An abnormal Ca²⁺ response in mutant sarcomere protein–mediated familial hypertrophic cardiomyopathy

Dominant-negative sarcomere protein gene mutations cause familial hypertrophic cardiomyopathy (FHC), a disease characterized by left-ventricular hypertrophy, angina, and dyspnea that can result in sudden death. We report here that a murine model of FHC bearing a cardiac myosin heavy-chain gene missense mutation (αMHC^403/+), when treated with calcineurin inhibitors or a K⁺-channel agonist, developed accentuated hypertrophy, worsened histopathology, and was at risk for early death. Despite distinct pharmacologic targets, each agent augmented diastolic Ca²⁺ concentrations in wild-type cardiac myocytes; αMHC^403/+ myocytes failed to respond. Pretreatment with a Ca²⁺-channel antagonist abrogated diastolic Ca²⁺ changes in wild-type myocytes and prevented the exaggerated hypertrophic response of treated αMHC^403/+ mice. We conclude that FHC-causing sarcomere protein gene mutations cause abnormal Ca²⁺ responses that initiate a hypertrophic response. These data define an important Ca²⁺-dependent step in the pathway by which mutant sarcomere proteins trigger myocyte growth and remodel the heart, provide definitive evidence that environment influences progression of FHC, and suggest a rational therapeutic approach to this prevalent human disease.

Cardiac hypertrophy, identified by increased ventricular wall thickness, is a prevalent finding that occurs in 1 of 500 healthy young individuals (1). Hypertrophy is recognized as a compensatory physiologic response to excessive hemodynamic burden (such as pressure or volume overload) or a pathologic state that compromises function and produces heart failure (reviewed in refs. 2, 3). Familial hypertrophic cardiomyopathy (FHC) is a heritable disorder of increased ventricular wall thickness that occurs in the absence of hemodynamic burden. Affected individuals can be asymptomatic or develop breathlessness, chest pain, congestive heart failure, or sudden death. Cardiac hypertrophy is the only finding of the disorder, and both the severity and anatomic distribution of this pathology varies considerably between affected individuals. Molecular genetic studies have demonstrated that dominant mutations in genes encoding β-cardiac myosin heavy chain, cardiac troponin T, myosin regulatory light chain, myosin essential light chain, cardiac actin, cardiac troponin I, α-tropomyosin, titin, or cardiac myosin-binding protein C cause hypertrophic cardiomyopathy (for a review see ref. 4). Although the genetic causes of FHC are now defined, the mechanisms by which sarcomere protein gene mutations lead to cardiac hypertrophy are not understood.

Cardiac hypertrophy can result from a variety of different causes, e.g., sarcomere protein gene mutations, pressure overload, or aberrant gene expression. To elucidate the cellular and molecular events that signal remodeling of the heart, researchers have investigated different models of cardiac hypertrophy (reviewed in ref. 5). Recent studies have exploited transgenic mice that overexpress target proteins in the heart (6, 7). Robust and unregulated transgenic expression of such unrelated proteins as α adrenergic receptors (8), calcineurin (9), and even green fluorescence protein (10) have been shown to increase myocardial mass. Significant information has also been obtained from analyses of rodent models of pressure overload produced by aortic banding. Analyses of these pressure-overload models have implicated Ca²⁺-dependent steps in the hypertrophic process leading to this form of hypertrophy (for a review see ref. 11). Whether all of these causes of hypertrophy are signaled via the same pathways remains uncertain. Studies in murine FHC models should help address this question.

The αMHC^403/+ mouse, a murine FHC model, bears a missense mutation in one allele of its endogenous α-cardiac myosin heavy-chain gene (12). This defect substitutes glutamine for arginine at residue 403 and is analogous to a well-characterized human myosin mutation (13, 14). As in the human disease, cardiac pathology in αMHC^403/+ mice evolves slowly; histopathology (myocyte hypertrophy and disarray) and increased ventricular wall thickness are absent in 6-week-old mice, are variably present at 15 weeks, and are established at 30 weeks. Although dissection of the hypertrophic process using pharmacologic agents has produced equivocal results in analyses of some models of hypertrophy, we hypothesized that treatment of αMHC^403/+ mice, which differ from wild-type mice by only a single nucleotide, would help define mechanisms by which a sarcomere protein defect triggers cardiac hypertrophy. Therefore, we screened for pharmacologic agents that augment or inhibit the hypertrophic process in αMHC^403/+ but not in wild-type mice. Three agents were identified that cause significant hypertrophy in mutant but not wild-type mice. These agents were used to unmask abnormal intracellular Ca²⁺ responses in both αMHC^403/+ mice and isolated myocytes and to define a critical role for this ion in triggering the hypertrophic response due to sarcomere protein defects. The demonstration that Ca²⁺ plays a role in modulating the hypertrophic response has implications for the management and treatment of FHC patients.

