I. INTRODUCTION

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Cardiomyopathies are diseases of heart muscle that are associated with cardiac dysfunction. Heart failure due to cardiomyopathy represents a major health problem in our society. Although increased emphasis on prevention programs and new therapeutic agents have resulted in improved survival from acute coronary artery disease, there has been a dramatic increase in morbidity and mortality from heart failure, which has been described as the emerging health epidemic of the 21st century.

Cardiomyopathies are classified traditionally according to morphological and functional criteria into four categories: hypertrophic cardiomyopathy, dilated cardiomyopathy, arrhythmogenic right ventricular dysplasia, and restrictive cardiomyopathy (135). These cardiomyopathies can be primary myocardial disorders or develop as a secondary consequence of a variety of conditions, including myocardial ischemia, inflammation, infection, increased myocardial pressure or volume load, and toxic agents. The pathogenesis of primary cardiomyopathies has been poorly understood. Over the last decade, the importance of gene defects in the etiology of primary cardiomyopathies has been recognized. Autosomal dominant, autosomal recessive, X-linked, and maternal patterns of inheritance have been observed. Families with inherited cardiomyopathies have provided a unique resource for studies of the genetic basis of these disorders. Molecular genetic studies performed to date have focused largely on monogenic inherited cardiomyopathies, i.e., caused by mutations in a single gene. Although relatively uncommon, these monogenic disorders enable pathophysiological processes applicable to a wide range of more commonly occurring heart diseases to be evaluated. The usefulness of studying monogenic disorders was first demonstrated by Brown and Goldstein (19) in their pioneering work on the role of the low-density lipoprotein receptor in atherosclerosis. In 1989, Christine and Jon Seidman and co-workers (63) reported the first association between an inherited gene defect and a primary cardiomyopathy. In a subsequent study, they showed that mutations in the -myosin heavy chain gene were responsible for familial hypertrophic cardiomyopathy (40). Since then, substantial progress has been made in elucidating further gene defects in hypertrophic cardiomyopathy. More recently, disease-causing genes have been identified in dilated cardiomyopathy, arrhythmogenic right ventricular dysplasia, and restrictive cardiomyopathy. Although these genetic studies have enabled molecular triggers of cardiomyopathies to be identified, the functional consequences of gene mutations and precise details of the signaling pathways that lead to hypertrophy, dilation, and contractile failure remain to be elucidated. Current concepts of the molecular genetic basis of inherited cardiomyopathies are summarized in this review.

	II. CARDIOMYOCYTE CELLULAR PHYSIOLOGY

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A.  Cardiac Myocyte Structure

1.  Cardiac muscle fibers

Cardiac muscle fibers are comprised of separate cellular units (myocytes) connected in series. In contrast to skeletal muscle fibers, cardiac muscle fibers do not assemble in parallel arrays but bifurcate and recombine to form a complex three-dimensional network and have centrally, rather than peripherally, positioned nuclei. Cardiac myocytes are joined at each end to adjacent myocytes by specialized areas of interdigitating cell membrane, the intercalated discs. The intercalated discs are comprised of adherens junctions (containing N-cadherin, catenins, and vinculin), desmosomes (containing desmin, desmoplakin, desmocollin, desmoglein), and gap junctions (containing connexins).

Cardiac myocytes are surrounded by a thin membrane, the sarcolemma. The interior of each myocyte contains bundles of longitudinally arranged myofibrils that have a characteristic striated appearance, similar to that of skeletal muscle, formed by repeating sarcomeres. The sarcomere is the fundamental structural and functional unit of cardiac muscle that is comprised of interdigitating thick and thin filaments (Fig. 1). The thick filaments are composed predominantly of myosin and also contain the myosin binding proteins C, H, and X. The thin filaments are composed of cardiac actin, -tropomyosin, and troponins C, I, and T. Each sarcomere contains an A band (comprised of overlapping thick filaments and thin filaments), with a central M line (comprised of thick filaments only). At each side of the A band are I bands (comprised of thin filaments only) bounded by Z lines.

View larger version (82K): [in this window] [in a new window]   Fig. 1. Schematic of the components of cardiac myocyte structure. The sarcomere, comprised of interdigitating thick and thin filaments, is the fundamental structural and functional unit of cardiac muscle. Sarcomeres are linked by a complex network of cytoskeletal proteins with the sarcolemma, extracellular matrix, and nucleus. The intermyofibrillar cytoskeleton is comprised of desmin intermediate filaments, actin-containing microfilaments, and microtubules. The subsarcolemmal cytoskeleton is comprised of costameres, sites of interconnection between the various pathways linking sarcomeres to sarcolemmal transmembrane proteins. Muscle contraction is an energy-requiring process that utilizes ATP supplied by mitochondria. The intracellular free Ca2+ concentration ([Ca2+]i) critically regulates muscle contraction and relaxation. Muscle contraction is initiated by an increase in [Ca2+]i. This results from depolarization-induced influx of a small amount of Ca2+ via voltage-gated dihydropyridine receptors (DHPR) in t tubules, which in turn triggers the release of a large quantum of Ca2+ from the sarcoplasmic reticulum (SR), a process called Ca2+-induced Ca2+ release. Muscle relaxation is initiated by the uptake of cytosolic Ca2+ into the SR via the high energy-dependent pump, the SR Ca2+-ATPase (SERCA2a) and by extrusion through the sarcolemmal Na+/Ca2+ exchanger. Pathophysiological mechanisms that have been implicated in hypertrophic and dilated cardiomyopathies include the following: defective force generation, due to mutations in sarcomeric protein genes; defective force transmission, due to mutations in cytoskeletal protein genes; myocardial energy deficits, due to mutations in ATP regulatory protein genes; and abnormal Ca2+ homeostasis, due to altered availability of Ca2+ and altered myofibrillar Ca2+ sensitivity. RyR, ryanodine receptor.

The sarcomeric cytoskeleton, comprised of titin and the myomesins, provides a scaffolding for the thick and thin filaments. Titin is a giant protein that spans from the Z line to the M line. As well as contributing to sarcomere assembly and organization, titin is a major determinant of the elastic properties of the cardiac myofibril. Myomesins 1 and 2 are titin-associated proteins. The Z disc (at the Z line) is formed by a lattice of interdigitating proteins that maintain myofilament organization by cross-linking antiparallel titin and thin filaments from adjacent sarcomeres. The Z-disc proteins include -actinin, filamin, nebulette, telethonin, and myotilin.

The extrasarcomeric cytoskeleton is a complex network of proteins linking the sarcomere with the sarcolemma and the extracellular matrix. It provides structural support for subcellular structures and transmits mechanical and chemical signals within and between cells. The extrasarcomeric cytoskeleton has intermyofibrillar and subsarcolemmal components (Fig. 1).

The intermyofibrillar cytoskeleton is comprised of intermediate filaments, microfilaments, and microtubules. Desmin intermediate filaments form a three-dimensional scaffold throughout the extrasarcomeric cytoskeleton. Desmin filaments surround the Z discs and form longitudinal connections to adjacent Z discs and lateral connections to subsarcolemmal costameres. Microfilaments composed of nonsarcomeric actin (mainly -actin) also form a complex network linking the sarcomere (via -actinin) to various components of the costameres. Tubulin is a cytosolic protein that is present in a polymerized form (as microtubules) and in an unpolymerized form. Changes in the total amount of tubulin and the proportion of the polymerized form are thought to influence cytoskeletal stiffness and hence contractile function.

