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The Ne w E n g l a nd Jo u r n a l o f Me d ic i ne

Review Articles

Mechanisms of Disease
F R A N K L I N H . E P S T E I N , M. D. , Editor





EART failure is a leading cause of mortality

in the United States. As a result of advances
in genetic technology, a molecular basis of
heart failure is emerging.1,2 This review highlights the
ways in which these insights are leading to new therapeutic targets in patients with acquired forms of
heart failure.

Myocardial hypertrophy is an early milestone during the clinical course of heart failure and an important risk factor for subsequent cardiac morbidity and
mortality. In response to a variety of mechanical, hemodynamic, hormonal, and pathologic stimuli, the
heart adapts to increased demands for cardiac work
by increasing muscle mass through the initiation of
a hypertrophic response. At the cellular level, cardiac
myocytes respond to biomechanical stress by initiating several different processes that lead to hypertrophy (Fig. 1). The so-called physiologic hypertrophy
that occurs in elite athletes is associated with proportional increases in the length and width of cardiac myocytes. By contrast, the assembly of contractile-protein units in series characterizes the eccentric
hypertrophy that occurs in patients with dilated cardiomyopathy, with a relatively greater increase in the
length than in the width of myocytes. During pressure overload, new contractile-protein units are assembled in parallel, resulting in a relative increase in

From the University of California San DiegoSalk Institute Program in

Molecular Medicine, Department of Medicine and Center for Molecular
Genetics, University of California San Diego School of Medicine, La Jolla,
Calif. Address reprint requests to Dr. Chien at the Department of Medicine, 0613-C, University of California San Diego, 9500 Gilman Dr., La
Jolla, CA 92093, or at
1999, Massachusetts Medical Society.


the width of individual cardiac myocytes and therefore in concentric hypertrophy. In hypertrophic cardiomyopathy, mutant contractile proteins lead to
myofibrillar disarray and secondary hypertrophy of
myocytes. In most forms of cardiac hypertrophy, there
is an increase in the expression of embryonic genes,
including the genes for natriuretic peptides and fetal
contractile proteins.3 The induction of the natriuretic peptide genes is a feature of hypertrophy in all
mammalian species and is a prognostic indicator of
clinical severity. Recently, evidence of the loss of myocytes as a result of programmed cell death (apoptosis) has also been reported in both experimental
and clinical cardiac hypertrophy (Fig. 1).

Cardiac hypertrophy and failure are highly complex

disorders that arise as a result of a combination of
genetic, physiologic, and environmental factors. The
identification of mutations involving a single gene
that are responsible for inherited forms of hypertrophic cardiomyopathy, dilated cardiomyopathy, and
ventricular arrhythmogenesis (the long-QT syndrome)
has allowed us to pinpoint several of the initiating
events that can lead to features of heart failure in humans.4,5 There is still a broad gap, however, between
identifying the defective gene and understanding how
this defect leads to the cardiac abnormalities. In this
regard, in vitro assays of cardiac muscle cells and studies of genetically engineered animals are beginning
to identify the points in cardiac growth signaling that
cause these distinct forms of cardiac hypertrophy and
Assays of Cardiac-Muscle Cells

The ability to culture primary cardiac myocytes

has resulted in the availability of a well-characterized
in vitro system in which to study the hypertrophic
response. Although they are based on neonatal cardiac-muscle cells, studies of these cultures have led
to the identification of signaling pathways that activate cellular responses known to occur during hypertrophy in vivo, including an increase in cell size,
an increase in the expression of embryonic genes, and
the accumulation and assembly of contractile proteins.6 By altering the expression of specific genes in
cultured cardiac myocytes, peptide hormones, growth
factors, and cytokines have been identified that can
activate specific features of the hypertrophic response
(Table 1).7 Among the most extensively characterized
of these substances are endothelin and angiotensin
II, insulin-like growth factor I, and other growth

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Physiologic hypertrophy

Normal muscle cell

Concentric hypertrophy

Increased expression7
of embryonic genes

Eccentric hypertrophy
Sarcomeric disorganization

Figure 1. Morphology of Ventricular Muscle Cells in Cardiac Hypertrophy and Failure.

Phenotypically distinct changes in the morphology of myocytes occur in response to various growth stimuli. The expression of
embryonic genes such as natriuretic peptides is increased in both eccentric and concentric hypertrophy, but not in physiologic hypertrophy, in response to exercise. Myofibrillar disarray (sarcomeric disorganization) is typical of hypertrophic cardiomyopathies;
this disorganization is focal and is accompanied by more widespread increases in the cross-sectional area of myocytes.