To identify the signaling pathways for hypertrophy that are triggered by mutant sarcomere proteins, αMHC403/+ and wild-type mice (aged 6 to 12 weeks) were treated with agents implicated previously in activating or repressing myocyte growth. Pharmacologic inhibition of calcineurin activity by the immunosuppressive agent CsA has been shown previously to prevent (9, 17) and cause regression (18) of cardiac hypertrophy in some rodent models. CsA was administered subcutaneously to mice at doses producing CsA serum levels of approximately 1.7 mg/ml; comparable levels are found in humans receiving immunosuppressant therapy (19). Surprisingly, approximately 40% of the αMHC403/+ mice, but no wild-type mice, died during treatment (Figure 1a). Left ventricular (LV) wall thickness was assessed in surviving mice using transthoracic echocardiography (15). Serial analyses demonstrated that LV wall thickness increased almost twofold with treatment of αMHC403/+ mice, but not wild-type mice (Figure 1b, Figure 2, Figure 3; Table 1). The extent of LV hypertrophy in αMHC403/+ mice correlated with the duration of treatment (Figure 1b; r = 0.83). CsA-induced LV hypertrophy was associated with significant reductions in LV chamber dimensions, increased LV fractional shortening, and left atrial dilation (Table 1).

Figure 1

(a) Survival of 13 CsA-treated wild-type (open circles) and 18 CsA-treated αMHC^403/+ (filled circles) mice. Two αMHC^403/+ mice died on the same day on two occasions. Wild-type mice were sacrificed at variable times to provide control specimens for αMHC^403/+ mice. (b) Serial assessment of LV wall thickness (measured by transthoracic echocardiography) of wild-type (open circles) and αMHC^403/+ (filled circles) mice treated with CsA. The coefficient of correlation is r = 0.83 for LV wall thickness and duration of treatment of αMHC^403/+ mice.

Figure 2

Echocardiographic assessment of CsA-treated and untreated αMHC^403/+ hearts. Upper panel: Transthoracic echocardiograms of untreated (a) and CsA-treated αMHC^403/+ mice (b, day 7; c, day 15). Lower panel: Cardiac morphology defined by echocardiograms of untreated (a) and CsA-treated αMHC^403/+ mice (b, day 7; c, day 15).

Figure 3

Gross morphology of hearts from CsA-treated αMHC^403/+ (left) and wild-type (right) mice. The αMHC^403/+ specimen exhibits marked left- and right-ventricular hypertrophy with cavity obliteration after 30 days of CsA treatment.

Table 1

The effects of CsA, FK506, minoxidil, and diltiazem on wild-type and αMHC^403/+ cardiac morphology assessed by echocardiography

Pathologic examination of CsA-treated αMHC403/+ hearts confirmed echocardiographic findings of markedly increased LV wall thickness and also demonstrated profound right ventricular hypertrophy (Figure 3). Reductions of ventricular cavity dimensions and massive left-atrial dilation associated with mural thrombus formation was also found in some hearts (Figure 3 and data not shown). Histologic study of myocardial sections from CsA-treated αMHC403/+ mice (Figure 4) demonstrated more extensive myocyte hypertrophy, myofibrillar disarray, and interstitial fibrosis than sections from untreated, age-matched, αMHC403/+ mice. No ultrastructural abnormalities were found in CsA-treated wild-type mice (Figure 4, a and b). Masson trichrome stain revealed increased collagen deposition and fibrosis in specimens derived from CsA-treated αMHC403/+ mice (Figure 4f) compared with hearts from untreated αMHC403/+ mice (Figure 4d); both were absent in untreated or CsA-treated wild-type mice (Figure 4a and data not shown). Tissue sections from hearts of wild-type and αMHC403/+ mice, with and without CsA treatment, were assessed using the immunohistochemical TUNEL assay. There were no significant differences in TUNEL staining between sections obtained from the four different heart samples (data not shown).

Figure 4

Cardiac histopathology in hypertrophic and normal mice. H&E-stained sections from CsA-treated wild-type mice (a) appear normal. Myocyte hypertrophy and disarray is mild in sections (×40 objective lens) from untreated αMHC^403/+ mice (b), but marked in sections from CsA-treated αMHC^403/+ mice (c). Masson trichrome was used to stain fibrosis (blue) in cardiac sections (×5 objective lens) from αMHC^403/+ mice receiving no treatment (d), CsA plus diltiazem (e), or CsA alone (f).

To determine whether the augmented pathology of CsA-treated αMHC403/+ mice represented an idiosyncratic response, the effects of another calcineurin inhibitor, FK506, were examined. Subcutaneous administration of FK506 to αMHC403/+ (n = 5) mice produced a comparable, exaggerated hypertrophic response. One αMHC403/+ mouse died suddenly 4 days after initiation of the treatment. Echocardiography of surviving mice after 1 week of treatment showed severe LV hypertrophy and histopathology similar to that observed in CsA-treated αMHC403/+ mice (Table 1). FK506 treatment of wild-type (n = 5) mice had no effect on LV wall thickness, cardiac hemodynamics (assessed by echocardiography; Table 1), or histopathology (data not shown).

