I MICRORNA NELLE DISTROFIE MIOTONICHE

Myotonic dystrophy (DM), the most common form of muscular dystrophy in adults, is a dominantly inherited disorder with a peculiar and rare pattern of multisystemic clinical features affecting skeletal muscle, the heart, the eye, and the endocrine system. Classical DM (first described by Steinert and called Steinert’s disease or DM1) has been identified as an autosomal dominant disorder associated with the presence of an abnormal expansion of a CTG trinucleotide repeat in the 3’ untranslated region of the dystrophia myotonica-protein kinase (DMPK) gene on chromosome 19q13.3. Recently, the expansion of a CCTG tetranucleotide repeat located in the intron of the zinc finger 9 (ZNF9) gene on chromosome 19q13.3 was identified as the mutation responsible for DM2. Both mutations lead to the production of mRNA transcripts containing expanded tri- or tetranucleotide repeats (CUG/CCUG) that are retained in muscle nuclei as ribonuclear inclusions and interact with RNA-binding proteins. These interaction are supposed to disrupt the regulation of alternative splicing of several transcripts. Clinical and molecular parallels strongly support that DM1 and DM2 physiopathology is in part the pathogenic consequence of an RNA gain of toxic function. MicroRNAs (miRNAs) are short non-coding RNAs (~22 nucleotides) regulating gene expression post-trascriptionally either via the degradation of target mRNAs or the inhibition of protein translation. MicroRNAs have been shown to be involved in a range of biological processes, including myogenesis and muscle regeneration. miRNAs are expressed in cardiac and skeletal muscle, and dysregulated miRNA expression has been correlated with muscle-related diseases, including cardiac hypertrophy, cardiac arrhythmias and muscular dystrophy. Given the emerging roles of microRNAs, we have performed miRNAs expression profiling in DM1 and DM2 patients on muscle biopsies and primary cell culture line. Using fast real time PCR, we report here the differences in miRNAs expression profiles between DM1 (n=15), DM2 (n=9) and control subjects (n=14) of 24 specific miRNAs. miRNAs expression profiles in muscle biopsies of DM1 showed up-regulation of miR-1 and miR-335 and down-regulation of miR-29b, miR-29c, miR-33, establishing a provisional DM1 miRNA signature. A similar trend in miRNA modulation was observed in DM2 patients. However, none of the differences reached statistical significance. In order to assess whether DM1 signature miRNA deregulations and DM2 were cell autonomous events, primary cultures of skeletal muscle satellite cells obtained from either DM1 patients (n=5), DM2 patients (n=5) or controls (n=5) were examined. Myoblasts were cultured in growth factor rich medium and then switched to differentiation medium for five days. DM1 and DM2 myoblasts did not display overt morphological alterations of differentiation. When DM1 miRNA signature was examined, we found that miR-29b was strongly down-modulated in differentiated DM1 myotubes. Conversely, miR-335 was enhanced in DM1 myoblasts in growth medium whereas, upon switching to differentiation medium, it increased to a similar level both in DM1 and control myoblasts. When DM2 myoblasts and myotubes was examined, we not found significance statistical differences in miRNAs expression compared with control myoblasts and myotubes. Furthermore, The cellular localization of DM1 signature miRNAs was assayed by in situ hybridization on cryostat muscle sections derived from DM1 (n=5) and control (n=5) biopsies using digoxigenin labelled LNA probes. We found that miR-29b, -29c, -33 and -335 were either barely detectable or did not show any overt abnormal localization in DM1 compared to control biopsies. Conversely, miR-1 was readily detectable and its intracellular distribution was disrupted. Specifically, in control samples, miR-1 displayed a peculiar enrichment in the perinuclear area. In DM1 sections, centrally nucleated myofibers, a hallmark of DM1, also exhibited a centralization of miR-1 localization. Very small fibers with nuclear clumps, a typical histopathological DM1 alteration, displayed intense miR-1 staining. Certain myofibers displayed an extremely intense and polarized miR-1 accumulation. Atrophic fibers in DM1 muscle are predominantly type I fibers (slow fibers). Aberrant miR-1 distribution was present both in type I and type II myofibers, as assessed by the myosin heavy chain slow isoform counterstaining. We also tested the cellular localization of two more muscle specific microRNAs, miR-133b and -206, albeit no overt deregulation of their expression was found in whole skeletal muscle RNAs. In control biopsies we found that miR-133b displayed a perinuclear distribution similar to that of miR-1; in keeping with previous findings, miR-206 was barely detectable. In DM1 patients, both miR-133b and -206 exhibited centralization in centrally nucleated myofibers and accumulated in association to small myofibers nuclear clumps. Finally, miRNAs have been shown not only to inhibit protein translation, but also to induce mRNA degradation, at least for certain targets. Thus, in order to assess whether miRNA deregulation was functionally relevant, we examined the impact of the identified miRNAs deregulation on the expression of their potential target genes in DM1 patients. Specifically, we focused on miR-29, that displayed the strongest deregulation, and miR-1, that plays a crucial role in muscle differentiation. Search of the potential targets was performed using Pictar and Targetscan prediction algorithms, given their reported specificity. Indeed, to maximize the accuracy, only targets identified by both softwares were considered. A sub-pool of the identified targets were analyzed, selected among these with a potential link to DM1 physio-pathology. Specifically, selected genes were previously demonstrated to be expressed in skeletal muscle and to be involved in events such as muscle development, atrophy, arrhythmia and splicing. Potential targets were assayed by qPCR and shows that both miR-29 and miR-1 targets were significantly up-regulated in DM1 patients. In conclusion, we identified a small subset of miRNA whose expression and/or localization were deregulated in DM. These findings may improve our understanding of the molecular mechanisms linking (CTG)n/(CCTG)n expansion to disease and may serve as potential prognostic/diagnostic markers.