Inhibition of pathologic hypertrophy




Antagonists of Gqa-dependent receptors

Inhibitors of intracellular kinase

Promotion of physiologic hypertrophy

Inhibition of neurohumoral overstimulation
Enhancement of contractile and
relaxation responses

Growth hormone
Insulin-like growth factor I



Angiotensin II receptor
Endothelin-1 receptor
? Novel receptors
Antagonists of ras, p38, and c-jun
N-terminal kinase (JNK)
? Novel kinases
Growth-hormone receptor
Insulin-like growth factor I receptor
b1-Adrenergic receptor

Relief of inhibition of sarcoplasmic retic- Phospholamban inhibitors

ulum calcium ATPase
Agents that counteract the desensitiza- Inhibitors of b-adrenergicreceptor kinase
tion of G proteincoupled receptor
Relief of energy deprivation
Angiogenic growth factors
Vascular endothelial-derived growth factor
Fibroblast growth factor 5
Agents involved in angiogenesis
? Others
Inhibition of pathways of apopto- Promoters of myocyte survival
gp130 ligands (e.g., cardiotrophin 1)
sis of myocytes
Inhibitors of apoptosis
Caspase inhibitors
Inhibitors of cytokines
Tumor necrosis factor a, ? others
*A more complete description of each of these classes has been published.7

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Pressure overload
Interleukin-6 family of cytokines

LIF receptor

Cardiac myocyte

Hypertrophic signals.
(ras, Gqa, p38b)

Organization of sarcomeres .
Increased expression of.
embryonic genes

Compensatory hypertrophy

Apoptotic signals.
(Gqa, p38a)


Heart failure

Figure 2. Pathways Involved in Hypertrophy, Apoptosis, and Survival of Myocytes during the Transition between Cardiac
Hypertrophy and Heart Failure in Response to Biomechanical Stress.
Biomechanical stress, such as chronic hypertension and pressure overload, activates multiple parallel and converging
signals for hypertrophy and apoptosis, which represent two distinct outcomes. At the same time, biomechanical stress
also leads to the induction of gp130-dependent ligands, such as cardiotrophin 1. This cytokine binds to its receptor,
which consists of gp130LIF (leukemia inhibitory factor) receptor heterodimers, resulting in the activation of downstream gp130 pathways that block the actions of apoptotic pathways. In the absence of gp130, the response of cardiac
myocytes to biomechanical stress is shifted toward apoptosis, resulting in the loss of functional myocytes and the onset
of heart failure. Thus, the outcome of biomechanical stress is dependent on the balance between these two contradictory signal-transduction pathways.

factors that activate either heteromeric (Gq) or lowmolecular-weight guanosine triphosphate (GTP)
binding protein (ras) signaling pathways, as well as
cardiotrophin 1 and other members of the interleukin-6 cytokine family that activate cellular responses
by means of the transmembrane signal transducer
gp130. A relatively distinct pattern of cardiac cellular
responses has been associated with each of these
substances, implying that their actions are specific.
To a certain extent, this specificity reflects the activation of different downstream intracellular kinase
cascades that stimulate the appearance of specific features of myocardial-cell hypertrophy (Fig. 2). Study
of these downstream signaling pathways has identified kinases that generate primarily hypertrophic, apoptotic, and anti-apoptotic signals,8-10 as well as kinases that regulate the assembly of myofilaments (rho
kinase).11 In addition, nuclear signaling proteins have
been found that activate and suppress various cardiac
genes during hypertrophy.7

Cardiac Hypertrophy and Failure

in Genetically Altered Animals

Mice have a heart rate of over 500 beats per

minute and an aorta that is 1 mm in diameter, but
they are a valid model for studying both pressureoverload hypertrophy and heart failure, because of
the similarities of these disorders in mice and humans.12-14 The ability to engineer precise mutations
in the heart, coupled with the ability to quantitate
the effects of these mutations on cardiac function
in vivo,15,16 has led to the recognition of a previously unsuspected set of signaling pathways and
molecules that stimulate specific aspects of cardiac
growth. The effects of both the overexpression and
the loss of individual cardiac genes in animals have
been studied, and we will describe examples of each.
These models are useful not simply because they
replicate human disease but also because they allow
the differentiation of the many different processes
that together cause such conditions as pressure-

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overload hypertrophy and congestive heart failure in

