Viral Agents and Systemic Levels of Inflammatory Cytokines in Vulnerable and Stable Atherosclerotic Carotid Plaques.

BACKGROUND: To investigate the presence of genetic material of viral agents and the serum level of inflammatory cytokines in patients submitted to carotid endarterectomy having vulnerable versus stable atherosclerotic plaques. METHODS: Data of patients consecutively submitted to carotid endarterectomy for a significant stenosis from July 2019 to December 2019 were prospectively collected. The genetic material of Epstein-Barr (EBV), CitoMegalo (CMV), Herpes Simplex (HSV), Varicella-Zoster (VZV) and Influenza (IV) Viruses was searched in the patient's plaques, both in the "mid" of the plaque and in an adjacent lateral portion of no-plaque area. The serum levels of TNF-α, IL-1ß, IL-6, IL10 and CCL5 were determined. The obtained results were then correlated to the histologic vulnerability of the removed carotid plaque. P values < 0.05 were considered statistically significant. RESULTS: Data of 50 patients were analyzed. A vulnerable plaque was found in 31 patients (62%). The genome of CMV, HSV, VZV and IV was not found in any of the vascular samples, while the EBV genome was found in the "mid" of 2 vulnerable plaques, but not in their respective control area. Eighty-two percent of patients who did not receive anti-IV vaccination (23/28) had vulnerable carotid plaque, compared with 36% of vaccinated patients (8/22, P = 0.001). Serum levels of TNF-α and IL-6 were higher in patients with a vulnerable plaque compared to patients with a stable plaque (73.6 ± 238.2 vs. 3.9 ± 13.1 pg/ml, P= 0.01, and 45.9 ± 103.6 vs. 10.1 ± 25.3 pg/ml, P= 0.01, respectively), independent of comorbidities, viral exposure or flu vaccination. CONCLUSIONS: The EBV genome was found in the "core" of 2 vulnerable carotid plaques, but not in their respective adjacent control. Influenza vaccination was associated with a lower incidence of carotid plaque vulnerability. Serum levels of TNF-α and IL-6 were higher in patients with a vulnerable plaque compared to patients with a stable plaque.

Co-occurrence of DMPK expansion and CLCN1 mutation in a patient with myotonia.

INTRODUCTION: Myotonic disorders are a group of diseases affecting the muscle, in different ways. Myotonic dystrophy type 1 (DM1) is related to (CTG)n expansion in the 3-untranslated region of the dystrophia myotonica protein kinase (DMPK) gene and is the most frequent and disabling form, causing muscular, visibility, respiratory, and cardiac impairment. Non-dystrophic myotonias (NDMs) affect the skeletal muscle alone. In particular, mutations in the chloride channel (CLCN1) gene cause myotonia congenita (MC), which can have autosomal dominant or recessive inheritance. CASE REPORT: We describe a patient with a family history of asymptomatic or paucisymptomatic myotonia, who presented handgrip myotonia which sharply reduced after mexiletine administration. Molecular analysis showed both a paternally inherited DMPK expansion and a maternally inherited CLCN1 mutation. CONCLUSIONS: Only one other similar case was reported so far; however, the segregation of the two mutations and the characteristics of the muscle were not studied. Since our patient lacked the classical phenotypical and muscle histopathological characteristics of DM1 and showed mild splicing alterations despite a pathogenic DMPK expansion and the nuclear accumulation of toxic RNA, we may speculate that the co-occurrence of a CLCN1 mutation could have attenuated the severity of DM1 phenotype.

Aberrant regulation of epigenetic modifiers contributes to the pathogenesis in patients with selenoprotein N-related myopathies.

