The L-type calcium channel inhibitor diltiazem prevents cardiomyopathy in a mouse model

Dominant mutations in sarcomere protein genes cause hypertrophic cardiomyopathy, an inherited human disorder with increased ventricular wall thickness, myocyte hypertrophy, and disarray. To understand the early consequences of mutant sarcomere proteins, we have studied mice (designated αMHC^403/+) bearing an Arg403Gln missense mutation in the α cardiac myosin heavy chain. We demonstrate that Ca²⁺ is reduced in the sarcoplasmic reticulum of αMHC^403/+ mice, and levels of the sarcoplasmic reticulum Ca²⁺-binding protein calsequestrin are diminished in advance of changes in cardiac histology or morphology. Further evidence for dysregulation of sarcoplasmic reticulum Ca²⁺ in these animals is seen in their decreased expression of the ryanodine receptor Ca²⁺-release channel and its associated membrane proteins and in an increase in ryanodine receptor phosphorylation. Early administration of the L-type Ca²⁺ channel inhibitor diltiazem restores normal levels of these sarcoplasmic reticular proteins and prevents the development of pathology in αMHC^403/+ mice. We conclude that disruption of sarcoplasmic reticulum Ca²⁺ homeostasis is an important early event in the pathogenesis of this disorder and suggest that the use of Ca²⁺ channel blockers in advance of established clinical disease could prevent hypertrophic cardiomyopathy caused by sarcomere protein gene mutations.

Hypertrophic cardiomyopathy is a prevalent autosomal dominant human disorder caused by mutations in contractile protein genes (for reviews, see refs. 1, 2). Over 200 DNA defects have been identified in ten disease genes that encode constituents of the cardiac muscle sarcomere. While much is known about these primary causes of hypertrophic cardiomyopathy, little is understood about the signaling events initiated by the mutant sarcomere that result in the hypertrophic response. Notably, there is a considerable latency period in the clinical expression of hypertrophic cardiomyopathy; ventricular hypertrophy evolves insidiously in genetically affected individuals, often beginning around pubescence. However, once established, the hypertrophic phenotype remains throughout life and causes a range of symptoms including chest pain, palpitations, and, in some, sudden death (3, 4). Sudden death can be the presenting manifestation of disease, and unrecognized hypertrophic cardiomyopathy remains the most frequent cause of sudden cardiac death in athletes (5, 6). Although a variety of pharmacologic agents are used to treat symptoms and arrhythmias in hypertrophic cardiomyopathy, therapies have not been identified that prevent expression of disease in individuals with sarcomere protein gene mutations. Study of animal models of hypertrophic cardiomyopathy (7–12) has indicated potential signaling pathways that may contribute to components of the cardiac pathology caused by sarcomere gene mutations. However, the identification of key early molecular events in disease pathogenesis has remained elusive. To define the early molecular steps in the pathogenesis of hypertrophic cardiomyopathy, αMHC^403/+ mice were studied. αMHC^403/+ mice carry an Arg403Gln missense mutation in one allele of the cardiac myosin heavy chain and model a severe human disease mutation (13). Like affected humans, αMHC^403/+ mice gradually develop myocardial hypertrophy, myocyte disarray, and fibrosis (14). Biochemical and biophysical studies indicate that αMHC^403/+ mice have abnormal Ca²⁺ homeostasis: myofibrillar preparations from mutant mice require higher Ca²⁺ concentrations to achieve the same contractile force as controls (15), and αMHC^403/+ mice treated with agents that affect myocyte Ca²⁺ levels exhibit dramatically accelerated hypertrophic pathology and decreased survival (16). Precise regulation of Ca²⁺ is essential for myocyte function (17). Cyclic fluxes of Ca²⁺ between three compartments — cytoplasm, sarcoplasmic reticulum (SR), and sarcomere — account for excitation-contraction coupling. Depolarization triggers entry of small amounts of Ca²⁺ through the L-type Ca²⁺ channels located on the cell membrane, which in turn prompts SR Ca²⁺ release by cardiac ryanodine receptors (RyR’s), a process termed calcium-induced Ca²⁺ release. The resulting rapid rise in cytosolic levels fosters Ca²⁺–troponin-C interactions and triggers sarcomere contraction. Activation of the ATP-dependent calcium pump (SERCA) recycles cytosolic Ca²⁺ into the SR to restore sarcomere relaxation. To understand the mechanism by which calcium dysregulation occurs in αMHC^403/+ myocytes, we studied Ca²⁺-binding proteins in specific myocyte compartments and monitored the hypertrophic response to the Arg403Gln missense mutation. We report that changes in Ca²⁺-binding protein levels occur in advance of disease, and we demonstrate that restoration of these protein levels by the L-type Ca²⁺ channel inhibitor diltiazem prevents clinical expression of hypertrophic cardiomyopathy in αMHC^403/+ mice.

