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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
2R01HL092097-05A1
Application #
8701650
Study Section
Electrical Signaling, Ion Transport, and Arrhythmias Study Section (ESTA)
Program Officer
Krull, Holly
Project Start
2008-04-01
Project End
2018-04-30
Budget Start
2014-05-01
Budget End
2015-04-30
Support Year
5
Fiscal Year
2014
Total Cost
$658,312
Indirect Cost
$168,584
Name
University of California Davis
Department
Pharmacology
Type
Schools of Medicine
DUNS #
047120084
City
Davis
State
CA
Country
United States
Zip Code
95618
Bartos, Daniel C; Morotti, Stefano; Ginsburg, Kenneth S et al. (2017) Quantitative analysis of the Ca2+ -dependent regulation of delayed rectifier K+ current IKs in rabbit ventricular myocytes. J Physiol 595:2253-2268
Rebbeck, Robyn T; Essawy, Maram M; Nitu, Florentin R et al. (2017) High-Throughput Screens to Discover Small-Molecule Modulators of Ryanodine Receptor Calcium Release Channels. SLAS Discov 22:176-186
Bers, Donald M (2017) CALMing Down Arrhythmogenic Calmodulinopathies via a Precision Medicine Approach. Circ Res 120:3-4
Rebbeck, Robyn T; Nitu, Florentin R; Rohde, David et al. (2016) S100A1 Protein Does Not Compete with Calmodulin for Ryanodine Receptor Binding but Structurally Alters the Ryanodine ReceptorĀ·Calmodulin Complex. J Biol Chem 291:15896-907
Barbagallo, Federica; Xu, Bing; Reddy, Gopireddy R et al. (2016) Genetically Encoded Biosensors Reveal PKA Hyperphosphorylation on the Myofilaments in Rabbit Heart Failure. Circ Res 119:931-43
Uchinoumi, Hitoshi; Yang, Yi; Oda, Tetsuro et al. (2016) CaMKII-dependent phosphorylation of RyR2 promotes targetable pathological RyR2 conformational shift. J Mol Cell Cardiol 98:62-72
Sato, Daisuke; Shannon, Thomas R; Bers, Donald M (2016) Sarcoplasmic Reticulum Structure and Functional Properties that Promote Long-Lasting Calcium Sparks. Biophys J 110:382-90
Gomez-Hurtado, Nieves; Boczek, Nicole J; Kryshtal, Dmytro O et al. (2016) Novel CPVT-Associated Calmodulin Mutation in CALM3 (CALM3-A103V) Activates Arrhythmogenic Ca Waves and Sparks. Circ Arrhythm Electrophysiol 9:
Wagner, Stefan; Maier, Lars S; Bers, Donald M (2015) Role of sodium and calcium dysregulation in tachyarrhythmias in sudden cardiac death. Circ Res 116:1956-70
Hammer, Karin P; Ljubojevic, Senka; Ripplinger, Crystal M et al. (2015) Cardiac myocyte alternans in intact heart: Influence of cell-cell coupling and ?-adrenergic stimulation. J Mol Cell Cardiol 84:1-9

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