Costameres are subsarcolemmal domains that are located in a periodic, gridlike pattern, flanking the level of the Z lines and overlying the I bands, along the cytoplasmic side of the sarcolemma. The costameres are sites of interconnection between various cytoskeletal networks linking the sarcomere and the sarcolemma. They are thought to function as anchor sites for stabilization of the sarcolemma and for integration of pathways involved in mechanical force transduction. Costameres contain three principal components: the focal adhesion-type complex, the spectrin-based complex, and the dystrophin/dystrophin-associated glycoprotein complex. The focal adhesion-type complex is comprised of cytoplasmic proteins, including vinculin, talin, tensin, paxillin, and zyxin, that connect with cytoskeletal actin filaments and with the transmembrane proteins - and -integrin. The extracellular domains of the integrins interact with collagens, laminin, and fibronectin in the extracellular matrix. The spectrin-based complex contains ankyrin, that cross-links actin and spectrin, and spectrin, a component of the sarcolemma that interacts with actin, ankyrin, and desmin. The dystrophin/dystrophin-associated glycoprotein complex is made up of the cytoskeletal protein dystrophin, which binds to actin filaments, and the dystrophin-associated glycoprotein complex (- and -dystroglycans; -, -, -, -sarcoglycans; dystrobrevin; and syntrophin). The dystrophin-associated glycoprotein complex has cytoplasmic, transmembrane, and extracellular components. The cytoplasmic domain binds to the COOH-terminal region of dystrophin; the extracellular domain binds to laminin. Several actin-associated proteins are located at sites of attachment of cytoskeletal actin filaments with costameric complexes, including -actinin and the muscle LIM protein MLP.

B.  Mechanisms of Contraction

1.  Actin-myosin interaction

Currently, the universally accepted concept for the process of muscle contraction is the "sliding filament" theory, proposed by Huxley in 1957 (58). In this model, force generation is achieved by the sliding movement of the thick filaments relative to thin filaments, which is mediated by the cyclical attachment and detachment of myosin "cross-bridges" to actin. These cross-bridges consist of myosin heavy chain (MHC) molecules that project from the thick filament. Muscle myosin contains two MHC. Each MHC contains a head, with an actin binding site and ATPase site. The heads are linked at a "hinge" region to a long rod; that is an -helical coiled-coil dimer. The rod contains an elastic element and also binds myosin light chains (MLC). The "swinging cross-bridge" model was a subsequent modification of the sliding filament model, which hypothesized that a swinging motion of the myosin head was a critical factor for progression along the thin filament (60, 84). In the first step of this model, myosin is bound strongly to actin, with the myosin head orientated in a 45° position relative to its tail. ATP binding at the ATPase site causes rapid dissociation of myosin from actin and the formation of ADP and P_i. Actin recombines weakly with this myosin-ADP-P_i complex, with the myosin head orientated at a 90° angle. Release of ADP and P_i enables the strong actin binding position to be regained. The power stroke is achieved in this last step by a rowing-like movement of the myosin head as it "walks" down the actin filament. Each cross-bridge cycle is associated with hydrolysis of one ATP molecule. The "lever-arm" hypothesis was a subsequent modification of the swinging cross-bridge model, in which structural changes in the catalytic domain of the myosin head are amplified by rotation of the myosin tail, acting as a lever arm (57, 59, 131). In this model, the power stroke is produced not by movement of the myosin head (at the point of actin attachment) but by the pivoting movement of the myosin tail (at the hinge region). The lever arm rotation has been attributed to the transition between two conformations of the myosin head, open and closed, that influence nucleotide affinity and hydrolysis and that are determined by actin binding. It has subsequently been proposed that altered orientation of both the myosin head and the -helical tail may contribute to the overall displacement of actin. Conflicting data have been reported for the extent of actin displacement, with estimates ranging from 4-5 nm to 15-20 nm (59, 178). The number of attached states may be an important determinant of actin displacement. It is possible that a single myosin head may attach at two points on an actin molecule or that one or two myosin heads may be attached at any time point. Differences in compliance and load in the various model systems studied may also contribute to this range of data.

The troponin-tropomyosin complex is a Ca²⁺-sensitive switch that regulates actin-myosin interaction. The backbone of the thin filament is formed by a double helical array of globular actin molecules. Tropomyosin proteins assemble as -helical coiled-coil dimers that lie in a head-to-tail orientation within the major groove of the actin filaments, spanning seven actin monomers. The troponin complex (troponins T, I, and C) is anchored to tropomyosin predominantly by troponin T, and to a lesser extent, by troponin I. Troponin C interacts with both troponins T and I. During diastole, actin-myosin interaction is inhibited by the binding of troponin I to actin-tropomyosin. Ca²⁺ binding to troponin C induces a conformational change that weakens the interaction between troponin I and actin-tropomyosin and strengthens the interaction between troponin I and troponin C. These changes release the thin filament from its inhibitory state, promoting actin-myosin interaction and force generation. A reduction in the intracellular Ca²⁺ concentration ([Ca²⁺]_i) causes dissociation of Ca²⁺ from troponin C and restores the relaxed state (148).

The importance of Ca²⁺ in regulation of contraction in normal myocytes and in cardiomyopathies has increasingly been recognized (Fig. 1). During the cardiac action potential, [Ca²⁺]_i is increased initially by direct Ca²⁺ entry into cells, via voltage-gated L-type channels and to a lesser extent via Na⁺/Ca²⁺ exchange. This influx of Ca²⁺ triggers Ca²⁺ release from stores in the sarcoplasmic reticulum via ryanodine receptors and inositol 1,4,5-trisphosphate receptors. Increases in [Ca²⁺]_i promote Ca²⁺ binding to multiple cytosolic buffers, including troponin C. The magnitude of the systolic rise in [Ca²⁺]_i is determined not only by Ca²⁺ entry from the extracellular fluid and the sarcoplasmic reticulum, but also by the buffering capacity of the cell. During relaxation, Ca²⁺ is removed from the cytosol by the sarcoplasmic reticulum Ca²⁺-ATPase (SERCA2a), sarcolemmal Na⁺/Ca²⁺ exchange, sarcolemmal Ca²⁺-ATPase, and mitochondrial Ca²⁺ uniporter. A complex network of positive and negative feedback mechanisms maintains the [Ca²⁺]_i (8).

The Ca²⁺ dependence of force is generally expressed as a function of the free [Ca²⁺]_i. This relationship does not directly indicate the total amount of Ca²⁺ required to activate myofilaments, since changes in the sensitivity to Ca²⁺ may be present. For example, myofilament Ca²⁺ sensitivity is decreased by protein kinase A phosphorylation of troponin I, low pH, and reduced sarcomere length (8). Ca²⁺ activation may not just exert an "on-off" action on actin-myosin interaction. It has been proposed that the thin filament exists in three activation states. In the "blocked" (off) state, Ca²⁺ is not bound to troponin C, and cross-bridge binding is unable to occur; in the "closed" (off) state, Ca²⁺ is bound to troponin C but there are no strong cross-bridges; in the "open" (on) state, Ca²⁺ is bound to troponin C and cross-bridges are strongly bound. According to the three-state model, Ca²⁺ may determine both whether or not cross-bridges bind and the strength of binding. It has also been proposed that changes in Ca²⁺ activation may alter the number of cross-bridges attached, as well as the kinetics of cross-bridge attachment and detachment (97, 148, 171).

MLC and cardiac myosin binding protein C (cMyBP-C) have been implicated as regulatory factors in muscle contraction. Essential and regulatory MLC bind to the -helical lever arm of the myosin cross-bridge and influence force production by modulating cross-bridge kinetics (125, 140). cMyBP-C is thought to participate in the adrenergic regulation of cardiac contractility (38). The MyBP-C motif, a highly conserved 100-residue region in the NH₂-terminal region of cMyBP-C, binds to the S2 segment near the lever arm of the myosin head (48). Ultrastructural studies have shown that phosphorylation of the MyBP-C motif by cAMP-dependent protein kinase extends myosin cross-bridges from the backbone of the thick filament and changes their orientation (176). Functional studies indicate that the phosphorylation status of cMyBP-C is a determinant of the stiffness and attachment rates of cross-bridges and Ca²⁺ sensitivity of force production (74, 176). In addition to these intrinsic sarcomere components, numerous extrinsic factors, including neurohumoral, endocrine, and hemodynamic factors, influence cardiac contractility in vivo.