In addition to the substantial cardiac hypertrophy found in αMHC403/+ mice treated with CsA and FK506, both the rate of development and the morphology of the drug-induced hypertrophy were unusual. Untreated 20-week-old αMHC403/+ mice exhibit only modest LV hypertrophy (<10% increase in maximum LV wall thickness; see ref. 12 and our unpublished data). In contrast, CsA or FK506 treatment of 8- to 10-week old mutant mice produced rapidly progressive severe LV hypertrophy (>50% increase in maximum LV wall thickness) within 16 days of treatment (Figures 1b, Figure 2, and Figure 3). The morphologic pattern of hypertrophy in untreated αMHC403/+ mice is uniformly concentric (data not shown), whereas CsA- or FK506-treated αMHC403/+ mice developed proximal thickening of anterior and posterior LV walls that caused dynamic midcavity obliteration and outflow tract obstruction (Figure 2). Such features are highly reminiscent of the pathology seen in humans with severe obstructive hypertrophic cardiomyopathy (4).

Long-term treatment with minoxidil, a K⁺-channel agonist, can cause midcavity hypertrophy in humans and rodents (20–22). To determine whether a molecule with distinct pharmacologic actions could also augment the hypertrophic response of αMHC403/+ mice, minoxidil was orally administered through drinking water to wild-type (n = 3) and mutant mice (n = 7). Echocardiographic studies at day 7 demonstrated significant LV hypertrophy only in αMHC403/+ mice that progressively became more severe through treatment day 14 (Table 1). The morphologic pattern of hypertrophy of minoxidil-treated αMHC403/+ mice mimicked that produced by CsA and FK506.

Molecular markers of cardiac hypertrophy were measured in the CsA-, FK506-, and minoxidil-treated αMHC403/+ mouse hearts. Northern blot analyses (15) demonstrated 14.4-, 2.57-, and 3.4-fold increases in atrial natriuretic factor, brain natriuretic factor, and skeletal α actin RNAs, respectively, in CsA-treated αMHC403/+ hearts compared with CsA-treated wild-type hearts. Both FK506 and minoxidil also elevated these RNA markers of cardiac hypertrophy at least twofold in hearts from αMHC403/+ compared with treated wild-type mice. Levels of these transcripts were the same in ventricles of untreated mutant mice (age less than 12 weeks) and wild-type mice receiving CsA or minoxidil (data not shown).

To determine whether increased hemodynamic load (due to systemic vasoconstriction) accounted for the accelerated hypertrophic response of αMHC403/+ mice, blood pressures were measured (23). Mean arterial blood pressures of untreated wild-type and αMHC403/+ mice were similar (117 ± 15 versus 112 ± 16 mmHg, respectively; P = NS). CsA is known to increase blood pressure in rats (24) and humans (19), an effect abrogated by coadministration of the nitric oxide donor L-arginine (24). CsA increased blood pressure in wild-type mice (days 1–10: 124 ± 15 mmHg, P < 0.05; days 11–30: 129 ± 15 mmHg, P < 0.05), but coadministration of L-arginine with CsA normalized arterial pressures (days 11–30: 119 ± 13 mmHg; P = NS, compared with untreated wild-type mice). Mean arterial pressures in αMHC403/+ mice did not increase during 1–10 days of CsA treatment (115 ± 12 mmHg; P = NS) and subsequently pressures significantly declined (days 11–30: 107 ± 13 mmHg, treated vs. untreated αMHC403/+ mice; P < 0.05), presumably because cardiac output was reduced by development of marked ventricular hypertrophy. Coadministration of CsA plus L-arginine had no effect on the augmented hypertrophic response of mutant mice. Minoxidil, an agent with antihypertensive properties, lowered the mean arterial pressures in all groups of mice (97 ± 3.8 mmHg, treated, vs. 117 ± 15 mmHg, untreated; P < 0.02).

CsA has been shown to alter Ca²⁺ concentration in multiple cell types (25, 26), although this effect has not been examined in cardiac myocytes. Since changes in the cyclical variation of Ca²⁺ concentrations that normally occur throughout the cardiac cycle have been proposed as one signal for LV hypertrophy (27), we examined whether diastolic or systolic Ca²⁺ levels were altered in wild-type and αMHC403/+ mice. No significant differences in diastolic or systolic Ca²⁺ concentration were found between cardiac myocytes isolated from untreated wild-type and αMHC403/+ adult mice (data not shown). However myocytes isolated from CsA-treated wild-type adult mice had elevated diastolic Ca²⁺ concentration (untreated, n = 16, 113 ± 8 nM vs. treated, n = 30, 78 ± 8 nM; P < 0.001). In contrast, diastolic Ca²⁺ concentrations were not increased in myocytes from CsA-treated αMHC403/+ mice (untreated, n = 11, 124 ± 11 nM vs. treated, n = 21, 114 ± 7 nM; P = NS).

