Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are autosomal dominant multisystemic disorders caused by expansion of microsatellite repeats. In both forms, the mutant transcripts accumulate in nuclear foci altering the function of alternative splicing regulators which are necessary for the physiological mRNA processing. Missplicing of insulin receptor (IR) gene (INSR) has been associated with insulin resistance, however, it cannot be excluded that post-receptor signalling abnormalities could also contribute to this feature in DM. We have analysed the insulin pathway in skeletal muscle biopsies and in myotube cultures from DM patients to assess whether downstream metabolism might be dysregulated and to better characterize the mechanism inducing insulin resistance. DM skeletal muscle exhibits alterations of basal phosphorylation levels of Akt/PKB, p70S6K, GSK3β and ERK1/2, suggesting that these changes might be accompanied by a lack of further insulin stimulation. Alterations of insulin pathway have been confirmed on control and DM myotubes expressing fetal INSR isoform (INSR-A). The results indicate that insulin action appears to be lower in DM than in control myotubes in terms of protein activation and glucose uptake. Our data indicate that post-receptor signalling abnormalities might contribute to DM insulin resistance regardless the alteration of INSR splicing.

Objective The excess of visceral adipose tissue might hinder and delay immune response. How people with abdominal obesity (AO) will respond to mRNA vaccines against SARS‐CoV‐2 is yet to be established. SARS‐CoV‐2‐specific antibody responses were evaluated after the first and second dose of the BNT162b2 mRNA vaccine, comparing the response of individuals with AO with the response of those without, and discerning between individuals with or without prior infection. Methods Immunoglobulin G (IgG)‐neutralizing antibodies against the Trimeric complex (IgG‐TrimericS) were measured at four time points: at baseline, at day 21 after vaccine dose 1, and at 1 and 3 months after dose 2. Nucleocapsid antibodies were assessed to detect prior SARS‐CoV‐2 infection. Waist circumference was measured to determine AO. Results Between the first and third month after vaccine dose 2, the drop in IgG‐TrimericS levels was more remarkable in individuals with AO compared with those without AO (2.44‐fold [95% CI: 2.22‐2.63] vs. 1.82‐fold [95% CI: 1.69‐1.92], respectively, p < 0.001). Multivariable linear regression confirmed this result after inclusion of assessed confounders (p < 0.001). Conclusions The waning antibody levels in individuals with AO may further support recent recommendations to offer booster vaccines to adults with high‐risk medical conditions, including obesity, and particularly to those with a more prevalent AO phenotype.

Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are multisystemic disorders linked to two different genetic loci and characterized by several features including myotonia, muscle weakness and atrophy, cardiac dysfunctions, cataracts and insulin-resistance. In both forms, expanded nucleotide sequences cause the accumulation of mutant transcripts in the nucleus deregulating the activity of some RNAbinding proteins and providing an explanation for the multisystemic phenotype of DM patients. However this pathogenetic mechanism does not explain some histopathological features of DM skeletal muscle like muscle atrophy. It has been observed that DM muscle shares similarities with the ageing muscle, where the progressive muscle weakness and atrophy is accompanied by a lower regenerative capacity possibly due to the failure in satellite cells activation. The aim of our study is to investigate if DM2 satellite cell derived myoblasts exhibit a premature senescence as reported for DM1 and if alterations in their proliferation potential and differentiation capabilities might contribute to some of the histopathological features observed in DM2 muscles. Our results indicate that DM myoblasts have lower proliferative capability than control myoblasts and reach in vitro senescence earlier than controls. Differentely from DM1, the p16 pathway is not responsible for the premature growth arrest observed in DM2 myoblasts which stop dividing with telomeres shorter than controls. During in vitro senescence, a progressive decrease in fusion index is observable in both DM and control myotubes with no significant differences between groups. Moreover, myotubes obtained from senescent myoblasts appear to be smaller than those from young myoblasts. Taken together, our data indicate a possible role of DM2 premature myoblast senescence in skeletal muscle histopathological alterations i.e., dystrophic changes and type 2 fibre atrophy.

Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are autosomal dominant multisystemic disorders linked to two different genetic loci and characterized by several features including myotonia, muscle atrophy and insulin resistance. The aberrant alternative splicing of insulin receptor (IR) gene and post-receptor signalling abnormalities have been associated with insulin resistance, however the precise molecular defects that cause metabolic dysfunctions are still unknown. Thus, the aims of this study were to investigate in DM skeletal muscle biopsies if beyond INSR missplicing, altered IR protein expression could play a role in insulin resistance and to verify if the lack of insulin pathway activation could contribute to skeletal muscle wasting. Our analysis showed that DM skeletal muscle exhibits a lower expression of the insulin receptor in type 1 fibers which can contribute to the defective activation of the insulin pathway. Moreover, the aberrant insulin signalling activation leads to a lower activation of mTOR and to an increase in MuRF1 and Atrogin-1/MAFbx expression, possible explaining DM skeletal muscle fiber atrophy. Taken together our data indicate that the defective insulin signalling activation can contribute to skeletal muscle features in DM patients and are probably linked to an aberrant specific-fiber type expression of the insulin receptor.

