertrophy (Hunter et al.,
1995; Wu et al., 2001). Furthermore, using a Rho-kinase inhibitor
(Fasudil), it was shown that Rho-kinase was critical for pressure
overload-induced pathological hypertrophy in rats but not swimming-induced physiological cardiac hypertrophy (Balakumar & Singh,
2006).
3.2.2. PI3K(p110γ) signaling
In contrast to the p110α isoform of PI3K (coupled to RTKs e.g.
IGF1R), PI3K(p110γ) is coupled to GPCRs (specific GPCR not all
definitively defined but thought to include Gαs/Gαi/Gαq e.g. adrenergic receptors, Ang II receptors and endothelin receptors) and
appears to have a detrimental effect in the heart (Oudit et al., 2004).
PI3K(p110γ) does not affect heart size under basal conditions but is a
negative regulator of cardiac contractility, as PI3K(p110γ) knockout
mice displayed enhanced contractile function (Crackower et al.,
2002). PI3K(p110γ) may have an impact on heart growth in settings
of pathological stress (Naga Prasad et al., 2000; Oudit et al., 2003),
although the role of PI3K(p110γ) in the diseased heart is complex and
appears to differ depending on the nature of the pathological
stimulus. PI3K(p110γ) knockout mice were protected from heart
failure induced by chronic activation of β-ARs, displaying less
hypertrophy and fibrosis, and better cardiac function than controls
(Oudit et al., 2003). However, PI3K(p110γ) knockout mice displayed
an accelerated progression to dilated cardiomyopathy in response to
pressure overload (Patrucco et al., 2004; Oudit & Kassiri, 2007). PI3K
(p110γ) might contribute to cardiac dysfunction via its effects on βAR internalization and regulation of phosphodiesterases (Fig. 5)
(Oudit & Kassiri, 2007; Pretorius et al., 2009b). Downregulation and
desensitization of β-ARs is detrimental for heart function (Bristow et
al., 1982; Perrino et al., 2007), and is dependent on the binding of
p110γ to β-ARK1 (Naga Prasad et al., 2001). Expression of a
catalytically inactive p110γ mutant or disruption of the interaction
between β-ARK1 and p110γ restored β-AR signaling and contractile
function in transgenic mice subjected to chronic β-AR stimulation
(Nienaber et al., 2003; Perrino et al., 2005; Perrino et al., 2007). PI3K
(p110γ) may also reduce cardiomyocyte contractility by regulating
the activity of phosphodiesterases (PDEs) (Patrucco et al., 2004;
Kerfant et al., 2007). PDEs hydrolyse cAMP, a second messenger which
plays a critical role in mediating Ca2+ release from the sarcoplasmic
reticulum to induce contraction. PDE inhibitors improve contractile
function by increasing intracellular cAMP levels, however the safety of
PDE inhibitors as therapeutic agents in patients with heart failure is
still being investigated (see Osadchii, 2007 for review).
3.2.3. Mitogen activated protein kinase (MAPK) pathways
MAPKs are divided into 3 subfamilies based on the terminal kinase
in the pathway: the extracellular signal-regulated kinases (ERKs), the
c-Jun amino-terminal kinase (JNKs), and the p38-MAPKs (Clerk &
Sugden, 1999; Widmann et al., 1999; Pearson et al., 2001). All three
types of MAPKs are activated in cultured cardiac myocytes in response
to GPCR agonists (couple to Gαq: AT1 receptors, endothelin receptors
and α1-ARs) and mechanical stress, as well as in pressure overloaded
hearts and failing human hearts, but the exact role of MAPKs has
remained unclear (Yamazaki et al., 1993; Sadoshima et al., 1995;
Komuro et al., 1996; Sugden & Clerk, 1998; Cook et al., 1999; Esposito
et al., 2001; Pearson et al., 2001; Takeishi et al., 2001; Purcell et al.,
2007).
3.2.3.1. ERK1/2. ERKs are protein kinases that phosphorylate a range of
cytosolic and nuclear substrates (see Chen et al., 2001b for review).
ERK1/2 are ubiquitously expressed (Boulton et al., 1991) and
activation has been reported in numerous settings of cardiac
hypertrophy and failure (see Muslin, 2008) however it is still unclear
whether ERK1/2 is a critical mediator of hypertrophic responses.
ERK1/2 was activated in response to agonists that induce pathological
heart growth, such as Ang II, ET-1 and NE, but not in response to the
physiological hypertrophic agonist IGF1 (Clerk et al., 2006). In isolated
cardiac myocytes, activation of ERK1/2 was essential for protein
synthesis (a requirement for cell growth) following stimulation with
hypertrophic agonists that signal via Gq protein coupled receptors
(Wang & Proud, 2002). Consistent with this finding, expression of a
dominant negative mutant of Raf-1 (a MAPK kinase kinase downstream of Gαq; Fig. 5) blunted cardiac hypertrophy in mice subjected
to pressure overload, implicating ERK1/2 in the development of
pathological cardiac hypertrophy (Harris et al., 2004). However,
transgenic mice expressing cardiac-specific constitutively active
MAPK kinase 1 (MEK1; a MAPK kinase immediately upstream of
ERK1/2; does not activate JNK or p38-MAPK) developed a physiological rather than a pathological phenotype, which was characterized by
concentric cardiac hypertrophy, enhanced systolic cardiac function
and no interstitial fibrosis (Bueno et al., 2000). Furthermore, loss of
ERK1 (global knockout mice) had no effect on heart size in mice
subjected to pressure overload or swim training, indicating that ERK1
is not a critical mediator of pathological or physiological hypertrophy,
or that the remaining ERK2 activity was sufficient to drive the
hypertrophic response (Purcell et al., 2007). Reduced expression of
ERK2 (ERK2+/− mice; ERK2−/− mice are embryonically lethal) alone
or in mice deficient for ERK1 (ERK1−/−ERK2+/− mice) also failed to
block hypertrophy induced by pressure overload or swim training
(Purcell et al., 2007). Together, these studies suggest that activation of
ERK1/2 is sufficient, but not critical, for inducing cardiac hypertrophy,
although the results of the latter study were confounded by the fact
that the ERK1−/− and ERK2+/− mice were not cardiac-specific.
Fig. 7. ERK1/2 appears to contribute to hypertrophic responses via two distinct
mechanisms. Stimulation of GPCRs leads to dissociation of Gq proteins. A) The Gα
subunit of Gq activates traditional MAP kinase signaling cascades, resulting in
phosphorylation and activation of ERK1/2 by MEK1/2. This in turn leads to protein
synthesis and cell growth. B) Association of Gβγ subunits with the Raf1/MEK/ERK1/2
complex is necessary for autophosphorylation of ERK1/2 at residue Thr188 and
localization of ERK1/2 in the nucleus. This leads to phosphorylation of nuclear targets
(such as Elk1, MSK1 and c-Myc) and transcription of hypertrophic genes.
B.C. Bernardo et al. / Pharmacology & Therapeutics 128 (2010) 191–227
Induction of ERK1/2 kinase activity requires phosphorylation of
the threonine and tyrosine residues within the TEY motif of the
activation loop by MEK1/2 (Anderson et al., 1990; Payne et al., 1991;
Robbins et al., 1993). A recent study reported that autophosphorylation of ERK2 at residue Thr188 promoted nuclear localization and
subsequent phosphorylation of hypertrophic factors, including Elk1,
MSK1 and c-Myc (Lorenz et al., 2009). This occurred following
association of the Raf/MEK/ERK complex with the βγ subunits of Gq
proteins (Lorenz et al., 2009). Thr188 phosphorylation appears to be a
key event in the development of ERK1/2-mediated cardiac hypertrophy, as transgenic mice with suppressed ERK2 Thr188 phosphorylation were resistant to cardiac hypertrophy induced by pressure
overload, while mice with enhanced ERK2 Thr188 phosphorylation
displayed more pronounced hypertrophy compared to wildtype mice
(Lorenz et al., 2009). Phosphorylation of Thr188 was also evident in
biopsies from human failing hearts, suggesting that ERK2 autophosphorylation of Thr188 is clinically relevant (Lorenz et al., 2009). Thus,
ERK1/2 appears to contribute to cardiac hypertrophic responses via
two distinct mechanisms (Fig. 7). Activation of the traditional MAPK
signaling cascade (Raf/MEK/ERK) following binding of Gαq proteins to
GPCRs results in phosphorylation of ERK1/2 within the TEY motif and
induction of ERK1/2 kinase activity. Subsequent phosphorylation of
substrates (such as p90 ribosomal S6 kinase (Lorenz et al., 2009)) may
contribute to cell hypertrophy by increasing protein synthesis. This
mechanism may be responsible for driving the physiological hypertrophy observed in the MEK1 transgenic mice. Secondly, interaction of
the Raf/MEK/ERK complex with Gβγ proteins causes autophosphorylation of ERK2 at Thr188. This results in nuclear localization, allowing
ERK1/2 to phosphorylate nuclear targets, which in turn promotes
transcription of hypertrophic genes. This event may be critical for
inducing the maladaptive phenotype associated with pathological
hypertrophic responses (Fig 7.)
3.2.3.2. ERK5. ERK5 (also known as big MAP kinase 1, BMK1) may play
a role in mediating pathological eccentric cardiac hypertrophy, as
transgenic mice expressing constitutively active MEK5 developed
eccentric cardiac hypertrophy which progressed to dilated cardiomyopathy and death (Nicol et al., 2001).
203
3.2.3.3. JNKs. The JNK family consists of at least ten isoforms, derived
from three genes: JNK1, JNK2 and JNK3 (Waetzig & Herdegen, 2005).
JNK1 and JNK2 are ubiquitously expressed, whereas JNK3 has a more
restricted expression profile in the heart, brain and testis (Waetzig &
Herdegen, 2005). JNK was activated in hearts from heart failure
patients (Cook et al., 1999) and in the remote myocardium of
infarcted rat hearts (Li et al., 1998). A number of in vitro studies have
suggested that JNKs may be important regulators of pathological
hypertrophy (Bogoyevitch et al., 1996; Ramirez et al., 1997a;
Choukroun et al., 1998; Wang et al., 1998b; Choukroun et al., 1999),
although in vivo studies have been more difficult to interpret
(described below and summarized in Table 2).
JNK is phosphorylated and activated by MAPK kinase 4 (MEK4) and
MEK7 (Fig. 5), and preferentially upregulated by MAPK kinase kinase 1
(MEKK1) (Fig 5). Transgenic mice with cardiac-specific activation of JNK
(via constitutive activation of MEK7) did not develop cardiac hypertrophy but died prematurely from congestive heart failure (Petrich et al.,
2003). Several loss of function approaches have also been utilized to
determine the role of JNKs in pathological cardiac hypertrophy. Pressure
overload-induced hypertrophy was attenuated in dnMEK4 transgenic
mice (Choukroun et al., 1999), and MEKK1 was essential for pathological
cardiac hypertrophy and dysfunction induced by cardiac-specific
transgenic expression of Gαq (Minamino et al., 2002). These data
suggest JNKs may be necessary regulators of pathological cardiac
hypertrophy. However, dnJNK1/2 transgenic mice and JNK1/2 genetargeted mice displayed an enhanced hypertrophic response to pressure
overload (Liang et al., 2003), suggesting JNKs antagonize cardiac
growth. Furthermore, cardiac-specific MEK4 knockout mice with
reduced JNK activity displayed normal cardiac growth and function
under basal conditions (Liu et al., 2009). However, following aorticbanding and chronic β-adrenergic stimulation, unlike the dnMEK4
transgenic mice, cardiac specific MEK4 knockout mice had enhanced
cardiac growth, increased hypertrophic gene transcription and ventricular fibrosis compared to wildtype aortic-banded controls. Following
swim training, the hypertrophic response was unchanged compared to
wildtype controls in this model (Liu et al., 2009). Null MEKK1 transgenic
mice had comparable heart weights to wildtype mice under basal
conditions. In response to aortic-banding, MEKK1−/− mice displayed an
enhanced hypertrophic response rather than a blunted response after
Table 2
JNK mouse models and their phenotype under basal conditions and in response to pressure overload as assessed by heart weight/body weight or left ventricular/body weight ratios.