	III. HYPERTROPHIC CARDIOMYOPATHY

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A.  Clinical Manifestations

1.  Disease characteristics

Hypertrophic cardiomyopathy (HCM) is a primary myocardial disorder with an autosomal dominant pattern of inheritance that is characterized by hypertrophy of the left (±right) ventricles with histological features of myocyte hypertrophy, myofibrillar disarray, and interstitial fibrosis. HCM is one of the most common inherited cardiac disorders, with a prevalence in young adults of 1 in 500 (91). Various names have been given to this disorder including hypertrophic obstructive cardiomyopathy and idiopathic subaortic stenosis. These names reflect "textbook" features of asymmetric septal hypertrophy and left ventricular outflow tract obstruction. This description of the disease is based primarily on patients with severe symptoms seen in tertiary hospital referral centers. Epidemiological studies now suggest that a wide spectrum of clinical manifestations of varying severity and prognosis is present in community populations. The generic name HCM is thus more appropriate and is used in this review. The first clinical description of HCM was reported in 1869 (51). HCM was recognized to be a genetic disorder in the late 1950s. Since then, numerous clinical and pathological studies of HCM have been performed. During the last decade, molecular genetic studies have given important insights into the pathogenesis of HCM and provide a new perspective for the diagnosis and management of patients with this disorder.

Affected individuals with HCM exhibit significant variability in their clinical presentation. Genotype-positive individuals may be asymptomatic or present with symptoms ranging from palpitations and dizziness to syncope and sudden death. Genotype-phenotype studies have shown that the age of onset of symptoms varies between different HCM disease genes. For example, individuals with -MHC mutations typically present in the first two decades of life, whereas those with cMyBP-C mutations may be asymptomatic until the fifth or sixth decades (116).

The primary modality for the diagnosis of HCM is transthoracic echocardiography. The hallmark diagnostic feature of HCM is asymmetric hypertrophy of the interventricular septum, with or without left ventricular outflow tract obstruction and systolic anterior motion of the mitral valve. It is now recognized that the obstructive form of HCM occurs in <25% of affected individuals. Studies of kindreds with HCM have shown that the distribution and severity of left ventricular hypertrophy may vary considerably and that asymmetric hypertrophy is no longer an essential requirement for the diagnosis of this disorder. The diagnosis of HCM generally requires exclusion of secondary causes of hypertrophy, such as hypertension or aortic stenosis; however, in some individuals, particularly in older age groups, these conditions may coexist. The differentiation of HCM from physiological left ventricular hypertrophy may be difficult, particularly in competitive athletes. The extent of left ventricular hypertrophy varies between different genes. For example, individuals with -MHC gene mutations usually develop moderate or severe hypertrophy with a high disease penetrance, whereas those with cardiac troponin T gene mutations generally have only mild or clinically undetectable hypertrophy (106, 173). Unusual forms of hypertrophy have been reported, localized to the left ventricular apex (cardiac troponin I mutations) (71) or midcavity (cardiac actin and MLC gene mutations) (119, 125). The extent of left ventricular hypertrophy may also vary between members of a single family with the same gene mutation. These observations may be explained by a modifying role of additional genetic and environmental factors, such as blood pressure, exercise, diet, and body mass.

The natural history of HCM is variable; some individuals remain asymptomatic throughout life, and others may develop progressive symptoms with or without heart failure or experience sudden death. Longitudinal echocardiographic studies have documented left ventricular remodeling with age. Progressive increases in left ventricular wall thickness have been reported in ungenotyped individuals during adolescence and early adult life. In some individuals, including those with cMyBP-C mutations, left ventricular wall thickness may increase in later life (116). Age-related reductions in left ventricular wall thickness, associated with myocyte loss and fibrosis, have also been described in individuals with long-standing disease ("burnt out" HCM). Ten to 20% of individuals with HCM may develop dilated cardiomyopathy. Ten to 16% of affected individuals develop atrial fibrillation. The risk of atrial fibrillation is increased in those with left atrial enlargement.

HCM is a frequent cause of sudden death, particularly in young individuals and competitive athletes. Estimates of the prevalence of sudden death vary according to the population studied, ranging from <1% in the general community to 3-6% in tertiary hospital referral centers. Various mechanisms for sudden death have been proposed, including ventricular bradyarrhythmias due to sinus node and atrioventricular conduction abnormalities and tachyarrhythmias triggered by reentrant depolarization pathways related to myofibrillar disarray and fibrosis, abnormal Ca²⁺ homeostasis, myocardial ischemia, left ventricular diastolic dysfunction, or left ventricular outflow tract obstruction. Various risk stratification algorithms based on clinical parameters have been proposed to identify individuals with an increased propensity for sudden death. Given the complexity of mechanisms that may precipitate sudden death, it is not surprising that no single risk factor has been identified. Conflicting results have been found for the positive predictive value of young age at diagnosis, history of syncope, severity of symptoms, left ventricular wall thickness, left ventricular outflow tract gradient, left atrial size, and atrial fibrillation. The majority of clinical risk factor studies have been performed in ungenotyped populations. In genotyped individuals, it has been demonstrated that prognosis varies considerably between different HCM genes and between different mutations in the same gene. For example, the Arg403Gln and Arg453Cys -MHC mutations, cardiac troponin T mutations, and some -tropomyosin mutations (Ala63Val, Lys70Thr) are "high risk" mutations with reduced life expectancy and high rates of sudden death, whereas the Val606Met -MHC mutation and cMyBP-C mutations have a relatively benign course (106, 116, 173, 174). The mechanisms whereby HCM gene mutations influence prognosis are unknown. Although some HCM mutations that alter the charge of the encoded amino acid have been associated with a poor outcome, other mutations that alter charge have a good prognosis (170, 174). Electrophysiological studies in mouse models may provide important insights into the differential propensity for sudden death between different HCM gene mutations.

B.  Chromosomal Loci and Disease Genes

1.  Chromosomal loci

HCM is a genetically heterogeneous disorder, with 12 distinct chromosomal loci mapped to date. Disease-causing genes have now been identified in all 12 loci: the first 10 of these genes encoded protein components of the cardiac sarcomere. Mutations have been found in four genes that encode components of the thick filament: -MHC (40), essential MLC (125), regulatory MLC (125), and cMyBP-C (14, 172); in five genes that encode thin filament proteins: cardiac actin (119), cardiac troponin T (165), cardiac troponin I (71), cardiac troponin C (56), and -tropomyosin (165); and in the sarcomeric cytoskeletal protein titin (143) (Table 1). Recently, mutations in two genes encoding nonsarcomeric proteins have been reported to cause HCM. Mutations in the ₂-regulatory subunit of an AMP-activated protein kinase (AMPK) were found to be responsible for a variant of HCM associated with ventricular preexcitation (Wolff-Parkinson-White syndrome) at the chromosome 7q36 locus (11). Mutations in the gene encoding the cytoskeletal muscle LIM protein have also been identified (39).

