An abnormal Ca(2+) response in mutant sarcomere protein-mediated familial hypertrophic cardiomyopathy

Abstract

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 (alphaMHC(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 Ca(2+) concentrations in wild-type cardiac myocytes; alphaMHC(403/+) myocytes failed to respond. Pretreatment with a Ca(2+)-channel antagonist abrogated diastolic Ca(2+) changes in wild-type myocytes and prevented the exaggerated hypertrophic response of treated alphaMHC(403/+) mice. We conclude that FHC-causing sarcomere protein gene mutations cause abnormal Ca(2+) responses that initiate a hypertrophic response. These data define an important Ca(2+)-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.

Figures

Figure 1

(a) Survival of 13 CsA-treated wild-type (open circles) and 18 CsA-treated αMHC403/+ (filled circles) mice. Two αMHC403/+ mice died on the same day on two occasions. Wild-type mice were sacrificed at variable times to provide control specimens for αMHC403/+ mice. (b) Serial assessment of LV wall thickness (measured by transthoracic echocardiography) of wild-type (open circles) and αMHC403/+ (filled circles) mice treated with CsA. The coefficient of correlation is r = 0.83 for LV wall thickness and duration of treatment of αMHC403/+ mice.

Figure 2

Echocardiographic assessment of CsA-treated and untreated αMHC403/+ hearts. Upper panel: Transthoracic echocardiograms of untreated (a) and CsA-treated αMHC403/+ mice (b, day 7; c, day 15). Lower panel: Cardiac morphology defined by echocardiograms of untreated (a) and CsA-treated αMHC403/+ mice (b, day 7; c, day 15).

Figure 3

Gross morphology of hearts from CsA-treated αMHC403/+ (left) and wild-type (right) mice. The αMHC403/+ specimen exhibits marked left- and right-ventricular hypertrophy with cavity obliteration after 30 days of CsA treatment.

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 αMHC403/+ mice (b), but marked in sections from CsA-treated αMHC403/+ mice (c). Masson trichrome was used to stain fibrosis (blue) in cardiac sections (×5 objective lens) from αMHC403/+ mice receiving no treatment (d), CsA plus diltiazem (e), or CsA alone (f).

Figure 5

Ca2+ concentrations, assessed in fura-2–loaded αMHC403/+ and wild-type myocytes, before and after CsA or minoxidil treatment. Isolated myocytes from wild-type (+/+) and αMHC403/+ (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 αMHC403/+ (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).

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.