Sub-Project #4

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Malignant hyperthermia (MH) and central core disease (CCD) arise from mutations in the skeletal muscle ryanodine receptor (RyR1). Although MH and CCD mutations in RyR1 alter mechanical coupling between sarcolemmal dihydropyridine receptors (DHPRs) and opposing Ca2+ release channels of the sarcoplasmic reticulum (SR), the integrated effects of these mutations on multiple subcellular Ca2+ transport processes are poorly understood. The long-term goal of this project is to determine the cellular/molecular mechanisms by which MH and CCD mutations in RyR1 alter Ca2+ signaling interactions between the sarcolemma, SR, and mitochondria (the "Ca2+ signaling triad"). Specifically, this project will test the hypothesis that "MH/CCD mutations in RyR1 enhance excitation coupled Ca2+ entry (ECCE) activity, sensitize voltage- & ligand-gated SR Ca2+ release, and alter mitochondrial Ca2+ uptake during EC coupling." Aim #1 will characterize effects of several common RyR1 MH/CCD mutations on bi-directional DHPR-RyR1 coupling in skeletal 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 of SR 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 clamp measurements to monitor changes in ECCE activity. Aim #3 will determine the degree to which mitochondrial triad 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 experiments will use confocal microscopy, high-speed Ca2+ imaging and mitochondrial-targeted ratiometric pericam to report 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 be conducted in human myotubes generated from muscle samples of control individuals and MHS patients harboring analogous mutations in RyR1 (e.g. R163C and G2435R) to those used to make knock-in mice. For these experiments, human muscle samples collected by Core C from control individuals and patients of known genotypes and IVCT results will be used by Core B to propagate human myoblasts required for generating myotube 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 by which MH/CCD mutations alter the function with the Ca2+ signaling triad.