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 to? create suitable models for human disease in mice and use them to study how mutations of RyR1 alter? intracellular Ca2+ homeostasis and to create a noninvasive diagnostic test for MH that has a high degree of? specificity 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 recently? created R163C (region 1) and T4826I (region 3) RyR1 MHS mice. A 1.2.To determine the relationship between? the clinical MH phenotype and myoplasmic resting free calcium ([Ca2+],) in vivo, with contracture properties? in vitro for each heterozygous MH/CCD mouse. A1.3 To determine the relationship between IVCT and? myoplasmic resting [Ca2+] in myotubes from human biopsy samples. A1.4 To determine if genetic? background can rescue the birth lethal phenotype seen in homozygous MH/CCD mice. A1.5 To determine if? genetic 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 alter? the 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 and? two forms of Ca2+ entry, ECCE and SOCE. A 2.2 To establish how halothane and dantrolene enhance and? diminish aberrant Ca2+ signaling in MH/CCD myotubes. A 2.3 To validate murine MH/CCD myotube model? results with myotubes obtained from humans with an analogous mutation.? Hypothesis III: The """"""""Funnel"""""""" approach can be used to create a non-invasive screening test for? MH 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 mutations? and use the Funnel approach to select a set of predictive markers to be used for future MHS screening.?
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