Basis of Muscle Dysfunction in Malignant Hyperthermia and Central Core Disease

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DESCRIPTION (provided by applicant): The skeletal muscle ryanodine receptor (RYR1) regulates Ca2+ release from the sarcoplasmic reticulum (SR) stores and is mutated in human central core disease (CCD) and in the pharmacogenetic syndrome, malignant hyperthermia (MH). Although MH and CCD mutations in RyR1 are thought to alter SR Ca2+ release channel function and muscle excitation-contraction (EC) coupling, the mechanisms by which these effects result in phenotypic changes in muscle characteristic of these disorders are unknown. This project will use transgenic MH and CCD knock-in mice to provide detailed analyses of the fundamental mechanisms by which RYR1 disease mutations alter in vivo muscle function. The long-term goal of this project is to define the cellular/molecular mechanisms and principles by which MH/CCD mutations alter Ca2+ homeostasis and excitation-contraction (EC) coupling in intact muscle. Our overall hypothesis is: MH and CCD mutations in MH/CCD regions 1 and 2 enhance voltage- and Ca2+-gated SR release by altering crucial intra and intermolecular interactions within RYR1 and between RYR1 and the voltage dependent Ca2+ channel in the t-tubule membrane, while CCD-selective mutations in the region 3 pore region of RyR1 disrupt Ca2+ permeation through the channel. To test this hypothesis, we propose to: 1. Create three new MH/CCD mouse lines and analyze the effects of the mutations on muscle contractile properties and RYR1 single channel properties in response to caffeine and temperature, 2. Map the location of MH/CCD regions 1 and 2 in the 3D structure and analyze the effects of the mutations on RYR1 structure, S.Analyze the effects of MH/CCD mutations in RyR1 on Ca2+ homeostasis and bi-directional DHPR-RyR1 coupling in myotubes and adult muscle fibers obtained from MH/CCD knock-in mice, and 4 Evaluate the effects of MH/CCD mutations on in situ release channel sensitivity to activation by RyR1 ligands and local increases in junctional Ca2+. This application brings together two collaborators, both highly committed to elucidating fundamental mechanisms of MH and CCD pathophysiology, but who approach the problems in very different, but complimentary ways. This union will result in a uniquely interdisciplinary project that will determine the mechanisms by which MH/CCD disease mutations alter RyR1 structure and regulation, subcellular Ca2+ transport/handling mechanisms, muscle EC coupling, and SR Ca2+ storage/sequestration. Results will have broad implications for other disorders of Ca2+ dysregulation in related clinical arenas including cardiac arrhythmias, heart failure, hypertension, and wide of central nervous system disorders including stroke and neurodegeneration.