Congenital myopathies are early onset, slowly progressive neuromuscular disorders of variable severity. They are genetically and phenotypically heterogeneous and caused by pathogenic variants in several genes. Multi-minicore Disease, one of the more common congenital myopathies, is frequently caused by recessive variants in either SELENON, encoding the endoplasmic reticulum glycoprotein selenoprotein N or RYR1, encoding a protein involved in calcium homeostasis and excitation-contraction coupling. The mechanism by which recessive SELENON variants cause Multiminicore disease (MmD) is unclear. Here, we extensively investigated muscle physiological, biochemical and epigenetic modifications, including DNA methylation, histone modification, and noncoding RNA expression, to understand the pathomechanism of MmD. We identified biochemical changes that are common in patients harboring recessive RYR1 and SELENON variants, including depletion of transcripts encoding proteins involved in skeletal muscle calcium homeostasis, increased levels of Class II histone deacetylases (HDACs) and DNA methyltransferases. CpG methylation analysis of genomic DNA of patients with RYR1 and SELENON variants identified >3,500 common aberrantly methylated genes, many of which are involved in calcium signaling. These results provide the proof of concept for the potential use of drugs targeting HDACs and DNA methyltransferases to treat patients with specific forms of congenital myopathies.

Dysregulation of Circular RNAs in Myotonic Dystrophy Type 1.

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.

The analysis of myotonia congenita mutations discloses functional clusters of amino acids within the CBS2 domain and the C-terminal peptide of the ClC-1 channel.

Myotonia congenita (MC) is a skeletal-muscle hyperexcitability disorder caused by loss-of-function mutations in the ClC-1 chloride channel. Mutations are scattered over the entire sequence of the channel protein, with more than 30 mutations located in the poorly characterized cytosolic C-terminal domain. In this study, we characterized, through patch clamp, seven ClC-1 mutations identified in patients affected by MC of various severities and located in the C-terminal region. The p.Val829Met, p.Thr832Ile, p.Val851Met, p.Gly859Val, and p.Leu861Pro mutations reside in the CBS2 domain, while p.Pro883Thr and p.Val947Glu are in the C-terminal peptide. We showed that the functional properties of mutant channels correlated with the clinical phenotypes of affected individuals. In addition, we defined clusters of ClC-1 mutations within CBS2 and C-terminal peptide subdomains that share the same functional defect: mutations between 829 and 835 residues and in residue 883 induced an alteration of voltage dependence, mutations between 851 and 859 residues, and in residue 947 induced a reduction of chloride currents, whereas mutations on 861 residue showed no obvious change in ClC-1 function. This study improves our understanding of the mechanisms underlying MC, sheds light on the role of the C-terminal region in ClC-1 function, and provides information to develop new antimyotonic drugs.

Myotonic dystrophy type 2 and modifier genes: an update on clinical and pathomolecular aspects.

Myotonic dystrophy (DM) is the most common adult muscular dystrophy, characterized by autosomal dominant progressive myopathy, myotonia, and multiorgan involvement. To date, two distinct forms caused by similar mutations in two different genes have been identified: myotonic dystrophy type 1 (DM1) and myotonic dystrophy type 2 (DM2). Aberrant transcription and mRNA processing of multiple genes due to RNA-mediated toxic gain-of function has been suggested to cause the complex phenotype in DM1 and DM2. However, despite clinical and genetic similarities, DM1 and DM2 may be considered as distinct disorders. This review is an update on the latest findings specific to DM2, including explanations for the differences in clinical manifestations and pathophysiology between the two forms of myotonic dystrophies.

Myotonic dystrophies: An update on clinical aspects, genetic, pathology, and molecular pathomechanisms.

Myotonic dystrophy (DM) is the most common adult muscular dystrophy, characterized by autosomal dominant progressive myopathy, myotonia and multiorgan involvement. To date two distinct forms caused by similar mutations have been identified. Myotonic dystrophy type 1 (DM1, Steinert's disease) is caused by a (CTG)n expansion in DMPK, while myotonic dystrophy type 2 (DM2) is caused by a (CCTG)n expansion in ZNF9/CNBP. When transcribed into CUG/CCUG-containing RNA, mutant transcripts aggregate as nuclear foci that sequester RNA-binding proteins, resulting in spliceopathy of downstream effector genes. However, it is now clear that additional pathogenic mechanism like changes in gene expression, protein translation and micro-RNA metabolism may also contribute to disease pathology. Despite clinical and genetic similarities, DM1 and DM2 are distinct disorders requiring different diagnostic and management strategies. This review is an update on the recent advances in the understanding of the molecular mechanisms behind myotonic dystrophies. This article is part of a Special Issue entitled: Neuromuscular Diseases: Pathology and Molecular Pathogenesis.