Attenuated Ca²⁺ responses were previously observed in αMHC^403/+ mice treated with two pharmacologically distinct agents, cyclosporin A and minoxidil (16). The hypothesis was therefore made that this deficient response indicated decreased SR Ca²⁺ stores caused by the sarcomere gene mutation. To test this hypothesis, isolated cardiac myocytes from wild-type and untreated αMHC^403/+ mice were fura-2–loaded and exposed to caffeine, which promotes SR Ca²⁺ release via RyR2. The amount of cytosolic Ca²⁺ was monitored by emission of fluorescence at specified wavelengths. A 10-mM caffeine bolus produced prompt and significant Ca²⁺ release into cytoplasm from wild-type myocytes (maximum height = 158.8 ± 55 units; n = 28) but a smaller response (maximum height = 133.1 ± 41 units; n = 25) from αMHC^403/+ myocytes (Figure 1a and data not shown; P = 0.007). These data suggest that the SRs of wild-type myocytes contain more Ca²⁺ than do those of mutant myocytes.

Figure 1

SR Ca²⁺ storage and altered Ca²⁺-related protein expression. (a) Ca²⁺ changes, assessed in wild-type (+/+) and αMHC^403/+ (403/+) myocytes, in response to a bolus administration of 10 mM caffeine (vertical arrows indicate time of administration). (b) Western blot analysis of calsequestrin (CSQ) and components of the quaternary complex in myofibrillar protein extracts from wild-type and αMHC^403/+ mice aged 30–50 weeks. Coomassie staining indicates loading of samples in each lane. Treatment of αMHC^403/+ mice with diltiazem led to normalization of all four components of the RyR2 complex — calsequestrin (8 days treatment), triadin, junctin, and RyR2 (30 weeks treatment). (c) Time course study of changes in calsequestrin protein expression in equal amounts of myofibrillar extracts from mice aged 2, 4, 6, 8, 12, and 30 weeks.

Ca²⁺-binding proteins from whole-cell preparations, myofibrillar extracts, and SR extracts were assessed by Western blot analysis. Several proteins were specifically reduced in myofibrillar extracts from mutant myocytes as compared with wild-type extracts. The amounts of calmodulin, calcineurin (PP2B-Aα and -Aβ), SERCA2, and phospholamban in myofibrillar extracts were comparable in wild-type and αMHC^403/+ myocytes (data not shown). However, calsequestrin, the primary Ca²⁺ storage protein in the SR, was markedly reduced in myofibrillar preparations and SR extracts of αMHC^403/+ ventricles. A 56% ± 19% reduction of normal calsequestrin levels was observed in myofibrillar preparations (n = 8) from mutant mice (Figure 1b). Comparable low calsequestrin levels were observed in 15- to 20-week-old and 30- to 50-week-old αMHC^403/+ mice (data not shown). Calsequestrin protein levels were then examined in younger αMHC^403/+ mice. Although calsequestrin levels in myofibrillar extracts from neonates and 2-week-old mutant mice (Figure 1c) were comparable to levels found in wild-type mice, calsequestrin was decreased in extracts from 4-week-old mutant mice. This decrease in calsequestrin level paralleled the augmented expression of the mutant α isoform of cardiac myosin heavy chain, which occurs 1–2 weeks after birth (24). Notably, this timeline precedes the onset of left ventricular hypertrophy by many weeks (14, 25). These data indicate that changes in calsequestrin levels are an early molecular event in the pathogenesis of hypertrophic cardiomyopathy.

Calsequestrin forms a quaternary complex in the SR membrane, with the cardiac RyR2 channel and two other membrane proteins, junctin and triadin (26). Levels of these proteins were examined by Western blotting. Each of these components was significantly decreased in myofibrillar preparations from the SR of αMHC^403/+ myocytes compared with wild-type cells (Figure 1b; decrease of 63% ± 20%, 48% ± 4%, and 38% ± 5% for RyR2, triadin, and junctin respectively; n = 3 in each group; P < 0.001 vs. wild-type). RyR2 channel phosphorylation was also assessed. (RyR2 was also decreased in SR extracts from αMHC^403/+ hearts; data not shown.) PKA phosphorylation of the RyR2 channel was increased almost threefold in SR preparations from αMHC^403/+ mouse hearts compared with wild-type (2.8 ± 0.2 units vs. 1.0 ± 0.1 units in wild-type; n = 3 in each group; P < 0.001). Consistent with the PKA hyperphosphorylation of RyR2 channels, coimmunoprecipitation assays showed that the amount of FKBP12.6 bound to the RyR2 channel was reduced by 40% (0.6 ± 0.1 units vs. 1.0 ± 0.1 units in wild-type; n = 3 in each group; P < 0.001). Collectively these data indicate that the RyR2 channels are both reduced in number and functionally modified in αMHC^403/+ mice, such that the sensitivity of the channels to Ca²⁺ and the channels’ gating properties are altered.

