? DESCRIPTION (provided by applicant): The long-term goal of this research is to determine the mechanisms by which mutations that alter intracellular Ca2+ homeostasis at rest cause fulminant MH after exposure to triggering agents. To achieve our objectives we will use the RyR1-R163C mouse which phenocopies human MH and CSQ1 nulls, which have an MH like phenotype. We hypothesize that because the amount of Ca2+ in internal stores is limited compared to the extracellular pool, after MH is triggered sarcolemmal channels are critical to its progression and that a primary mechanism for this is increased ROS production promoted by increased [Ca2+]i causing activation of TRPCs, increasing RCaE and supporting the MH syndrome. In addition to using in vitro mechanistic analyses and in vivo pharmacological approaches to reduce RyR1 leak, ROS production via NOX2 and sarcolemmal Ca2+ entry to abrogate the MH phenotype, because TRPCs exist as heteromultimers, we will molecularly suppress function of all TRPC3/6/7 family members by expression of a dominant negative non-conducting TRPC6 channel in MH muscles. Finally we will use similar approaches to discover the mechanism(s) by which the syndrome is both prevented and aborted by dantrolene, which has heretofore been thought to act only through inhibition of RyR1 Ca2+ release. Within this framework we will investigate the transformative concept that while RyR1 leak is a common convergent pathway leading to all MHS, the RyR1 leak is the proximate driver of a mitochondrial Ca2+ overload and augmented ROS and NO production, which in turn activates TRPC dependent sarcolemmal Ca2+ and Na+ entry that initiates and sustains the fulminant MH episode. Such a mechanism can not only explain responses to MH triggering agents through dysregulated EC coupling in MH, but also more chronic adaptations and progression of muscle damage ascribed to a vicious cycle of increased RyR1 Ca2+ leak, sarcolemmal Ca2+ and Na+ entry, ROS, RNS, TRPC activity and mitochondrial impairments in other muscle diseases. Hypothesis 1: MH susceptibility is the result of a conformational change in RyR1 caused either by a RyR1 mutation, or induced indirectly by mutations in other proteins closely associated with RyR1 which results in increased RyR1 SR Ca2+ leak and sarcolemmal Na+ and Ca2+ entry. Specific Aim 1. To determine the filling state of the SR and rate of SR Ca2+ leak in MH muscle fibers. Specific Aim 2. To determine the role of TRPCs in causing abnormalities in sarcolemmal Na+ and Ca2+ entry RyR1-R163C and CSQ1 null muscles and then to determine if skeletal muscle specific over-expression of a dominant negative non-conducting TRPC6 channel can modify RyR1-R163C's MH phenotype. Hypothesis 2: In addition to blocking RyR1 SR Ca2+ release, dantrolene abrogates the MH phenotype by modulating RyR1 SR Ca2+ leak and sarcolemmal Na+ and Ca2+ entry. Specific Aim 3. To determine the mechanisms by which dantrolene diminishes aberrant Ca2+ signaling Successful completion of these specific aims will provide a more comprehensive understanding of MH.
|Effective start/end date||5/1/16 → 4/30/21|
- National Institutes of Health: $345,125.00
Ryanodine Receptor Calcium Release Channel