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 #
5P01AR052354-08
Application #
8667315
Study Section
Special Emphasis Panel (ZAR1-MLB)
Project Start
Project End
Budget Start
2014-06-01
Budget End
2015-05-31
Support Year
8
Fiscal Year
2014
Total Cost
$251,604
Indirect Cost
$57,794
Name
University of California Davis
Department
Type
DUNS #
047120084
City
Davis
State
CA
Country
United States
Zip Code
95618
Lavorato, Manuela; Iyer, V Ramesh; Dewight, Williams et al. (2017) Increased mitochondrial nanotunneling activity, induced by calcium imbalance, affects intermitochondrial matrix exchanges. Proc Natl Acad Sci U S A 114:E849-E858
Holland, Erika B; Goldstone, Jared V; Pessah, Isaac N et al. (2017) Ryanodine receptor and FK506 binding protein 1 in the Atlantic killifish (Fundulus heteroclitus): A phylogenetic and population-based comparison. Aquat Toxicol 192:105-115
Zhang, Rui; Pessah, Isaac N (2017) Divergent Mechanisms Leading to Signaling Dysfunction in Embryonic Muscle by Bisphenol A and Tetrabromobisphenol A. Mol Pharmacol 91:428-436
Perni, Stefano; Lavorato, Manuela; Beam, Kurt G (2017) De novo reconstitution reveals the proteins required for skeletal muscle voltage-induced Ca2+ release. Proc Natl Acad Sci U S A 114:13822-13827
Linsley, Jeremy W; Hsu, I-Uen; Groom, Linda et al. (2017) Congenital myopathy results from misregulation of a muscle Ca2+ channel by mutant Stac3. Proc Natl Acad Sci U S A 114:E228-E236
Lavorato, Manuela; Gupta, Pawan K; Hopkins, Philip M et al. (2016) Skeletal Muscle Microalterations in Patients Carrying Malignant Hyperthermia-Related Mutations of the e-c Coupling Machinery. Eur J Transl Myol 26:6105
Ronjat, Michel; Feng, Wei; Dardevet, Lucie et al. (2016) In cellulo phosphorylation induces pharmacological reprogramming of maurocalcin, a cell-penetrating venom peptide. Proc Natl Acad Sci U S A 113:E2460-8
Franzini-Armstrong, Clara (2016) Can the Arrangement of RyR2 in Cardiac Muscle Be Predicted? Biophys J 110:2563-5
Hopkins, Philip M; Fiszer, Dorota; Shaw, Marie-Anne et al. (2016) In Reply. Anesthesiology 124:511
Polster, Alexander; Nelson, Benjamin R; Olson, Eric N et al. (2016) Stac3 has a direct role in skeletal muscle-type excitation-contraction coupling that is disrupted by a myopathy-causing mutation. Proc Natl Acad Sci U S A 113:10986-91

Showing the most recent 10 out of 73 publications