To further evaluate SR binding proteins in αMHC^403/+ myocytes, immunohistochemical studies were performed. In wild-type myocytes, antibody-labeled SR calsequestrin exists in close association with the sarcomere (27) (Figure 2a). Immunocytochemistry of calsequestrin, like myosin and other sarcomere proteins, exhibited a highly registered pattern of cross-striations, demarcated by the two nuclei characteristic of cardiac myocytes (Figure 2, a and b). αMHC^403/+ myocytes are more irregularly shaped than wild-type cells and exhibited a nonuniform pattern of calsequestrin immunohistochemistry (Figure 2a). The normal alignment of calsequestrin along sarcomere cross-striations observed in wild-type cardiac myocytes was observed in approximately half of αMHC^403/+ myocytes (data not shown). The remaining αMHC^403/+ myocytes demonstrated no association between calsequestrin and sarcomere cross-striations, despite having normal sarcomere structure based on normal sarcomeric patterns with myosin (Figure 2, a and b) and actin antibodies (data not shown). High-power magnification of αMHC^403/+ myocytes confirmed a disordered alignment of SR calsequestrin but did not show sarcomere disarray: cross-striations of the myosin-decorated sarcomere appeared normal in αMHC^403/+ myocytes (Figure 2, a and b). Together with the reduction of calsequestrin levels in myofibrillar extracts, this indicates that both structural and functional alteration of SR Ca²⁺-binding proteins occurs in αMHC^403/+ myocytes.

Figure 2

Calsequestrin localization within myocytes. (a) Immunostaining of isolated wild-type (+/+) and αMHC^403/+ (403/+) myocytes with calsequestrin (CSQ) and myosin primary antibodies. Calsequestrin primary antibodies were detected with a fluoro-conjugated secondary antibody, and myosin antibodies were visualized with a rhodamine-red secondary antibody (confocal images at ×40 magnification). (b) Corresponding images analyzed at higher magnification (×200), demonstrating loss of normal distribution of calsequestrin within αMHC^403/+ myocytes.

In an attempt to correct aberrant Ca²⁺ handling in αMHC^403/+ myocytes, several pharmacologic agents were administered. Diltiazem, an L-type Ca²⁺ channel inhibitor; atenolol, a β-adrenergic inhibitor; enalapril, an angiotensin-converting enzyme inhibitor; and fludrocortisone, a mineralocorticoid that alters cardiac volume, were orally administered (see Methods) to young (age 6–8 weeks) male wild-type and αMHC^403/+ mice (n = 6 in diltiazem treatment group; n = 2 each for other three agents) for 7–10 days. In addition to specific effects of each drug, these pharmacologic agents alter cardiac hemodynamics. Both diltiazem and atenolol decrease heart rate; these and enalapril can also reduce blood pressure. Fludrocortisone can alter circulatory volume. Only the L-type Ca²⁺ channel inhibitor diltiazem affected levels of the calsequestrin complex in mutant mice, suggesting that the effect of diltiazem was independent of its effects on heart rate or blood pressure. Levels of calsequestrin, triadin, junctin, and RyR2 from myofibrillar extracts of diltiazem-treated αMHC^403/+ mice were higher than those of untreated αMHC^403/+ extracts (Figure 1b) and comparable to levels found in myofibrillar extracts from wild-type mice. Diltiazem restoration of calsequestrin levels was brisk, occurring within 8 days of treatment, and sustained during 30 weeks of treatment (Figure 1b). Furthermore, the amount of Ca²⁺ in the SR of myocytes, as measured by caffeine release (23), from diltiazem-treated wild-type and mutant hearts was not different (n = 16 wild-type myocytes and 29 mutant myocytes; P = 0.23).

