Epigenetic changes as a common trigger of muscle weakness in congenital myopathies

Congenital myopathies are genetically and clinically heterogeneous conditions causing severe muscle weakness, and mutations in the ryanodine receptor gene (RYR1) represent the most frequent cause of these conditions. A common feature of diseases caused by recessive RYR1 mutations is a decrease of ryanodine receptor 1 protein content in muscle. The aim of the present investigation was to gain mechanistic insight into the causes of this reduced ryanodine receptor 1. We found that muscle biopsies of patients with recessive RYR1 mutations exhibit decreased expression of muscle-specific microRNAs, increased DNA methylation and increased expression of class II histone deacetylases. Transgenic mouse muscle fibres over-expressing HDAC-4/HDAC-5 exhibited decreased expression of RYR1 and of muscle-specific miRNAs, whereas acute knock-down of RYR1 in mouse muscle fibres by siRNA caused up-regulation of HDAC-4/HDAC-5. Intriguingly, increased class II HDAC expression and decreased ryanodine receptor protein and miRNAs expression were also observed in muscles of patients with nemaline myopathy, another congenital neuromuscular disorder. Our results indicate that a common pathophysiological pathway caused by epigenetic changes is activated in some forms of congenital neuromuscular disorder

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 functio

Variable Myopathic Presentation in a Single Family with Novel Skeletal RYR1 Mutation

We describe an autosomal recessive heterogeneous congenital myopathy in a large consanguineous family. The disease is characterized by variable severity, progressive course in 3 of 4 patients, myopathic face without ophthalmoplegia and proximal muscle weakness. Absence of cores was noted in all patients. Genome wide linkage analysis revealed a single locus on chromosome 19q13 with Zmax = 3.86 at theta = 0.0 and homozygosity of the polymorphic markers at this locus in patients. Direct sequencing of the main candidate gene within the candidate region, RYR1, was performed. A novel homozygous A to G nucleotide substitution (p.Y3016C) within exon 60 of the RYR1 gene was found in patients. ARMS PCR was used to screen for the mutation in all available family members and in an additional 150 healthy individuals. This procedure confirmed sequence analysis and did not reveal the A to G mutation (p.Y3016C) in 300 chromosomes from healthy individuals. Functional analysis on EBV immortalized cell lines showed no effect of the mutation on RyR1 pharmacological activation or the content of intracellular Ca(2+) stores. Western blot analysis demonstrated a significant reduction of the RyR1 protein in the patient's muscle concomitant with a reduction of the DHPRalpha1.1 protein. This novel mutation resulting in RyR1 protein decrease causes heterogeneous clinical presentation, including slow progression course and absence of centrally localized cores on muscle biopsy. We suggest that RYR1 related myopathy should be considered in a wide variety of clinical and pathological presentation in childhood myopathies

RyR1 Deficiency in Congenital Myopathies Disrupts Excitation-Contraction Coupling

In skeletal muscle, excitation-contraction (EC) coupling is the process whereby the voltage-gated dihydropyridine receptor (DHPR) located on the transverse tubules activates calcium release from the sarcoplasmic reticulum by activating ryanodine receptor (RyR1) Ca(2+) channels located on the terminal cisternae. This subcellular membrane specialization is necessary for proper intracellular signaling and any alterations in its architecture may lead to neuromuscular disorders. In this study, we present evidence that patients with recessive RYR1-related congenital myopathies due to primary RyR1 deficiency also exhibit downregulation of the alfa 1 subunit of the DHPR and show disruption of the spatial organization of the EC coupling machinery. We created a cellular RyR1 knockdown model using immortalized human myoblasts transfected with RyR1 siRNA and confirm that knocking down RyR1 concomitantly downregulates not only the DHPR but also the expression of other proteins involved in EC coupling. Unexpectedly, this was paralleled by the upregulation of inositol-1,4,5-triphosphate receptors; functionally however, upregulation of the latter Ca(2+) channels did not compensate for the lack of RyR1-mediated Ca(2+) release. These results indicate that in some patients, RyR1 deficiency concomitantly alters the expression pattern of several proteins involved in calcium homeostasis and that this may influence the manifestation of these diseases

Epigenetic changes as a common trigger of muscle weakness in congenital myopathies

Item does not contain fulltextCongenital myopathies are genetically and clinically heterogeneous conditions causing severe muscle weakness, and mutations in the ryanodine receptor gene (RYR1) represent the most frequent cause of these conditions. A common feature of diseases caused by recessive RYR1 mutations is a decrease of ryanodine receptor 1 protein content in muscle. The aim of the present investigation was to gain mechanistic insight into the causes of this reduced ryanodine receptor 1. We found that muscle biopsies of patients with recessive RYR1 mutations exhibit decreased expression of muscle-specific microRNAs, increased DNA methylation and increased expression of class II histone deacetylases. Transgenic mouse muscle fibres over-expressing HDAC-4/HDAC-5 exhibited decreased expression of RYR1 and of muscle-specific miRNAs, whereas acute knock-down of RYR1 in mouse muscle fibres by siRNA caused up-regulation of HDAC-4/HDAC-5. Intriguingly, increased class II HDAC expression and decreased ryanodine receptor protein and miRNAs expression were also observed in muscles of patients with nemaline myopathy, another congenital neuromuscular disorder. Our results indicate that a common pathophysiological pathway caused by epigenetic changes is activated in some forms of congenital neuromuscular disorders