The goal of our proposed research is to understand how the cardiac Ca2+ release channel (ryanodine receptor, RyR2) regulates cardiac function. The RyR2s are 2,200 kDa ion channels that release Ca2+ ions in response to an action potential from the sarcoplasmic reticulum. The RyR2 ion channel is composed of four RyR2 560 kDa subunits that bind calmodulin (CaM), and four small 12.6 kDa FK506 binding proteins. Our proposed studies make use of genetically modified mice with mutations in the CaM binding domain of RyR2 to elucidate signaling mechanisms associated with cardiac hypertrophy. Homozygous mice expressing a mutant form of RyR2 (RyR2-W3587A/L3591D/F3603A or RyR2ADA), that is not inhibited by CaM at diastolic and systolic Ca2+ concentrations, show signs of cardiac hypertrophy as early as 1 day after birth. There is up- regulation of genes and proteins associated with class II histone deacetylase(HDAC)/myocyte enhancer factor- 2(MEF2) and calcineurin signaling pathways, and the homozygous mutant mice die within two weeks of birth. Genetically modified mice deficient in CaM regulation of RyR2 by CaM at diastolic or diastolic and systolic Ca2+ concentrations will be used to test the hypothesis that a defective sarcoplasmic reticulum Ca2+ release activates signaling mechanisms in the embryonic heart, and ensuing major alterations in signaling and Ca2+ handling proteins contribute to the rapid progression of cardiac hypertrophy in newborn mice. We will determine heart and cardiomyocyte function and morphology, and temporal changes in relative abundance and activity of signaling molecules by microarray, quantitative RT-PCR, immunoblot and enzymatic analysis. The functional significance of class II HDAC/MEF2 and calcineurin signaling will be probed using class II HDAC mutants, and by crossing mice impaired in CaM regulation of RyR2 with mice deficient in calcineurin A2, and with mice that carry the luciferase transgene driven by NFAT-dependent promoter. Ca2+ handling by wild type and mutant mice will be assessed using whole cell patch clamp techniques, Ca2+ imaging, cell homogenates and membrane preparations.
The proposed research will make use of genetically modified mice with mutations in the calmodulin binding domain of the cardiac sarcoplasmic reticulum Ca2+ release channel to reveal new regulatory mechanisms in cardiac hypertrophy. Our studies will provide new approaches to minimize the risks of cardiac hypertrophy and heart failure, one of the most frequent causes of death in humans.
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