For each disease gene, a variety of different mutations have been reported. Single nucleotide substitutions ("missense" mutations) and deletion or insertion of nucleotides have been identified. In some cases, the encoded protein is of normal size. In other cases, the mutation may result in a premature termination codon or cause a shift of the reading frame with truncation of the encoded protein. Mutations located at intron-exon boundaries can result in abnormal splicing. In general, each affected family has a unique mutation, although several mutation "hot spots" have been found. Methylated CpG dinucleotides within the genome are particularly prone to point mutations (26). Mutations in the 12 known disease genes account for ~50-70% of all cases of HCM. It is possible that families in which mutations have not been found have undiscovered abnormalities in one of the known disease-causing genes. Alternatively, a significant number of novel genes may remain to be identified. Generally, individuals with HCM-causing mutations are heterozygous at the disease locus, i.e., one copy (allele) of the gene is mutated and the other allele has the normal DNA sequence. Two cases of homozygous HCM mutations have recently been reported; one of these was an Arg869Gly point mutation in the -MHC gene (55), and the other was a Ser179Phe mutation in the cardiac troponin T gene (136). Both mutations caused a particularly severe phenotype with onset in childhood and premature death.

Myosin exists as a hexameric protein with two heavy chains and two sets of light chains. Cardiac MHC exists as two isoforms: - and -MHC. In humans, -MHC is present in the embryonic heart and the adult atrium and is the predominant isoform expressed in the adult ventricle. -MHC is also expressed in slow skeletal muscles, such as the soleus. In rodents, -MHC is the principal isoform expressed in the embryonic ventricle with a switch to predominance of -MHC in the adult ventricle. The MYH7 gene (encoding -MHC) and the MYH6 gene (encoding -MHC) are located in tandem on chromosome 14, ~4 kb apart. MYH7 is comprised of 23 kb of genomic DNA, with 41 exons, 38 of which encode a protein of 1,935 amino acids. Seventy-three mutations in the MYH7 gene have been reported, accounting for ~30-35% of cases of HCM (32, 40). The majority of mutations are missense mutations. Deletions and premature termination codons have also been identified. MYH7 mutations appear to be distributed throughout the gene-coding sequence. Three-dimensional structural studies of the MHC molecule have demonstrated, however, that these mutations cluster predominantly in four domains in the MHC head: 1) the actin binding surface, 2) the nucleotide binding pocket, 3) adjacent to two reactive cysteines in the hinge region, and 4) in the -helical tail near the essential MLC binding site (130). A small number of mutations occur in the MHC rod. Although the functional consequences have not been defined precisely, it has been proposed that rod mutations may result in altered transmission of force from the head to the body of the thick filament (170).

Myosin binding protein C has three isoforms: slow skeletal, fast skeletal, and cardiac. The cardiac MyBP-C isoform is expressed exclusively in cardiac tissue. It is located in the sarcomere A bands and forms a series of seven to nine transverse bands spaced at 43-nm intervals. MYBPC3 is comprised of 24 kb of genomic DNA with 37 exons that encode a protein of 1,274 amino acids. The protein has multiple immunoglobulin C2-like and fibronectin type 3 domains, as well as a cardiac-specific region, a phosphorylation region, and overlapping myosin and titin binding sites. MYPBC3 mutations are found in ~15-20% individuals with HCM. Thirty mutations have been reported (14, 32, 172). The majority of mutations are insertions, deletions, or splice site mutations that result in truncation of the cMyBP-C protein with loss of the myosin and titin binding sites. Missense mutations that preserve the myosin and titin binding sites have also been found. None of these mutations has been located in the cardiac-specific region.

Cardiac troponin T is expressed in embryonic and adult heart and in developing skeletal muscle. TNNT2 is comprised of 17 kb of genomic DNA and has 17 exons. A number of different cardiac troponin T isoforms are produced by alternate splicing. The principal isoform in the adult heart consists of 288 amino acids and has two major domains: an NH₂-terminal domain that interacts with tropomyosin and a COOH-terminal domain that binds to tropomyosin, troponin C, and troponin I. TNNT2 mutations account for ~5-10% of cases of HCM. Fourteen mutations have been reported: 12 missense mutations located in both the NH₂-terminal and COOH-terminal domains, 1 splice site mutation, and 1 single codon deletion (32, 165).

Cardiac troponin I is expressed solely in cardiac tissue. TNNI3 is comprised of 6.2 kb of genomic DNA and has 8 exons that encode a protein of 210 amino acids. Cardiac troponin I has an inhibitory region (residues 129-149) that, at low Ca²⁺ concentrations, depresses contraction by inhibition of actomyosin ATPase (mediated by protein kinase C phosphorylation). Increased binding of cardiac troponin I to the thin filament (mediated by protein kinase A) also contributes to inhibition of contraction. Under activating conditions, the inhibitory region of cardiac troponin I binds to troponin C-Ca²⁺, permitting actin-myosin interaction. Two binding sites for actin-tropomyosin and a second troponin C site are located more proximal to the COOH terminus. Troponin I forms an antiparallel dimer with troponin C. Eight TNNI3 mutations have been reported (<5% cases of HCM): seven missense mutations and one single codon deletion (32, 71). Two mutations are located in the inhibitory region, with one mutation found in each of the second troponin C and actin-tropomyosin sites, respectively.

Troponin C has two isoforms that are expressed in cardiac and skeletal muscle. The TNNC1 gene encodes the isoform that is present in the heart and in slow skeletal muscle. TNNC1 consists of 3 kb of genomic DNA with 6 exons that encode a protein of 161 amino acids. Ca²⁺ binding to cardiac troponin C induces conformational changes in the troponin-tropomyosin complex that initiate muscle contraction. Cardiac troponin C has two Ca²⁺ binding sites, only one of which is functional. Recently, one missense mutation in the TNNC1 gene was reported in an individual with HCM (56).

-Tropomyosin is expressed in ventricular myocardium and in fast skeletal muscle. TPM1 consists of 15 exons, with multiple isoforms resulting from alternate splicing. Five exons are present in all -tropomyosin transcripts with the remaining 10 exons variably present in different tissues. The cardiac -tropomyosin isoform is comprised of 10 exons and 284 amino acids. -Tropomyosin has two binding sites for troponin T, one of which is Ca²⁺ sensitive and the other Ca²⁺ insensitive. Six TPM1 missense mutations have been reported; three of these are located in the Ca²⁺-sensitive troponin T site (32, 165).

The MLC belong to a superfamily of Ca²⁺-binding proteins, which is characterized by helix-loop-helix of Ca²⁺-binding sites (EF hands). Other members of this family included troponin C and calmodulin. Cardiac muscle has two regulatory (or phosphorylatable) MLC isoforms. The MLC-2 slow isoform is expressed in the ventricle and in slow skeletal muscle; the MLC-2 atrial isoform is expressed in atrial myocardium. MYL2 encodes the MLC2 slow isoform. It has seven exons that encode a protein of 166 amino acids. Eight MYL2 missense mutations have been reported (32, 125).

Two of the five essential (or alkali) MLC isoforms are present in cardiac muscle. The MLC-1 slow/ventricular isoform is expressed in the ventricle and slow skeletal muscle. MYL3 is comprised of 7 exons, 6 of which encode a protein of 195 amino acids. Two MYL3 missense mutations have been reported (32, 125).

Twenty actin genes are present in the human genome, four of which are found in cardiac, skeletal, and smooth muscle. The cardiac and skeletal actin isoforms are both expressed in the heart and skeletal muscle. -Skeletal actin is the predominant actin isoform in the embryonic heart but is downregulated in the adult heart. ACTC has 6 exons that encode 375 amino acids. The NH₂-terminal domain of cardiac actin is the site of myosin cross-bridge attachment; the COOH-terminal domain has binding sites for -actinin and dystrophin. Five ACTC mutations have been identified; two of these were missense mutations located close to the myosin binding region (32, 119).

