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 tailor the design of potential therapeutic interventions (initially informed by the collective properties of the HH and MH cohorts) to the phenotype (molecular, cellular, or organismal) of individual patients. In future iterations, the top therapeutic paradigms will join clinical testing already going on at the MHIU. (Rev. 11/02/16)
) A large number of inheritable skeletal muscle diseases alter the intracellular signals that drive contraction. These diseases are caused by changes in the structure and function of the molecules (proteins) that control the frequency, size and duration of those signals. CHCT testing on biopsied muscle is the gold standard means for diagnosis of Malignant Hyperthermia (MH). MH patients are generally symptom-free until exposed to ?triggering? agents (certain anesthetics and muscle relaxants). The CHCT, however, identifies other patients, a larger population than the MH, whom we call the HH and are the focus of this grant. In contrast to the typical MH patient, said to have a ?susceptibility? rather than a disease, a large majority of the HH are chronically sick, suffering from various symptoms of a muscle disease (weakness, muscle cramps and pain, fatigability, bone deformities, low tolerance to stress, or heat, or statin sensitivity). MH patients often know the protein mutation that causes their disease, and benefit from having a well- established therapeutic intervention that limits triggered MH episodes should one ever occur. The chronically ill HH often do not know what causes their disease and have few (if any) options for treatment. This proposal refines HH diagnosis, defines the underlying molecular mechanisms and tests a set of drugs newly synthesized by us, with the potential to normalize the abnormal HH muscle function. The close collaboration of a molecular lab, a cellular lab and a specialized muscle disease clinic enables a multi-level experimental approach, needed to fuel future clinical trials with prospective anti-HH interventions. Estimates of MH prevalence are as high as 1/500. Our preliminary testing of 82 subjects indicates that HH prevalence is at least 2 times greater than that of MH. Consequently, this project stands to benefit a large group of underserved patients. (Rev. 11/02/16)
| Ríos, Eduardo (2018) Calcium-induced release of calcium in muscle: 50 years of work and the emerging consensus. J Gen Physiol 150:521-537 |
| Ferreira Gregorio, Juan; Pequera, Germán; Manno, Carlo et al. (2017) The voltage sensor of excitation-contraction coupling in mammals: Inactivation and interaction with Ca2. J Gen Physiol 149:1041-1058 |