To determine whether the altered Ca²⁺ levels in myocytes reflected a direct effect of CsA on myocyte physiology versus indirect, systemic effects of this agent, myocytes were isolated, treated ex vivo with CsA, and Ca²⁺ transients were measured (Figure 5). Within 3 minutes of exposure to CsA, wild-type myocytes exhibited a 30% increase in diastolic Ca²⁺ concentrations (Figure 6), maximum systolic Ca²⁺ concentrations remained unchanged (before CsA, 222.9 ± 36 nM; after CsA, 233.3 ± 50 nM; n = 10, P = NS). The Ca²⁺ concentration differential between diastole and systole was therefore diminished by 23%. In contrast, αMHC403/+ myocytes exhibited little ex vivo response to CsA; diastolic Ca²⁺ concentration increased less than 8% and neither systolic Ca²⁺ concentrations nor the systolic/diastolic differential changed (Figures 5, 6).

Figure 5

Ca²⁺ concentrations, assessed in fura-2–loaded αMHC^403/+ and wild-type myocytes, before and after CsA or minoxidil treatment. Isolated myocytes from wild-type (+/+) and αMHC^403/+ (403/+) mice have comparable calcium concentrations at base line. Addition of CsA (15 μg/ml) or minoxidil (200 μg/ml) (vertical arrows) increases diastolic Ca²⁺ concentrations (vertical bars) in wild-type, but not mutant, myocytes.

Figure 6

The change (%) in diastolic Ca²⁺ concentrations in myocytes derived from wild-type (open symbols, dashed lines) or αMHC^403/+ (closed symbols, solid lines) mice treated with minoxidil (triangles), CsA (circles), or CsA plus diltiazem (squares). Diltiazem (28 μg/ml) administration began 20 seconds before addition of CsA. Each data point represents the average Ca²⁺ concentration from ten myocytes. After 2 and 3 minutes of treatment with either CsA or minoxidil, the Ca²⁺ concentration in wild-type myocytes was significantly different from the Ca²⁺ concentration in mutant myocytes treated with the same drug (P < 0.02) and was significantly different from the Ca²⁺ concentration in wild-type myocytes treated with diltiazem and CsA or minoxidil (P < 0.01).

The effects of ex vivo minoxidil on myocyte Ca²⁺ transients were also examined. Surprisingly, the responses of wild-type and αMHC403/+ myocytes to minoxidil mirrored responses to CsA. That is, wild-type myocytes increased diastolic Ca²⁺ concentrations in response to minoxidil, whereas diastolic Ca²⁺ concentrations remained unchanged in mutant myocytes (Figure 5 and Figure 6).

To determine if aberrant Ca²⁺ responses accounted for the dramatic hypertrophic response of αMHC403/+ mice to CsA, FK506, or minoxidil, animals were pretreated with the L-type Ca²⁺-channel blocker, diltiazem (28–30). After 2 weeks of diltiazem pretreatment, coadministration of CsA, FK506, or minoxidil for 3 weeks had no effect on αMHC403/+ mouse hearts. Hearts from αMHC403/+ mice treated with diltiazem and CsA, FK506, or minoxidil had LV wall thickness, myocyte hypertrophy, and fibrosis comparable to that found in untreated mutant mouse hearts (Table 1, Figure 1b, and Figure 4e). No deaths occurred in αMHC403/+ mice pretreated with diltiazem (data not shown). Ex vivo coadministration of diltiazem and CsA abrogated the diastolic Ca²⁺ increases in isolated wild-type myocytes (Figure 6).

Human genetic studies defined the molecular cause of hypertrophic cardiomyopathy as heterozygous mutations in genes encoding sarcomere proteins. The development of cell and animal model systems to study the cellular consequences of these gene defects have shown incorporation of mutant peptides into the contractile apparatus with dominant-negative effects (12, 31–33). However, the mechanism by which sarcomere mutations trigger hypertrophic growth of the myocardium remains unknown.

We demonstrate here that minoxidil, FK506, and CsA dramatically increase the hypertrophic response of αMHC403/+ mice (Figures 1–3). We have compared the mutant and wild-type myocyte response to these agents as a first step toward defining the mechanism by which the hypertrophic response is signaled in these cells. Myocytes from wild-type mice increase their diastolic Ca²⁺ concentration in response to either minoxidil or CsA (Figure 5 and 6) by approximately 30%. However, αMHC403/+-derived myocytes increased their diastolic Ca²⁺ concentration in response to these drugs by less than 10%. We conclude that mutant myocytes do not regulate their Ca²⁺ concentration appropriately.

