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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Program Projects (P01)
Project #
5P01AR052354-07
Application #
8478053
Study Section
Special Emphasis Panel (ZAR1-MLB)
Project Start
Project End
Budget Start
2013-06-01
Budget End
2014-05-31
Support Year
7
Fiscal Year
2013
Total Cost
$245,876
Indirect Cost
$56,169
Name
University of California Davis
Department
Type
DUNS #
047120084
City
Davis
State
CA
Country
United States
Zip Code
95618
Yang, Zhong; Liu, Qiang; Mannix, Robert J et al. (2014) Mononuclear cells from dedifferentiation of mouse myotubes display remarkable regenerative capability. Stem Cells 32:2492-501
Burr, Adam R; Millay, Douglas P; Goonasekera, Sanjeewa A et al. (2014) Na+ dysregulation coupled with Ca2+ entry through NCX1 promotes muscular dystrophy in mice. Mol Cell Biol 34:1991-2002
Bannister, Roger A; Beam, Kurt G (2013) Impaired gating of an L-Type Ca(2+) channel carrying a mutation linked to malignant hyperthermia. Biophys J 104:1917-22
Brandom, Barbara W; Bina, Saiid; Wong, Cynthia A et al. (2013) Ryanodine receptor type 1 gene variants in the malignant hyperthermia-susceptible population of the United States. Anesth Analg 116:1078-86
Altamirano, Francisco; Lopez, Jose R; Henriquez, Carlos et al. (2012) Increased resting intracellular calcium modulates NF-ýýB-dependent inducible nitric-oxide synthase gene expression in dystrophic mdx skeletal myotubes. J Biol Chem 287:20876-87
Boncompagni, Simona; Protasi, Feliciano; Franzini-Armstrong, Clara (2012) Sequential stages in the age-dependent gradual formation and accumulation of tubular aggregates in fast twitch muscle fibers: SERCA and calsequestrin involvement. Age (Dordr) 34:27-41
Barrientos, Genaro C; Feng, Wei; Truong, Kim et al. (2012) Gene dose influences cellular and calcium channel dysregulation in heterozygous and homozygous T4826I-RYR1 malignant hyperthermia-susceptible muscle. J Biol Chem 287:2863-76
Yuen, Benjamin; Boncompagni, Simona; Feng, Wei et al. (2012) Mice expressing T4826I-RYR1 are viable but exhibit sex- and genotype-dependent susceptibility to malignant hyperthermia and muscle damage. FASEB J 26:1311-22
Giulivi, Cecilia; Ross-Inta, Catherine; Omanska-Klusek, Alicja et al. (2011) Basal bioenergetic abnormalities in skeletal muscle from ryanodine receptor malignant hyperthermia-susceptible R163C knock-in mice. J Biol Chem 286:99-113
Feng, Wei; Barrientos, Genaro C; Cherednichenko, Gennady et al. (2011) Functional and biochemical properties of ryanodine receptor type 1 channels from heterozygous R163C malignant hyperthermia-susceptible mice. Mol Pharmacol 79:420-31

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