The overall goal of this project is to continue the Investigator's work on the molecular mechanism of excitation- contraction coupling in normal and diseased cardiac muscle focusing on the role of the ryanodine receptor. The importance of the ryanodine receptor (RyR) is shown by the fact that mutations in the RyR lead to various cardiac disorders. In congenital cardiac diseases, mutations are localized in the four regions of each subunit of the RyR tetramer. The Investigator has shown that two of these regions form a Domain Switch. Their zipping closes the calcium channel, while unzipping opens it;incomplete domain zipping in the resting state causes calcium channel dysfunctions, resulting in heart failure (arrhythmias and hypertrophy). The investigator's recent data suggest that the same type of channel regulation is operating also within the other two regions, pointing to their involvement in the channel gating mechanism, serving as Gate Manager. It appears that the Domain Switch and the Gate Manager are coupled by mediation of yet another set of domains, Transmitter. Thus, defect of the inter-domain interaction within each of these regions of the receptor will cause RyR-linked cardiac diseases. To elucidate the spatial relationship of interacting domains in each of these three key regions, the Investigator plans to perform site-directed fluorescence labeling of the target domain using peptides as a site- direction carrier and identify the sites of domain-domain interaction using the peptide mapping and mass spectroscopic techniques. To establish the role of these domains in channel regulation, (i) a local conformational change induced by domain peptides or site-specific antibodies in these domains will be detected using the protein-bound fluorescence probe, and (ii) the resultant activation of the channel activity will be followed by assays of [3H]ryanodine binding, single channel conductance and intra-cellular Ca2+ transients, to determine correlation between (i) and (ii). Further, in collaboration with researchers working on model systems of cardiac arrhythmias and hypertrophy, the Investigator will examine how the defective domain- domain interaction in the in vivo RyR causes disease states, and how one can pharmacologically control the domain interaction and channel function. The new information derived from this program will permit a better understanding of the pathogenic mechanisms of calcium channel dysfunction occurring in some cardiac diseases and will offer new opportunities for therapeutic interventions. The ryanodine receptor is a calcium channel protein playing a central role in the beat to beat regulation of the cellular calcium level in cardiac muscle. Defective channel function causes various heart diseases, such as ventricular tachycardia (sudden cardiac death) and hypertrophy. This program is designed to identify molecular mechanisms (inter-domain interactions in particular), whose defect causes channel dysfunction, and to develop new methods of therapeutic treatment of these disorders.

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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Przywara, Dennis
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Boston Biomedical Research Institute
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Hino, Akihiro; Yano, Masafumi; Kato, Takayoshi et al. (2012) Enhanced binding of calmodulin to the ryanodine receptor corrects contractile dysfunction in failing hearts. Cardiovasc Res 96:433-43
Gangopadhyay, Jaya P; Ikemoto, Noriaki (2011) Aberrant interaction of calmodulin with the ryanodine receptor develops hypertrophy in the neonatal cardiomyocyte. Biochem J 438:379-87
Suetomi, Takeshi; Yano, Masafumi; Uchinoumi, Hitoshi et al. (2011) Mutation-linked defective interdomain interactions within ryanodine receptor cause aberrant Caýýýýýrelease leading to catecholaminergic polymorphic ventricular tachycardia. Circulation 124:682-94
Gangopadhyay, Jaya Pal; Ikemoto, Noriaki (2010) Intracellular translocation of calmodulin and Ca2+/calmodulin-dependent protein kinase II during the development of hypertrophy in neonatal cardiomyocytes. Biochem Biophys Res Commun 396:515-21
Uchinoumi, Hitoshi; Yano, Masafumi; Suetomi, Takeshi et al. (2010) Catecholaminergic polymorphic ventricular tachycardia is caused by mutation-linked defective conformational regulation of the ryanodine receptor. Circ Res 106:1413-24
Xu, Xiaojuan; Yano, Masafumi; Uchinoumi, Hitoshi et al. (2010) Defective calmodulin binding to the cardiac ryanodine receptor plays a key role in CPVT-associated channel dysfunction. Biochem Biophys Res Commun 394:660-6
Liu, Zheng; Wang, Ruiwu; Tian, Xixi et al. (2010) Dynamic, inter-subunit interactions between the N-terminal and central mutation regions of cardiac ryanodine receptor. J Cell Sci 123:1775-84
Ono, Makoto; Yano, Masafumi; Hino, Akihiro et al. (2010) Dissociation of calmodulin from cardiac ryanodine receptor causes aberrant Ca(2+) release in heart failure. Cardiovasc Res 87:609-17
Kobayashi, Shigeki; Yano, Masafumi; Suetomi, Takeshi et al. (2009) Dantrolene, a therapeutic agent for malignant hyperthermia, markedly improves the function of failing cardiomyocytes by stabilizing interdomain interactions within the ryanodine receptor. J Am Coll Cardiol 53:1993-2005
Tateishi, Hiroki; Yano, Masafumi; Mochizuki, Mamoru et al. (2009) Defective domain-domain interactions within the ryanodine receptor as a critical cause of diastolic Ca2+ leak in failing hearts. Cardiovasc Res 81:536-45

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