Multimodal Single Cell profiling of Duchenne Muscular Dystrophy

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miR-708-5p and miR-34c-5p are involved in nNOS regulation in dystrophic context

International audienceBackground: Duchenne (DMD) and Becker (BMD) muscular dystrophies are caused by mutations in the DMD gene coding for dystrophin, a protein being part of a large sarcolemmal protein scaffold that includes the neuronal nitric oxide synthase (nNOS). The nNOS was shown to play critical roles in a variety of muscle functions and alterations of its expression and location in dystrophic muscle fiber leads to an increase of the muscle fatigability. We previously revealed a decrease of nNOS expression in BMD patients all presenting a deletion of exons 45 to 55 in the DMD gene (BMDd45-55), impacting the nNOS binding site of dystrophin. Since several studies showed deregulation of microRNAs (miRNAs) in dystrophinopathies, we focused on miRNAs that could target nNOS in dystrophic context.Methods: By a screening of 617 miRNAs in BMDd45-55 muscular biopsies using TLDA and an in silico study to determine which one could target nNOS, we selected four miRNAs. In order to select those that targeted a sequence of 3′UTR of NOS1, we performed luciferase gene reporter assay in HEK393T cells. Finally, expression of candidate miRNAs was modulated in control and DMD human myoblasts (DMDd45-52) to study their ability to target nNOS.Results: TLDA assay and the in silico study allowed us to select four miRNAs overexpressed in muscle biopsies of BMDd45-55 compared to controls. Among them, only the overexpression of miR-31, miR-708, and miR-34c led to a decrease of luciferase activity in an NOS1-3′UTR-luciferase assay, confirming their interaction with the NOS1-3′UTR. The effect of these three miRNAs was investigated on control and DMDd45-52 myoblasts. First, we showed a decrease of nNOS expression when miR-708 or miR-34c were overexpressed in control myoblasts. We then confirmed that DMDd45-52 cells displayed an endogenous increased of miR-31, miR-708, and miR-34c and a decreased of nNOS expression, the same characteristics observed in BMDd45-55 biopsies. In DMDd45-52 cells, we demonstrated that the inhibition of miR-708 and miR-34c increased nNOS expression, confirming that both miRNAs can modulate nNOS expression in human myoblasts.Conclusion: These results strongly suggest that miR-708 and miR-34c, overexpressed in dystrophic context, are new actors involved in the regulation of nNOS expression in dystrophic muscle

Dystrophin threshold level necessary for normalisation of nNOS, iNOS and RyR1 nitrosylation in GRMD dystrophinopathy

International audienceCurrently, the clinically most advanced strategy to treat Duchenne muscular dystrophy (DMD) is the exon skipping strategy. Whereas antisense oligonucleotide-based clinical trials are underway for DMD, it is essential to determine a dystrophin restoration threshold needed to ensure improvement of muscle physiology at the molecular level. A preclinical trial was recently conducted in golden retriever muscular dystrophy (GRMD) dogs treated in a forelimb by locoregional delivery of rAAV8-U7snRNA to promote exon skipping on the canine dystrophin messenger. Here, we exploited the rAAV8-U7snRNA transduced GRMD muscle samples, well-characterized for their percentage of dystrophin-positive fibers, in the aim to define a threshold of dystrophin rescue necessary for normalization of the status of the neuronal nitric oxide synthase mu (nNOSµ), the inducible nitric oxide synthase (iNOS), and the ryanodine receptor-calcium release channel type 1 (RyR1), crucial actors for an efficient contractile function. Results showed that the restoration of dystrophin in 40% of muscle fibers is needed to decrease the abnormal cytosolic nNOSµ expression and to reduce the overexpression of iNOS, these two parameters leading to a reduction of the NO level into the muscle fiber. Furthermore, the same percentage of dystrophin-positive fibers of 40 % was associated with the normalization of the RyR1 nitrosylation status and to a stabilization of the RyR1/calstabin1 complex that is required to facilitate coupled gating. We concluded that a minimal threshold of 40% of dystrophin-positive fibers is necessary for the reinstatement of central proteins needed for a proper muscle contractile function, and thus identified a rate of dystrophin expression significantly improving, at the molecular level, the dystrophic muscle physiology