Mouse model
caMEK7
(cardiac-specific)
dnMEK4
(cardiac-specific by adenovirus-mediated gene transfer)
dnJNK1/2
(cardiac-specific)
Jnk1+/−/Jnk2−/−
(targeted deletion, global)
MEK4−/−
(cardiac-specific)
MEKK1−/−
(targeted deletion, global)
Jnk1−/−
(targeted deletion, global)
Jnk2−/−
(targeted deletion, global)
Jnk3−/−
(targeted deletion, global)
Basal phenotype
Pressure overload
Versus Ntg/WT
Hypertrophic response versus Ntg/WT
↔
n/a
Petrich et al., 2004
n/a
↓
Choukroun et al., 1999
↑
↑
Liang et al., 2003
↑
↑
Liang et al., 2003
↔
↑
Liu et al., 2009
↔
↑
Sadoshima et al., 2002
↔
↔
Tachibana et al., 2006
↔
↔
Tachibana et al., 2006
↔
↔
Tachibana et al., 2006
Key:
↔ = JNK has no effect on heart growth, possibly due to redundancy of isoforms.
↑ = Indicates JNK antagonizes pathological growth.
↓ = JNK is essential for pathological growth.
n/a = not examined/assessed.
Reference
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Table 3
p38 mouse models and their phenotype under basal conditions and in response to pressure overload as assessed by heart weight/body weight or left ventricular/body weight ratios.
Mouse model
dn-p38α
(cardiac-specific, Black Swiss background)
dn-p38α
(cardiac-specific, FVB/N background)
dn-p38α
(cardiac-specific females compared to males)
p38α CKO
(cardiac-specific KO)
dn-p38β
(cardiac-specific)
dnMEK3
(cardiac-specific)
dnMEK6
(cardiac-specific)
MEK3
(cardiac-specific)
MEK6
(cardiac-specific)
Basal phenotype
Pressure overload
Versus Ntg/WT
Hypertrophic response versus Ntg/WT
Reference
↔
↔
Zhang et al., 2003a
↑
↑
Braz et al., 2003
↔
↑
Liu et al., 2006
↔
↔
Nishida et al., 2004
↔
↔
Zhang et al., 2003a
↑
↑
Braz et al., 2003
↑
↑
Braz et al., 2003
↔
n/a
Liao et al., 2001
↔
n/a
Liao et al., 2001
Key:
↔ = p38 has no effect on heart growth.
↑ = p38 is essential for pathological hypertrophy.
n/a = not examined/assessed.
14 days compared to wildtype controls (Sadoshima et al., 2002).
Furthermore, aortic-banded MEKK1−/− mice had a higher mortality
rate and congestive heart failure compared to wildtype banded mice.
This study implies that cardiac hypertrophy induced by pressure
overload occurs in the absence of JNK activation. In further support of
the latter, mice with selective deletion of the three JNK genes (Jnk1−/−,
Jnk2−/− and Jnk3−/−) subjected to aortic-banding developed cardiac
hypertrophy that was comparable to wildtype mice (Tachibana et al.,
2006). Thus, it appears individual members of the JNK family are not
required to induce cardiac growth, or that the JNK isoforms are
functionally redundant.
3.2.3.4. p38-MAPK. p38-MAPK is an important mediator of numerous
biological functions including cell growth, cell proliferation, cell cycle
and cell death, and is considered a critical component of stress
response pathways (Wilson et al., 1996; Bassi et al., 2008). In the
heart, p38-MAPK is known to be activated during ischemia, and p38MAPK activity was increased in the myocardium from patients with
ischemic heart disease (Cook et al., 1999). p38-MAPK has been
implicated in the regulation of cardiac gene expression, cardiac
myocyte apoptosis, myocyte hypertrophy, contractility, remodeling
and metabolism (Liao et al., 2001; Petrich & Wang, 2004; Baines &
Molkentin, 2005; Wang, 2007).
The p38-MAPK family consists of four isoforms (α, β, δ and γ),
though it appears only α and β are expressed in the heart (Jiang et al.,
1997; Clark et al., 2007). The p38α isoform is predominately
expressed in the human and rodent myocardium (Lemke et al.,
2001; Braz et al., 2003). The role of p38α and p38β in the heart has
been examined extensively using both transgenic and knockout
mouse models. As with JNK, many of the studies appear to be
contradictory (summarized in Table 3). Cardiac-specific transgenic
mice expressing a dominant negative form of p38α were generated
and analyzed by two independent groups (Braz et al., 2003; Zhang et
al., 2003a). Zhang et al. (2003a) reported no basal phenotype in dnp38α transgenics. In contrast, Braz et al. (2003) found that dn-p38α
transgenics developed concentric hypertrophy associated with a fall
in cardiac function and elevated fetal gene expression (ANP and BNP),
with the majority of mice dying prematurely from cardiomyopathy by
8 months of age. Both groups subjected the dn-p38α transgenics to
pressure overload and reported a significant increase in heart size.
Zhang et al. reported a hypertrophic response (to pressure overload)
in the dn-p38α transgenics similar to control animals but, interestingly, the transgenics had significantly less fibrosis (Zhang et al.,
2003a). In contrast, Braz et al. (2003) reported an exaggerated
hypertrophic response in dn-p38α transgenics compared with
controls in response to pressure overload or 14 day minipump
infusions of phenylephrine, Ang II and isoproterenol. Finally,
cardiac-specific p38α knockout mice displayed normal cardiac
structure and function under basal conditions (Nishida et al., 2004).
In response to pressure overload, knockout mice developed a similar
degree of cardiac hypertrophy to controls, but displayed greater
cardiac dysfunction, more fibrosis and apoptosis (Nishida et al., 2004).
The authors concluded that p38α plays a critical role in protecting the
heart in a setting of pressure overload.
Cardiac-specific dn-p38β transgenic mice had no cardiac hypertrophy under basal conditions but seemed to have reduced systolic
function (Zhang et al., 2003a). The hypertrophic response to pressure
overload was not different from that observed in non-transgenic mice,
though the dn-p38β transgenics were reported to display less fibrosis
(Zhang et al., 2003a).
MEK3 and MEK6 are regulators of p38-MAPK (Fig 5). Under basal
conditions dnMEK3 and dnMEK6 transgenic mice developed pathological hypertrophy by 2 and 8 months of age, respectively. Both
models displayed cardiac dysfunction, and fibrosis was also reported
in dnMEK3 transgenics at 4 months of age. The majority of dnMEK3
mice died by 8 months of age due to cardiomyopathy (Braz et al.,
2003). In response to pressure overload, dnMEK3 and dnMEK6
transgenic mice displayed an exacerbated cardiac hypertrophic
response, increased fibrosis and depressed cardiac function, similar
to the observations in dn-p38α transgenic mice (Braz et al., 2003).
Activation of p38-MAPK in either MEK3 or MEK6 transgenic hearts
under baseline conditions led to the increased expression of the fetal
gene program, substantial induction of interstitial fibrosis, and loss of
contractility (Liao et al., 2001). Both transgenic mouse models developed
heart failure, although this was not associated with hypertrophy of
cardiac myocytes (Liao et al., 2001). In this set of studies, p38-MAPK does
not appear to promote hypertrophy, but seems to contribute to fibrosis,
loss of contractility and the development of dilated cardiomyopathy.
Explanations for the possible discrepancies between many of these
models (see Table 3) may be attributed to the generation of transgenic
mice on different genetic backgrounds (Braz et al., 2003; Zhang et al.,
2003a), gender differences (Liu et al., 2006) (discussed in detail in
B.C. Bernardo et al. / Pharmacology & Therapeutics 128 (2010) 191–227
Section 7), p38-MAPK having an anti-apoptotic function that is
independent of kinase activity or a biphasic dose response curve to
p38α-MAPK (Muslin, 2008), as well as distinct roles of p38α and β that
may be differentially regulated by MEK3 and MEK6 (Wang et al., 1998a).
3.2.3.5. Protein kinases. Extracellular stimuli such as pressure overload
activate PKC and PKD via GPCRs to trigger hypertrophic responses
(Dorn & Force, 2005; Harrison et al., 2006) (Fig 5).
3.2.3.5.1. PKC. PKC is considered a critical signal transducer
downstream of Gq. There are at least 12 isoforms of PKC and at least
4 (α, β, δ, and ε) have been implicated in the induction of cardiac
hypertrophy (see Dorn & Force, 2005). PKCα and PKCβ are
conventional Ca2+-dependent isoforms whereas PKCε and PKCδ are
novel Ca2+-independent isoforms (Mackay & Mochly-Rosen, 2001;
Sabri & Steinberg, 2003). Mice null for PKCα, β, δ, or ε had no obvious
cardiac phenotype under basal conditions (see Dorn & Force, 2005),
however it has been suggested that the role of specific PKC isoforms
may be masked by compensatory signaling by other PKC isoforms
(Dorn & Force, 2005). Cardiac-specific PKCβ transgenic mice developed cardiac hypertrophy associated with cardiac dysfunction,
fibrosis and premature death (Bowman et al., 1997; Wakasaki et al.,
1997; Chen et al., 2001a), but knockout mice displayed a typical
hypertrophic response to a GPCR agonist (PE) or aortic-banding
(Roman et al., 2001). Thus, it appears that PKCβ is not required for the
pathological hypertrophic response. Cardiac-specific transgenic mice
over-expressing PKCε or PKCδ displayed mild concentric hypertrophy
with a physiological phenotype (no evidence of fibrosis, and normal
cardiac function) (Takeishi et al., 2000; Chen et al., 2001a). However,
in response to a cardiac insult (ischemia-induced damage), PKCε had
a protective role, whereas activation of PKCδ exacerbated the damage
(Chen et al., 2001a). PKCα appears to be critical for regulating cardiac
contractility but not cardiac hypertrophy (Braz et al., 2002; Hahn et
al., 2003; Braz et al., 2004). Transgenic mice with over-expression of
PKCα had diminished cardiac contractility while PKCα−/− mice had
improved cardiac contractility (Braz et al., 2004). Furthermore,
inhibition of PKCα activity in a model of pathological hypertrophy
(Gαq transgenic mice) improved cardiac contractility, whereas
activation of PKCα resulted in a lethal cardiomyopathy (Hahn et al.,
2003). There were no apparent effects on heart size under basal
conditions or in response to cardiac stress (e.g. pressure overload)
with the PKCα mouse models.
3.2.3.5.2. PKD. Cardiac-specific transgenic mice expressing a
constitutively active form of PKD1 developed pathological hypertrophy and died prematurely (Harrison et al., 2006). In contrast, mice
with conditional cardiac-specific deletion of PKD1 had no phenotype
under basal conditions but displayed a blunted hypertrophic response
to various pathological models (pressure overload, Ang II-dependent
hypertrophy and isoproterenol-dependent hypertrophy) which was
associated with better cardiac function, less fibrosis, and less fetal
gene activation compared to control mice (Fielitz et al., 2008).
3.2.4. Calcium signaling
Each heartbeat is associated with entry of calcium into cardiac
myocytes. Calcium is central to the control of contractile function and
cardiac growth. Calcium/calmodulin is an important second messenger for GPCR agonists and biomechanical stress (Frey et al., 2000; Aoki
& Izumo, 2001; Sugden, 2001). The best described calcium-dependent
signaling proteins include calcineurin and calcium/calmodulin-dependent protein kinases (CaMKs). Calcineurin is a serine–threonine
phosphatase that consists of a catalytic A subunit and a regulatory B
subunit. Two regulatory subunit genes (B1, B2) have been identified,
and three genes encode the A catalytic subunit, calcineurinAα (CnAα),
calcineurinAβ (CnAβ) and calcineurinAγ (CnAγ). Only the α and β
genes have been shown to be expressed in human, mouse and rat
hearts (Klee et al., 1998; Molkentin & Dorn, 2001). Calcineurin
dephosphorylates nuclear factor of activated T cells (NFAT) transcrip-
205
tion factors which promotes nuclear translocation and activation of
gene transcription (Fig. 5).
Following stimulation with GPCR hypertrophic agonists (Ang II
and PE) in cultured rat neonatal cardiac myocytes, calcineurin
enzymatic activity, CnAβ (but not CnAα or CnAγ) mRNA and protein
levels were increased (Taigen et al., 2000). Calcineurin activity was
also increased in hypertrophied and failing hearts from human
patients (Haq et al., 2001), and human failing heart ventricular
muscle exposed to ET-1, Ang II and urotensin II (Li et al., 2005).
Furthermore, calcineurin activity was upregulated in hypertrophied
hearts following aortic-banding in rodents (Shimoyama et al., 1999;
Lim et al., 2000; De Windt et al., 2001; Zou et al., 2001; Saito et al.,
2003). Finally, transgenic mice expressing an activated form of
calcineurin in the heart developed profound cardiac hypertrophy
which rapidly progressed to dilated cardiomyopathy, with extensive
interstitial fibrosis, congestive heart failure and sudden death, often
by 3 months of age (Molkentin et al., 1998). Taken together, these
studies imply that elevated calcineurin induces pathological cardiac
hypertrophy.