Previous studies had demonstrated that diltiazem abrogated the accelerated and exaggerated hypertrophic response of αMHC^403/+ mice to cyclosporin A and minoxidil (16). To ascertain the effects of diltiazem on the natural progression of disease, this calcium channel inhibitor was orally administered to male, prehypertrophic αMHC^403/+ mice (n = 8 wild-type and 12 αMHC^403/+) for 30 weeks. No differences were observed between treatment initiated in mice of age 6–8 weeks and in mice of age 10–12 weeks. Cardiac hemodynamics were profiled in diltiazem-treated wild-type and αMHC^403/+ mice. ECG telemetry demonstrated a 13.4% reduction in heart rates of both wild-type and mutant unanesthetized mice (582 ± 25 beats per minute in untreated vs. 504 ± 1 beats per minute in diltiazem-treated; P < 0.05). Diltiazem did not change atrioventricular (PR interval) or intraventricular (QRS) conduction as compared with untreated mice, and no ventricular arrhythmias were observed (data not shown). Tail-cuff blood pressure measurements (n > 20) were compared in four conscious treated and untreated mutant mice; no differences were found in the mean systolic blood pressure (treated, 121 ± 6 mmHg; untreated, 120 ± 7 mmHg).

Invasive cardiac hemodynamic monitoring in diltiazem-treated αMHC^403/+ mice demonstrated improvements in contractile (systolic) properties. Increases in end-diastolic and end-systolic volumes compared with untreated αMHC^403/+ mice were observed, indicating overall improved cardiac function (Table 1). Maximal change in pressure over change in time (dP/dt_max) values and end-systolic elastance (EES) also indicated enhanced ventricular contraction with diltiazem (P < 0.001 vs. untreated αMHC^403/+ mice). Measures of ventricular relaxation, e.g., time to peak filling, dP/dt_min, and tau, were unchanged in diltiazem-treated compared with untreated αMHC^403/+ mice.

Table 1

Left ventricular function of wild-type and αMHC^403/+ mice aged 30–50 weeks with or without diltiazem treatment

Ventricular hypertrophy is demonstrable by two-dimensional echocardiogram at 30 weeks in αMHC^403/+ mice (14, 25). We performed echocardiography in diltiazem-treated wild-type and αMHC^403/+ mice at ages 30 and 39 weeks and compared left ventricular wall measurements in untreated age-, sex-, and strain-matched wild-type and αMHC^403/+ mice (n = 23 total untreated). Ventricular chamber volumes, left atrial dimensions, and fractional shortening in αMHC^403/+ mice were not affected by diltiazem treatment (data not shown). Left ventricular wall thickness of wild-type mice was not affected by diltiazem treatment (0.89 ± 0.03 vs. 0.87 ± 0.06 mm in untreated; P = not significant; Figure 3a). In contrast, the maximum left ventricular wall thickness of diltiazem-treated αMHC^403/+ mice was significantly less than that of untreated age-matched αMHC^403/+ mice at 30 weeks and at 39 weeks (1.01 ± 0.05 vs. 1.12 ± 0.07 mm; P < 0.001; Figure 3a). Wall thickness reduction was seen uniformly throughout the left ventricle, with comparable diminution of anterior and posterior left ventricular wall hypertrophy. Molecular markers of cardiac hypertrophy were also assessed. Untreated adult αMHC^403/+ mice had a three- to fivefold increase in RNA expression of atrial natriuretic factor (ANF) and α-skeletal actin compared with wild-type mice (Figure 3b). In contrast, diltiazem-treated αMHC^403/+ mice had approximately 50% less ANF and α-skeletal actin RNA expression (Figure 3b) than untreated mutant mice.

Figure 3

Prevention of cardiac hypertrophy in αMHC^403/+ mice. (a) Left ventricular anterior wall thickness (LVAW) was evaluated in wild-type and αMHC^403/+ mice at 30–50 weeks of age using transthoracic echocardiography. A left ventricular anterior wall thickness greater than 1.00 mm, in mice 30–50 weeks of age, is considered to be hypertrophied. White bars indicate wild-type mice; black bars represent αMHC^403/+ mice. *P < 0.01 vs. untreated αMHC^403/+ mice. Two mice (one wild-type, one αMHC^403/+ mouse) died during the study and were not included in the analysis. (b) Changes in RNA molecular markers of cardiac hypertrophy. RNA expression in hearts from αMHC^403/+ mice (30–50 weeks old) compared with expression in age-matched wild-type (WT) hearts, with and without diltiazem treatment. Northern blots of atrial natriuretic factor (ANF) and α-skeletal actin expression are shown. RNA levels were standardized to 28S RNA band stained with ethidium bromide. The intensity of hybridizing species was assessed by densitometry of the Northern blots, and the RNA levels were normalized to the ANF level in wild-type left ventricle.

