The molecular dysregulation of excitation contraction coupling in patients with congenital muscle disorders

Excitation contraction coupling (ECC) is the process whereby an action potential spreading throughout the muscle membrane activates muscle contraction, by releasing Ca2+ from the Sarcoplasmic Reticulum (SR). Ca2+ release from the SR is mediated by the Ryanodine Receptor located on the SR membrane. Any alterations in the architecture of the intercellular muscle membrane compartments or mutations in the RYR1 gene are associated with neuromuscular disorders such as Central core disease, Multi minicore disease, Central nuclear myopathy or congenital fiber type disproportion.
In the last few decades, ECC characteristics were extensively investigated in our lab, on myotubes originating from patient’s muscle satellite cells. In the 1st paper entitled “Establishment of human skeletal muscle- derived cell line: biochemical, cellular and electrophysiological characterization”, we studied the ECC in an immortalized human muscle cell line (HMCL-7304), which helps to overcome many of the technical limitations of working with primary muscle cells from human patients. ECC in HMCL-7304 was characterized with qPCR and western blotting as well as super resolution microscopy (SIM), Ca2+ imaging and electrophysiological measurements. We discovered that HMCL-7304 have a phenotype closer to slow twitch muscles than fast twitch muscles. HMCL-7304 can be used as a platform to investigate genetic mechanisms of muscle disorders, as shown in our 2nd publication; “RyR1 deficiency in congenital myopathies disrupts excitation contraction coupling”, where we simulated the downregulation of RyR1 expression as seen in patients with recessive RYR1 mutations, by silencing RyR1 expression in the HMCL-7304.
Patients with recessive RYR1 mutations have been shown to downregulate RyR1 expression in skeletal muscles. This is in contrast to what is observed in patients with dominant RYR1 mutations, in whom we could not find reduction in the RyR1 expression. In patient’s muscle biopsies where RyR1 expression is reduced, all isoforms of InsP3R Receptors (ITPR1-ITPR3) were found to be up-regulated. Ca2+ release was not altered by the reduction of RyR1 expression using siRNA in HMCL or by blocking of IP3Rs using Xestospongin, rejecting the possibility for InsP3R functional compensation for the downregulation of RyR1.
The potential mechanisms causing downregulation of RyR1 in patients with recessive RYR1 mutations is addressed in our 3rd publication; “Epigenetic changes as a common trigger of muscle weakness in congenital myopathies”. Patients with downregulation of RyR1, exhibit decreased expression of muscle specific microRNAs and increased expression of HDAC4 and HDAC5. Additionally hyper-methylation of CpG Island in the RYR1 gene was observed. Down regulation of RyR1, downregulation microRNAs and upregulation of HDAC4 and HDAC5 was also observed in patients with Nemaline myopathy, reflecting common epigenetic changes activated in congenital myopathies. Using HDAC or DNMT inhibitors can target common downstream pathways activated in muscles of patients with congenital myopathies offers an interesting new approach for the amelioration of muscle function