Consistent with the idea that calcineurin/NFAT coupling induces
pathological cardiac growth, when NFAT-luciferase reporter mice
were subjected to both physiological stimuli (exercise training,
growth hormone-IGF1 infusion) and pathological stimuli (pressure
overload, myocardial infarction), NFAT luciferase reporter activity was
upregulated in both pathological models but not in the physiological
models (Wilkins et al., 2004). In addition, transgenic mice with
targeted inactivation of calcineurin Aβ displayed an impaired
hypertrophic response to pressure overload and infusion of GPCR
agonists (Bueno et al., 2002), and cardiac hypertrophy in transgenic
mice expressing a dominant negative form of calcineurin A displayed
a blunted hypertrophic response to pressure overload compared to
wildtype mice (Zou et al., 2001). Finally, pharmacological inhibition of
calcineurin activity prevented cardiac hypertrophy in constitutively
active calcineurin A transgenic mice (Molkentin et al., 1998).
As noted previously, calcineurin is considered to regulate the
pathological hypertrophic response via dephosphorylation of NFAT
(Fig. 5)(Olson & Williams, 2000). NFAT translocates to the nucleus,
where it associates with other transcription factors such as GATA4 and
myocyte enhancer factor 2 (MEF2), to regulate the expression of
cardiac genes (Wilkins et al., 2002; Frey & Olson, 2003). In support of
this, Molkentin et al. (1998) showed that cardiac-specific transgenic
mice expressing a constitutively activate mutant form (nuclear
localized) of NFAT3 developed cardiac hypertrophy and heart failure,
whereas expression of the wildtype protein did not lead to
hypertrophy (Molkentin et al., 1998).
The contribution of calcineurin in mediating cardiac hypertrophy
has also been examined utilizing myocyte-enriched calcineurininteracting protein 1 (MCIP1) transgenic mice. MCIP is able to inhibit
calcineurin signaling by binding directly to the catalytic subunit
(CnA), which inactivates its ability to dephosphorylate NFAT and
MEF2 (Fig. 5). Forced over-expression of MCIP1 selectively in the
heart of transgenic mice was able to attenuate hypertrophy and
prevent progression to dilated cardiomyopathy in response to various
pathological stimuli, including aortic-banding, transgenic expression
of CnA, and the β-adrenergic receptor agonist isoproterenol (Rothermel et al., 2001; Hill et al., 2002). This is consistent with the idea that
enhanced calcineurin signaling mediates pathological growth.
Though, interestingly, transgenic expression of MCIP also inhibited
exercise-induced hypertrophy (voluntary running wheel) (Rothermel
et al., 2001). Studies utilizing MCIP1 null mice have also been
complicated to interpret. MCIP−/− mice had a normal cardiac
phenotype under basal conditions, displayed an exaggerated hypertrophic response to transgenic expression of CnA, but a blunted
hypertrophic response to pressure overload and adrenergic stimulation (isoproterenol) (Vega et al., 2003). The authors concluded that
MCIP1 may have a dual role in hypertrophic signaling, acting as a
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suppressor or activator, depending on the initiating stimulus (Vega et
al., 2003).
Calcium/calmodulin-dependent protein kinase II (CaMKII) is a
serine/threonine protein kinase that has been implicated in cardiac
hypertrophy and heart failure. Upregulation of CaMKII has been
reported in hearts of patients and animal models with heart failure
(Hoch et al., 1999; Kirchhefer et al., 1999; Bossuyt et al., 2008). Four
isoforms (α, β, δ and γ) of CaMKII exist, which are encoded by
separate genes. CaMKIIδ is the predominant isoform in the heart and
has distinct splice variants (δA, δB, δC); γ is also expressed in the heart
(Tobimatsu & Fujisawa, 1989; Edman & Schulman, 1994; Srinivasan et
al., 1994; Baltas et al., 1995; Mayer et al., 1995; Ramirez et al., 1997b).
Transgenic studies have provided evidence for the involvement of
CaMKII in pathological cardiac hypertrophy. Over-expression of
CaMKIIδB (nuclear isoform) in the mouse heart induced cardiac
hypertrophy and dilated cardiomyopathy (Zhang et al., 2002b).
Similarly, cardiac-specific transgenic mice over-expressing CaMKIIδC
(cytoplasmic isoform) developed cardiac hypertrophy (with mild
fibrosis), dilated cardiomyopathy and heart failure (lung congestion,
atrial dilation and severe edema) (Zhang et al., 2003b). Both CaMKIIδB
and CaMKIIδC transgenic expression induced a genetic program
associated with hypertrophy and heart failure (i.e., ANP, β-MHC and
α-skeletal actin mRNA were increased, α-MHC, SERCA2a and
phospholamban (PLB) were decreased). Further supporting this
role, a genetic mouse model of CaMKII inhibition prevented cardiac
dilation and dysfunction resulting from myocardial infarction and βAR stimulation (Zhang et al., 2005). Under basal conditions, these
transgenic mice had a stable cardiac phenotype (no hypertrophy),
normal cardiac development and function (Zhang et al., 2005). More
recently, CaMKIIδ-null mice (generated using a Cre-loxP approach)
were shown to be protected against hypertrophy and fibrosis in
response to pressure overload caused by aortic-constriction (Backs et
al., 2009). Fetal gene expression (ANP, BNP, β-MHC) was also
attenuated in this model. In contrast, germline ablation of CaMKIIδ
did not affect the development of hypertrophy following pressure
overload induced by two weeks of aortic constriction, which may be
explained by the upregulation of CaMKIIγ compensating for the loss of
CaMKIIδ (Ling et al., 2009). However, after long term aortic banding
(6 weeks), cardiac function was maintained, enlargement of the heart
was attenuated, pulmonary congestion and lung edema were
reduced, and survival rates improved in the knockout model
compared to wildtype mice, suggesting that CaMKIIδ deletion inhibits
the development of heart failure induced by long term pressure
overload (Ling et al., 2009).
CaMKII signaling is thought to exert its effect on cardiac hypertrophy
by causing phosphorylation of class II histone deacetylase 4 (HDAC4),
which in turn dissociates from the MEF2 transcription factor, and is
translocated from the nucleus to the cytoplasm. This causes activation of
MEF2 which is sufficient to promote pathological hypertrophy (Backs &
Olson, 2006; Backs et al., 2006; Kim et al., 2008a). In further support of
this role, CaMKIIδ-null mice demonstrated a clear reduction of HDAC4 in
ventricular lysates (Backs et al., 2009).
3.3. Complexities and future work in relation to signaling cascades
The most frequently used animal models of pathological hypertrophy (e.g. aortic-banding, hypertension) represent a chronic
pressure load that results in concentric hypertrophy. In contrast,
models of physiological hypertrophy (e.g. treadmill, voluntary freewheel, swimming) represent an intermittent volume load that results
in eccentric hypertrophy. Thus, it has been argued that differences in
phenotypes and signaling observed in models of pathological and
physiological hypertrophy may be a consequence of the duration of
the insult (constant versus intermittent) or type of load (volume
versus pressure). These issues have been addressed to some extent by
the following studies: i) mouse hearts subjected to intermittent
pressure overload displayed pathological features including diastolic
dysfunction, reduced capillary density, histological and cellular
abnormalities, and a fall in SERCA2a (Perrino et al., 2006); ii)
cardiac-specific chronic transgenic expression of IGF1R or caPI3K
was associated with a physiological phenotype that did not progress
to a pathological phenotype (Shioi et al., 2000; McMullen et al.,
2004b); iii) transgenic mice with enhanced ERK5 activation developed eccentric hypertrophy that progressed to dilated cardiomyopathy (Nicol et al., 2001), iv) a pathological model of eccentric
hypertrophy (myocardial infarction) and a physiological model of
eccentric hypertrophy (voluntary exercise wheel) in rats were
associated with differential regulation of signaling proteins (Gosselin
et al., 2006). Together these data suggest that it is not whether a
stimulus is chronic or intermittent, or whether the initiating
hypertrophic stimuli represent a pressure or volume overload that
determines whether the resultant cardiac hypertrophy is pathological
or physiological.
Further studies are required to determine whether postnatal
cardiac growth, pregnancy-induced growth, and exercise-induced
hypertrophy are mediated by similar molecular mechanisms. PI3K
(p110α) is critical for postnatal heart growth and exercise-induced
growth (Shioi et al., 2000; McMullen et al., 2003); whereas the Gαq
signaling pathway was critical for pathological growth but not
postnatal heart growth (Wettschureck et al., 2001). Signaling
cascades implicated in mediating pregnancy-induced heart growth
have not been extensively studied but may be coupled to Kv4.3, c-Src,
cGMP and estrogen receptors. Consistent with the classification of
pregnancy-induced growth as physiological, pregnancy does not
trigger changes in classic markers of pathological hypertrophy
including β-MHC or ANP (Eghbali et al., 2005; Eghbali et al., 2006).
Examination of signaling pathways that specifically induce and
differentiate between eccentric or concentric hypertrophy have not
been clearly defined. Patients and animal models with eccentric
hypertrophy have a particularly poor prognosis (Berenji et al., 2005).
An understanding of eccentric hypertrophy at the molecular level is
likely to provide important insight into why compensated hypertrophy can progress to heart failure. It will also be important to
differentiate between physiological eccentric hypertrophy and pathological eccentric hypertrophy. For instance, hypertrophy in response
to isotonic exercise is classified as eccentric but is quite different to
eccentric hypertrophy associated with decompensation of the heart.
Furthermore, pregnancy-induced hypertrophy due to volume overload is not associated with recapitulation of the fetal gene program
whereas a similar volume overload in response to aortocaval shunt
caused an increase in ANP and β-MHC gene expression (Sopontammarak et al., 2005; Eghbali et al., 2006). Gp130-mediated signals
contribute to the development of eccentric hypertrophy. Both CT-1
and LIF cause elongation of myocytes due to assembly of sarcomeric
units in series rather than in parallel (Wollert et al., 1996). But as
previously noted, activation of this pathway is considered protective.
In contrast, MEK5 induces eccentric hypertrophy and heart failure
with no report of intervening concentric hypertrophy (Nicol et al.,
2001). An intact sensing apparatus that can detect changes in
functional load appears essential in the compensatory response to
pathological insults. Mutations or deletions of sensor proteins have
been associated with dilated cardiomyopathy/eccentric hypertrophy
characterized by ventricular chamber dilation, fibrosis and heart
failure. Integrins (heterodimeric transmembrane receptors), link the
extracellular matrix to the intracellular cytoskeleton. Interacting
proteins/downstream effectors of integrins (e.g. melusin, focal
adhesion kinase (FAK), small GTPases) as well as proteins at the
level of the Z-disc within sarcomeres (e.g. muscle LIM protein) are
considered biomechanical stretch sensors. Control mice typically
develop concentric hypertrophy in response to pressure overload
(aortic banding) that may later progress to eccentric hypertrophy and
heart failure. In contrast, mice lacking stretch-sensor proteins (e.g. β-
B.C. Bernardo et al. / Pharmacology & Therapeutics 128 (2010) 191–227
integrin, melusin, FAK, and muscle LIM protein) developed eccentric
hypertrophy and cardiac dysfunction under basal conditions or in
response to pressure overload, i.e. no intermediate concentric
hypertrophy (Arber et al., 1997; Shai et al., 2002; Brancaccio et al.,
2003; Peng et al., 2006). On the other hand, melusin expression is
increased during compensated hypertrophy induced by pressure
overload, and over-expression of melusin in transgenic mice
prolonged concentric hypertrophy and protected against the transition to eccentric hypertrophy and failure (De Acetis et al., 2005).
4. Molecular mechanisms associated with structural
features of pathological and physiological hypertrophy
4.1. Molecular mechanisms associated with protein synthesis
An essential feature of both physiological and pathological cardiac
hypertrophy is increased protein synthesis. Ribosomal S6 kinases (S6Ks:
S6K1 and S6K2) are considered critical regulators of protein synthesis in
response to hypertrophic stimuli. Activation of S6K1 was increased in
hearts of transgenic mouse models of physiological hypertrophy (mice
with increased activation of the IGF1-PI3K(p110α) pathway) and
pathological hypertrophy (pressure overload) (Shioi et al., 2000; Shioi
et al., 2003; McMullen et al., 2004a,b). Rapamycin, an inhibitor of the
mammalian target of rapamycin (mTOR; a proximal effector of S6Ks)
inhibited S6K1 and attenuated and regressed pathological hypertrophy
induced by pressure overload (Shioi et al., 2003; McMullen et al.,
2004a). Unexpectedly, deletion of S6Ks (utilizing global S6K knockout
mice) did not attenuate cardiac hypertrophy induced by exercise
training, transgenic expression of IGF1R/caPI3K or pressure overload
(McMullen et al., 2004c). Together, these data suggest that S6Ks are not
essential for the induction of physiological or pathological cardiac
hypertrophy, or that other proteins were upregulated in the knockout
models.