The pathologic hallmarks of hypertrophic cardiomyopathy — myocyte hypertrophy, disarray, and fibrosis — were also assessed in diltiazem-treated mice. Thirty-week-old untreated αMHC^403/+ mice demonstrate typical histopathology of hypertrophic cardiomyopathy (13, 14). Sections that were reacted with Masson’s Trichrome stain exhibited significant fibrosis (Figure 4a). The fractional area of myocardium that reacted with Masson’s Trichrome stain was compared in histologic sections from diltiazem-treated and untreated αMHC^403/+ hearts (Figure 4a). Twenty pathologic sections were analyzed from ten anatomic levels in the left ventricles of hearts. Whereas 4.07% ± 0.18% of the left ventricle was fibrotic in untreated αMHC^403/+ hearts (n = 3), significantly less fibrosis (0.23% ± 0.04%) was present in diltiazem-treated αMHC^403/+ hearts (n = 4; P < 0.001 vs. untreated αMHC^403/+ mice). Comparison of hematoxylin and eosin–stained myocardial sections also revealed less myocyte hypertrophy and disarray in 30-week-old diltiazem-treated than in untreated αMHC^403/+ mice (Figure 4b).

Figure 4

Histopathologic analyses of diltiazem-treated mice. (a) Low-magnification (×2.5) images of whole-heart sections from wild-type and αMHC^403/+ mice cut transversely through the left and right ventricles, and stained with Masson’s Trichrome. Areas of fibrosis (blue) are seen in αMHC^403/+ hearts that are absent in diltiazem-treated hearts. (b) High-magnification (×100) images of left ventricular sections, stained with Masson’s Trichrome. Myocyte hypertrophy, disarray, and fibrosis are present in untreated αMHC^403/+ hearts; all changes are prevented with diltiazem treatment.

This study demonstrates that abnormal SR Ca²⁺ responses and reduced Ca²⁺-binding proteins are early events in the pathogenesis of hypertrophic cardiomyopathy in αMHC^403/+ mice. Caffeine-induced release of SR Ca²⁺ stores and levels of calsequestrin, the Ca²⁺ channel RyR2, and associated peptides junctin and triadin were decreased in advance of histopathology or echocardiographic evidence of cardiac hypertrophy in αMHC^403/+ mice. Restitution of normal Ca²⁺ storage protein levels occurred in response to diltiazem-mediated inhibition of L-type Ca²⁺ channels, and when diltiazem was administered early to αMHC^403/+ mice, the cardiac histopathology and hemodynamic features of this sarcomere protein mutation were significantly abated.

Previous studies demonstrated that levels of cytosolic Ca²⁺ in αMHC^403/+ myocytes are comparable to those found in wild-type cells (16). However, responses to pharmacologic agents such as cyclosporin A or minoxidil, which produce a brisk (30%) increase in diastolic Ca²⁺ levels in wild-type myocytes, are attenuated (<8% increases) in αMHC^403/+ myocytes (16). This diminutive response by αMHC^403/+ myocytes is explained by the current studies. In response to the potent activation of RyR2 channels by caffeine, the maximum and total Ca²⁺ release from the SR was less in αMHC^403/+ myocytes than in wild-type myocytes. This suggests that the SRs of αMHC^403/+ myocytes contain lower levels of Ca²⁺ than do the SRs of wild-type myocytes (Figures 1 and 2).

Because αMHC^403/+ myocyte SRs contained reduced amounts of Ca²⁺, we hypothesized that SR Ca²⁺ storage proteins would correspondingly be diminished. Decreased levels of calsequestrin and RyR2 in the SRs of αMHC^403/+ myocytes were found. Calsequestrin is a 55-kDa high-capacity Ca²⁺-binding protein that both serves as a reservoir for SR Ca²⁺ and, with junctin and triadin, forms a complex that regulates Ca²⁺ release via RyR2 (26, 28, 29). Downregulation of calsequestrin and the other components of this quaternary RyR2 complex in αMHC^403/+ myocytes is consistent with decreased SR Ca²⁺ levels. SR Ca²⁺ levels may further be depleted by increased phosphorylation of the RyR2 channel in αMHC^403/+ myocytes. Phosphorylation decreases the amount of RyR2-associated FKBP12.6, which makes the channel more sensitive to calcium-induced activation and increases the probability of channel opening (21, 30, 31). Whereas only one of three potential sites was phosphorylated in wild-type myocytes, most RyR2 channels from αMHC^403/+ myocytes had two or more phosphorylated sites.

