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

Abstract

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 alphaMHC(403/+)) bearing an Arg403Gln missense mutation in the alpha cardiac myosin heavy chain. We demonstrate that Ca(2+) is reduced in the sarcoplasmic reticulum of alphaMHC(403/+) mice, and levels of the sarcoplasmic reticulum Ca(2+)-binding protein calsequestrin are diminished in advance of changes in cardiac histology or morphology. Further evidence for dysregulation of sarcoplasmic reticulum Ca(2+) in these animals is seen in their decreased expression of the ryanodine receptor Ca(2+)-release channel and its associated membrane proteins and in an increase in ryanodine receptor phosphorylation. Early administration of the L-type Ca(2+) channel inhibitor diltiazem restores normal levels of these sarcoplasmic reticular proteins and prevents the development of pathology in alphaMHC(403/+) mice. We conclude that disruption of sarcoplasmic reticulum Ca(2+) homeostasis is an important early event in the pathogenesis of this disorder and suggest that the use of Ca(2+) channel blockers in advance of established clinical disease could prevent hypertrophic cardiomyopathy caused by sarcomere protein gene mutations.

Figures

Figure 1

SR Ca2+ storage and altered Ca2+-related protein expression. (a) Ca2+ changes, assessed in wild-type (+/+) and αMHC403/+ (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 αMHC403/+ mice aged 30–50 weeks. Coomassie staining indicates loading of samples in each lane. Treatment of αMHC403/+ 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.

Figure 2

Calsequestrin localization within myocytes. (a) Immunostaining of isolated wild-type (+/+) and αMHC403/+ (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 αMHC403/+ myocytes.

Figure 3

Prevention of cardiac hypertrophy in αMHC403/+ mice. (a) Left ventricular anterior wall thickness (LVAW) was evaluated in wild-type and αMHC403/+ 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 αMHC403/+ mice. *P < 0.01 vs. untreated αMHC403/+ mice. Two mice (one wild-type, one αMHC403/+ 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 αMHC403/+ 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.

Figure 4

Histopathologic analyses of diltiazem-treated mice. (a) Low-magnification (×2.5) images of whole-heart sections from wild-type and αMHC403/+ mice cut transversely through the left and right ventricles, and stained with Masson’s Trichrome. Areas of fibrosis (blue) are seen in αMHC403/+ 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 αMHC403/+ hearts; all changes are prevented with diltiazem treatment.

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 (αMHC403/+) 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.