4.2. Molecular mechanisms associated with sarcomeric organization
The formation of sarcomeres is a complex process involving the
synthesis of numerous proteins (Zak, 1984; Vigoreaux, 1994). These
proteins aggregate into filaments, which are organized into specific
three-dimensional arrays and aligned with other contractile elements
already present in the cardiac myocyte (Zak, 1984; Sanger et al., 2000).
Cardiac hypertrophy facilitates the increased workload (e.g. strain of
pressure or volume overload) by increasing contractile capacity.
Organization of sarcomeres, and thereby an increase in the contractile
units, is an essential component to maximize force generation. The
transition from pathological hypertrophy to heart failure has been
associated with loss of contractile filaments in the presence of
microtubule densification and desmin disorganization, and loss of
proteins of the sarcomeric skeleton (titin, α-actin, myomesin) (Tsutsui
et al., 1993; Hein et al., 2000; Hamdani et al., 2008). Defects or mutations
of sarcomeric proteins, including cardiac troponin I or T, β-MHC, αMHC, myosin light chain (MLC), α-tropomyosin, titin, and actin, have
been associated with familial hypertrophic cardiomyopathy in humans
(Margulies & Houser, 2004; Lind et al., 2006; Morimoto, 2008;
Tsoutsman et al., 2008). Signaling proteins and regulators of gene
expression that have been implicated in sarcomere organization include
Rac1, RhoA (Aoki et al., 1998; Hoshijima et al., 1998; Pracyk et al., 1998),
FAK and p130Cas (substrates for the non-receptor tyrosine kinase Src)
(Kovacic-Milivojevic et al., 2001), multiple kinases (see Seguchi et al.,
2007; Solaro 2008), Nkx2.5/Csx (Kasahara et al., 2003), and HDAC4
(Gupta et al., 2008).
4.3. Molecular mechanisms associated with fibrosis
Cardiac fibrosis is a common feature in animal models of
pathological hypertrophy and patients with advanced heart failure.
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In response to a pathological insult, cardiac fibroblasts and extracellular matrix proteins accumulate disproportionately and excessively.
This leads to mechanical stiffness which contributes to diastolic
dysfunction and can progress to systolic dysfunction (Weber & Brilla,
1991; Villarreal & Dillmann, 1992; Weber et al., 1993; Brower et al.,
2006). It is recognized that anti-fibrotic therapies may be useful in
improving cardiac function of the diseased heart. However, the
development of such therapies has been limited by an incomplete
understanding of the source of fibroblasts and mechanisms responsible for the induction of excessive collagen accumulation. Traditionally, adult fibroblasts are considered to be derived directly from
embryonic mesenchymal stem cells and to increase in number in a
setting of pathological stress due to proliferation of resident
fibroblasts (Weber & Brilla, 1991; Maric et al., 1997; Weber, 1997;
Lang and Fekete 2001). More recently, it has been demonstrated that
bone marrow derived fibroblasts, as well as endothelial cell derived
fibroblasts (via endothelial–mesenchymal transition) contribute to
the population of cardiac fibroblasts (Zeisberg et al., 2007).
Genetic mouse models have provided some insight in relation to
the molecular mechanisms responsible of fibrosis associated with
pathological hypertrophy. Triggers, receptors, signaling proteins, and
transcription factors implicated in the development of cardiac fibrosis
include: Ang II, AT1 receptor, TGF-β, tumor necrosis factor (TNF)-α, G
proteins (Gαq, Gαs), PKCβ2, RhoA, calcineurin, calsequestrin, NFATs,
Csx/Nkx2.5, and serum response factor (see Manabe et al., 2002;
Gunasinghe & Spinale, 2004; Rosenkranz, 2004).
TGF-β1 is probably the best described mediator of cardiac fibrosis. In
the heart, TGF-β1 is secreted by cardiac fibroblasts in response to stimuli
such as Ang II (Khan & Sheppard, 2006). The effects of TGF-β1 on cardiac
fibrosis appear to be mediated in part via Smads and TGF-β-activated
kinase-1 (TAK1) leading to increased transcription of extracellular
matrix proteins (Rosenkranz, 2004; Khan & Sheppard, 2006). Transgenic mice over-expressing TGF-β1 developed cardiac hypertrophy that
was associated with interstitial fibrosis and increased myocyte size
(Rosenkranz et al., 2002). In contrast, heterozygous TGF-β1+/− deficient
mice displayed less cardiac fibrosis with aging (Brooks & Conrad, 2000).
TGF-β1 was also responsible for endothelial cells undergoing endothelial–mesenchymal transition and the induction of increased fibrosis in a
setting of pathological hypertrophy induced by pressure overload
(Zeisberg et al., 2007). Furthermore, inhibition of TGF-β1 with
recombinant bone morphogenic protein-7 (member of the TGF-β
superfamily of growth factors) was able to inhibit endothelial–
mesenchymal transition and fibrosis in mice with pressure overloadinduced hypertrophy (Zeisberg et al., 2007).
Myocyte death by apoptosis or necrosis is typically observed in
parallel with the onset of fibrosis. Debate remains as to whether
fibrosis is simply a non-specific response to myocyte loss or whether
the release of secretory factors from myocytes promote or inhibit
collagen synthesis also plays a significant role (Benjamin et al., 1989).
It was shown that rat cardiac myocytes induce secretion of active TGFβ in the presence of Ang II and that a paracrine action of TGF-β
induced cytokines in fibroblasts to promote collagen synthesis (Sarkar
et al., 2004).
4.4. Molecular mechanisms associated with cell death
Pathological hypertrophy is typically associated with increased cell
death (apoptosis and necrosis) whereas physiological hypertrophy is
not. Thus, differential activation of pro-survival and pro-death signals
is likely to be an important factor that contributes to the distinct
phenotypes of these two forms of heart growth. Low levels of
apoptosis were sufficient to induce heart failure in mice, and
inhibition of cell death with a polycaspase inhibitor largely prevented
the heart failure phenotype (Wencker et al., 2003). In another study,
inhibition of apoptosis with a caspase inhibitor improved cardiac
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and anti-apoptotic effects (De Windt et al., 2000; Saito et al., 2000; Pu
et al., 2003). JNK, p38-MAPK, and loss of gp130 have been associated
with increased apoptosis in cardiac myocytes (Wang et al., 1998a;
Hirota et al., 1999; Kang et al., 2002).
Rats with pathological hypertrophy (Dahl salt sensitive rats on a
high salt diet) were more sensitive to apoptotic stimulation than
cardiac myocytes from rats with exercise-induced physiological
hypertrophy (treadmill). The physiological hypertrophic model was
associated with changes in Bcl-2 family members and caspases
favoring survival, whereas the pathological model was associated
with changes in mitochondrion- and death receptor-mediated pathways (e.g. a decreased Bcl-xL/Bax ratio, increase in Fas) that have
previously been associated with pathological hypertrophy and heart
failure (Kang et al., 2004).
Fig. 8. Alterations in substrate utilization in pathological and physiological cardiac
hypertrophy. Pathological hypertrophy is associated with a switch from fatty acid to
glucose utilization, although glucose metabolism also decreases with the progression to
heart failure. In contrast, physiological hypertrophy is associated with enhanced rates
of fatty acid and glucose oxidation.
function and prevented mortality in pregnant cardiac-specific Gαq
transgenic mice (Hayakawa et al., 2003).
Growth factors (e.g. IGF1), and cytokines (e.g. CT-1) have antiapoptotic effects, in part, via PI3K and/or ERK signaling (Parrizas et al.,
1997; Sheng et al., 1997; Haunstetter and Izumo 1998; Kang et al.,
2002). In contrast, signaling via Gαq or Gαs in transgenic mice was
shown to promote cardiac myocyte apoptosis (Adams et al., 1998;
Geng et al., 1999). Calcineurin has been reported to have both pro-
4.4.1. Autophagy
Autophagy is the process by which cells degrade and recycle aged
proteins and damaged organelles. Inhibition of autophagy triggers
apoptosis, indicating that autophagy plays a key role in cell survival
(Boya et al., 2005). Autophagy is upregulated in numerous models of
pathological hypertrophy and in failing hearts (see Nishida et al.,
2008; Nishida et al., 2009). This may be a protective mechanism,
preventing the accumulation of cytoplasmic components that disrupt
cardiac function (Nishida et al., 2009). Little is known about the role of
autophagy in settings of physiological cardiac hypertrophy, however
autophagy is upregulated by exercise and appears to be important for
amino acid turnover and protein synthesis in skeletal muscle post
exercise (see Gottlieb et al., 2009).
Fig. 9. Overview of energy metabolism in cardiac myocytes. Circulating fatty acids, glucose and lactate are the main fuel sources utilized by cardiac myocytes to generate ATP.
Following uptake into cardiac myocytes, fatty acids are transported to the mitochondria by carnitine palmitoyltransferase I (CPTI) and II (CPTII) for β-oxidation, entry into the
tricarboxylic acid (TCA) cycle and oxidative phosphorylation. Glucose and lactate are converted to pyruvate before entering the mitochondria and converging with fatty acid
oxidation pathways. Asterisks denote proteins and processes that can induce cardiac hypertrophy when altered or defective. ATP: adenosine triphosphate, CoA: coenzyme A, HIF1α:
hypoxia inducible factor 1α, NADPH: nicotinamide adenine dinucleotide phosphate, PPAR: peroxisome proliferator-activated receptor, ROS: reactive oxygen species.
B.C. Bernardo et al. / Pharmacology & Therapeutics 128 (2010) 191–227
5. Molecular mechanisms associated with differences in
energy metabolism in pathological and physiological hypertrophy
As described earlier, pathological and physiological hypertrophy
are associated with distinct metabolic profiles. Substrate utilization in
pathological hypertrophy resembles that of the fetal heart (decreased
fatty acid oxidation, increased glycolysis), while physiological
hypertrophy induced by exercise training is associated with enhanced
fatty acid and glucose oxidation (see Lehman & Kelly, 2002; Fig. 8).
Whether alterations in energy metabolism are a cause or consequence
of cardiac hypertrophy and failure is controversial, however there is a
growing body of evidence to suggest that switches in substrate
utilization and other alterations in energy metabolism do contribute
to the development of pathological hypertrophy and failure. The
accumulation of lipids in cardiac myocytes is deleterious and can lead
to pathological hypertrophy and heart failure, although the mechanisms responsible for lipotoxic cardiomyopathy are still unknown.
Reactive oxygen species (ROS), a by-product of mitochondrial energy
metabolism, may also induce hypertrophy in pathological settings
(Fig. 9).
5.1. Peroxisome proliferator-activated receptors (PPARs)
The PPAR family of transcription factors are key metabolic
regulators that have been implicated in the development of cardiac
hypertrophy (see Robinson & Grieve, 2009 for review). PPARα is a
nuclear receptor that regulates lipid metabolism by increasing
transcription of genes involved in fatty acid oxidation, such as
muscle-type carnitine palmitoyltransferase 1 (mCPT-1) and medium
chain acyl coenzyme A dehydrogenase (MCAD) (Fig. 9) (see Barger &
Kelly, 2000). PPARα expression was elevated in hearts of trained rats
(Rimbaud et al., 2009) and downregulated in several models of
pathological hypertrophy (see Barger et al., 2000; Lehman & Kelly,
2002; Akki et al., 2008) as well as in the fetal heart (Sack et al., 1997).
Transgenic mice deficient for PPARα developed greater hypertrophy
and had worse cardiac function compared to wildtype mice when
subjected to pressure overload (Smeets et al., 2008). In addition,
administration of a PPARα agonist attenuated hypertrophy induced
by aldosterone treatment in mice (Lebrasseur et al., 2007) and in two
rat models of pathological hypertrophy (Ichihara et al., 2006; Linz et
al., 2009). These studies suggest that downregulation of PPARα (and
therefore fatty acid oxidation) contributes to the development of
pathological hypertrophy, although a study in cardiac-specific
transgenic mice has demonstrated that enhanced PPARα protein
expression can also induce hypertrophy and cardiac dysfunction
(Finck et al., 2003).
