Mutation and dysregulation of the cardiac ryanodine receptor (RyR2) or sarcoplasmic reticulum (SR) calcium release channel contributes directly to catecholaminergic polymorphic ventricular tachycardia (CPVT) and heart failure (HF) in humans. Affected RyRs are more active than normal, leaking Ca from the SR during diastole causing arrhythmias and dysfunction. FKBP12.6 and calmodulin (CaM) are proteins that bind tightly with and may stabilize RyR2. We have developed novel quantitative methods to assess (in adult cardiac myocytes) how FKBP, CaM and other peptides bind to and modulate RyR2 gating, and are structurally positioned on the myocyte RyR2. While FKBP12.6 binds RyR2 with high affinity, it is not a critical RyR2 regulator. Here we examine how CaM binding and altered domain-domain interaction in RyR2 are involved in disease-related RyR2 dysfunction, using fluorescent tagged proteins and novel targeted sensors in adult ventricular myocyte confocal imaging to address 4 aims. We will: 1) Test whether HF-related RyR alterations decrease CaM binding and increase the access of the structural unzipping peptide DPc10. 2) Test whether known RyR inhibitors work on pathological RyR2 by altering FKBP12.6, CaM or DPc10 binding. 3) Measure Cleft [Ca] using novel targeted Ca sensors. 4) Enhance RyR2 structural model by mapping sites of S100A1, CaMKII, IPTx and Sorcin. This is a highly collaborative project between two groups with shared interests in cardiac calcium regulation in HF and arrhythmias and with complementary technical expertise in physiology, pharmacology, biochemistry, molecular biology, fluorescence spectroscopy and confocal imaging. This will greatly enhance our understanding of RyR2 structure and function in cardiac myocytes in health and disease and provide novel strategies for the development of therapeutics for pathologically modulated RyR2 in the heart.

Public Health Relevance

Heart failure and arrhythmias are major human health issues, afflicting millions of Americans. It has become clear that leak of calcium from the sarcoplasmic reticulum within the cardiac myocyte between normal heart beats contributes importantly to both of these major cardiac problems. Here we will elucidate the structural and functional problem at the molecular level and develop novel therapeutic strategies.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Electrical Signaling, Ion Transport, and Arrhythmias Study Section (ESTA)
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Krull, Holly
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University of California Davis
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Bers, Donald M (2014) Cardiac sarcoplasmic reticulum calcium leak: basis and roles in cardiac dysfunction. Annu Rev Physiol 76:107-27
Hwang, Hyun Seok; Nitu, Florentin R; Yang, Yi et al. (2014) Divergent regulation of ryanodine receptor 2 calcium release channels by arrhythmogenic human calmodulin missense mutants. Circ Res 114:1114-24
Mahalingam, Mohana; Girgenrath, Tanya; Svensson, Bengt et al. (2014) Structural mapping of divergent regions in the type 1 ryanodine receptor using fluorescence resonance energy transfer. Structure 22:1322-32
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
Gruber, Simon J; Cornea, Razvan L; Li, Ji et al. (2014) Discovery of enzyme modulators via high-throughput time-resolved FRET in living cells. J Biomol Screen 19:215-22
Despa, Sanda; Shui, Bo; Bossuyt, Julie et al. (2014) Junctional cleft [Ca²?]i measurements using novel cleft-targeted Ca²? sensors. Circ Res 115:339-47
Oda, Tetsuro; Yang, Yi; Nitu, Florentin R et al. (2013) In cardiomyocytes, binding of unzipping peptide activates ryanodine receptor 2 and reciprocally inhibits calmodulin binding. Circ Res 112:487-97
Girgenrath, Tanya; Mahalingam, Mohana; Svensson, Bengt et al. (2013) N-terminal and central segments of the type 1 ryanodine receptor mediate its interaction with FK506-binding proteins. J Biol Chem 288:16073-84
Bers, Donald M; Shannon, Thomas R (2013) Calcium movements inside the sarcoplasmic reticulum of cardiac myocytes. J Mol Cell Cardiol 58:59-66
Saucerman, Jeffrey J; Bers, Donald M (2012) Calmodulin binding proteins provide domains of local Ca2+ signaling in cardiac myocytes. J Mol Cell Cardiol 52:312-6

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