Reducing contrast agent residuals in hospital wastewater: the GREENWATER study protocol.

The potential enviromental impact of iodinated (ICAs) and gadolinium-based contrast agents (GBCAs) have recently come under scrutiny, considering the current nonselective wastewater treatment. However, their rapid excretion after intravenous administration could allow their potential recovery by targeting hospital sewage. The GREENWATER study aims to appraise the effective quantities of ICAs and GBCAs retrievable from patients' urine collected after computed tomography (CT) and magnetic resonance imaging (MRI) exams, selecting ICA/GBCA per-patient urinary excretion and patients' acceptance rate as study endpoints. Within a prospective, observational, single-centre, 1-year framework, we will enrol outpatients aged ≥ 18 years, scheduled to perform contrast-enhanced CT or MRI, willing to collect post-examination urine in dedicated canisters by prolonging their hospital stay to 1 h after injection. Collected urine will be processed and partially stored in the institutional biobank. Patient-based analysis will be performed for the first 100 CT and 100 MRI patients, and then, all analyses will be conducted on the pooled urinary sample. Quantification of urinary iodine and gadolinium will be performed with spectroscopy after oxidative digestion. The evaluation of the acceptance rate will assess the "environmental awareness" of patients and will aid to model how procedures to reduce ICA/GBCA enviromental impact could be adapted in different settings. Key points • Enviromental impact of iodinated and gadolinium-based contrast agents represents a growing point of attention.• Current wastewater treatment is unable to retrieve and recycle contrast agents.• Prolonging hospital stay may allow contrast agents retrieval from patients' urine.• The GREENWATER study will assess the effectively retrievable contrast agents' quantities.• The enrolment acceptance rate will allow to evaluate patients' "green sensitivity".

Impact of BNT162b2 Booster Dose on SARS-CoV-2 Anti-Trimeric Spike Antibody Dynamics in a Large Cohort of Italian Health Care Workers.

Accurate studies on the dynamics of Pfizer-Biontech BNT162b2-induced antibodies are crucial to better tailor booster dose administration depending on age, comorbidities, and previous natural infection with SARS-CoV-2. To date, little is known about the durability and kinetics of antibody titers months after receiving a booster dose. In this work, we studied the dynamic of anti-Trimeric Spike (anti-TrimericS) IgG titer in the healthcare worker population of a large academic hospital in Northern Italy, in those who had received two vaccine doses plus a booster dose. Blood samples were collected on the day of dose 1, dose 2, then 1 month, 3 months, and 6 months after dose 2, the day of the administration of the booster dose, then 1 month and 3 months after the booster dose. The vaccination immunogenicity was evaluated by dosing anti-TrimericS IgG titer, which was further studied in relation to SARS-CoV-2 infection status, age, and sex. Our results suggest that after the booster dose, the anti-TrimericS IgG production was higher in the subjects that were infected only after the completion of the vaccination cycle, compared to those that were infected both before and after the vaccination campaign. Moreover, the booster dose administration exerts a leveling effect, mitigating the differences in the immunogenicity dependent on sex and age.

BNT162b2 Booster Dose Elicits a Robust Antibody Response in Subjects with Abdominal Obesity and Previous SARS-CoV-2 Infection.