PPARα is a target of ERK1/2 (Barger et al., 2000), a signaling
protein which is activated in settings of pathological cardiac
hypertrophy (see Section 3.2.3.1). Deactivation of PPARα by ERK1/2
was responsible for the reduction in fatty acid oxidation observed in
cardiac myocytes stimulated with the hypertrophic agonist PE (Barger
et al., 2000), indicating that signaling pathways involved in the
development of cardiac hypertrophy can also exert changes in cellular
metabolism.
The role of PPARγ in the development of cardiac hypertrophy is
less clear. Several studies suggest that PPARγ is protective, as mice
lacking PPARγ in the heart developed cardiac hypertrophy and
dysfunction (Ding et al., 2007), and treatment with a PPARγ agonist
reduced cardiac remodeling and fibrosis in a rat model of hypertension (Henderson et al., 2007). However, cardiac-specific overexpression of PPARγ in mice also resulted in cardiac dysfunction
(Son et al., 2007), indicating that PPARγ activation is not always
protective (Fig. 9). A recent study demonstrated that transgenic mice
with enhanced PPARγ activity (due to elevated levels of ventricular
hypoxia-inducible factor 1α, HIF1α) developed concentric hypertrophy which progressed to dilated cardiomyopathy at around 5–
209
6 months of age (Fig. 9) (Krishnan et al., 2009). This was attributed
to PPARγ-dependent lipid accumulation and cardiac myocyte apoptosis. HIF1α is a key regulator of genes involved in glycolytic
metabolism and was found to be highly expressed in ventricular
biopsies from patients with hypertrophic cardiomyopathy. HIF1α was
also highly expressed in a mouse model of pathological hypertrophy
induced by pressure overload, but not in physiological hypertrophy
induced by exercise training. This study provides strong evidence that
a switch from fatty acid to glycolytic metabolism leads to pathological
hypertrophy and is ultimately detrimental for cardiac function.
5.2. Lipid accumulation and pathological hypertrophy
Lipid accumulation appears to act as a trigger for cardiac myocyte
hypertrophy (Chiu et al., 2001) and is evident in the failing human
heart (Sharma et al., 2004), however mechanisms responsible for
lipotoxic cardiomyopathy are unclear (see McGavock et al., 2006 and
Park et al., 2007 for reviews). Lipid accumulation can result from
alterations in fatty acid uptake, transport or oxidation. For example,
carnitine is responsible for transporting long-chain fatty acids from
the cytosol into the mitochondria for subsequent β-oxidation (Fig. 9).
Carnitine deficiency impairs fatty acid oxidation and has been
associated with the development of left ventricular hypertrophy and
cardiomyopathy in humans (Koizumi et al., 1999). Mice with systemic
carnitine deficiency (juvenile visceral steatosis (JVS) mice) displayed
significant cardiac hypertrophy at eight weeks of age (Horiuchi et al.,
1993; Kuwajima et al., 1998) and were more susceptible to pressure
overload (Takahashi et al., 2007). Treatment with carnitine attenuated the basal hypertrophic phenotype in JVS mice (Horiuchi et al.,
1993), and improved cardiac function in JVS mice subjected to
pressure overload (Takahashi et al., 2007). Furthermore, limiting
dietary lipid intake attenuated hypertrophy in JVS mice and this was
associated with reduced triglyceride accumulation in the ventricles
(Jalil et al., 2006).
Enhanced fatty acid uptake (e.g. due to excess dietary lipids, as
occurs in obesity) has also been linked with the development of
pathological hypertrophy (Kankaanpaa et al., 2006; Koonen et al.,
2007) and may play a role in the development of diabetic
cardiomyopathy (see Carley & Severson, 2005), although high-fat
feeding attenuated hypertrophy in rats with hypertension (Okere et
al., 2005).
5.3. Reactive oxygen species
(ROS)/oxidative stress and pathological hypertrophy
ROS are a natural by-product of mitochondrial energy production
(Fig. 9). Oxidative stress occurs when ROS production outweighs the
antioxidant capabilities of the cell, and has been implicated in the
pathogenesis of cardiac hypertrophy and heart failure (McMurray
et al., 1993). Hypertrophic stimuli, such as Ang II, ET-1 and
catecholamines, are capable of stimulating ROS production in cardiac
myocytes (Liu et al., 2004; Laskowski et al., 2006). Defective NADPH
oxidase activity is another source of ROS that has been implicated in
the development of pathological hypertrophy (see Murdoch et al.,
2006). A study in transgenic mice demonstrated that NADPH oxidase
plays an important role in the development of cardiac hypertrophy, as
mice deficient for the gp91phox subunit of NADPH oxidase were
protected from developing pathological hypertrophy induced by
chronic infusion of Ang II (Bendall et al., 2002).
ROS production also increases during exercise although this
appears to have a preconditioning effect, reducing susceptibility to
oxidative stress-related disorders (see Radak et al., 2008). Exerciseinduced ROS production is much lower in the heart compared with
liver and skeletal muscle (Traverse et al., 2006). Thus it seems unlikely
that ROS play a role in the development of physiological hypertrophy.
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5.4. Changes in energy metabolism
associated with physiological cardiac hypertrophy
There are clear differences in energy metabolism between
pathological and physiological hypertrophy. While pathological
hypertrophy is typically associated with a switch from fatty acid to
glucose utilization, physiological hypertrophy is accompanied by
enhanced rates of fatty acid and glucose oxidation. This has been
demonstrated in numerous animal models, as well as in humans. For
example, the expression of genes encoding enzymes involved in fatty
acid oxidation (i.e. PPARα, mCPT-1, and MCAD) was significantly
increased in the hearts of trained rats but not hypertensive rats,
despite a similar degree of hypertrophy (Rimbaud et al., 2009). In
another study, phosphofructokinase and lactate dehydrogenase, key
regulators of glycolytic metabolism, were upregulated in pathological
hypertrophy induced by hypertension but not in physiological
hypertrophy induced by exercise training (Iemitsu et al., 2003). As
mentioned previously, HIF1α (a key regulator of glycolytic metabolism) was elevated in mice and human patients with pathological
hypertrophy, but not in a mouse model of physiological hypertrophy
(Fig. 9) (Krishnan et al., 2009). Rats with exercise-induced hypertrophy had lower rates of glycolysis compared to sedentary controls,
while glucose and palmitate (i.e. fatty acid) oxidation increased by
45% and 50–65%, respectively (Burelle et al., 2004). The enhanced
oxidative capacity of hearts that have undergone physiological
hypertrophy suggests a protected phenotype.
better understood models of cardiac hypertrophy are probably best
defined based on functional and structural parameters.
Transcription factors including GATA4, GATA6, Csx/Nkx2.5, MEF2,
c-jun, c-fos, c-myc, nuclear factor-κB, and NFAT have been implicated
for the activation of cardiac genes in response to hypertrophic stimuli
(Sadoshima & Izumo, 1997; Aoki & Izumo, 2001; Akazawa & Komuro,
2003). Of note, Gata4 has been implicated in the regulation of a
number of genes associated with pathological hypertrophy (e.g. ANP,
BNP, α-skeletal actin, and β-MHC) but may also be important for
physiological growth. A recent study demonstrated that mice with
reduced GATA4 had mild cardiac dysfunction and reduced heart
weight under basal conditions. In response to pressure overload,
GATA4 deficient mice developed eccentric hypertrophy and heart
failure associated with marked apoptosis and fibrosis (Bisping et al.,
2006).
NGF1A-binding protein (Nab1) is a transcriptional repressor for
early growth response transcription factors. Nab1 appears to be a
specific regulator of pathological cardiac hypertrophy. Cardiacspecific Nab1 transgenic mice displayed a blunted hypertrophic
response to adrenergically-induced and pressure overload-induced
hypertrophy whereas physiological growth (development or exercise-induced hypertrophy) was not affected (Buitrago et al., 2005).
Profiling studies are beginning to provide a more global view of
differences in gene expression in various hypertrophic models (see
Section 6.2).
6.1. Chromatin-modifying enzymes
6. Characteristic gene expression changes
associated with pathological and physiological hypertrophy
Intracellular signaling pathways are coupled with transcription
factors in the nucleus to regulate the long-term alterations in gene
expression that are associated with cardiac hypertrophy. Classically,
pathological hypertrophy has been associated with upregulation of
fetal genes including ANP, BNP, α-skeletal actin, atrial MLC-1, and βMHC; and downregulation of genes normally expressed at higher
levels in the adult than in the fetal ventricle, such as α-MHC and
SERCA2a (Izumo et al., 1988; Chien et al., 1991; MacLellan &
Schneider, 2000). MHC is the major component of myosin, the
protein complex responsible for driving contraction in muscle cells. In
rodents, β-MHC is the predominant isoform present in the prenatal
heart, but is down-regulated soon after birth when α-MHC is
expressed (Morkin, 2000). During pathological hypertrophy the
increased expression of β-MHC and fall in α-MHC may represent an
adaptive response, as β-MHC is slower than α-MHC at catalyzing the
hydrolysis of ATP (the chemical reaction driving myocyte contraction)
leading to slower, more economical, contractile function (Swynghedauw, 1986; Izumo et al., 1987; Dorn et al., 1994; Swynghedauw,
1999). Recapitulation of the fetal gene program and switch in
contractile protein composition does not commonly occur in models
of physiological hypertrophy e.g. exercise-induced hypertrophy
(McMullen et al., 2003) (Fig. 4). The direct impact of changes in
fetal gene expression on cardiac growth, function and fibrosis remain
unclear. While fetal genes are often upregulated in models of
pathological hypertrophy, this may be a compensatory response to
protect the heart. ANP and BNP signaling have antihypertrophic
actions within cardiac myocytes (Woods, 2004). Furthermore, some
transgenic models of physiological hypertrophy have been associated
with a modest upregulation of fetal genes (e.g. IGF1R (McMullen et al.,
2004b) and MEK1 (Bueno et al., 2000)), whereas a model of
intermittent pressure overload was associated with a pathological
phenotype but no upregulation of the fetal gene program (Perrino et
al., 2006). Finally, some studies have demonstrated that a small/
normal heart size phenotype can be associated with activation of the
fetal gene program (Shioi et al., 2000; Antos et al., 2002). Thus, until
the biological significance of changes in the fetal gene program is
Histone-dependent packaging of genomic DNA into chromatin is a
central mechanism for gene regulation. Nucleosomes (the basic unit
of chromatin) interact to create a highly compact structure that limits
access of genomic DNA to transcription factors, thus repressing gene
expression (McKinsey et al., 2002). Chromatin-remodeling enzymes
have been implicated in the re-expression of the fetal gene program
(McKinsey et al., 2002). The chromatin-modifying enzymes, HDACs,
promote chromatin condensation and thus repress transcription
(McKinsey et al., 2002).
HDACs have been grouped into three classes (I–III). Class IIa
HDACs (4, 5, 7 and 9) which are regulated by signaling proteins
including PKC and CaMK have been implicated as suppressors of
pathological hypertrophy (Bush & McKinsey, 2009). HDAC5 and
HDAC9 knockout mice developed hypertrophy with increased age and
showed an exaggerated hypertrophic response to pressure overload
(aortic constriction), however postnatal heart growth was unaffected
in the knockout models (Zhang et al., 2002a; Chang et al., 2004). By
Fig. 10. Venn diagram showing the shared and distinct gene clusters (as determined
from microarray analysis) that have been shown to be associated with pathological and
physiological hypertrophy. HSF1 is a heat shock protein transcription factor that has
recently been shown to be involved in physiological hypertrophy. Data collected from
(Kong et al., 2005; Mirotsou et al., 2006; Dorn 2007).
B.C. Bernardo et al. / Pharmacology & Therapeutics 128 (2010) 191–227
contrast, class I HDACs (1 and 2) are reported to promote
hypertrophy. Cardiac-specific HDAC2 transgenic mice developed
cardiac hypertrophy associated with increased fetal gene expression
(Trivedi et al., 2007). Class III HDACs (also known as sirtuins) are also
reported to inhibit pathological hypertrophy, including apoptosis (see
Bush & McKinsey, 2009).
6.2. Profiling studies of cardiac hypertrophy
In another approach to identify transcriptome changes associated
with pathological and physiological cardiac hypertrophy, a number of
investigators have conducted comprehensive microarray gene expression profiling studies in hearts from rodent models of pathological and physiological cardiac hypertrophy, as well as tissue from
heart failure patients (Friddle et al., 2000; Hwang et al., 2000; Yang et
al., 2000; Barrans et al., 2001; Hwang et al., 2002b; Diffee et al., 2003;
McMullen et al., 2004b; Kong et al., 2005; Strom et al., 2005). More
recent studies have used proteomic methods, such as 2-dimensional
polyacrylamide gel electrophoresis (2-D PAGE) in conjunction with
mass spectrometry to catalogue exercise-induced changes in the
cardiac proteome (Boluyt et al., 2006; Burniston, 2009).