The conclusion that inappropriate myocyte regulation of Ca²⁺ concentration plays a role in the hypertrophic response of αMHC403/+ mice is confirmed by the observation that diltiazem, an L-type Ca²⁺-channel inhibitor blocks this response (Figures 5 and 6). Further, diltiazem blocks the wild-type myocytes’ increase in diastolic Ca²⁺ concentration due to CsA and minoxidil (Figure 6). We conclude that sarcomere protein defects trigger hypertrophic remodeling of the heart through a novel Ca²⁺-dependent event.

Previous studies have suggested a role for calcineurin-dependent dephosphorylation of the transcription factor NFAT in hypertrophic growth of the heart (9, 17). Our data indicate this inhibition of calcineurin does not prevent, and indeed severely enhances, hypertrophy caused by the cardiac myosin heavy-chain Arg403Gln mutation. CsA has been reported to provide variable degrees of benefit in transgenic models of heart disease (for a review see ref. 34). However, we demonstrate here that CsA treatment of mice with sarcomere defects similar to those found in the human disease showed definitive adverse effects, including dramatic worsening of histopathology and sudden death. The histopathologic changes are not due to CsA-induced apoptosis (see Results). Several findings indicate this to be a direct effect on myocyte physiology. First, significant increases in arterial blood pressure were not observed in CsA-treated αMHC403/+ mice, and coadministration of L-arginine, an agent that inhibits multiple systemic effects of CsA, failed to block the enhanced hypertrophic effect. Second, minoxidil, an antihypertensive agent that reduced blood pressure in wild-type mice, also exacerbated the pathology of αMHC403/+ mice. Third, ex vivo CsA or minoxidil treatment of wild-type, but not αMHC403/+, myocytes produced the same intracellular response. We also hypothesize, because both minoxidil and CsA appear to alter the hypertrophic response through a Ca²⁺-mediated response and because myocyte alterations in Ca²⁺ concentration occur within a few minutes after administration of these agents, that these agents are not acting through the dephosphorylation of the transcription factor NFAT.

Studies of CsA effects on other cells types (25–27) demonstrated that an early event was increased cytosolic Ca²⁺ concentrations. Our data show that myocytes respond similarly to this agent by augmenting cytosolic Ca²⁺. In the myocyte, as in other contractile cells, this should prompt sarcoplasmic reticulum release of Ca²⁺ (termed calcium-induced calcium release) predominantly through ryanodine receptors, perhaps with some contribution from inositol triphosphate receptors (Figure 7; see refs. 35, 36). Like CsA, the K⁺-channel agonist minoxidil also elevated myocyte Ca²⁺ concentrations. Activation of the ryanodine receptor by K⁺ influx is one mechanism by which this might occur (Figure 7; ref. 37).

Figure 7

The pathway leading from sarcomere protein gene mutation to hypertrophic cardiomyopathy and the role of an abnormal Ca²⁺ response. CsA and FK506 may increase cytoplasmic Ca²⁺ through interaction with calcineurin or by activation of the L-type Ca²⁺ channel. Cytoplasmic Ca²⁺ enters the sarcoplasmic reticulum by way of an ATPase-dependent calcium pump (SERCA) and exits by way of the inositol triphosphate receptor (InsP₃R) and the ryanodine receptor (RyR). Small increases in Ca²⁺ trigger Ca²⁺-induced Ca²⁺ release (CICR) primarily through the RyR. Considerable Ca²⁺ is stored in the sarcomere. We hypothesize that sarcomeres containing mutant myosins (denoted by asterisks) store more Ca²⁺ than normal sarcomeres, causing a reduction in sarcoplasmic reticulum Ca²⁺ that signals a hypertrophic response. Most Ca²⁺ in the sarcomere and sarcoplasmic reticulum is bound to carrier proteins, whereas most Ca²⁺ in the cytoplasm is free. Diltiazem is an inhibitor of the L-type Ca²⁺ channel, whereas minoxidil is an activator of the K⁺ channel.

Ca²⁺ flux is essential for excitation-contraction coupling in the heart and skeletal muscle (reviewed in 38). Depolarization triggers entry of small amounts of Ca²⁺ through L-type Ca²⁺ channels located on the cell membrane. Calcium-induced calcium release from the sarcoplasmic reticulum then rapidly raises cytosolic Ca²⁺, which fosters Ca²⁺ -troponin-C interactions and triggers sarcomere contraction. Ca²⁺ recycling into the sarcoplasmic reticulum then occurs by an ATPase-dependent calcium pump (SERCA), resulting in sarcomere relaxation.