Circular RNAs (circRNAs) constitute a recently re-discovered class of non-coding RNAs functioning as sponges for miRNAs and proteins, affecting RNA splicing and regulating transcription. CircRNAs are generated by “back-splicing”, which is the linking covalently of 3′- and 5′-ends of exons. Thus, circRNA levels might be deregulated in conditions associated with altered RNA-splicing. Significantly, growing evidence indicates their role in human diseases. Specifically, myotonic dystrophy type 1 (DM1) is a multisystemic disorder caused by expanded CTG repeats in the DMPK gene which results in abnormal mRNA-splicing. In this investigation, circRNAs expressed in DM1 skeletal muscles were identified by analyzing RNA-sequencing data-sets followed by qPCR validation. In muscle biopsies, out of nine tested, four transcripts showed an increased circular fraction: CDYL, HIPK3, RTN4_03, and ZNF609. Their circular fraction values correlated with skeletal muscle strength and with splicing biomarkers of disease severity, and displayed higher values in more severely affected patients. Moreover, Receiver-Operating-Characteristics curves of these four circRNAs discriminated DM1 patients from controls. The identified circRNAs were also detectable in peripheral-blood-mononuclear-cells (PBMCs) and the plasma of DM1 patients, but they were not regulated significantly. Finally, increased circular fractions of RTN4_03 and ZNF609 were also observed in differentiated myogenic cell lines derived from DM1 patients. In conclusion, this pilot study identified circRNA dysregulation in DM1 patients.

Myotonic dystrophy type 2 (DM2) is an autosomal dominant disorder caused by the expansion of the tetranucleotidic repeat (CCTG)n in the first intron of the Zinc Finger Protein-9 gene. In DM2 tissues, the expanded mutant transcripts accumulate in nuclear focal aggregates where splicing factors are sequestered, thus affecting mRNA processing. Interestingly, the ultrastructural alterations in the splicing machinery observed in the myonuclei of DM2 skeletal muscles are reminiscent of the nuclear changes occurring in age-related muscle atrophy. Here, we investigated in vitro structural and functional features of satellite cell-derived myoblasts from biceps brachii, in the attempt to investigate cell senescence indices in DM2 patients by ultrastructural cytochemistry. We observed that in satellite cell-derived DM2 myoblasts, cell-senescence alterations such as cytoplasmic vacuolization, reduction of the proteosynthetic apparatus, accumulation of heterochromatin and impairment of the pre-mRNA maturation pathways occur earlier than in myoblasts from healthy patients. These results, together with preliminary in vitro observations on the early onset of defective structural features in DM2 myoblast derived-myotubes, suggest that the regeneration capability of DM2 satellite cells may be impaired, thus contributing to the muscular dystrophy in DM2 patients.

Myotonic dystrophy type 2 (DM2) is a common adult onset muscular dystrophy caused by a dominantly transmitted (CCTG)( n ) expansion in intron 1 of the CNBP gene. In DM2 there is no obvious evidence for an intergenerational increase of expansion size, and no congenital cases have been confirmed. We describe the clinical and histopathological features, and provide the genetic and molecular explanation for juvenile onset of myotonia in a 14-year-old female with DM2 and her affected mother presenting with a more severe phenotype despite a later onset of symptoms. Histological and immunohistochemical findings correlated with disease severity or age at onset in both patients. Southern blot on both muscle and blood samples revealed only a small increase in the CCTG repeat number through maternal transmission. Fluorescence in situ hybridization, in combination with MBNL1 immunofluorescence on muscle sections, showed the presence of mutant mRNA and MBNL1 in nuclear foci; the fluorescence intensity and its area appeared to be similar in the two patients. Splicing analysis of the INSR, CLCN1 and MBNL1 genes in muscle tissue demonstrates that the level of aberrant splicing isoforms was lower in the daughter than in the mother. However, in the CLCN1 gene, a heterozygous mutation c.501C>G p.F167L was present in the daughter's DNA and found to be maternally inherited. Biomolecular findings did not explain the unusual young onset in the daughter. The co-segregation of DM2 with a recessive CLCN1 mutation provided the explanation for the unusual clinical findings

Myotonic dystrophy type 1 (DM1) is a multi-systemic disorder caused by abnormally expanded stretches of CTG DNA triplets in the DMPK gene, leading to mutated-transcript RNA-toxicity. MicroRNAs (miRNAs) are short non-coding RNAs that, after maturation, are loaded onto the RISC effector complex that destabilizes target mRNAs and represses their translation. In DM1 muscle biopsies not only the expression, but also the intracellular localization of specific miRNAs is disrupted, leading to the dysregulation of the relevant mRNA targets. To investigate the functional alterations of the miRNA/target interactions in DM1, we analyzed by RNA-sequencing the RISC-associated RNAs in skeletal muscle biopsies derived from DM1 patients and matched controls. The mRNAs found deregulated in DM1 biopsies were involved in pathways and functions relevant for the disease, such as energetic metabolism, calcium signaling, muscle contraction and p53-dependent apoptosis. Bioinformatic analysis of the miRNA/mRNA interactions based on the RISC enrichment profiles, identified 24 miRNA/mRNA correlations. Following validation in 21 independent samples, we focused on the couple miR-29c/ASB2 because of the role of miR-29c in fibrosis (a feature of late-stage DM1 patients) and of ASB2 in the regulation of muscle mass. Luciferase reporter assay confirmed the direct interaction between miR-29c and ASB2. Moreover, decreased miR-29c and increased ASB2 levels were verified also in immortalized myogenic cells and primary fibroblasts, derived from biopsies of DM1 patients and controls. CRISPR/Cas9-mediated deletion of CTG expansions rescued normal miR-29c and ASB2 levels, indicating a direct link between the mutant repeats and the miRNA/target expression. In conclusion, functionally relevant miRNA/mRNA interactions were identified in skeletal muscles of DM1 patients, highlighting the dysfunction of miR-29c and ASB2.