6.2.1. Gene expression profiling (microarray)
Microarray technology is a powerful technique and quite often the
method of choice to study mRNA expression differences between a
cohort of samples, different transgenic mouse models, or tissues
subjected to different conditions. This technology has been used to
identify molecular differences between physiological and pathological
hypertrophy in a number of transgenic rodent models (Friddle et al.,
2000; McMullen et al., 2004b; Kong et al., 2005; Strom et al., 2005), as
well as in human tissue samples of non-failing hearts versus failing
hearts (Hwang et al., 2000; Yang et al., 2000). The outcomes of these
studies have been reviewed in depth (Barrans et al., 2001; Hwang et
al., 2002a; Dorn 2007). In brief, these studies have identified shared
and distinct gene cluster expression profiles of physiological and
pathological hypertrophy (Fig. 10). Genes associated with pathological hypertrophy were largely from inflammation, apoptotic, cardiac
fetal gene, and oxidative stress clusters. On the other hand, genes that
were associated with physiological hypertrophy were predominately
involved in cell survival, fatty acid oxidation, insulin signaling,
epidermal growth factor signaling and HSF1 expression (Fig. 10)
(Aronow et al., 2001; McMullen et al., 2004b; Kong et al., 2005; Strom
et al., 2005; Mirotsou et al., 2006). Cardiac apoptosis has been shown
to be involved in various human and animal models of heart failure
(Kang & Izumo, 2000), including the Gαq transgenic mouse which
develops hypertrophy and heart failure (Aronow et al., 2001).
Expression of genes that are involved with fatty acid oxidation in
physiological models are consistent with the known increase in fatty
acid oxidation in response to exercise (see Section 5.4). These studies,
in addition to the data obtained from genetic mouse models, lend
further support to the notion that distinct molecular mechanisms can
regulate pathological and physiological cardiac hypertrophy. Members of the IGF1/EGF signaling pathway showed statistically significant changes in a rat model of physiological hypertrophy, in
comparison to compensated pathological hypertrophy (Dahl salt
sensitive rats on a high salt diet) (Kong et al., 2005). Another
application of microarray technology is the discovery of new drug
targets. Identification of novel genes that play important roles in
mediating physiological hypertrophy and protection may open up
new avenues for treating patients with heart failure (discussed
further in Section 8). In general, research and therapy has concentrated on identifying and inhibiting pathological processes. Another
therapeutic strategy may be to activate novel regulators of physiological hypertrophy that have been identified from gene expression
profiling studies (McMullen et al., 2004b; Kong et al., 2005; Lin et al.,
2010).
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Gene expression profiling has also been utilized to examine
differences between pathological eccentric and concentric hypertrophy. Rat models of concentric hypertrophy (aortic constriction for
pressure overload) and eccentric hypertrophy (aortocaval shunt for
volume overload) with similar degrees of cardiac hypertrophy were
associated with different gene expression patterns (Miyazaki et al.,
2006). Sixty-four genes behaved similarly between the 2 models but
93 genes were altered only in the pressure overload model and 134
genes were differentially expressed in the volume overload model. In
another study, comparisons were made between a model of
pathological hypertrophy (pressure overload by ascending aortic
banding for 1 week) and physiological hypertrophy (chronic swim
training for 4 weeks) in mice. Only a small percentage (approximately
3%) of genes were regulated in a similar manner between the 2
models (Cardiogenomics, 2001–2003).
6.2.2. Proteomics
Investigators have also used proteomic techniques to identify
changes in expression, splice variation and post translational
modifications in models of cardiac hypertrophy (Boluyt et al., 2006;
Burniston, 2009). Treadmill exercise-trained rats showed cardiac
hypertrophy, with a 14–18% increase in heart weight to body weight
ratio compared to sedentary controls (Boluyt et al., 2006). Analysis of
2-dimensional electrophoresis gels revealed protein spots that were
decreased, increased, or detected exclusively in exercise trained
hearts compared to controls. Heat shock protein 20 (Hsp20)
represented a protein exclusively expressed in exercise trained hearts
(identified by mass spectrometry and immunoblotting). A later study
corroborated this finding in another rat model, but in a setting of
moderate exercise that is similar to physical activity guidelines for
humans (Burniston, 2009).
Hsp20 belongs to the subfamily of small heat shock proteins in
which there are 10 known members in mammalian species (Fan et al.,
2005a). Hsp20 contains three phosphorylation sites, serine 16, serine
59, and serine 157, but conclusions on the role of Hsp20 have mostly
been based on serine 16 phosphorylation (Fan et al., 2005a). Heat
shock proteins have been involved in the preservation of myocardial
function after ischemia/reperfusion (Kingma, 1999). Studies have
shown that Hsp20 enhances myocardial contraction in vitro (Chu et
al., 2004), and cardiac-specific transgenic mice over-expressing
Hsp20 had enhanced cardiac function in the absence of pathological
abnormalities (Fan et al., 2005c). Furthermore, following coronary
artery occlusion and reperfusion (24 h), Hsp20 transgenic hearts
exhibited better cardiac functional recovery, and reduced infarct area
compared to wildtype hearts (Fan et al., 2005c). Finally, Hsp20
transgenic mice displayed an attenuated hypertrophic response to βAR agonist-induced cardiac hypertrophy, while retaining enhanced
cardiac function. Transgenic expression also prevented the β-AR
agonist-induced increase in fetal genes (ANP, BNP), fibrosis, and
reduced apoptosis (Fan et al., 2006). In vitro experiments from this
study suggest that Hsp20 is able to inhibit apoptosis signal regulating
kinase 1, which in turn leads to protection from β-AR agonist-induced
cardiac hypertrophy/remodeling (Fan et al., 2006). Together, this
suggests that proteins that play unique roles in mediating exerciseinduced physiological hypertrophy may be attractive targets for the
treatment of heart disease.
6.3. microRNAs — role in cardiac hypertrophy
MicroRNAs (miRNAs) are a relatively recently discovered family of
small, endogenous, single-stranded RNAs that are approximately 20–
25 nucleotides in length. miRNAs have emerged rapidly as a major
new direction in several different fields of research. miRNAs are
partially complementary to their mRNA targets and have an
important role in the regulation of target genes by hybridizing to 3′
untranslated regions of messenger transcripts to repress their
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translation or regulate degradation (Bartel, 2004; Griffiths-Jones et al.,
2006). An average miRNA is estimated to affect expression of
hundreds of mRNA targets, and up to 30% of human protein coding
genes may be regulated by miRNAs (Lewis et al., 2005; Rajewsky
2006).
Although miRNAs were first described in 1993 by Victor Ambrose
and colleagues (Lee et al., 1993), since then, 500–1000 different
miRNAs have been identified, however, an understanding of their
diverse biological functions remains in its infancy. It is known that
miRNAs play a role in a number of biological processes including
development, cell proliferation, differentiation, and apoptosis
(reviewed in Sassen et al., 2008; Cordes & Srivastava, 2009).
Furthermore, changes in miRNA levels have been correlated with
disease processes, which has sparked numerous academic research
and commercial interest, particularly for cancer (reviewed by Sassen
et al., 2008), viral diseases (Jopling et al., 2005; Lecellier et al., 2005),
and more recently kidney disease (Kato et al., 2009; Liang et al., 2009).
A number of studies have demonstrated the involvement of several
miRNAs in cardiac development (reviewed in (Thum et al., 2008a;
Catalucci et al., 2009; Cordes & Srivastava, 2009)) and in cardiac
pathology (van Rooij et al., 2006; Ikeda et al., 2007; Tatsuguchi et al.,
2007; van Rooij & Olson, 2007; Divakaran & Mann, 2008; Thum et al.,
2008b; Rao et al., 2009; Ren et al., 2009; Tang et al., 2009). More
recently, cardiac specific over-expression of miRNA-208a was shown
to cause pathological cardiac hypertrophy, as shown by thickening of
ventricular walls, depressed cardiac function and increased expression of β-MHC (Callis et al., 2009). In contrast, miRNA biology of
physiological cardiac hypertrophy has received little attention.
miRNA-1 and miRNA-133 were decreased in two models of
physiological cardiac hypertrophy (exercised trained rats and cardiac
specific Akt transgenic mice), however, these miRNAs were also
decreased in a model of pathological hypertrophy (pressure overload), and in patients with heart disease (Care et al., 2007). Thus, it is
recognized that further studies outlining similarities and differences
between the regulation of miRNAs in physiological and pathological
hypertrophy are needed (Latronico et al., 2008). To address this gap in
miRNA biology, we recently identified miRNAs that are differentially
regulated in a setting of physiological hypertrophy and cardiac
protection versus a model of cardiac stress associated with pathological growth (Lin et al., 2010). We first selected miRNAs that were
differentially expressed in the dnPI3K and caPI3K transgenic mouse
models. We then selected those miRNAs that were differentially
regulated in a setting of physiological hypertrophy and cardiac
protection (caPI3K model) and cardiac stress (myocardial infarction
model).
Silencing of miRNAs in vivo with antagomiRNAs (chemically
modified, cholesterol-conjugated single-stranded RNA analogues
complementary to miRNAs) is considered a powerful approach that
may represent a new therapeutic strategy for targeting cardiac
disease. These molecules have been shown to be effective in vivo,
capable of passing through cellular membranes to inhibit miRNA
action by sequestering it from its targets (Krutzfeldt et al., 2005).
7. Gender differences in cardiac hypertrophy
An area of research that has received increasing attention is that of
differential hypertrophic responses associated with gender (Du et al.,
2006). It is recognized that women typically develop heart disease
later than men. One feature that is considered to help explain the
better prognosis in females than males, is the smaller degree of
cardiac hypertrophy and/or type (concentric versus eccentric) in
females in response to cardiovascular complications such as pressure
overload (demonstrated in animal and human studies) (Du et al.,
2006).
Prior to adolescence, there are no significant differences in heart
size between males and females, suggesting a similar number of
cardiac myocytes at birth, as myocytes are terminally differentiated
(Zak, 1974; de Simone et al., 1995; Sugden & Clerk, 1998; Luczak &
Leinwand, 2009). Following puberty however, males have 15–30%
larger hearts than females, suggesting a significantly larger degree of
hypertrophy in males (de Simone et al., 1995). Men also lose
approximately 1 g of cardiac mass per year following puberty,
which leads to compensatory hypertrophy to maintain adequate
cardiac mass. Females, however, appear to maintain their myocyte
number and size with aging (Grandi et al., 1992; Olivetti et al., 1995;
Luczak & Leinwand, 2009).
7.1. Pathological hypertrophy
From animal studies there are clear differences in the degree and/
or type of pathological hypertrophy in response to cardiac insults
between males and females (Du et al., 2006; Podesser et al., 2007)
(Table 4). Male spontaneously hypertensive rats developed more
cardiac hypertrophy and left ventricular dysfunction than females.
Subsequent heart failure also occurred earlier in males than females
(Tamura et al., 1999). Similar findings were reported in response to
pressure overload (aortic-banding) (Douglas et al., 1998; Weinberg et
al., 1999; Skavdahl et al., 2005). Gender differences have also been
reported in humans, though the data are generally less conclusive
owing to confounding factors (e.g. treatments, lifestyle), relatively
small sample sizes, and the limited studies in patients with pure
pressure overload (i.e. in the absence of coronary heart disease).
Furthermore, in human studies there is the question of the best
normalization of left ventricular mass data (body surface area, body
mass index, height2.7 etc). Animal data is tighter because left
ventricular mass can be accurately assessed at autopsy (rather than
relying on echocardiography). However, in spite of these limitations,
gender differences in cardiac hypertrophy in response to similar
pressure loads (in the absence of coronary heart disease) are
apparent. In normotensive men and women (age matched b69),
normalized left ventricular (LV) mass was higher in men than women
(Levy et al., 1987; Laufer et al., 1989). In a population of premenopausal and post-menopausal women with mild essential
hypertension matched with men (in regard to mean arterial pressure,
age and race), pre-menopausal women had smaller left ventricular
mass and higher LV performance indices than men. These gender
differences were most pronounced before menopause and tended to
disappear after menopause (Garavaglia et al., 1989). Normalized LV
mass was also greater in men than women with aortic stenosis
(Carroll et al., 1992). In other studies, males were found to display
eccentric hypertrophy, while females displayed concentric hypertrophy, suggesting the male heart may decompensate quicker (Krumholz
et al., 1993; Douglas et al., 1995). Data from the Framingham Heart
Study suggest that different degrees of hypertrophy in males and
females have an impact on clinical outcomes. Women with LV
hypertrophy (free of coronary heart disease) had better clinical
outcomes than men [age-adjusted prevalence of ventricular arrhythmias higher in men than women (28% versus 17%), 6 year cumulative
mortality higher in men than women (38% versus 22%)] (Bikkina et
al., 1993). Women were also shown to have less cardiac myocyte
apoptosis in normal and in failing hearts compared with males at
autopsy (Guerra et al., 1999; Patten & Karas, 2006). Interestingly,
however, in settings of diabetes or hypertension women have a
greater risk than men to develop cardiovascular disease (RegitzZagrosek, 2006), though the reasons for this remain unclear.
