Central Core Disease (CCD), the most common congenital myopathy in humans, is characterized by fetal hypotonia, proximal muscle weakness, and inert core regions that lack functional mitochondria and oxidative enzyme activity in type 1 skeletal muscle fibers. Although CCD is known to be caused by mutations in the skeletal muscle Ca2+ release channel (RyR1), diagnosis still depends on painful and invasive muscle biopsies and there are currently no effective treatments. The long-term goal of this project is to characterize the pathophysiological mechanisms of muscle weakness in CCD and to develop novel therapeutic and diagnostic strategies. Motivated by this goal, we aim to drive discovery of novel and fundamental aspects of altered Ca2+ signaling in CCD, as well as test the validity of potential therapeutic (allele-specific gene silencing) and diagnostic (superoxide flashes as a CCD biomarker) strategies.
Aim #1 will test the hypothesis that CCD mutations in different structural regions of RyR1 alter Ca2+ release during muscle contraction via fundamentally distinct molecular and biophysical mechanisms. Experiments will determine if CCD mutations in the M6 and pore/TM10 transmembrane regions of RyR1 alter Ca2+ permeation, channel gating, and/or regulation by specific RyR1 binding proteins.
Aim #2 will assess the utility of allele-specific gene silencing approaches to correct defects in Ca2+ homeostasis, Ca2+ release, and mitochondrial function observed in knock-in mice heterozygous for either the Y522S or I4895T CCD mutations in RyR1. An important conceptual innovation of this project is to use shRNA-mediated mutant RyR1 allele-specific knockdown to reverse toxic gain-of-function effects produced by CCD mutations in RyR1 in heterozygous Y522S and I4895T knock-in mice. An important technological innovation is to take advantage of our recently developed mitochondrial-targeted superoxide sensor (mt-cpYFP) to assess changes in mitochondrial function (reflected in the incidence and properties of mitochondrial superoxide flashes) in normal and RyR1 knock-in mice. The fundamental pathophysiologic mechanisms identified and discoveries made as a result of this venture will provide extraordinary promise for the development of novel approaches, therapeutics, and diagnostics not only for CCD, but also other disorders of Ca2+ dysregulation and altered oxidative stress in related clinical arenas including cardiac arrhythmias, heart failure, hypertension, diabetes, neuro-degeneration and stroke.
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