Project Summary Intracellular Ca signals reach great intensity in muscle, where they are key to the “Excitation- Contraction Coupling” (ECC) process. In striated muscles they are produced by a supramolecular assembly that we named the couplon, which crucially includes ryanodine receptors (RyRs), channels of the sarcoplasmic reticulum (SR). Multiple diseases arise from abnormal ECC; among them, the paradigmatic Malignant Hyperthermia is diagnosed by the “CHCT”, a conventional challenge with caffeine and halothane. In a 72- patient sample, we have found that roughly 20% tested positive, 40% were negative and 40% tested equivocally, meaning that they Hyper-reacted to Halothane, but not to caffeine. Clinical work found that these patients, which we call the “HH”, are sick, suffering from muscle pain, weakness, high sensitivity to stress, or heat, or statins, and experience rhabdomyolysis and other setbacks. This is in stark contrast with most MH- positive patients, who have a susceptibility to well-known triggers, but otherwise no active disease phenotype. Here, two physiology labs have teamed with the clinic that studies the greatest number of congenital non- dystrophic myopathies in the hemisphere (the MHIU) to propose a comprehensive study of approximately 300 patients. A detailed clinical and genetic picture of each tested patient will be matched by: (1) a cell-level quantification of Ca handling (from measurements of steady and stimulated Ca ion concentration in cytosol and SR, as well as steady and stimulated fluxes between these compartments in adult and cultured cells derived from patients’ biopsies), and (2) a matching molecular description, from measurements of function of single SR Ca release RyR1 channels derived from the patients. Many of these measurements will be the first done in human cells. The results will be interpreted in terms of “pathogenic pathways”, which track the causal chain, starting from a primary defect (e.g. an excessive tendency for RyR to open) to account for and predict the multiple changes that occur downstream. This mechanistic knowledge will then be used to devise therapeutic interventions, tailored rationally to offset the primary defect or the main drivers of the established pathogenic pathways. These may include steady changes in ion composition of the extracellular medium, the classic drug dantrolene and/or application of a large set of newly synthesized RyR-inhibiting drugs, carvedilol derivatives modified from the parent drug to eliminate its beta-blocking action. Among the novel derivatives, 34 were prescreened favorably in a RyR expression system. The best of these, identified based on affinity, efficacy and RyR-isoform specificity, will be applied to single human RyR1 channels, myotubes and myofibers; their outcomes will be compared to those of dantrolene and interpreted within the mechanistic context established in this project. The close bench-clinical correlation of our study makes it possible to tai...