7.2. Physiological hypertrophy
Gender differences in physiological cardiac hypertrophy have not
been examined widely but seem to exist based on animal studies
(Table 4). For example, female rats that underwent chronic swim
training showed an increased hypertrophic response compared with
B.C. Bernardo et al. / Pharmacology & Therapeutics 128 (2010) 191–227
213
Table 4
Differential hypertrophic responses to pathological and physiological stimuli in males and females.
Pathological/
physiological
Model
Subject Age
Female
Male
Reference
Pathological
C57BL/6 mice → Transverse
aortic constriction
Mice
2 months
Aortic banding (ascending aortic Rats
constriction — 4 months)
6 months
↑↑ Hypertrophy
HW/BW 64%
increase
↑↑ Hypertrophy
↑ Progression to
heart failure
↑ Chamber
dilation
Skavdahl et al., 2005
Pathological
↑ Hypertrophy
HW/BW 31%
increase
↑ Hypertrophy
Pathological
Ascending aortic stenosis
Rats
Not specified
Dahl salt-sensitive hypertensive
rats
Rats
3–4 months
Pathological
Spontaneously hypertensive
heart failure rats
Rats
6 months, and failing hearts (18 months
— male; 24 months — female)
Physiological
Voluntary cage wheel running
Mice
4 months
↑ Hypertrophy
↑↑ Fetal gene
expression
↑↑ ANP
↑↑ β-MHC
↓ SERCA2a
↓ Contractile
reserve
↑ Hypertrophy
(eccentric)
↑ LVPW
↑↑ Myocyte crosssectional area
↓ Cardiac function
↑ Progression to
heart failure
↑ Mortality
↑ Hypertrophy
Weinberg et al., 1999
Pathological
↑ Hypertrophy
↑ Fetal gene
expression
↑ ANP
↑ β-MHC
↔ SERCA2a
↔ Contractile
reserve
↑ Hypertrophy
(concentric)
↑ Septal thickness
↑ Myocyte crosssectional area
↔ Cardiac
function
Physiological
Swim training
Rats
4 months
their male counterparts (Schaible & Scheuer, 1979, 1981; Luczak &
Leinwand, 2009). Female mice have increased exercise capacity for
both voluntary wheel or treadmill running, with females running
more on a cage wheel than males independent of the strain or age.
Furthermore, female mice performed better in endurance tests,
indicative of increased cardiovascular performance (Konhilas et al.,
2004; Luczak & Leinwand, 2009). While cage wheel running induces
significant cardiac hypertrophy in both genders, females displayed a
greater percent increase in cardiac mass (Konhilas et al., 2004). To
date, there appears to be no supporting data in humans.
7.3. Molecular mechanisms associated
with gender differences in cardiac physiology
Based on clinical trials in which both genders have been included
in significant numbers, it is clear that males and females respond
differently to cardiovascular drugs. For example, ACEI and β-blockers
are less effective in women and show more side effects (RegitzZagrosek, 2006). Digitalis also causes more deaths in women (RegitzZagrosek, 2006). Furthermore, the prevalence of cardiac disease is
declining in men, but not in women (Zipes et al., 2005). Thus, it is of
great importance to understand the molecular mechanisms responsible for the different hypertrophic responses in males and females.
The molecular mechanisms underlying gender dimorphism are
complex and are still not well understood (Babiker et al., 2002;
Turgeon et al., 2004; Edwards, 2005; Mendelsohn & Karas, 2005;
Luczak & Leinwand, 2009). In general, pre-menopausal women tend
to be protected against cardiovascular disease compared with agematched men, but this protection is abolished following menopause.
Thus, it has been suggested that estrogen has protective properties
and activation of signaling cascades downstream of estrogen may
explain gender-related differences in the heart (Kannel, 2002;
↑↑ Hypertrophy
↑ Exercise
capacity
↑↑ Hypertrophy
↑ Hypertrophy
Douglas et al., 1998
Podesser et al., 2007
Tamura et al., 1999
Konhilas et al., 2004.
Schaible, T.F. and Scheuer, J.,
1979. [male animals]
Schaible, T.F. and Scheuer, J.,
1981. [female animals]
Mikkola & Clarkson, 2002; Wenger, 2002; Sullivan, 2003; Zipes et
al., 2005; Luczak & Leinwand, 2009).
The sex steroid hormones (estrogen, progesterone and testosterone) and their respective receptors are thought to mediate, at least in
part, gender differences in the heart (Mendelsohn & Karas, 2005; Du
et al., 2006; Luczak & Leinwand, 2009). Estrogen and estrogen
receptors (ERs) have been most extensively characterized. Both men
and women produce estrogen, but circulating levels of estrogen are
10–20 fold lower in men (Luczak & Leinwand, 2009). Cardiac
expression of ERα is similar in both genders, whereas ERβ expression
is significantly higher in males (Mahmoodzadeh et al., 2006).
Downregulation of ERs in response to ovariectomy was associated
with adverse cardiac remodeling and cardiac enlargement in animal
studies, suggesting ERs are important for inhibition of pathological
hypertrophy associated with aging (Xu et al., 2003). Early studies in
knockout mice suggested that ERβ rather than ERα was important for
the development of pathological hypertrophy induced by pressure
overload and cardioprotection in ischemia–reperfusion studies (Gabel
et al., 2005; Pelzer et al., 2005; Skavdahl et al., 2005). However, these
findings have been questioned and require further investigation.
Estrogen is able to initiate both 1) genomic responses through
binding to steroid hormone nuclear receptors, which when bound to
estrogen, modulate the transcriptional activity of target genes; as well
as 2) rapid membrane-initiated, estrogen-triggered signaling
responses (non-genomic) via a plasma membrane-associated form
of the receptor (Fig. 11) (de Jager et al., 2001; Konhilas et al., 2004;
Deroo & Korach, 2006; Du et al., 2006; Moriarty et al., 2006). The
protective properties of estrogen on hypertrophy appear to be
mediated in part by its ability to activate Akt, and inhibit GPCR, PKC,
p38-MAPK, and degrade calcineurin (Fig. 11) (Simoncini et al., 2000;
Konhilas et al., 2004; Du et al., 2006; Liu et al., 2006; Donaldson et al.,
2009). Estrogen has also been shown to initiate anti-hypertrophic
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Fig. 11. Signaling cascades involved in estrogen-mediated cardiac hypertrophic responses. Estrogen acts via the estrogen receptor which interacts with the p85 regulatory subunit of
PI3K to induce physiological hypertrophy and attenuate pathological signaling cascades. Nuclear estrogen receptors alter gene expression to regulate cardiac hypertrophic
responses. Estrogen is also able to induce ubiquitination of calcineurin, which leads to degradation of calcineurin by the proteasome. Ang II: angiotensin II, ANP: Atrial natriuretic
peptide, BNP: B-type natriuretic peptide, CN: calcineurin, ER: estrogen receptor, ERK: extracellular regulated kinase, ET-1: endothelin-1, GPCR: G protein-coupled receptor, IGF1:
insulin-like growth factor 1, IRS1: insulin receptor substrate 1, PI3K: phosphoinositide 3-kinase, PKC: protein kinase C, Ub: ubiquitin.
signaling, via increased ANP and BNP expression (Fig. 11) (van Eickels
et al., 2001; Jankowski et al., 2005). Activation of Akt is reported to
occur through a direct, non-nuclear pathway involving the regulatory
subunit of PI3K (Fig. 11) (Simoncini et al., 2000). Finally, estrogen is
also thought to have a positive impact on energy metabolism (upregulate lipid utilization and down regulate glucose oxidation), in
part, through interaction with PPARs and PPARα-activated γ
coactivator-1 (Keller et al., 1995; Nunez et al., 1997; Ma et al., 1998;
Tcherepanova et al., 2000; Kamei et al., 2003; Schreiber et al., 2003;
Schreiber et al., 2004; Bourdoncle et al., 2005; Du et al., 2006).
Testosterone is also able to initiate genomic responses via nuclear
androgen receptors that modulate transcription, as well as nongenomic responses. Adult men have approximately 10 fold higher
circulating testosterone levels than females but androgen receptors
are present in both male and female hearts, suggesting testosterone
has a role in both sexes (see Luczak & Leinwand, 2009). In contrast to
estrogen, testosterone is reported to have pro-hypertrophic properties (Ikeda et al., 2005; Du et al., 2006). Signaling cascades activated by
testosterone in the heart requires further examination (Du et al.,
2006).
only male mice null for both α1A/C and α1B adrenergic receptors or
muscle specific (cardiac and skeletal) transgenic mice over-expressing myostatin showed reduced cardiac size (O'Connell et al., 2003;
Reisz-Porszasz et al., 2003). Mutations of cardiac troponin T are
associated with different hypertrophic responses that are dependent
on gender under basal conditions and in response to pathological
stimuli (e.g. Ang II and isoproterenol) (Maass et al., 2004).
7.4. Gender differences in cardiac
hypertrophy — molecular mechanisms based on genetic mouse models
8.1. Current drug therapy
A further understanding of the different hypertrophic responses in
males and females at the molecular level has come from studies of
both male and female genetic mouse models. In the past, studies on
genetically modified mouse models have typically focused on males
though there have been some studies which have examined both
genders. Cardiac-specific over-expression of the β2-adrenergic receptor, TNF-α, and phospholamban was associated with greater
pathological hypertrophy in males compared to females (Kadokami et
al., 2000; Dash et al., 2003; Gao et al., 2003). By contrast, cardiac
specific transgenic expression of dn-p38α leads to more hypertrophy
in females than in males under basal conditions and in response to
pressure overload (Liu et al., 2006). Disruption of FKBP12.6 caused
pathological hypertrophy in male but not in female mice (Xin et al.,
2002), and loss of CD38 (regulator of calcium homeostasis) led to
hypertrophy only in male mice (Takahashi et al., 2003). In contrast,
8. Therapeutic strategies for the treatment of heart failure
Current therapies for heart failure include drug therapy, implantation of devices and surgery (see Krum & Abraham, 2009 for detailed
review). Palliative care and exercise training regimes are important
non-pharmacological approaches that can be implemented to
alleviate symptoms and improve quality of life (Flynn et al., 2009;
Goodlin, 2009). This section gives an overview of current drug
therapies and the signaling proteins they target, followed by new
potential therapeutic strategies that may be implemented in the
future based on differences between physiological and pathological
cardiac hypertrophy.
Traditionally, research and therapy has focused on identifying and
inhibiting processes associated with pathological hypertrophy, cardiac dysfunction, and the transition to heart failure. It is not uncommon
for heart failure patients to be prescribed between two to seven
medications alone, including medications to treat the side effects.
Heart failure medications include ACEI, diuretics, β-blockers, ARBs,
hydrolazine, and nitrates (McMurray & Pfeffer, 2005). Side effects can
include coughing, fluid retention, joint pain, hypotension, fatigue,
depression, renal insufficient, anemia and headaches. ACEI and ARBs
are typically the first line of defence for patients with heart failure
(Hunt et al., 2009). As outlined previously, Ang II signaling via GPCRs
is a stimulus for pathological hypertrophy and fibrosis. ACEI and ARBs
reduce blood pressure and attenuate LV remodeling by reducing
signaling via the Ang II receptors.
β-blockers are another class of drug that are used in combination
with ACEI/ARBs to treat patients with heart failure (Hunt et al., 2009).
Table 5
Mouse models which highlight the protective effects of IGF1-PI3K-Akt signaling in settings of heart disease.
