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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL073051-09
Application #
8429359
Study Section
Cardiac Contractility, Hypertrophy, and Failure Study Section (CCHF)
Program Officer
Krull, Holly
Project Start
2003-04-15
Project End
2014-12-31
Budget Start
2013-01-01
Budget End
2014-12-31
Support Year
9
Fiscal Year
2013
Total Cost
$348,718
Indirect Cost
$113,098
Name
University of North Carolina Chapel Hill
Department
Biochemistry
Type
Schools of Medicine
DUNS #
608195277
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Yang, Yi; Guo, Tao; Oda, Tetsuro et al. (2014) Cardiac myocyte Z-line calmodulin is mainly RyR2-bound, and reduction is arrhythmogenic and occurs in heart failure. Circ Res 114:295-306
Yamaguchi, Naohiro; Chakraborty, Asima; Huang, Tai-Qin et al. (2013) Cardiac hypertrophy associated with impaired regulation of cardiac ryanodine receptor by calmodulin and S100A1. Am J Physiol Heart Circ Physiol 305:H86-94
Eu, Jerry P; Meissner, Gerhard (2012) Detection of calcium release via ryanodine receptors. Methods Mol Biol 798:373-82
Yamaguchi, Naohiro; Chakraborty, Asima; Pasek, Daniel A et al. (2011) Dysfunctional ryanodine receptor and cardiac hypertrophy: role of signaling molecules. Am J Physiol Heart Circ Physiol 300:H2187-95
Ramachandran, Srinivas; Serohijos, Adrian W R; Xu, Le et al. (2009) A structural model of the pore-forming region of the skeletal muscle ryanodine receptor (RyR1). PLoS Comput Biol 5:e1000367
Meissner, Gerhard; Pasek, Daniel A; Yamaguchi, Naohiro et al. (2009) Thermodynamics of calmodulin binding to cardiac and skeletal muscle ryanodine receptor ion channels. Proteins 74:207-11
Meissner, Gerhard; Wang, Ying; Xu, Le et al. (2009) Silencing genes of sarcoplasmic reticulum proteins clarifies their roles in excitation-contraction coupling. J Physiol 587:3089-90
Sun, Junhui; Yamaguchi, Naohiro; Xu, Le et al. (2008) Regulation of the cardiac muscle ryanodine receptor by O(2) tension and S-nitrosoglutathione. Biochemistry 47:13985-90
Yamaguchi, Naohiro; Takahashi, Nobuyuki; Xu, Le et al. (2007) Early cardiac hypertrophy in mice with impaired calmodulin regulation of cardiac muscle Ca release channel. J Clin Invest 117:1344-53
Yamaguchi, Naohiro; Meissner, Gerhard (2007) Does Ca2+/calmodulin-dependent protein kinase deltac activate or inhibit the cardiac ryanodine receptor ion channel? Circ Res 100:293-5

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