Titin is a giant (3 kDa) protein that is the largest known polypeptide. It comprises 10% of vertebrate striated muscle. Titin spans half sarcomeres, with an extensible portion within the I band and a stiffer portion within the A band. Ninety percent of titin's mass is comprised of up to 298 repeating immunoglobulin and fibronectin 3 domains. The I-band region contains tandemly arranged immunoglobulin domains, as well as a PEVK segment, composed of a sequence rich in proline, glutamine, valine, and lysine residues, and a cardiac-specific N2B region. Titin contributes to the maintenance of sarcomere organization and myofibrillar elasticity. Titin may also participate in myofibrillar cell signaling. Tissue-specific expression of various titin isoforms results in differential tissue elasticity. One HCM-causing TTN missense mutation has been identified in a single individual (143). This mutation was located in the Z-disc binding region of titin.

AMPK is a heterotrimeric protein comprised of a catalytic subunit () and two regulatory subunits ( and ). The -subunit has three isoforms (₁, ₂, ₃) that vary in length and tissue expression. The PRKAG2 gene encodes the ₂-subunit, which is the predominant -isoform present in the heart. PRKAG2 is comprised of >280 kb of genomic DNA. Two isoforms have been identified: a longer transcript (PRKAG2b) with 16 exons that encodes 569 amino acids, and a shorter transcript (PRKAG2a) with 12 exons that encodes tissue-specific proteins of 352 and 328 amino acids, respectively. The shorter transcript results from an alternate transcription initiation site in intron 4. The ₂-AMPK protein consists primarily of four consecutive cystathionine--synthase (CBS) domains. AMPK acts as a "metabolic sensor" in cells, responding to ATP depletion by regulating diverse intracellular pathways that utilize and generate ATP. In the absence of metabolic stress, AMPK activity is suppressed by an autoinhibitory region on the -subunit that blocks the catalytic site. During periods of hypoxic or metabolic stress, consumption of ATP results in an increase in the AMP/ATP ratio. Rising levels of AMP activate AMPK by interactions with both the autoinhibitory region and the -subunit, as well as activating an upstream kinase, AMPKK. In addition to its protein kinase activity, AMPK is postulated to have a transcriptional regulatory role, since it is homologous to the SNF1 transcription factor complex that regulates glucose metabolism in yeast. Five PRKAG2 mutations have recently been reported in families with HCM associated with preexcitation (Wolff-Parkinson-White syndrome); four missense mutations and one in-frame single codon insertion (2, 11, 47).

Cardiac muscle LIM protein is expressed in cardiac and slow-twitch skeletal muscle. CLP has 4 exons that encode a protein of 194 amino acids. Cardiac muscle LIM protein consists of 2 LIM domains, linked by a spacer of 50 residues. LIM domains are highly conserved cysteine-rich structures that contain two zinc fingers. Cardiac muscle LIM protein is present in embryonic muscle and has been shown to be an important regulator of myogenic differentiation. Cardiac muscle LIM protein is also thought to act as a scaffold for protein assembly in the actin-based cytoskeleton. The first of the two LIM domains interacts with -actinin, a component of the Z discs, whereas the second LIM domain interacts with actin filaments and spectrin. Four CLP missense mutations have been reported in individuals with HCM (39).

A) ARG403GLN -MHC STUDIES. Based on their structural location in the myosin head (see sect. IIIB2), the majority of MYH7 gene mutations can be predicted to disrupt both mechanical and catalytic components of actin-myosin interaction leading to a reduction of force generation. The Arg403Gln missense mutation in -MHC has been studied the most extensively. The Arg-403 residue is located in the myosin head at the base of a loop that interacts with actin. A Glu substitution at this residue results in a loss of charge. Studies of the structural and functional consequences of the Arg403Gln mutation in -MHC have been performed. Sarcomere assembly was disrupted when human Arg403Gln -MHC was transfected into adult feline cardiomyocytes (87) but was unchanged when this mutation was introduced into neonatal rat cardiomyocytes (7). Functional studies of the Arg403Gln -MHC mutation using soleus muscle biopsy specimens from patients with HCM have shown that mutant muscle fibers have depressed velocity of shortening, reduced force/stiffness ratio, and reduced power output when compared with control muscle (77). A number of in vitro motility assays performed with human and recombinant mutant -MHC have shown reduced velocity of actin translocation (23, 142, 155).

B) ARG403GLN -MHC MOUSE MODEL. Geisterfer-Lowrance et al. (41) generated a mouse model of HCM in which an Arg403Gln point mutation was introduced into the murine -MHC gene using the "hit-and-run" technique. In contrast to transgenic models in which the location and level of expression of a mutant transgene can be highly variable, the hit-and-run technique results in precise targeting of a single allele at a specific locus, analogous to human heterozygous HCM mutations. Heterozygous Arg403Gln -MHC (designated -MHC^403/+) mutant mice demonstrated progressive left ventricular hypertrophy on transthoracic echocardiography, together with histological evidence of myocyte hypertrophy, myofibrillar disarray, and fibrosis (35, 41). Left ventricular sections from -MHC^403/+ mice showed normal sarcomere structure on electron microscopy (12).

Systolic function in -MHC^403/+ mouse hearts has been evaluated in detail using several techniques. Serial in vivo hemodynamic studies demonstrated that young (6 wk) -MHC^403/+ mice had normal myocardial histology but altered contraction kinetics with accelerated systolic pressure rise. By 20 wk of age, -MHC^403/+ mice had developed myocardial histopathology as well as hyperdynamic left ventricular contraction, increased end-systolic chamber stiffness, increased left ventricular outflow tract pressure gradient, and lower cardiac index (43). It was proposed that the fast systolic kinetics in young mice represented primary effects of the Arg403Gln mutation with later exacerbation of left ventricular dysfunction attributable to altered left ventricular wall composition and chamber geometry. Two groups of investigators have studied isolated papillary muscles from -MHC^403/+ mice. In one study, -MHC^403/+ and control left ventricular trabeculae and papillary muscles were able to generate similar peak force during twitch contractions, but mutant muscle required higher intracellular Ca²⁺ concentrations to achieve equivalent force. Increased Ca²⁺ mobilization in mutant muscle was insufficient to maintain force, however, at high stimulation rates (37). In a second study, sinusoidal length perturbation analysis was used to generate oscillatory work and evaluate cross-bridge function in -MHC^403/+ papillary muscle strips. Ca²⁺ dependence of -MHC^403/+ muscle was demonstrated. At maximal or near-maximal Ca²⁺ activation, peak isometric tension and oscillatory power were reduced in mutant muscle strips; at submaximal Ca²⁺ concentrations, tension and power output were shown to have increased Ca²⁺ sensitivity. Compared with wild-type muscle strips, -MHC^403/+ muscle strips had depressed cross-bridge kinetics (12). -MHC^403/+ myocytes have been shown to have depressed velocity of contraction but equivalent sarcomere length, fractional shortening, and peak amplitude of Ca²⁺ transients when compared with wild-type myocytes (70). Finally, elegant single molecule studies of MHC isolated from -MHC^403/+ mouse hearts have shown dose-related increases in actin-activated ATPase activity, force generation, and actin filament sliding velocity (169).