Mouse model
Expression/activity
Evidence of protective effect in settings of cardiovascular disease
↑ IGF1 signaling
IGF1 transgenic (Reiss et al., 1996)
Cardiac-specific
84% increase in circulating IGF1 due
to increased secretion from cardiac myocytes
IGF1R transgenic (McMullen et al., 2004b)
Cardiac-specific
20-fold increase in IGF1R expression
in cardiac myocytes
caPI3K transgenic (Shioi et al., 2000)
Cardiac-specific
6.5-fold increase in PI3K(p110α)
activity in cardiac myocytes
dnPI3K transgenic (Shioi et al., 2000)
Cardiac-specific
77% reduction in PI3K(p110α)
activity in cardiac myocytes
Akt-nuc (Shiraishi et al., 2004)
Increased accumulation and activation
of Akt in cardiac myocyte nuclei
Loss of Akt1 mRNA and protein
expression due to targeted disruption
of Akt1 gene (whole body)
Dilated cardiomyopathy
– Attenuated LV remodeling and cardiac dysfunction, prevented apoptosis (Welch et al., 2002)
Diabetic cardiomyopathy
– Reduced cardiac dysfunction (Kajstura et al., 2001)
Eccentric cardiac hypertrophy
– Attenuated cardiomyocyte necrosis (Li et al., 1999)
Myocardial infarction
– Attenuated ventricular dilation and cell death (Li et al., 1997)
Pressure overload
– Blunted pathological hypertrophy and fibrosis (McMullen et al., 2004b)
Diabetic cardiomyopathy
– Reduced cadiac dysfunction and fibrosisi (Huynh et al., 2010)
Pressure overload
– Prevented cardiac dysfunction, blunted pathological hypertrophy and fibrosis (McMullen et al., 2007)
Dilated cardiomyopathy
– Prolonged lifespan (McMullen et al., 2007)
Pathological hypertrophy/heart failure
– Prevented fibrosis and improved cardiac function in mice overexpressing PKCβ2 (Rigor et al., 2009)
Pressure overload
– Reduced PI3K(p110α) activity exacerbated fibrosis and cardiac dysfunction, indicating that
PI3K(p110α) is important for maintaining cardiac structure and function in settings of cardiac stress
(McMullen et al., 2003; McMullen et al., 2007)
Dilated cardiomyopathy
– Reduced lifespan (McMullen et al., 2007)
– Induced atrial fibrillation, reduced cardiac function, increased fibrosis (Pretorius et al., 2009a)
Pressure overload
– Attenuated LV remodeling and cardiac dysfunction, improved survival (Tsujita et al., 2006)
Pressure overload
– Loss of Akt1 exacerbated hypertrophy and cardiac dysfunction, indicating that Akt is
important for protecting the heart in settings of cardiac stress (DeBosch et al., 2006b)
Hypertrophic cardiomyopathy
– Reversed expression of hypertrophic markers, reduced fibrosis, inhibited apoptosis
(Konhilas et al., 2006)
Dilated cardiomyopathy
– Prolonged lifespan (McMullen et al., 2007)
↑ PI3K activity
↑ Akt activity
Akt1−/− (Cho et al., 2001)
Exercise (activates
IGF1-PI3K signaling)
Voluntary cage wheel running
Swim training
B.C. Bernardo et al. / Pharmacology & Therapeutics 128 (2010) 191–227
Protective mechanism
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The use of β-blockers to treat heart failure patients was first
introduced by Waagstein et al. (1975). This was subsequently
confirmed in later studies (Swedberg et al., 1979, 1980; Waagstein
et al., 1989). Since these discoveries, several randomized, controlled
clinical trials (e.g. CIBIS-II, MERIT-HF) and smaller studies have shown
that administration of β-blockers improved survival rates and/or was
associated with improvement in left ventricular ejection fraction
(reviewed by Molenaar & Parsonage, 2005). β-blockers reduce
symptoms and improve survival by reducing signal transduction via
β-ARs (see Bristow, 2000).
As heart failure progresses, administration of diuretics is essential
for alleviating fluid retention (Hunt et al., 2009). Despite the success
of pharmacological agents that reduce pathological remodeling,
mortality remains high, with approximately one third of patients
with heart failure dying within a year of diagnosis (McMurray &
Pfeffer, 2005). Thus, novel therapeutic strategies are needed to further
reduce mortality due to heart failure.
8.2. Novel therapeutic strategies for the treatment of heart failure
Current drug treatments usually delay heart failure progression
rather than regressing it. Thus, there is an urgent need for the
development of therapies that have the potential to improve function
of the failing heart. Identification of the molecular distinction between
pathological and physiological cardiac hypertrophy has provided a
new avenue for tackling this problem. An alternate strategy to directly
inhibiting pathological heart growth is to activate regulators of
physiological heart growth.
8.3. Benefits of activating physiological
signaling cascades in a setting of cardiac disease
delayed the onset of heart failure, and improved lifespan. Conversely,
decreasing PI3K(p110α) activity dramatically accelerated the progression of heart failure and lifespan was shortened (McMullen et al.,
2007). In a more recent study, reduction of PI3K(p110α) activity in
another transgenic mouse model of dilated cardiomyopathy (mammalian sterile 20-like kinase 1; (Yamamoto et al., 2003)) caused atrial
fibrillation that was associated with heart failure and premature death
(Pretorius et al., 2009a). Furthermore, PI3K(p110α) activity was
reduced in atrial appendages from patients who developed atrial
fibrillation, but not patients in sinus rhythm (Pretorius et al., 2009a).
Akt1 has also been shown to protect the heart in a setting of pressure
overload-induced hypertrophy. Akt1−/− displayed more hypertrophy
and cardiac dysfunction in response to aortic-banding (DeBosch et al.,
2006b). Finally, the PI3K-Akt pathway may also play a role in
protecting the heart against dysfunction in a setting of pressure
overload via an interaction with an intercalated disc protein, nectin-2
(McMullen 2009; Satomi-Kobayashi et al., 2009). Taken together,
these studies suggest that the IGF1-PI3K(p110α)-Akt1 pathway
protects the heart against cardiac insults (summarized in Table 5).
Mechanisms via which activation of the IGF1-PI3K(p110α)-Akt
cascade leads to protection against cardiac dysfunction and progression to heart failure include: anti-fibrotic properties, anti-apoptotic
properties, maintenance of proteins associated with cardiac contractile function (e.g. SERCA2a), and inhibition of pathological signaling
cascades (see Section 8.4) (McMullen, 2008).
Other novel therapeutic approaches that are currently under
investigation include statin therapy, vasopressin-receptor antagonists, inhibition of oxidative stress, improved Ca2+ handling,
prevention of apoptosis, and cardiac regeneration (Landmesser and
Drexler, 2005).
8.4. Possible advantages of activating physiological signaling cascades
Induction of physiological cardiac hypertrophy may be a potential
therapeutic strategy for the treatment of heart failure (see Pretorius et
al., 2008; Owen et al., 2009). Regular physical activity protects against
cardiovascular disease, and exercise training in stable chronic heart
failure patient groups is safe and beneficial (Scheuer et al., 1982;
Jennings et al., 1986; Nelson et al., 1986; Schaible et al., 1986;
Jennings, 1995; Orenstein et al., 1995; Coats, 2000; Konhilas et al.,
2006). In animal studies, exercise training was shown to reverse
functional and molecular abnormalities associated with cardiac
pathology (Scheuer et al., 1982; Schaible et al., 1986; Orenstein et
al., 1995; McMullen et al., 2003; Konhilas et al., 2006; McMullen et al.,
2007). As described in Section 3.1.1, the IGF1-PI3K(p110α)-Akt
pathway is a critical mediator of exercise-induced physiological
hypertrophy. Studies from genetic mouse models have highlighted
the beneficial effects of this pathway in settings of cardiac stress
(Table 5).
Over-expression of IGF1 or IGF1R was beneficial in models of
pressure overload (McMullen et al., 2004b), dilated cardiomyopathy
(Welch et al., 2002), myocardial infarction (Li et al., 1997), decompensated eccentric hypertrophy (Li et al., 1999), and diabetic
cardiomyopathy (Kajstura et al., 2001; Huynh et al., 2010). caPI3K
mice maintain normal cardiac function and are protected from
developing pathological hypertrophy and fibrosis when subjected to
pressure overload (McMullen et al., 2007). In contrast, dnPI3K mice
with decreased cardiac PI3K(p110α) activity had depressed cardiac
function and increased fibrosis after pressure overload (McMullen et
al., 2003; McMullen et al., 2007). Further supporting evidence of a
protective role of PI3K(p110α) came from genetically crossing caPI3K
and dnPI3K mice with a transgenic mouse model of dilated
cardiomyopathy (DCM-Tg) (McMullen et al., 2007). Under basal
conditions, DCM-Tg mice had ventricular dilation, impaired systolic
function, congestive heart failure, and premature death (Buerger et al.,
2006; McMullen et al., 2007). Increasing PI3K(p110α) activity in
hearts of this model by crossing DCM-Tg with caPI3K transgenic mice,
A dual action of physiological signaling was recently identified.
Studies utilizing PI3K(p110α) transgenic and Akt1 knockout mice
demonstrated that the PI3K(p110α)-Akt1 pathway can inhibit
pathological growth in addition to promoting physiological growth.
dnPI3K transgenic mice and Akt1 knockout mice showed an
exaggerated hypertrophic response to pressure overload whereas
IGF1R and caPI3K showed a blunted response (McMullen et al., 2004a;
DeBosch et al., 2006b; McMullen et al., 2007). The PI3K(p110α)-Akt1
pathways appear to inhibit pathological growth by inhibiting
signaling proteins downstream of GPCR, including ERK1/2 and PKCβ
(Fig. 5) (DeBosch et al., 2006b; McMullen et al., 2007; Rigor et al.,
2009).
8.5. Activating the IGF1-PI3K pathway in a clinical setting
Epidemiological studies in the general population suggest that
serum IGF1 levels in the lower normal range are associated with
increased risk of acute myocardial infarction, ischemic heart disease
and heart failure (Juul et al., 2002; Vasan et al., 2003; Laughlin et al.,
2004). This is consistent with findings from patients with growth
hormone deficiency (see review (Colao, 2008)). Patients with
hypopituitarism had a 2-fold higher risk of dying from cardiovascular
disease compared with healthy controls (Rosen and Bengtsson, 1990;
Tomlinson et al., 2001). Furthermore, growth hormone deficiency has
been associated with decreased cardiac size and impairment in
cardiac function (Colao et al., 2002; Shulman et al., 2003; Salerno et
al., 2004). In support of the suggestion that these cardiac effects are
due to growth hormone deficiency, multiple studies have reported
that growth hormone replacement in these patients increases heart
size and preserves/improves cardiac function (see review (Shulman et
al., 2003; Salerno et al., 2004; Colao, 2008)). However, of note,
patients with acromegaly (growth hormone excess) develop cardiac
hypertrophy that has been associated with cardiac dysfunction.
B.C. Bernardo et al. / Pharmacology & Therapeutics 128 (2010) 191–227
Furthermore, while growth hormone and IGF1 have been considered
as potential therapeutic agents in patients with heart failure, results
have been conflicting (see Colao et al., 2001).
While substantial evidence shows the benefits of activating the
IGF1-PI3K(p110α) pathway in the heart (see Section 8.3), there are
challenges in targeting PI3K(p110α) directly because of its numerous
actions in various cell types. Of particular note, PI3K(p110α) permits
cancer cells to bypass normal growth-limiting controls (McMullen &
Jay, 2007). Thus, it is of interest to identify downstream targets
regulated by PI3K(p110α) that may represent more specific therapeutic targets. We have recently identified cardiac-selective miRNAs
and mRNAs regulated by PI3K(p110α) (Lin et al., 2010). Modulation
of these targets are likely to be better tolerated in patients than
activating PI3K(p110α) directly.
9. Summary
The generation and characterization of transgenic and knockout
mice have allowed investigators to use a reductionist approach to
delineate molecular mechanisms that are responsible for mediating
distinct forms of heart growth. Further progress has been accomplished by subjecting these models to pathological and physiological
stimuli. Recognition of distinct mechanisms responsible for the
induction of pathological and physiological cardiac hypertrophy has
provided new possibilities for drug discovery. Identification of unique
regulators of physiological heart growth may lead to the development
of innovative pharmacotherapies in the clinical management of heart
failure. A better understanding of the mechanisms responsible for the
development of concentric and eccentric hypertrophy, gender
differences, and the key mechanisms responsible for the transition
from hypertrophy to heart failure will also be important for the
development of new and improved therapeutics with an ability to
improve function of the failing heart as opposed to delaying disease
progression.
Acknowledgments
We acknowledge funding support from the National Health and
Medical Research Council of Australia (NHMRC) and the National
Heart Foundation of Australia. KLW and LP are supported by
Australian Postgraduate Awards and Baker IDI Foundation Postgraduate Awards. JRM is supported by an Australian Research Council
Future Fellowship and holds an Honorary NHMRC Research Fellowship. We also thank Nelly Cemerlang and Joon Win Tan for
administrative support.
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