In the tissue-restricted approach to overexpression,
the regulatory region from a cardiac-specific gene is
fused to a candidate gene of interest and used to
produce transgenic mice that express the candidate
gene specifically in cardiac-muscle cells because of
the ability of the regulatory sequences to restrict expression to the heart.17 The availability of well-characterized regulatory regions of cardiac-specific genes
has allowed the expression of candidate signaling molecules in the heart and even in the ventricles alone.
For example, transgenic mice have been produced
that express an active mutant of ras, a protein that
mediates many growth-related responses in cardiac
myocytes as well as in cancer cells.18 Since the regulatory sequences of a ventricle-specific gene control
the expression of this active form of ras, the heart is
the only tissue in the animals in which the ras pathways are activated.19,20 High levels of expression of
ras result in hypertrophic cardiomyopathy, including
massive cardiac hypertrophy, heart failure, and sudden death, but not the dilatation of any heart chamber (unpublished data). Increased concentrations of
ras messenger RNA were recently described in endomyocardial-biopsy specimens from humans with
familial hypertrophic cardiomyopathy.21
It is possible to disrupt, or target, a gene of interest in a mouse by replacing it with a mutated sequence
early in embryogenesis.22 Mice that are heterozygous
for the mutated allele can be mated to produce homozygous mice that do not have a functional copy
of the targeted gene. For example, mice with deletions of a muscle-restricted cytoskeletal protein have
features of dilated cardiomyopathy,23 a finding that
supports a causative role for disrupted cytoskeleton
components in the pathogenesis of cardiomyopathy.
Since many structural and signaling components of
cardiac myocytes are common to other tissues, gene
targeting may be lethal; the animals may die from
defects in other tissues before the role of the gene
in the heart can be studied. This difficulty can now
be avoided by techniques to engineer heart-specific
gene deletions.24,25

The extent of ventricular hypertrophy in patients

is a powerful predictor of adverse events. Accordingly, identifying the signals that mediate the pathways
from mechanical stress to downstream cellular events
has been a major area of interest. Both myocytes and
nonmyocytes are direct biomechanical sensors of hemodynamic load. Growth signals are generated by
the release of growth factors and cytokines, which
lead to a regionally localized response. The factors
that have been implicated in this response include
peptides that stimulate G proteincoupled receptors

(endothelin-126,27), angiotensin II,28,29 interleukin-6

related cytokines (cardiotrophin 130,31), and growth
factors that activate receptor tyrosine kinases (insulin-like growth factor I32,33).
One of the first genetically defined models of concentric ventricular hypertrophy resulted from cardiacdirected overexpression of the a1b-adrenergic receptor.34 This confirmed previous work in cultured
cardiac myocytes demonstrating that a-adrenergic
stimulation induced a hypertrophic response. a-Adrenergic receptors share common intracellular signaling pathways with other hypertrophic growth factors, including angiotensin II and endothelin-1. In
each of these pathways, signaling that results in hypertrophy proceeds by means of the Gqa subunit
of heteromeric G protein, which was found to be
both necessary and sufficient to cause hypertrophy
in cultured cardiac-muscle cells.35 Subsequently, overexpression of Gqa itself was found to induce both
a hypertrophic and an apoptotic response.36,37 Furthermore, a protein inhibitor of Gqa, whose expression was also targeted to the heart by transgenic
techniques, had no effect on cardiac structure or function in unstressed mice, but it prevented hypertrophy when pressure overload was induced by constricting the ascending aorta.38 Taken together, these
results suggest that Gqa-dependent pathways have a
critical role in the development of myocardial hypertrophy (Fig. 2).
The activation of cell-surface receptors and their
immediate signaling targets, such as ras and Gqa, by
cardiac growth factors is the first step in initiating the
growth of myocytes (Fig. 2). Increases in intracellular calcium concentrations in response to these growth
factors may also activate calmodulin-dependent pathways.39-41 According to in vitro and in vivo results,
the primary downstream effectors are the mitogenactivated protein kinases, including c-jun N-terminal
kinase and p38.7,9,10,31 These kinases are particularly
important switches in the pathways between apoptosis and adaptive hypertrophy. For example, in mice
p38 mitogen-activated protein kinases are strongly
activated by pressure overload, and upstream kinases
that specifically activate p38 cause the growth of cultured myocytes. However, the activation of p38 is
also accompanied by an increase in the rate of apoptosis.9 The two isoforms of p38, a and b, have opposite effects on apoptosis when stimulated by upstream
activators: p38a increases apoptosis, whereas p38b
inhibits it (Fig. 2).9

Dilated cardiomyopathy represents a final common

pathway of the myocardium in response to many different pathologic conditions. This has led to the obvious conclusion that there are common pathways to
cardiac dilatation and failure. Local myocardial injuVol ume 341