Little is known about the long-term durability of the induced immune response in subjects with obesity, particularly in those with an abdominal distribution of adipose tissue. We evaluated SARS-CoV-2-specific antibody responses after BNT162b2 vaccine booster dose, comparing individuals with and without abdominal obesity (AO), discerning between individuals previously infected or not. IgG-TrimericS were measured in 511 subjects at baseline, on the 21st day after vaccine dose 1, and at 1, 3, 6, and 9 months from dose 2, and at 1 and 3 months following the booster dose. To detect SARS-CoV-2 infection, nucleocapsid antibodies were measured at baseline and at the end of the study. Multivariable linear regression evaluated the three-month difference in the absolute variation in IgG-TrimericS levels from booster dose, showing AO and SARS-CoV-2 infection status interactions (p = 0.016). Regardless of possible confounding factors and IgG-TrimericS levels at the booster dose, AO is associated with a higher absolute change in IgG-TrimericS in prior infected individuals (p = 0.0125). In the same regression model, no interaction is highlighted using BMI (p = 0.418). The robust response in the development of antibodies after booster dose, observed in people with AO and previous infection, may support the recommendations to administer a booster dose in this population group.

Rationale and design of the CV-PREVITAL study: an Italian multiple cohort randomised controlled trial investigating innovative digital strategies in primary cardiovascular prevention.

INTRODUCTION: Prevention of cardiovascular disease (CVD) is of key importance in reducing morbidity, disability and mortality worldwide. Observational studies suggest that digital health interventions can be an effective strategy to reduce cardiovascular (CV) risk. However, evidence from large randomised clinical trials is lacking. METHODS AND ANALYSIS: The CV-PREVITAL study is a multicentre, prospective, randomised, controlled, open-label interventional trial designed to compare the effectiveness of an educational and motivational mobile health (mHealth) intervention versus usual care in reducing CV risk. The intervention aims at improving diet, physical activity, sleep quality, psycho-behavioural aspects, as well as promoting smoking cessation and adherence to pharmacological treatment for CV risk factors. The trial aims to enrol approximately 80 000 subjects without overt CVDs referring to general practitioners' offices, community pharmacies or clinics of Scientific Institute for Research, Hospitalization and Health Care (Italian acronym IRCCS) affiliated with the Italian Cardiology Network. All participants are evaluated at baseline and after 12 months to assess the effectiveness of the intervention on short-term endpoints, namely improvement in CV risk score and reduction of major CV risk factors. Beyond the funded life of the study, a long-term (7 years) follow-up is also planned to assess the effectiveness of the intervention on the incidence of major adverse CV events. A series of ancillary studies designed to evaluate the effect of the mHealth intervention on additional risk biomarkers are also performed. ETHICS AND DISSEMINATION: This study received ethics approval from the ethics committee of the coordinating centre (Monzino Cardiology Center; R1256/20-CCM 1319) and from all other relevant IRBs and ethics committees. Findings are disseminated through scientific meetings and peer-reviewed journals and via social media. Partners are informed about the study's course and findings through regular meetings. TRIAL REGISTRATION NUMBER: NCT05339841.

Cell environment shapes TDP-43 function with implications in neuronal and muscle disease.

TDP-43 (TAR DNA-binding protein 43) aggregation and redistribution are recognised as a hallmark of amyotrophic lateral sclerosis and frontotemporal dementia. As TDP-43 inclusions have recently been described in the muscle of inclusion body myositis patients, this highlights the need to understand the role of TDP-43 beyond the central nervous system. Using RNA-seq, we directly compare TDP-43-mediated RNA processing in muscle (C2C12) and neuronal (NSC34) mouse cells. TDP-43 displays a cell-type-characteristic behaviour targeting unique transcripts in each cell-type, which is due to characteristic expression of RNA-binding proteins, that influence TDP-43's performance and define cell-type specific splicing. Among splicing events commonly dysregulated in both cell lines, we identify some that are TDP-43-dependent also in human cells. Inclusion levels of these alternative exons are altered in tissues of patients suffering from FTLD and IBM. We therefore propose that TDP-43 dysfunction contributes to disease development either in a common or a tissue-specific manner.

Association of miR-144 levels in the peripheral blood with COVID-19 severity and mortality.

