The long-term goal of Project 1 is to define the mechanisms responsible for the ryanodine receptor (RyR1)myopathies, malignant hyperthermia and central core disease (MH/CCD). Our immediate objectives are tocreate suitable models for human disease in mice and use them to study how mutations of RyR1 alterintracellular Ca2+ homeostasis and to create a noninvasive diagnostic test for MH that has a high degree ofspecificity and sensitivity.HYPOTHESIS I: Heterozygous MH 'knock-in' mice model Human MH susceptibility.A 1.1. To create a 'hot-spot' region 2 MH 'knock in' mouse line (RyR1 G2434R). In addition to our recentlycreated R163C (region 1) and T4826I (region 3) RyR1 MHS mice. A 1.2.To determine the relationship betweenthe clinical MH phenotype and myoplasmic resting free calcium ([Ca2+],) in vivo, with contracture propertiesin vitro for each heterozygous MH/CCD mouse. A1.3 To determine the relationship between IVCT andmyoplasmic resting [Ca2+] in myotubes from human biopsy samples. A1.4 To determine if geneticbackground can rescue the birth lethal phenotype seen in homozygous MH/CCD mice. A1.5 To determine ifgenetic background will lower or raise [Ca2+]i or sensitivity to halothane in heterozygous MH/CCD mice.HYPOTHESIS II: Mutations responsible for human MH/CCD increase passive RyR1 'leak' and alterthe dynamics of EC coupling by enhancing ECCE and SOCE.A 2.1 To analyze heterozygous and homozygous MH/CCD myotubes for abnormalities in EC coupling andtwo forms of Ca2+ entry, ECCE and SOCE. A 2.2 To establish how halothane and dantrolene enhance anddiminish aberrant Ca2+ signaling in MH/CCD myotubes. A 2.3 To validate murine MH/CCD myotube modelresults with myotubes obtained from humans with an analogous mutation.Hypothesis III: The 'Funnel' approach can be used to create a non-invasive screening test forMH that has both a high degree of specificity and a high degree of sensitivity.A 3.1. We will analyze whole blood transcriptional profiles from patients with known MHS RyR1 mutationsand use the Funnel approach to select a set of predictive markers to be used for future MHS screening.
Showing the most recent 10 out of 78 publications