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ry can cause progressive and sometimes deleterious

dilatation and thinning of the ventricular wall.
In about 25 percent of patients with idiopathic dilated cardiomyopathy, the disorder is familial and genetic, and it is likely to be genetic in some nonfamilial cases as well.4 Indeed, the first example of familial
dilated cardiomyopathy for which the genetic basis
was defined was Duchennes muscular dystrophy. In
this and related muscular dystrophies, the molecular
defect is in the dystrophindystroglycanlaminin
transmembrane complex that connects the actin cytoskeleton of the muscle cells to structural proteins
that are synthesized by fibroblasts surrounding the
myocytes (Fig. 3). In these dystrophies, there is an
impairment of the normal linkage by which force
generated by individual myocytes is translated into
work done by the muscle tissue as a unit, and excessive stresses on individual myocytes cannot be spread
across that muscle.
The recent demonstration that the molecular defect in Syrian hamsters with cardiomyopathy lies in
the d-sarcoglycan component of this complex42 further implicates the linkage between the myocyte cy-

toskeleton and the extracellular matrix in the pathogenesis of cardiomyopathy. Moreover, a molecular
defect involved in familial dilated cardiomyopathy in
humans has been mapped to the cytoskeletal region
of the cardiac actin gene.43 One of the first examples
of a genetic link between the cytoskeleton and dilated cardiomyopathy was provided by studies of mice
that have a deficiency in a muscle-specific LIM (lin-1,
ISL-1, and mec-3) domain protein23 and have many
features of the dilated cardiomyopathy that occurs in
humans. This cytoskeletal protein may be a component of a biomechanical sensor pathway that transduces hemodynamic force into specific signaling responses. Disruption of other cytoskeletal proteins,
such as desmin, plakoglobin, and N-cadherin, results
in cardiac dilatation and impaired cardiac function
during fetal development or after birth. In summary,
increased biomechanical stress on cardiac myocytes,
either through genetic abnormalities or through excessive stress on the chamber wall due to myocyte loss
or severe hemodynamic loading, generates a persistent signal for ventricular growth and hypertrophy.2,44
By contrast, mutations in sarcomeric proteins cause

Extracellular matrix




g a b






Figure 3. Primary Structural Components of the Linkage between the Cytoskeleton and the Extracellular Matrix, Including Actin, the DystrophinGlycoprotein Complex, and Laminin-2 (Merosin).
Genetic defects in these components lead to dilated cardiomyopathy, with or without associated skeletal myopathy.
This complex is physically associated with the Z-disk of cardiac myocytes, the Z-disk components desmin (associated
with dilated cardiomyopathy in humans and mice) and a-actinin, and a muscle-specific cytoskeletal protein (MLP) (associated with dilated cardiomyopathy in mice).23 The question mark indicates an unknown factor.


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hypertrophic cardiomyopathy but do not affect ventricular systolic function.

Apoptosis of Cardiac Myocytes

Apoptosis is a mechanism by which cells can be

eliminated without an inflammatory response. Evidence of an increased rate of apoptosis has been detected in failing hearts at the time of transplantation
in humans, as well as in hearts from animals with experimentally induced hypertrophy and cardiomyopathy. Unlike necrosis, apoptosis leaves little or no
histologic trace of the lost cells. Accordingly, documenting its occurrence and estimating the extent of
the loss of myocytes as a result of apoptosis have
been problematic; therefore, the importance of apoptosis in the transition from compensatory hypertrophy to heart failure has been unclear.
At the cellular level, there is normally a balance between apoptotic and anti-apoptotic signals, and cell
death occurs in response to a persistent shift in this
balance. The cytokine tumor necrosis factor a, acting through its receptor, activates both apoptotic
and anti-apoptotic signals, with a tendency toward
promoting apoptosis. Similarly, p21 ras induces both
apoptotic c-jun N-terminal kinase and anti-apoptotic
1-phosphatidylinositol 3-kinase signals. Among mitogen-activated protein kinases, the extracellular signal
regulated kinases tend to be anti-apoptotic, c-jun
N-terminal kinase promotes apoptosis,8 and as mentioned, the a and b isoforms of p38 have opposing

effects (Fig. 2).9 Cell death may occur when the apoptotic forces exceed a certain threshold. The activation of apoptotic signals during the hypertrophic
response of myocytes may explain the risk of death
associated with ventricular hypertrophy in humans.
In support of this concept, mice with a loss-of-function mutation in the cytokine receptor gp130 of the
ventricular chamber have normal cardiac structure but
have massive cardiac apoptosis accompanied by rapidly progressive dilated cardiomyopathy when subjected to pressure overload.25 These studies indicate
that the inhibition of apoptosis by gp130-dependent
pathways in myocytes has a critical role in the transition between compensatory hypertrophy and overt
heart failure and suggest that the balance between apoptotic and hypertrophic pathways determines whether chamber dilatation will occur (Fig. 2).25
Cardiac Function and Contractility