Does the Arg403Gln MHC mutation cause hypo- or hypercontractile function? Although several studies have suggested that Arg403Gln MHC augments cross-bridge kinetics and force generation, a large body of data derived from in vitro studies has suggested that Arg403Gln MHC perturbs actin-myosin interaction and depresses motor function. Several pathophysiological mechanisms for these findings have been proposed, including altered myosin binding affinity for actin, reductions in cross-bridge cycling rates, reduction in the extent of actin displacement per cross-bridge cycle, a drag effect on normal cross-bridges by mutant cross-bridges, or abnormal interactions between heterodimeric myosin heads. Observations of depressed contractile function, at the cellular level, however, cannot be readily reconciled with the clinical finding of preserved, or enhanced, systolic function in patients with HCM. These apparently conflicting findings highlight the importance of the methods used to evaluate contractile performance. Studies of the interaction between single myosin and actin molecules might intuitively appear to be the optimal technique for determining the fundamental consequences of a mutant protein. However, these studies do not take into account the effects of loading or the regulatory influences of other protein components of the sarcomere. In the intact heart, the presence of hypertrophy, myofibrillar disarray, fibrosis, and hemodynamic changes will further influence left ventricular systolic and diastolic performance. Differences in contractile "environment" may explain the seemingly paradoxical findings related to effects of mutant protein "dose": in single molecule studies, increasing amounts of mutant myosin cause progressive increases in systolic function, whereas in vivo studies in homozygous Arg403Gln -MHC mice demonstrate reduced systolic function with a rapidly progressive dilated cardiomyopathy (33).

Studies of left ventricular diastolic function in -MHC^403/+ mouse hearts have uniformly shown prolongation of relaxation, analogous to that observed in patients with HCM (41, 43, 149). It has been proposed that diastolic dysfunction may be a direct mechanical consequence of slowed cross-bridge cycling rates and thus a fundamental consequence of HCM mutations. The detection of prolonged decay of Ca²⁺ transients in -MHC^403/+ myocytes raises the additional possibility that Ca²⁺ dysregulation or altered Ca²⁺ sensitivity may contribute to impaired relaxation (70). Myocardial energetic studies suggest that an energy-requiring process may also prolong diastolic relaxation (149). The development with age of left ventricular structural changes may increase diastolic stiffness and further exacerbate left ventricular diastolic filling abnormalities. Further studies are required to determine the molecular and cellular consequences of diastolic dysfunction and its relationship with the development of hypertrophy.

cMyBP-C is thought to have structural and regulatory roles in the sarcomere. Experimental studies have focused on the effects of cMyBP-C mutations, in particular, those that result in truncated proteins, on sarcomere assembly. Transfection of a range of COOH-terminal truncation mutants into skeletal myoblasts demonstrated a minimal region of 372 amino acids was required for correct localization into the sarcomere A band; one construct, which lacked the major myosin binding domain, strongly inhibited myofibril assembly. It was proposed that truncated protein might compete with endogenous cMyBP-C at the myosin binding site. The truncated proteins were all detected by Western blot (45). In another study, truncated cMyBP-C protein in cardiac tissue from a patient with a splice donor site mutation was unable to be detected by Western blot, despite the presence of a mRNA transcript. It was suggested that the truncated protein may have undergone rapid proteolysis and that a reduction in cMyBP-C protein might alter the stoichiometry of sarcomere proteins with consequent impairment of sarcomere assembly (138).

Western blot analyses of myocardium from a transgenic mouse expressing a truncated cMyBP-C lacking myosin and titin binding domains demonstrated endogenous regulation of total cMyBP-C levels with overexpression of the mutant protein compensated by a reduction in wild-type protein (180). Histological analyses of myocardial sections from 25-wk-old mice showed foci of degenerate myocytes and sarcomeric disorganization. The mutant protein was incorporated into the sarcomere but was also present diffusely throughout the cytoplasm. A second mouse model with a similar cMyBP-C truncation was generated using homologous recombination techniques (95). In contrast to -MHC^403/+ mice, heterozygous mice (MyBP-C^t/+) did not develop left ventricular hypertrophy until after 125 wk of age. This late onset of hypertrophy is analogous to that observed in patients with HCM caused by cMyBP-C mutations. Total cMyBP-C protein expression in the left ventricle of MyBP-C^t/+ mice was slightly reduced (90% wild-type levels). In contrast to the transgenic mouse studies, myocardial histology of MyBP-C^t/+ mice aged 125 wk was indistinguishable from age-matched wild-type mice. In particular, sarcomere assembly was normal. Differences between these in vitro and in vivo studies in the effects of truncated cMyBP-C on sarcomere organization and protein localization may result from differences in the relative proportions of mutant and wild-type protein, with abnormalities detected only with overexpression models. Endogenous regulation of protein levels or posttranslational modification may reduce the levels of wild-type protein below a threshold level required for maintenance of normal sarcomere relationships. Alternatively, truncated protein may competitively inhibit wild-type protein function. Dose-related effects are suggested by the findings of normal myocardial histology in heterozygous MyBP-C^t/+ mice but prominent left ventricular hypertrophy, disarray, and fibrosis in homozygous MyBP-C^t/t mice (95, 96).

Few studies have examined the functional consequences of cMyBP-C truncations. Hemodynamic analyses in isolated heart preparations from transgenic cMyBP-C mice aged 25 wk showed no differences in contraction or relaxation parameters between mutant and wild-type hearts (180). Similarly, in vivo hemodynamic studies showed no differences in systolic or diastolic parameters between MyBP-C^t/+ and wild-type mice (95). Studies of ventricular fibers from transgenic mice demonstrated a leftward shift in the pCa-force relationship and a reduction of maximum power (180). It is not clear whether these changes represent primary effects of the mutant protein on sarcomere function or are a consequence of the deranged sarcomere structure present in this model.

A) ARG92GLN, ILE79ASN MUTATIONS. The Arg-92 and Ile-79 residues are located in the NH₂-terminal domain of cardiac troponin T. The functional consequences of the Arg92Gln and Ile79Asn missense mutations have varied according to the techniques employed. In one study, transfection of Arg92Gln and Ile79Asn cardiac troponin T into quail myotubes resulted in increased myotube shortening velocity. The Ile79Asn mutation, but not Arg92Gln, reduced maximum force (154). In a second study, in vitro motility assays performed using recombinant embryonic rat cardiac troponin T bearing an Ile79Asn substitution demonstrated a 50% increase in velocity of translocation of thin filaments over myosin (83). In contrast, transfection of Arg92Gln cardiac troponin T in adult feline cardiac myocytes showed reductions in fractional shortening and peak velocity of shortening (88). Ca²⁺ sensitivity of force production was increased in Arg92Gln and Ile79Asn cardiac troponin T in skinned cardiac muscle preparations (108, 157) but was decreased in transfected rat adult cardiac myocytes (139) and quail myotubes (154). Ca²⁺ sensitivity of myofibrillar ATPase was increased in transfected rabbit cardiac myofibrils (177). No abnormalities of sarcomere structure have been reported in any of these studies.

An Arg92Gln cardiac troponin T transgenic mouse model with transgene expression levels ranged from 1 to 10% has been characterized. Adult mice exhibited systolic and diastolic left ventricular dysfunction with variable myocyte degeneration, necrosis, myofibril disarray, and fibrosis (82, 117). Another group of investigators have found that Arg92Gln cardiac troponin T transgenic mice with 30, 67, and 92% transgene expression levels exhibited dose-related myocardial disarray and fibrosis (162). Surprisingly, mutant hearts in the latter study were smaller than control hearts, with a reduction in both the number and size of myocytes. Isolated working hearts from the mice with 67% transgene expression were hypercontractile with stepwise increases in cardiac work load. Single myocytes from these mice had reduced shortening and prolonged contraction times. Both isolated heart and myocyte studies showed impaired diastolic relaxation. Skinned papillary muscle fibers from transgenic mice expressing Ile79Asn cardiac troponin T showed increased Ca²⁺ sensitivity of ATPase activity and force development with accelerated kinetics of force activation and relaxation. A computer simulation of intracellular Ca²⁺ and force generation predicted that the Ile79Asn cardiac troponin T mutation altered cross-bridge kinetics and increased cardiac troponin T Ca²⁺ affinity (103).