Reduced SR Ca²⁺ levels but normal cytosolic Ca²⁺ levels in αMHC^403/+ myocytes are consistent with the model that the mutant sarcomere functions like an ion trap (Figure 5). Altered biophysical properties of a mutated myosin heavy chain could enhance Ca²⁺ retention by the sarcomere and deplete SR Ca²⁺ stores. Observations of αMHC^403/+ muscle physiology support this model: mutant fibers show greater-than-normal isometric tension development at submaximal Ca²⁺ levels, and mutant hearts exhibit decreased rates of relaxation (15, 32).

Figure 5

Model of Ca²⁺ cycling in cardiac myocytes and the effects of a hypertrophic cardiomyopathy–causing mutation. (a) Wild-type myocytes showing normal Ca²⁺ regulation. Ca²⁺ enters the myocyte through L-type Ca²⁺ channels. Small entry of Ca²⁺ stimulates Ca release (calcium-induced Ca²⁺ release; CICR) from the SR via cardiac ryanodine receptors (RyR2) to the sarcomere. Ca²⁺ returns to the SR via the sarcoplasmic/endoplasmic Ca²⁺ ATPase (SERCA) pump, which is regulated by phospholamban (PLB). Ca²⁺ cycling is “balanced” between the sarcomere and SR. (b) Mutant (αMHC^403/+) myocytes have a mutation in the sarcomere (represented by an asterisk). The defective sarcomere acts as an ion trap, resulting in accumulation of Ca²⁺. Less Ca²⁺ returns to the SR, resulting in decreased SR Ca²⁺ stores, decreased SR calsequestrin (CSQ), and reduced expression of RyR2. The net effect is a Ca²⁺ shift within the cell, with a relative Ca²⁺ excess in the sarcomere, and Ca²⁺ depletion in the SR.

The development of SR Ca²⁺ dysregulation follows mutant peptide expression and predates the hypertrophic response in αMHC^403/+ mice. By 4 weeks after birth, when αMHC^403/+ myocytes express equal amounts of mutant and wild-type peptide (24), dysregulation of SR calcium was apparent, and if it went uninterrupted, phenotypic manifestations of hypertrophic cardiomyopathy ensued (13, 14). L-type channel inhibition by diltiazem most likely interrupts this process by chronic attenuation of calcium-induced SR Ca²⁺ release, so as to limit Ca²⁺ sequestration by the mutant sarcomere. The concurrent restoration of normal RyR2 and SR Ca²⁺-binding proteins levels and reduction of cardiac hypertrophy and fibrosis in diltiazem-treated αMHC^403/+ mice further support a mechanistic link between SR Ca²⁺ dysregulation and disease phenotype.

The L-type Ca²⁺ channel is the primary target of diltiazem. However, diltiazem is known to have other cellular targets, such as the mitochondrial Na⁺/Ca²⁺ exchanger. Alteration in mitochondrial Ca²⁺ concentration might change αMHC^403/+ myocyte energetics and thereby alter the hypertrophic response of the heart. Diltiazem might reduce the hypertrophic response by improving cardiac energetics. However, under normal conditions, diltiazem-treated αMHC^403/+ hearts appear to do less work (lower heart rate, reduced stiffness [EES], and reduced dP/dt_max; see Table 1) than the untreated αMHC^403/+ heart, suggesting that even if cardiac energetics remain unaltered in αMHC^403/+ hearts, the hearts should benefit from diltiazem-mediated work reduction.

Other investigators have identified several important intracellular Ca²⁺ sensors that can activate several signaling pathways to effect myocyte growth and function (for review, see ref. 33). Based on its association with the SR and function in RyR2 channel phosphorylation, calcium/calmodulin–dependent protein kinase (CamKII) is a particularly intriguing candidate for signaling myocyte growth (34). Levels of two well-characterized signaling molecules, calmodulin and the calcium/calmodulin–activated phosphatase calcineurin (PP2B-Aα and -Aβ), were not altered in αMHC^403/+ mice (data not shown). Whether transient or compartmentalized changes in these or Ca²⁺ activation of other signaling molecules triggers hypertrophy in αMHC^403/+ mice remains unknown.

An important conclusion from these data is that early restoration of SR Ca²⁺ decreases the hypertrophic response to a sarcomere mutation. Administration of diltiazem, in doses resulting in only modest hemodynamic effects, to prehypertrophic αMHC^403/+ mice improved cardiac function and provided long-term (39 weeks) attenuation of hypertrophic pathology. Improvement in cardiac function was almost exclusively in systolic function parameters, with little effect on diastolic function (Table 1). This may suggest that altering Ca²⁺ handling is important for contractile function, but that diastolic function is mediated by alternate mechanisms. In humans with clinical evidence of hypertrophic cardiomyopathy, L-type Ca²⁺ channel inhibitors such as diltiazem are used to treat symptoms. Our findings indicate that greater benefit should occur if these inhibitors are administered in advance of disease expression. In conjunction with gene-based, preclinical diagnosis of human sarcomere protein mutations, evaluation of the potential for diltiazem, and other pharmacologic agents that restore SR Ca²⁺ levels, to inhibit development of hypertrophic cardiomyopathy appears warranted.