The b-adrenergicreceptor pathway is a critical

point of control for cardiac contractility in both normal and failing hearts (Fig. 4). The primary functional disturbance in dilated cardiomyopathy is impaired contractility, yet when contractility is decreased
in mice by overexpression of the calcium-regulatory
protein phospholamban, the mass and volume of the
cardiac chamber are no different from those in normal mice.46 Moreover, when contractility is decreased,
as in mice with mutations in the myosin heavy chain,
the result is hypertrophic cardiomyopathy, without

b-Adrenergic receptor

Adenylyl cyclase


Cyclic AMP.




receptor kinase

Calcium pump
Figure 4. Regulation of the Contractile Function of Myocytes.
The contractile function of myocytes is regulated by changes in calcium flux into and out of the sarcoplasmic reticulum.
Activation of b-adrenergic receptors leads to increased uptake of calcium into the sarcoplasmic reticulum by the calcium
pump; the phosphorylation (P) of phospholamban by cyclic AMP-dependent protein kinase (PKA) removes its tonic inhibition of the calcium pump. b-Adrenergic receptors are desensitized in both heart failure and maladaptive hypertrophy; a substantial component in this desensitization is up-regulation of the b-adrenergicreceptor kinase (bARK). A deficiency of phospholamban has recently been shown to halt the progression of heart failure and dilated cardiomyopathy
in a genetically based animal model.45

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chamber dilatation.47 These observations, as well as

clinical and experimental studies of b-adrenergic
blocking drugs in patients with heart failure, suggest
that impaired contractility in certain forms of dilated
cardiomyopathy may be a secondary phenomenon,
perhaps resulting from alterations in energy metabolism or intracellular calcium handling.
Animals have been developed that have increased
ventricular contractile function as a primary feature.
These include mice that overexpress b1- and b 2-adrenergic receptors48,49 or the Gsa protein to which it is
coupled50; mice that overexpress a peptide inhibitor
of the b-adrenergicreceptor kinase, the principal
desensitizer of b-adrenergic receptors51; and mice in
which the phospholamban gene has been disrupted.52
The increased risk of death among patients with heart
failure that is associated with chronic stimulation of
b-adrenergic agonists can be replicated in mice with
dilated cardiomyopathy due to a cytoskeletal gene
mutation.23 The offspring of genetic crosses between
these mice 23 and those overexpressing b 2-adrenergic
receptors49 have a very high mortality rate.53 However, a genetic cross between the cardiomyopathic
mice 23 and those overexpressing the peptide inhibitor of the b-adrenergicreceptor kinase results in a
mouse with decreased chamber dimensions and improved contractile function.53 This suggests that the
deleterious effect of long-term exposure to inotropic
drugs, such as phosphodiesterase inhibitors, in patients with heart failure may not be due to changes
in contractility alone.53 The different effects of overexpression of the b 2-adrenergic receptor and overexpression of the inhibitor of the b-adrenergicreceptor kinase might also reflect differing downstream
effects on cardiac relaxation,54 or pathologic effects
of chronic b-adrenergic overstimulation48 as compared
with those resulting from relief of desensitization.53
In this regard, phospholamban negatively regulates
the uptake of calcium by the sarcoplasmic reticulum
(Fig. 4), and a deficiency of phospholamban can halt
progression of dilated cardiomyopathy and heart failure.54 b-Adrenergic pathways lead to the phosphorylation of phospholamban, which reduces its activity and increases ATPase activity in the sarcoplasmic

The decrease in cardiac performance in the failing

heart may be a consequence of alterations in specific
signaling molecules and their downstream pathways
in individual myocytes (Table 1). By analogy to carcinogenesis, heart failure may be viewed as a progressive, multistep process involving physiologic and
molecular initiators, promoters, suppressors, and effectors of the chronic course to heart-muscle failure.7
Further unraveling of the signals that cause specific
features of heart failure, coupled with the growing
human genome data base, should ultimately lead to

the identification of targets whose actions could be

interrupted, thereby halting or perhaps reversing clinical deterioration.
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Vol ume 341

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