Altered Type 1 Ryanodine Receptor Activity and Functional Rescue In a Mouse Model of Central Core Disease

In the human population, two RyR1 mutations, I4898T (IT) and Y522S (YS), result in CCD and MH with cores, respectively. Providing a comprehensive comparison of the proposed pathogenic mechanisms in skeletal muscle of IT and YS knock-in mice is the central goal of this thesis.;Heterozygous IT (IT/+) and YS (YS/+) mice were crossed to generate compound heterozygous mice (YS/IT) in order to evaluate potential rescue of RyR1 activity due to functional RyR1 complementation. The foundation of this hypothesis is the opposing mechanistic defects for the IT and YS mutations (reduced Ca 2+ permeation vs increased Ca2+ leak, respectively). Results from compound heterozygous YS/IT myotubes demonstrate a partial rescue of both electrically-evoked (21.4% rescue) and 4-CMC-induced (42.5% rescue) Ca2+ release compared to IT/IT myotubes. These results indicate that co-expression of YS and IT mutant monomers leads to functional complementation that partially rescues RyR1 Ca2+ release, SR Ca2+ content, and EC coupling. These results buttress the hypothesis that the two mutations act via fundamentally distinct mechanisms.;Alterations in RyR1 function, EC coupling, and Ca2+ homeostasis in skeletal muscle fibers from IT/+ mice was assessed to provide further insight into CCD pathogenesis. Adult (4-6 month old) IT/+ mice exhibited reduced performance compared to age-matched WT mice during in vivo hanging task and grip strength tests, consistent with significant skeletal muscle weakness. The peak magnitude and maximum rate of electrically-evoked (Magnitude (F/F 0) WT: 0.39 +/- 0.02; IT/+: 0.24 +/- 0.01 and Rate (d(F/F 0)/dt) WT: 0.17 +/- 0.01; IT/+: 0.11 +/- 0.01) and 4-CMC-induced (Magnitude (DeltaR) WT: 0.68 +/- 0.03; IT/+: 0.54 +/- 0.03 and Rate (dR/dt) WT: 0.39 +/- 0.05; IT/+: 0.18 +/- 0.03) Ca2+ release were significantly reduced in single intact FDB fibers from IT/+ mice in the absence of a detectable change in SR Ca2+ content. Together, these findings are consistent with the hypothesis that the IT mutation significantly reduces Ca2+ flux through the channel in muscle fibers from IT/+ mice.;The final area of research addressed herein is to provide proof-of-principle for mutant allele-specific gene silencing (ASGS) as a potential therapeutic strategy in autosomal dominantly-inherited RyR1 disorders. Using RT-PCR and confocal microscopy based assays, candidate IT- and YS-selective siRNAs were evaluated for efficacy and specificity. Local delivery of the most promising siRNAs into FDB muscles was achieved by injection/electroporation (1/week for 2-4 weeks) of footpads of 4-6 month old IT/+ and YS/+ mice. Results indicate that 2-4 weeks of treatment in IT/+ mice results in modest but significant functional rescue of the deficits in rate (38.5% rescue) and magnitude (78.6% rescue) of 4-CMC-induced Ca2+ release without a significant effect on electrically-evoked Ca2+ transients. More impressively, enhanced RyR1 sensitivity to activation by caffeine (EC50 164% rescued) and temperature-dependent increase in resting Ca2+ (fold increase 124% rescued) in YS/+ mice was significantly rescued to WT levels at the two and four week timepoints. As determined by RT-PCR, the degree of functional rescue in both the YS/+ and IT/+ mice correlates well with the relative shift of fractional allele expression from the same muscles.;Taken together, the work presented here supports the hypothesis that the IT mutation reduces RyR1 Ca2+ release in a manner that does not reduce SR Ca2+ content or increase channel sensitivity to activation. Results also confirm that the YS mutation operates by a mechanistically distinct pathway---sensitization to activation by voltage, caffeine, and temperature, as well as promoting RyR1 Ca2+ leak and SR Ca 2+ store depletion. Though the mechanisms of these mutations are distinct, the concept of ASGS is applicable to both the IT and YS mutations and results in significant in vivo functional rescue that depends strongly on mutant allele silencing efficiency. (Abstract shortened by UMI.)...