We thank Larry R. Jones for providing the RyR2 and junctin antibodies, and Julie Zhang for myocyte isolation. The Howard Hughes Medical Institute supported these studies. C. Semsarian is the recipient of a National Heart Foundation of Australia grant. U. Mende is supported by an NIH Specialized Centers of Research grant and an American Heart Association Scientist Development grant. I. Ahmad is supported by a Sarnoff Fellowship.

1. Fatkin, D., Seidman, J.G., and Seidman, C.E. 2000. Hypertrophic cardiomyopathy. In Cardiovascular medicine. J.T. Willerson and J.N. Cohn, editors. W.B. Saunders Co. Philadelphia, Pennsylvania, USA. 1055–1074.
2. Seidman, C.E., and Seidman, J.G. 2001. Hypertrophic cardiomyopathy. In The metabolic and molecular bases of inherited disease. C.R. Scriver, A.L. Beaudet, W.S. Sly, and D. Valle, editors. McGraw-Hill. New York, New York, USA. 5433–5452.
3. Maron, BJ, Bonow, RO, Cannon (III), RO, Leon, MB, Epstein, SE. Hypertrophic cardiomyopathy. Interrelations of clinical manifestations, pathophysiology, and therapy (1). N Engl J Med 1987. 316:844-852.
   View this article via: PubMed Google Scholar
4. Spirito, P, Seidman, CE, McKenna, WJ, Maron, BJ. The management of hypertrophic cardiomyopathy. N Engl J Med 1997. 336:775-785.
   View this article via: PubMed CrossRef Google Scholar
5. Maron, BJ, Epstein, SE, Roberts, WC. Causes of sudden death in competitive athletes. J Am Coll Cardiol 1986. 7:204-214.
   View this article via: PubMed Google Scholar
6. Maron, BJ, et al. Sudden death in young competitive athletes. Clinical, demographic, and pathological profiles. JAMA 1996. 276:199-204.
   View this article via: PubMed CrossRef Google Scholar
7. Vikstrom, KL, Factor, SM, Leinwand, LA. Mice expressing mutant myosin heavy chains are a model for familial hypertrophic cardiomyopathy. Mol Med 1996. 2:556-567.
   View this article via: PubMed Google Scholar
8. Kittleson, MD, et al. Familial hypertrophic cardiomyopathy in maine coon cats: an animal model of human disease. Circulation 1999. 99:3172-3180.
   View this article via: PubMed Google Scholar
9. Marian, AJ, et al. A transgenic rabbit model for human hypertrophic cardiomyopathy. J Clin Invest 1999. 104:1683-1692.
   View this article via: JCI PubMed CrossRef Google Scholar
10. Oberst, L, et al. Dominant-negative effect of a mutant cardiac troponin T on cardiac structure and function in transgenic mice. J Clin Invest 1998. 102:1498-1505.
    View this article via: JCI PubMed CrossRef Google Scholar
11. Patel, R, et al. Simvastatin induces regression of cardiac hypertrophy and fibrosis and improves cardiac function in a transgenic rabbit model of human hypertrophic cardiomyopathy. Circulation 2001. 104:317-324.
    View this article via: PubMed CrossRef Google Scholar
12. Lim, DS, et al. Angiotensin II blockade reverses myocardial fibrosis in a transgenic mouse model of human hypertrophic cardiomyopathy. Circulation 2001. 103:789-791.
    View this article via: PubMed Google Scholar
13. Geisterfer-Lowrance, AA, et al. A mouse model of familial hypertrophic cardiomyopathy. Science 1996. 272:731-734.
    View this article via: PubMed CrossRef Google Scholar
14. McConnell, BK, et al. Comparison of two murine models of familial hypertrophic cardiomyopathy. Circ Res 2001. 88:383-389.
    View this article via: PubMed Google Scholar
15. Blanchard, E, Seidman, C, Seidman, JG, LeWinter, M, Maughan, D. Altered crossbridge kinetics in the αMHC^403/+ mouse model of familial hypertrophic cardiomyopathy. Circ Res 1999. 84:475-483.
    View this article via: PubMed Google Scholar
16. Fatkin, D, et al. An abnormal Ca2+ response in mutant sarcomere protein-mediated familial hypertrophic cardiomyopathy. J Clin Invest 2000. 106:1351-1359.
    View this article via: JCI PubMed CrossRef Google Scholar
17. Bers, DM. Calcium fluxes involved in control of cardiac myocyte contraction. Circ Res 2000. 87:275-281.