Coronavirus disease-2019 (COVID-19) can be asymptomatic or lead to a wide symptom spectrum, including multi-organ damage and death. Here, we explored the potential of microRNAs in delineating patient condition and predicting clinical outcome. Plasma microRNA profiling of hospitalized COVID-19 patients showed that miR-144-3p was dynamically regulated in response to COVID-19. Thus, we further investigated the biomarker potential of miR-144-3p measured at admission in 179 COVID-19 patients and 29 healthy controls recruited in three centers. In hospitalized patients, circulating miR-144-3p levels discriminated between non-critical and critical illness (AUCmiR-144-3p = 0.71; p = 0.0006), acting also as mortality predictor (AUCmiR-144-3p = 0.67; p = 0.004). In non-hospitalized patients, plasma miR-144-3p levels discriminated mild from moderate disease (AUCmiR-144-3p = 0.67; p = 0.03). Uncontrolled release of pro-inflammatory cytokines can lead to clinical deterioration. Thus, we explored the added value of a miR-144/cytokine combined analysis in the assessment of hospitalized COVID-19 patients. A miR-144-3p/Epidermal Growth Factor (EGF) combined score discriminated between non-critical and critical hospitalized patients (AUCmiR-144-3p/EGF = 0.81; p < 0.0001); moreover, a miR-144-3p/Interleukin-10 (IL-10) score discriminated survivors from nonsurvivors (AUCmiR-144-3p/IL-10 = 0.83; p < 0.0001). In conclusion, circulating miR-144-3p, possibly in combination with IL-10 or EGF, emerges as a noninvasive tool for early risk-based stratification and mortality prediction in COVID-19.

Antibody responses to BNT162b2 mRNA vaccine: Infection-naïve individuals with abdominal obesity warrant attention.

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.

RNA processing is altered in skeletal muscle nuclei of patients affected by myotonic dystrophy.

Myotonic dystrophies (DMs) are characterised by highly variable clinical manifestations consisting of muscle weakness and atrophy, and a wide spectrum of extramuscular manifestations. In both DM1 and DM2 forms, expanded nucleotide sequences cause the accumulation of mutant transcripts in the nucleus, thus deregulating the function of some RNA-binding proteins and providing a plausible explanation for the multifactorial phenotype of DM patients. However, at the skeletal muscle level, no mechanistic explanation for the muscle wasting has so far been proposed. We therefore performed a study in situ by immunoelectron microscopy on biceps brachii biopsies from DM1, DM2 and healthy subjects, providing the first ultrastructural evidence on the distribution of some nuclear ribonucleoprotein (RNP)-containing structures and molecular factors involved in pre-mRNA transcription and maturation in dystrophic myonuclei. Our results demonstrated an accumulation of splicing and cleavage factors in myonuclei of both DM1 and DM2 patients, suggesting an impairment of post-transcriptional pre-mRNA pathways. The transcription of the expanded sequences in DM myonuclei would therefore hamper functionality of the whole splicing machinery, slowing down the intranuclear molecular trafficking; this would reduce the capability of myonuclei to respond to anabolic stimuli thus contributing to muscle wasting.

First Family of MATR3-Related Distal Myopathy From Italy: The Role of Muscle Biopsy in the Diagnosis and Characterization of a Still Poorly Understood Disease.

Mutations in the MATR3 gene are associated to distal myopathy with vocal cord and pharyngeal weakness (VCPDM), as well as familiar and sporadic motor neuron disease. To date, 12 VCPDM families from the United States, Germany, Japan, Bulgary, and France have been described in the literature. Here we report an Italian family with a propositus of a 40-year-old woman presenting progressive bilateral foot drop, rhinolalia, and distal muscular atrophy, without clinical signs of motor neuron affection. Her father, deceased some years before, presented a similar distal myopathy phenotype, while her 20-year-old son is asymptomatic. Myopathic changes with vacuolization were observed in muscle biopsy from the propositus. These results, together with the peculiar clinical picture, lead to MATR3 gene sequencing, which revealed a heterozygous p.S85C mutation in the propositus. The same mutation was found in her son. Over a 5-year follow-up, progression is mild in the propositus, while her son remains asymptomatic. Clinical, radiological, and pathological data of our propositus are presented and compared to previously reported cases of VCPDM. VCPDM turns out to be a quite homogenous phenotype of late-onset myopathy associated to p.S85C mutation in MATR3 gene. MATR3-related pathology, encompassing myopathy and motor neuron disease, represents an illustrative example of multisystem proteinopathy (MSP), such as other diseases associated to mutations in VCP, HNRNPA2B1, HNRNPA1, and SQSTM1 genes. The present report contributes to a further characterization of this still poorly understood pathology and points out the diagnostic utility of muscle biopsy in challenging cases.

