Thursday, May 1, 2025
5/1/2025
ROLE OF RYRS, DHPRS AND CA2+ ENTRY IN THE INCLUSION BODY MYOSITIS - DESCRIPTION (provided by applicant): The principal investigator of this proposal, Dr. Alexander Shtifman, is an Associate Investigator/Assistant Professor in Neurology at St. Elizabeth's Medical Center/Tufts University School of Medicine. The work outlined in this proposal will lead to an application for R01 level funding. The environment in which the proposed work will be carried out at St. Elizabeth's Medical Center/Tufts University School of Medicine, a world class scientific institution, where biomedical research is performed at the highest level, with intimate associations between clinical and basic science disciplines. In this proposal Dr. Shtifman will investigate the effects of 2-amyloid proteins on Ca2+ dysregulation in skeletal muscle as it relates to a skeletal muscle disorder, the Inclusion Body Myositis (IBM). Dr. Shtifman has already established that transient overexpression of a 42-amino acid form of 2-amyloid (A21 42) in skeletal myotubes results in a two-fold increase in resting [Ca2+]i. He has also demonstrated that chronically elevated levels of 2- amyloid in the muscle fibers from the transgenic mouse models of IBM also exhibit a substantially elevated myoplasmic [Ca2+]i. Based on the preliminary findings the proposed study will determine whether the intracellular deposits of 2-amyloid proteins lead to Ca2+ dysregulation by pathologic modulation of active and passive Ca2+ release through the ryanodine receptors (RyR), Ca2+ influx through dihydropyridine receptors (11sDHPR) and Ca2+ entry via store-operated and excitation-coupled pathways. The goals of this proposal are to: 1) investigate the roles of RyRs, DHPRs and Ca2+ entry in A21-42-mediated, passive elevation in resting, myoplasmic Ca2+ concentration and 2) determine the effect of A2 on depolarization-induced Ca2+ release from RyRs and on Store Operated (SOC) and Excitation Contraction-Coupled (ECC) Ca2+ Entry in muscle fibers from the IBM transgenic mice. The roles of RyRs and DHPRs in the amyloid-mediated Ca2+ elevation will be established by determining the ability of heterologously expressed A21-42 to elevate [Ca2+]i in the absence of RyRs in the RyR-knockout myogenic cells lines and the absence of 11sDHPR in the DHPR-knockout myogenic cells lines. To determine the effects of chronic exposure of muscle to 2-amyloid, isolated muscle fibers from a transgenic mouse model of IBM will be used. Effects of 2-amyloid on numerous aspects of the depolarization- induced Ca2+ release, as well as different Ca2+ entry mechanisms in the transgenic muscle will be determined with high-speed fluorescence imaging. The results of these findings will provide new insights into the effects of the intracellular deposition of 2-amyloid proteins in IBM and could lead to design of the future therapeutic agents. Inclusion Body Myositis (IBM) is the most common muscle disease people over 50 years of age. There is evidence that derangement of calcium homeostasis due to an abnormal accumulation beta- amyloid proteins is one of the early events that lead to the pathology of IBM, however, the detailed information on the subject is severely lacking. The goal of this proposal is to investigate the contribution of key intracellular calcium regulatory components to alterations in calcium homeostasis associated with beta-amyloid accumulation in the IBM.
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