B) INT15G TO A SUBSTITUTION. The Int15G to A substitution results in truncation of the COOH terminus of the cardiac troponin T protein. Transfection of a truncated cardiac troponin T in quail myotubes resulted in decreased Ca²⁺ sensitivity of force production, similar to the findings in the Arg92Gln experiments (175). In recombinant cardiac muscle preparations, this truncation mutant reduced Ca²⁺ activation and inhibition of force and reduced activation and inhibition of actin-tropomyosin-activated myosin ATPase activity (157). In in vitro motility assays, truncated cardiac troponin T increased the velocity of actin-tropomyosin filaments to a greater extent than wild-type cardiac troponin T at low pCa (activating conditions) but failed to decrease the velocity of filaments and had negligible inhibition of actin-tropomyosin-activated myosin ATPase at high pCa (relaxing conditions). Varying proportions of wild-type and mutant cardiac troponin T significantly influenced the results: at pCa 5, the inhibitory effect of wild-type protein on thin filament velocity was enhanced at lower protein levels (10-50%) (132). A transgenic mouse with a truncated cardiac troponin T exhibited similar findings to the Arg92Gln cardiac troponin T transgenic mouse, with myocyte disarray, fibrosis, and a reduction in the number and size of myocytes. Echocardiographic studies in the truncated cardiac troponin T mouse showed a mild reduction in systolic contraction and a relatively greater extent of impaired diastolic relaxation (161).

Cardiac troponin T mutations would be predicted to influence the inhibitory regulatory effect of the tropomyosin-troponin complex. In vitro studies showing increased contraction kinetics and increased Ca²⁺ sensitivity of force generation are consistent with this concept. The observation of hypocontractility in some studies has also been attributed to increased Ca²⁺ sensitivity, with higher basal levels of sarcomeric activation resulting in initiation of contraction at shorter, less optimal sarcomere lengths against a stronger passive restoring force (162). This may also partly explain the small size of myocytes. Myocyte degeneration and loss may further depress contractile performance. Despite myocardial histopathology and functional changes, none of the cardiac troponin T mouse models has exhibited left ventricular hypertrophy. Individuals with HCM caused by cardiac troponin T mutations typically have minimal left ventricular hypertrophy.

The Arg-145 residue is located in the inhibitory region of cardiac troponin I that is required for inhibition of actin-tropomyosin-activated myosin ATPase and Ca²⁺-mediated binding to troponin C. In vitro functional analyses have shown that the Arg145Gly cardiac troponin I mutation results in reduced inhibition of actin-tropomyosin-activated myosin ATPase and increased Ca²⁺ sensitivity of ATPase regulation (30, 158). A transgenic mouse overexpressing Arg145Gly cardiac troponin I (1.2-fold) had normal life expectancy and no overt phenotype, with the exception that females stressed by pregnancy developed histological evidence of cardiomyocyte hypertrophy and patchy myocardial fibrosis. Isolated working heart preparations from unstressed mutant mice showed significantly enhanced contractility (+dP/dt) and prolonged relaxation (dP/dt). Mice with 3.5-fold transgene overexpression died by 17 days. At 10 days after birth, the ventricles of these mice showed myocyte disarray, nuclear degeneration, and interstitial fibrosis. Skinned papillary muscle strips from 10-day-old mutant mice showed no differences in unloaded shortening velocity, maximum shortening velocity, or maximum relative power but increased Ca²⁺ sensitivity and reduced maximum tension (61). It has been proposed that mutations in the inhibitory region of cardiac troponin I disrupt the Ca²⁺-sensitive switch and cause the thin filament to remain in an "on" position analogous to the changes observed with cardiac troponin T gene mutations. The differences in contractile performance between mice with 1.2- and 3.5-fold transgene expression may reflect dose-related differences in basal sarcomere length or more severe myocardial histopathology.

Asp175 is located in the Ca²⁺-sensitive troponin T binding domain of -tropomyosin. The substitution of Asp to Asn at this residue causes the loss of one negative charge, which alters the mobility of -tropomyosin on gel electrophoresis (16) and results in local unfolding of the encoded protein. In in vitro motility assays, Asp175Asn -tropomyosin had a relatively greater increase in velocity than wild-type -tropomyosin, following addition of troponin. All other parameters were unchanged, suggesting that the effects of the mutation were due predominantly to altered interaction with troponin T (9). Addition of N-ethylmaleimide-conjugated myosin subfragment-1 (NEM-S1), a strongly binding myosin analog that cooperatively enhances thin filament activation, failed to further increase thin filament velocity, indicating that the Asp175Asn substitution switched -tropomyosin to a fully on state (133).

Mechanical properties of skeletal muscle fibers from patients with HCM due to Asp175Asn -tropomyosin have been studied (16). The levels of mutant and wild-type -tropomyosin protein were similar (~50%), and other -tropomyosin isoforms were not upregulated. The mutant fibers demonstrated no differences in shortening velocity or maximal force generation but did have increased Ca²⁺ sensitivity of force production, compared with controls. Transgenic mice expressing Asp175Asn -tropomyosin exhibited varying extent of myocyte hypertrophy, disarray, and fibrosis by 20 wk of age. Both endogenous and mutant -tropomyosin protein incorporated into myofibrils. Normal -tropomyosin stoichiometry was maintained with overexpression of the mutant protein compensated by a reciprocal reduction in the level of endogenous protein. Transthoracic echocardiography showed no significant differences between transgenic and control mice in left ventricular diameters, fractional shortening, or wall thickness. After an 8-wk swimming program, left ventricular fractional shortening increased in control mice but was unchanged in transgenic mice. In isolated heart preparations from mice expressing 60% Asp175Asn protein, both the rates of contraction and relaxation were reduced. These functional changes were not observed in mice expressing <40% mutant protein. Skinned fiber preparations from transgenic mouse hearts showed a leftward shift in the pCa²⁺-force relationship (112).

These in vitro and in vivo data indicate that the Asp175Asn mutation increases the basal level of activation of -tropomyosin. This may result from structural changes, such as altered protein conformation, or functional changes mediated by changes in binding of the troponin complex or altered Ca²⁺ affinity. Variability in functional assays has been observed, as found with cardiac troponins T and I. Downregulation of endogenous -tropomyosin suggests that mutations may act by haploinsufficiency. However, similar (50%) levels of mutant and endogenous protein have been found in human HCM, consistent with a dominant negative mechanism.

MLC are thought to influence the mechanical efficiency of cross-bridge cycling and the speed of contraction. No specific functional domains in the MLC have been recognized. In vitro motility assays have shown that actin sliding velocities were increased by the Met149Val essential MLC substitution but were unchanged by the Glu22Lys regulatory MLC substitution (125). These two missense mutations have also been evaluated in transgenic mouse models (141). Mice with Met158Val essential MLC (analogous to human Met149Val essential MLC) exhibited dose-related myofibril disarray and fibrosis. While this mutation is associated with the development of papillary muscle hypertrophy in humans, transgenic mice had lower left ventricular mass and smaller myocytes than observed in control mice. Functional analyses in isolated working heart preparations showed hypercontractility and impaired relaxation in the mutant mice. Studies of skinned ventricular fibers and in vitro motility assays showed no differences in shortening velocity or actin translocation, respectively, between mutant and wild-type mice. Met158Val essential MLC fibers did demonstrate leftward shifts in the ATPase activity and pCa-force curves and reduced power output. Histological, cellular, and molecular analyses of transgenic mice with the Glu22Lys RLC mutation were indistinguishable from wild-type mice. Skeletal muscle fibers from an individual with a Glu22Lys regulatory MLC mutation were found to have a leftward shift in the pCa-force relationship (78). These data suggest that the essential MLC and regulatory MLC may regulate power output through a Ca²⁺-dependent mechanism.