Wild-type myocytes treated with CsA or minoxidil promptly responded with an elevation of diastolic Ca²⁺. Because this response caused no demonstrable effect on either cardiac hemodynamics or cardiac morphology in wild-type mice, we suspect that Ca²⁺ reservoirs in healthy myocytes adequately compensate to accommodate this demand. In contrast, αMHC403/+ myocytes failed to raise diastolic Ca²⁺ concentration in response to these agents. We interpret this result to indicate a relative depletion of intracellular Ca²⁺ reserves. The finding that wild-type myocytes respond to CsA or minoxidil in the same fashion, even when extracellular Ca²⁺ is significantly reduced, supports this interpretation (B. McConnell and J.G. Seidman, unpublished data). In addition to the cytoplasm, myocyte Ca²⁺ is enriched in the sarcoplasmic reticulum and the sarcomere. Since αMHC403/+ myocytes differ from wild-type only by expression of a single amino acid difference in the myosin heavy-chain polypeptide, the mutant sarcomere is likely to account for the observed Ca²⁺ deficit. We suggest that the contractile apparatus in αMHC403/+ myocytes functions like an unregulated ion trap (Figure 7). Two previous observations of αMHC403/+ muscle physiology support this model: (a) mutant fibers show greater than normal isometric tension development at submaximal Ca²⁺ levels (39), and (b) mutant hearts exhibit decreased rates of relaxation (40). Chronic elevation in sarcomere Ca²⁺ levels could account for both findings. The beneficial response of L-type channel inhibition by diltiazem is also explained by this model; chronic reduction of calcium-induced calcium release might limit Ca²⁺ sequestration by the mutant sarcomere.

A corollary to this hypothesis is that altered Ca²⁺ is the inciting trigger for the hypertrophic response. We presume this effect requires interaction with a protein that functions like a Ca²⁺ thermostat. Of the many known Ca²⁺-binding proteins within myocytes (reviewed in ref. 38), those located in the sarcoplasmic reticulum are particularly well positioned for this function. The unaltered levels of cytoplasmic Ca²⁺ levels in αMHC403/+ myocytes (Figure 7) after either calcineurin inhibition or K⁺-channel agonist treatment, despite the induction of dramatic hypertrophy, suggests a compartmentalized location for this putative Ca²⁺ sensor. Furthermore, model systems that alter expression of sarcoplasmic reticulum proteins such as calsequestrin have been shown to cause marked cardiac hypertrophy (41).

The conclusion that FHC is mediated through a Ca²⁺-dependent event has implications for the management and treatment of patients with this condition. First, these studies explain, at least in part, the variable severity of disease, long recognized in patients with the same mutation (4, 42, 43). The finding that agents that modify myocyte intracellular Ca²⁺ concentration can dramatically augment the hypertrophic response in FHC provides the first demonstration that an environmental factor can alter the disease severity. Given that agents as diverse as calcineurin inhibitors and a K⁺-channel agonist affect this ion, environmental factors such as lifestyle, diet, exercise, and pharmacologic agents are likely to substantially influence responses to a sarcomere protein gene mutation. Second, a rational therapeutic approach to FHC can now be considered. Patients with hypertrophic cardiomyopathy are routinely treated with Ca²⁺-channel blockers and/or beta-adrenergic receptor blockers because these drugs alter heart rate and/or cardiac contractility. The results presented here suggest the mechanism by which Ca²⁺-channel blockers may alter symptoms in FHC patients. Third, these studies may suggest a therapy for a recently identified class of patients—individuals bearing sarcomere protein gene mutations who do not yet have clinical signs of FHC, but who are genetically programmed to develop hypertrophic cardiomyopathy. Perhaps treatment of “presymptomatic” FHC patients (i.e., individuals bearing sarcomere protein gene mutations but who do not yet display signs of their disease) with diltiazem should be considered.

These studies have significant implications for understanding muscle biology and the pathology of hypertrophic cardiomyopathy. A central role for the sarcomere is defined in which the contractile apparatus regulates cell growth through Ca²⁺. In the healthy myocyte, Ca²⁺ equilibrium between the sarcomere, sarcoplasmic reticulum, and cytoplasm is critically maintained. In genetic and possibly acquired forms of sarcomere dysfunction, Ca²⁺ balance shifts (Figure 7), triggering events that remodel the myocyte and ultimately perturb cardiac structure. For patients with hypertrophic cardiomyopathy the immediate ramifications of these data are that treatment with CsA, as has been proposed elsewhere (refs. 17, 18; see also www.clinicaltrials.gov), should be reconsidered. Further, environmental factors that alter Ca²⁺ homeostasis should be considered modifiers of disease expression. Finally, Ca²⁺ channel-blocking agents may have beneficial effects, particularly as a “preventative” treatment, in contrast to the common use of such agents in the management of symptoms. Identification of the effectors through which Ca²⁺ modulates the hypertrophic response should provide further insights into the mechanism by which sarcomere gene defects produce the striking pathophysiology of hypertrophic cardiomyopathy.

