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

S IGNALING P ATHWAYS FOR C ARDIAC H YPERTROPHY AND F AILURE

AND

JOHN J. HUNTER, M.D., KENNETH R. CHIEN, M.D., PH.D.

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).
GENETIC METHODS OF STUDYING CARDIAC HYPERTROPHY AND FAILURE

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.
MORPHOLOGIC CLASSIFICATION OF CARDIAC HYPERTROPHY

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

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 failure.
Assays of Cardiac-Muscle Cells

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 kchien@ucsd.edu. 1999, Massachusetts Medical Society.

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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Apoptosis

Physiologic hypertrophy

Growth stimuli Normal muscle cell Concentric hypertrophy

Increased expression 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.

TABLE 1. POTENTIAL THERAPEUTIC

AND

MOLECULAR TARGETS IN HEART FAILURE.*

TYPE DRUGS

IN THE

SIGNALING PATHWAYS INVOLVED

GOAL

OF

MOLECULAR TARGET

Inhibition of pathologic hypertrophy

Antagonists of Gqa-dependent receptors Inhibitors of intracellular kinase cascades

Promotion of physiologic hypertrophy Inhibition of neurohumoral overstimulation Enhancement of contractile and relaxation responses

Growth hormone Insulin-like growth factor I Beta-blockers

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 kinases 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 Neuregulin 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 gp130

Cardiac myocyte

LIF receptor

Hypertrophic signals (ras, Gqa, p38b)

Apoptotic signals (Gqa, p38a)

Organization of sarcomeres Increased expression of embryonic genes

Apoptosis

Compensatory hypertrophy

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
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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 humans. 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
PRESSURE OVERLOAD AND CONCENTRIC HYPERTROPHY

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
CHAMBER DILATATION AND DILATED CARDIOMYOPATHY

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 Numb e r 17

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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

Laminin-2

Sarcoglycans

a
Dystroglycans

g a b

a-Actinin Cytoskeletal a-actin

MLP ?

Z-disk
Dystrophin

Syntrophins

Desmin

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

GSa

Adenylyl cyclase

Sarcoplasmic reticulum

Cyclic AMP

PKA

P
Phospholamban

b-Adrenergic receptor kinase

Calcium

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 reticulum.
CONCLUSIONS

the identification of targets whose actions could be interrupted, thereby halting or perhaps reversing clinical deterioration.
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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
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