D.  Triggers and Effectors of Left Ventricular Hypertrophy

1.  Triggers of left ventricular hypertrophy

The development of left ventricular hypertrophy has been generally considered to be an adaptive response to biomechanical stress that enables cardiac work to be maintained. Despite these generally accepted principles of hypertrophy development and its consequences, it is of note that a recent study using genetically engineered mice with a markedly blunted growth response to pressure overload questions the adaptive value of load-induced hypertrophy (31). Surprisingly, despite failing to correct wall stress after acute partial transthoracic aortic constriction, cardiac function in these animals with either inhibition of G_q signaling or disruption of catecholamine synthesis was well maintained. Indeed, function was even better maintained than in wild-type mice whose wall stress was normalized as a result of hypertrophy development. This study also highlights the primacy, not only of the G_q signaling pathway in load-induced hypertrophy, but somewhat more surprisingly, that of the sympathetic nervous system.

Left ventricular hypertrophy may be "physiological" in elite athletes or occur in pathological states, such as hypertension, myocardial infarction, cardiomyopathies, valvular heart disease, and a variety of systemic disorders. The hypertrophic process is thought to be initiated by factors extrinsic and intrinsic to the cardiac myocyte. Extrinsic stimuli include vasoactive peptides (e.g., angiotensin II, endothelin-1), ₁-adrenergic agonists (e.g., norepinephrine, epinephrine, phenylephrine), activators of protein kinase C (e.g., tumor-producing phorbol esters), peptide growth factors (e.g., insulin-like growth factor, fibroblast growth factor), cytokines (e.g., cardiotrophin-1), arachidonate metabolites (e.g., prostaglandin F₂), mechanical stretch, and cell contact. Intrinsic stimuli include elevated [Ca²⁺]_i, the heterotrimeric G protein G_q, as well as activated small G proteins, kinases, phosphatases, and transcriptional factors (151). These extrinsic and intrinsic factors trigger a complex cascade of intracellular pathways, termed the "hypertrophic response," which results in increased myocardial mass, altered spatial relationships between myocytes and other cellular and extracellular components of the myocardium, reprogramming of myocardial gene expression, and apoptosis. A rapidly growing list of genes has been found to elicit cardiac hypertrophy when overexpressed in transgenic mice. Determination of which of these genes are clinically relevant in human hypertrophy will be important.

A) HCM. Despite a decade of research, the stimulus for hypertrophy in HCM has not been definitively identified. Collectively, data from in vitro and in vivo studies demonstrate that sarcomere protein gene mutations perturb sarcomere structure and/or function and thus are likely to be disease causing. The precise consequences of sarcomere protein gene mutations differ according to the type of model studied with both reduced and augmented motor function described for individual HCM disease genes. Mutations in the various sarcomere protein genes also have diverse effects on motor function. It appears, however, that mechanical dysfunction of the sarcomere is a common feature and, hence, a potential stimulus for hypertrophy (Fig. 2). Whether a threshold level of sarcomere dysfunction is required before the hypertrophic response is triggered is unknown. Observations of hypo- and hypercontractility in different studies of a single gene highlight the importance of the model used and the effects of the contractile "milieu," i.e., the presence or absence of thin filament regulatory elements, myocyte loading, myocardial histopathology, etc. What is the most physiologically relevant method for studying contractile function of mutant sarcomere proteins? This will remain unanswered until we understand the origin (i.e., in the intact organ or at the cellular or molecular level) and the nature of the signaling processes that initiate the hypertrophic response.

View larger version (14K): [in this window] [in a new window]   Fig. 2. Potential pathophysiological mechanisms underlying hypertrophic cardiomyopathy (A) and dilated cardiomyopathy (B). LV, left ventricular; SERCA2a, sarcoendoplasmic reticulum Ca2+-ATPase; SNSA, sympathetic nervous system activity.

B) INTRACELLULAR CALCIUM. Several years ago, the observation that transgenic mice expressing constitutively activated calcineurin developed left ventricular hypertrophy that was prevented by administration of the calcineurin inhibitors cyclosporin and FK506 initiated intense interest in the role of elevated [Ca²⁺]_i and the calcineurin signaling pathway in the pathogenesis of left ventricular hypertrophy (105). Calcineurin is a cytoplasmic protein phosphatase that is activated by sustained [Ca²⁺]_i. Activated calcineurin dephosphorylates NFAT3 (nuclear factor of activated T cells) transcription factors, which causes their translocation to the nucleus where they combine with the cardiac-restricted zinc finger transcription factor GATA4, resulting in synergistic activation of embryonic cardiac genes and a hypertrophic response. Although the transgenic mouse studies suggested that the calcineurin pathway might be critical in the development of myocardial hypertrophy, studies in a variety of different models have suggested that multiple signaling pathways are likely to be involved (118, 151). Recently, the effects of calcineurin inhibition in HCM were examined in -MHC^403/+ mice (35). Paradoxically, cyclosporin and FK506 administration both dramatically increased the severity of left ventricular hypertrophy and accelerated sudden death in the mutant mice. Cyclosporin-induced hypertrophy was prevented by coadministration of the L-type Ca²⁺ channel blocker diltiazem. Studies of -MHC^403/+ and wild-type myocytes showed no differences in Ca²⁺ transients at baseline. Diastolic Ca²⁺ levels increased in wild-type myocytes but were unchanged in -MHC^403/+ myocytes after acute administration of cyclosporin, and after chronic oral treatment with cyclosporin. Similar results were also found in response to acute administration of minoxidil, a K⁺ channel agonist that has been shown to cause left ventricular hypertrophy in humans and rodents. These data suggest first that abnormal Ca²⁺ regulation may play an important role in the hypertrophic response in HCM, and second, that administration of calcineurin inhibitors such as cyclosporin may actually be harmful to patients with HCM.

What is the basis for the abnormalities of Ca²⁺ regulation observed in HCM? The inability to elevate diastolic Ca²⁺ in response to cyclosporin in -MHC^403/+ myocytes might be indicative of a relative depletion of intracellular Ca²⁺ reserves that might result from sequestration of Ca²⁺ by the contractile apparatus due to reciprocal feedback of cross-bridge formation on troponin C/Ca²⁺ binding affinity. This might also explain the increased Ca²⁺ sensitivity observed in mutant myofibrils and contribute to delayed diastolic relaxation (Fig. 2). These data raise intriguing questions about the mechanisms by which intracellular Ca²⁺ might influence hypertrophic signaling pathways. In contrast to the traditional concept, cytoplasmic free [Ca²⁺] did not appear to be the stimulus for left ventricular hypertrophy in -MHC^403/+ mice. It is possible that the [Ca²⁺] in another intracellular compartment or total cellular Ca²⁺ might be the critical Ca²⁺ sensor. Calcium may provide a critical link between mechanical dysfunction of the sarcomere and induction of the hypertrophic gene program. Despite these considerations, it should be noted that recent studies of mice in which calcineurin was inhibited not by drugs, which can affect unrelated pathways as well as being toxic, but by genetic engineering, provide compelling evidence for an important role of the calcineurin/NFAT3/GATA4 pathway in load-induced hypertrophy (28, 137, 182). It will be of interest to see if inhibition of this pathway also prevents the hypertrophy observed in the various genetically induced HCM models, which should be evident when they are crossed with the calcineurin-inhibited animals.

C) MYOCARDIAL ENERGETICS. The recent finding of mutations in the PRKAG2 gene (see sect. IIIB12) was somewhat unexpected, given the traditional dogma that sarcomere dysfunction was the fundamental defect in HCM. These PRKAG2 mutations raise the intriguing possibility that defective myocardial energetics may be a common feature of HCM mutations. Alterations of myocardial metabolism had been observed previously in patients with HCM (66) and in -MHC^403/+ mice (149). Although these energe