The Howard Hughes Medical Institute supported these studies. J.O. Mudd was a Fellow of the Stanley J. Sarnoff Endowment for Cardiovascular Science Inc. C. Semsarian is the recipient of a National Heart Foundation of Australia scholarship.

Diane Fatkin, Bradley K. McConnell, James O. Mudd, and Christopher Semsarian contributed equally to this work.

1. Maron, BJ, et al. Prevalence of hypertrophic cardiomyopathy in a general population of young adults. Echocardiographic analysis of 4111 subjects in the CARDIA Study. Coronary Artery Risk Development in (Young) Adults. Circulation 1995. 92:785-789.
   View this article via: PubMed Google Scholar
2. Colluci, W.S., and Braunwald, E.B. 1997. Pathophysiology of heart failure. In Heart disease. E. Braunwald, editor. W.B. Saunders Co. Philadelphia, Pennsylvania, USA. 394–420.
   View this article via: PubMed Google Scholar
3. Benjamin, EJ, Levy, D. Why is left ventricular hypertrophy so predictive of morbidity and mortality? Am J Med Sci 1999. 317:168-175.
   View this article via: PubMed CrossRef Google Scholar
4. 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. 1055–1075.
   View this article via: PubMed Google Scholar
5. Hunter, J.J., Grace, A., and Chien, K.R. 1999. Molecular and cellular biology of cardiac hypertrophy and failure. In Molecular basis of cardiovascular disease. K.R. Chien, editor. W.B. Saunders Co. Philadelphia, Pennsylvania, USA. 211–250.
   View this article via: PubMed Google Scholar
6. Subramaniam, A, et al. Tissue-specific regulation of the alpha-myosin heavy chain gene promoter in transgenic mice. J Biol Chem 1991. 266:24613-24620.
   View this article via: PubMed Google Scholar
7. James, JF, Hewett, TE, Robbins, J. Cardiac physiology in transgenic mice. Circ Res 1998. 82:407-415.
   View this article via: PubMed Google Scholar
8. Gaudin, C, et al. Overexpression of Gs alpha protein in the hearts of transgenic mice. J Clin Invest 1995. 95:1676-1683.
   View this article via: JCI PubMed CrossRef Google Scholar
9. Molkentin, JD, et al. A calcineurin-dependent transcriptional pathway for cardiac hypertrophy. Cell 1998. 93:215-228.
   View this article via: PubMed CrossRef Google Scholar
10. Huang, W-Y, Aramburu, J, Douglas, PS, Izumo, S. Transgenic expression of green fluorescence protein can cause dilated cardiomyopathy. Nat Med 2000. 6:482-483.
    View this article via: PubMed CrossRef Google Scholar
11. Chien, KR. Meeting Koch’s postulates for calcium signaling in cardiac hypertrophy. J Clin Invest 2000. 105:1339-1342.
    View this article via: JCI PubMed CrossRef Google Scholar
12. 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
13. Jarcho, JA, et al. Mapping a gene for familial hypertrophic cardiomyopathy to chromosome 14q1. N Engl J Med 1989. 321:1372-1378.
    View this article via: PubMed Google Scholar
14. Geisterfer-Lowrance, AA, et al. A molecular basis for familial hypertrophic cardiomyopathy: a beta cardiac myosin heavy chain gene missense mutation. Cell 1990. 62:999-1006.
    View this article via: PubMed CrossRef Google Scholar
15. 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
16. Kim, SJ, et al. An alpha-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
17. Sussman, MA, et al. Prevention of cardiac hypertrophy in mice by calcineurin inhibition. Science 1998. 1281:1690-1693.
18. Lim, HW, et al. Reversal of cardiac hypertrophy in transgenic disease models by calcineurin inhibition. J Mol Cell Cardiol 2000. 32:697-709.
    View this article via: PubMed CrossRef Google Scholar
19. Ventura, HO, Malik, FS, Mehra, MR, Stapleton, DD, Smart, FW. Mechanisms of hypertension in cardiac transplantation and the role of cyclosporine. Curr Opin Cardiol 1997. 12:375-381.
    View this article via: PubMed Google Scholar
20. Moravec, CS, Ruhe, T, Cifani, JR, Milovanovic, M, Khairallah, PA. Structural and functional consequences of minoxidil-induced cardiac hypertrophy. J Pharmacol Exp Ther 1994. 269:290-296.
    View this article via: PubMed Google Scholar
21. Pogatsa-Murray, G, et al. Changes in left ventricular mass during treatment with minoxidil and cilazapril in hypertensive patients with left ventricular hypertrophy. J Hum Hypertens 1997. 11:149-156.
    View this article via: PubMed CrossRef Google Scholar
22. van der Velden, J, Borgdorff, P, Stienen, GJ. Minoxidil-induced cardiac hypertrophy in guinea pigs. Cell Mol Life Sci 1999. 55:788-798.