    View this article via: PubMed Google Scholar
18. Kim, SJ, et al. An α-cardiac myosin heavy chain gene mutation impairs contraction and relaxation function of cardiac myocytes. Am J Physiol 1999. 276:H1780-H1787.
    View this article via: PubMed Google Scholar
19. McConnell, BK, Moravec, CS, Bond, M. Troponin I phosphorylation and myofilament calcium sensitivity during decompensated cardiac hypertrophy. Am J Physiol 1998. 274:H385-H396.
    View this article via: PubMed Google Scholar
20. McConnell, BK, et al. Dilated cardiomyopathy in homozygous myosin-binding protein-C mutant mice. J Clin Invest 1999. 104:1235-1244.
    View this article via: JCI PubMed CrossRef Google Scholar
21. Marx, SO, et al. PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts. Cell 2000. 101:365-376.
    View this article via: PubMed CrossRef Google Scholar
22. Georgakopoulos, D, et al. The pathogenesis of familial hypertrophic cardiomyopathy: early and evolving effects from an α-cardiac myosin heavy chain missense mutation. Nat Med 1999. 5:327-330.
    View this article via: PubMed CrossRef Google Scholar
23. Palmer, BM, Lynch, JM, Snyder, SM, Moore, RL. Renal hypertension prevents run training modification of cardiomyocyte diastolic Ca2+ regulation in male rats. J Appl Physiol 2001. 90:2063-2069.
    View this article via: PubMed Google Scholar
24. Fatkin, D, et al. Neonatal cardiomyopathy in mice homozygous for the Arg403Gln mutation in the α-cardiac myosin heavy chain gene. J Clin Invest 1999. 103:147-153.
    View this article via: JCI PubMed CrossRef Google Scholar
25. Semsarian, C, et al. A polymorphic modifier gene alters the hypertrophic response in a murine model of familial hypertrophic cardiomyopathy. J Mol Cell Cardiol 2001. 33:2055-2060.
    View this article via: PubMed CrossRef Google Scholar
26. Zhang, L, Kelley, J, Schmeisser, G, Kobayashi, YM, Jones, LR. Complex formation between junctin, triadin, calsequestrin, and the ryanodine receptor. Proteins of the cardiac junctional sarcoplasmic reticulum membrane. J Biol Chem 1997. 272:23389-23397.
    View this article via: PubMed CrossRef Google Scholar
27. Scriven, DR, Dan, P, Moore, ED. Distribution of proteins implicated in excitation-contraction coupling in rat ventricular myocytes. Biophys J 2000. 79:2682-2691.
    View this article via: PubMed Google Scholar
28. Jones, LR, et al. Regulation of Ca2+ signaling in transgenic mouse cardiac myocytes overexpressing calsequestrin. J Clin Invest 1998. 101:1385-1393.
    View this article via: JCI PubMed CrossRef Google Scholar
29. Niki, I, Yokokura, H, Sudo, T, Kato, M, Hidaka, H. Ca2+ signaling and intracellular Ca2+ binding proteins. J Biochem (Tokyo) 1996. 120:685-698.
    View this article via: PubMed Google Scholar
30. Szegedi, C, Sarkozi, S, Herzog, A, Jona, I, Varsanyi, M. Calsequestrin: more than ‘only’ a luminal Ca2+ buffer inside the sarcoplasmic reticulum. Biochem J 1999. 337:19-22.
    View this article via: PubMed CrossRef Google Scholar
31. Yano, K, Zarain-Herzberg, A. Sarcoplasmic reticulum calsequestrins: structural and functional properties. Mol Cell Biochem 1994. 135:61-70.
    View this article via: PubMed CrossRef Google Scholar
32. Spindler, M, et al. Diastolic dysfunction and altered energetics in the αMHC^403/+ mouse model of familial hypertrophic cardiomyopathy. J Clin Invest 1998. 101:1775-1783.
    View this article via: JCI PubMed CrossRef Google Scholar
33. Frey, N, McKinsey, TA, Olson, EN. Decoding calcium signals involved in cardiac growth and function. Nat Med 2000. 6:1221-1227.
    View this article via: PubMed CrossRef Google Scholar
34. Passier, R, et al. CaM kinase signaling induces cardiac hypertrophy and activates the MEF2 transcription factor in vivo. J Clin Invest 2000. 105:1395-1406.
    View this article via: JCI PubMed CrossRef Google Scholar