Malignant hyperthermia (MH) and central core disease (CCD) arise from mutations in the skeletal muscleryanodine receptor (RyR1). Although MH and CCD mutations in RyR1 alter mechanical coupling betweensarcolemmal dihydropyridine receptors (DHPRs) and opposing Ca2+ release channels of the sarcoplasmicreticulum (SR), the integrated effects of these mutations on multiple subcellular Ca2+ transport processes arepoorly understood. The long-term goal of this project is to determine the cellular/molecular mechanisms bywhich MH and CCD mutations in RyR1 alter Ca2+ signaling interactions between the sarcolemma, SR, andmitochondria (the 'Ca2+ signaling triad'). Specifically, this project will test the hypothesis that 'MH/CCDmutations in RyR1 enhance excitation coupled Ca2+ entry (ECCE) activity, sensitize voltage- & ligand-gatedSR Ca2+ release, and alter mitochondrial Ca2+ uptake during EC coupling.' Aim #1 willcharacterize effects of several common RyR1 MH/CCD mutations on bi-directional DHPR-RyR1 coupling inskeletal myotubes and fully differentiated muscle fibers derived from MH knock-in mice generated by Core B.
Aim #2 will test if MH/CCD mutations in RyR1 elevate steady-state resting Ca2+ by promoting a depletion ofSR Ca2+ and increasing the activity of sarcolemmal ECCE channels. Experiments will use SR-targeted, Ca2+-sensitive fluorescent 'cameleons' to directly report changes in SR Ca2+ and whole-cell patch clampmeasurements to monitor changes in ECCE activity.
Aim #3 will determine the degree to which mitochondrialtriad targeting and local SR-mitochondrial Ca2+ signaling is altered by MH/CCD mutations in RyR1.Experiments in collaboration with Core D will use electron microscopy to assess mitochondrial morphology,localization, and triad targeting in FOB fibers of normal and MH/CCD knock-in mice. Functional experimentswill use confocal microscopy, high-speed Ca2+ imaging and mitochondrial-targeted ratiometric pericam toreport effects of MH mutations on the magnitude, kinetics, and voltage-dependence of mitochondrial Ca2+changes during EC coupling. Additionally, parallel experiments to those described in Aims 1-3 will beconducted in human myotubes generated from muscle samples of control individuals and MHS patientsharboring analogous mutations in RyR1 (e.g. R163C and G2435R) to those used to make knock-in mice. Forthese experiments, human muscle samples collected by Core C from control individuals and patients of knowngenotypes and IVCT results will be used by Core B to propagate human myoblasts required for generatingmyotube cultures in Project 4. This project will combine the tools of molecular biology, mouse genetics,electrophysiology, confocal/electron microscopy, and high-speed Ca2+ imaging to asses the mechanisms bywhich MH/CCD mutations alter the function with the Ca2+ signaling triad.
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