    View this article via: PubMed CrossRef Google Scholar
23. Krege, JH, Hodgin, JB, Hagaman, JR, Smithies, O. A noninvasive computerized tail-cuff system for measuring blood pressure in mice. Hypertension 1995. 25:1111-1115.
    View this article via: PubMed Google Scholar
24. Bartholomeusz, B, Hardy, KJ, Nelson, AS, Phillips, PA. Modulation of nitric oxide improves cyclosporin A-induced hypertension in rats and primates. J Hum Hypertens 1998. 12:839-844.
    View this article via: PubMed CrossRef Google Scholar
25. Chou, YC, Fong, JC. Cyclosporin A induces a biphasic increase in KCl-induced calcium influx in GH3 pituitary cells. Biochem Biophys Res Commun 1999. 254:169-173.
    View this article via: PubMed CrossRef Google Scholar
26. Misra, UK, Gawdi, G, Pizzo, SV. Cyclosporin A inhibits inositol 1,4,5-trisphosphate binding to its receptors and release of calcium from intracellular stores in peritoneal macrophages. J Immunol 1998. 161:6122-6127.
    View this article via: PubMed Google Scholar
27. Devereux, RB. Therapeutic options in minimizing left ventricular hypertrophy. Am Heart J 2000. 139(Suppl.):S9-S14.
    View this article via: PubMed CrossRef Google Scholar
28. Libersan, D, Marchand, R, Montplaisir, S, Chartrand, C, Dumont, L. Cardioprotective effects of diltiazem during acute rejection on heterotopic heart transplants. Eur Surg Res 1997. 29:229-236.
    View this article via: PubMed CrossRef Google Scholar
29. Abernethy, DR, Schwartz, JB. Calcium-antagonist drugs. N Engl J Med 1999. 341:1447-1457.
    View this article via: PubMed CrossRef Google Scholar
30. Fassi, A, et al. Beneficial effects of calcium channel blockade on acute glomerular hemodynamic changes induced by cyclosporine. Am J Kidney Dis 1999. 33:267-275.
    View this article via: PubMed CrossRef Google Scholar
31. Watkins, H, Seidman, CE, Seidman, JG, Feng, HS, Sweeney, HL. Expression and functional assessment of a truncated cardiac troponin T that causes hypertrophic cardiomyopathy. Evidence for a dominant negative action. J Clin Invest 1996. 98:2456-2461.
    View this article via: JCI PubMed CrossRef Google Scholar
32. Marian, AJ, Zhao, G, Seta, Y, Roberts, R, Yu, QT. Expression of a mutant (Arg92Gln) human cardiac troponin T, known to cause hypertrophic cardiomyopathy, impairs adult cardiac myocyte contractility. Circ Res 1997. 81:76-85.
    View this article via: PubMed Google Scholar
33. 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
34. Force, T, Rosenzweig, A, Choukroun, G, Hajjar, R. Calcineurin inhibitors and cardiac hypertrophy. Lancet 1999. 353:1290-1292.
    View this article via: PubMed CrossRef Google Scholar
35. Banijamali, HS, ter Keurs, MH, Paul, LC, ter Keurs, HE. Excitation-contraction coupling in rat heart: influence of cyclosporin A. Cardiovasc Res 1993. 27:1845-1854.
    View this article via: PubMed CrossRef Google Scholar
36. Cameron, AM, et al. Calcineurin associated with the inositol 1,4,5-trisphosphate receptor- FKBP12 complex modulates Ca²⁺ flux. Cell 1995. 83:463-472.
    View this article via: PubMed CrossRef Google Scholar
37. Hasselbach, W, Migala, A. Cations and anions as modifiers of ryanodine binding to the skeletal muscle calcium release channel. J Membr Biol 1998. 164:215-227.
    View this article via: PubMed CrossRef Google Scholar
38. MacKrill, JJ. Protein-protein interactions in intracellular Ca²⁺-release channel function. Biochem J 1999. 337:345-361.
    View this article via: PubMed CrossRef Google Scholar
39. Blanchard, EM. Targeted ablation of the murine alpha-tropomyosin gene. Circ Res 1997. 81:1005-1010.
    View this article via: PubMed Google Scholar
40. Georgakopoulos, D. The pathogenesis of familial hypertrophic cardiomyopathy: early and evolving effects from an alpha-cardiac myosin heavy chain missense mutation. Nat Med 1999. 5:327-330.
    View this article via: PubMed CrossRef Google Scholar
41. 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
42. 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. 316:780-789.
    View this article via: PubMed Google Scholar
43. Maron, BJ, Bonow, RO, Cannon (III), RO, Leon, MB, Epstein, SE. Hypertrophic cardiomyopathy. Interrelations of clinical manifestations, pathophysiology, and therapy (2). N Engl J Med 1987. 316:844-852.
    View this article via: PubMed Google Scholar