The long-term goal of Project 1 is to define the mechanisms responsible for the malignant hyperthermia syndrome caused by mutations in RyR1 and Cav1 .1 as well as leveraging new discovery of other gene linkage in humans using new and proven mouse models of human disease and human myotubes to study how mutations of RyR1 and Cav1.1alter intracellular Ca2+ homeostasis. Hypothesis I: MH """"""""knock-in"""""""" mice model Human MH susceptibility.
Aim 1. To phenotype heterozygous and if viable homozygous RyR1 R2435H and Cav1 .1 R174W mice.'A1.1 Do they trigger the MH syndrome in response to volatile anesthetics or heat stress? Is susceptibility affected by age or gender? A1.2 Determine [ Ca2+]I and [Na+]i in vivo A1.3 Determine sensitivity to KCI, 4CmC and halothane A1.4 Western blot, immunohistochemistry and EM for pathology (Core D).Hypothesis II: Mutations responsible for human MH increase passive RyR1 """"""""leak"""""""" and alter the dynamics of sarcolemmal d Ca2+ entry. A2.1. Analyze heterozygous and homozygous MH muscles for abnormalities in EC coupling and sarcolemmal Na+ and Ca2+ entry both at rest and after exposure to triggering agents. Determine the role(s) of TRPCs 1,3, and 6 and their control by DAG and PKC. A2.2. Explore how azumolene (dantrolene) diminishes aberrant Ca2+ signaling. A2.3. Validate abnormalities seen in murine MH models in myotubes obtained from humans with MH mutations supplied by Core B. Hypothesis III: Deleterious changes in Ca2+ homeostasis that are sequelae of MHS mutations can be reduced/prevented by genetic/pharmacological manipulations that decrease sarcolemmal Ca2+ entry, reduce RyR1 leak, increase SR Ca2+ load or scavenge lipid peroxides resulting from ROS production. A3.1: We will study the above paradigms in 3-6 month old male Het RyR1-T4826l MHS mice that have been crossed with mice over-expressing SERCA1 (enhanced SR Ca2+ filling), dnTPRCG (reduced SOCE), or A3.2 have been administered 4-OH-BDE49 (reduced RyR1 leak) or salicylamine (yKA scavenger). Hypothesis IV: Discovery - new mutations will provide new insights into the pathogenesis of MH.
Aim 4 New mutations will be expressed in WT or null myotubes as they are discovered and we will determine how they disturb [ Ca2+]i, [Na+]i, RcaE and SR Ca2+ load complementing experiments in Projects 2 and 3.
Project 1 will characterize two new MH mouse models that will then be shared with the other projects through Core B. Based on its discovery that Ca2+entry caused by transient receptor potential channels (TRPCs) it will investigate the importance of these channels in the MH syndrome and their regulation. In collaboration with Projects 2 and 3 and Cores C and D the Project 1 will be able to determine how people who are susceptible to MH are able to live otherwise normal lives until exposed to anesthesia.
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