A 14-Year Italian Experience in DM2 Genetic Testing: Frequency and Distribution of Normal and Premutated CNBP Alleles.

Myotonic dystrophy type 2 (DM2) is a multisystemic disorder caused by a (CCTG) n in intron 1 of the CNBP gene. The CCTG repeat tract is part of a complex (TG) v (TCTG) w (CCTG) x (NCTG) y (CCTG) z motif generally interrupted in CNBP healthy range alleles. Here we report our 14-year experience of DM2 postnatal genetic testing in a total of 570 individuals. The DM2 locus has been analyzed by a combination of SR-PCR, TP-PCR, LR-PCR, and Sanger sequencing of CNBP alleles. DM2 molecular diagnosis has been confirmed in 187/570 samples analyzed (32.8%) and is mainly associated with the presence of myotonia in patients. This set of CNBP alleles showed unimodal distribution with 25 different alleles ranging from 108 to 168 bp, in accordance with previous studies on European populations. The most frequent CNBP alleles consisted of 138, 134, 140, and 136 bps with an overall locus heterozygosity of 90%. Sequencing of 103 unexpanded CNBP alleles in DM2-positive patients revealed that (CCTG)5(NCTG)3(CCTG)7 and (CCTG)6(NCTG)3(CCTG)7 are the most common interruption motifs. We also characterized five CNBP premutated alleles with (CCTG) n repetitions from n = 36 to n = 53. However, the molecular and clinical consequences in our cohort of samples are not unequivocal. Data that emerged from this study are representative of the Italian population and are useful tools for National and European centers offering DM2 genetic testing and counseling.

Proteome profile in Myotonic Dystrophy type 2 myotubes reveals dysfunction in protein processing and mitochondrial pathways.

Myotonic Dystrophy type 2 (DM2) is caused by a DNA microsatellite expansion within the Zinc Finger Protein 9 gene leading to an abnormal splicing pattern largely responsible for the pathological condition. To better define the functional changes occurring in human DM2 myotubes we performed a quantitative proteome comparison between myotubes of DM2 and control patients using two-dimensional gel electrophoresis followed by mass spectrometry. Our results indicate that the proteins, altered in DM2 cultures, belong to two major functional categories: i) mitochondrial components, with a reduction of EFTu, HSP60, GRP75 and Dienoyl-CoA-Isomerase, an enzyme involved in fatty acids degradation; ii) the ubiquitin proteasome system with increase of the 26S proteasome regulatory subunit 13 and a reduction of Proteasome subunit Alfa6 and of Rad23B homolog. Altered ubiquitin-proteasomal activity is supported by a global reduction of cytosolic ubiquitinated proteins. Although future work is required to clarify how these changes affect the degradation machinery and mitochondrial function and to evaluate if these changes also occur in the biopsies of DM2 patients, these results identify the mitochondrial proteins and the ubiquitin-proteasomal system as candidates potentially relevant to DM2 pathogenesis.

Differences in aberrant expression and splicing of sarcomeric proteins in the myotonic dystrophies DM1 and DM2.

Aberrant transcription and mRNA processing of multiple genes due to RNA-mediated toxic gain-of-function has been suggested to cause the complex phenotype in myotonic dystrophies type 1 and 2 (DM1 and DM2). However, the molecular basis of muscle weakness and wasting and the different pattern of muscle involvement in DM1 and DM2 are not well understood. We have analyzed the mRNA expression of genes encoding muscle-specific proteins and transcription factors by microarray profiling and studied selected genes for abnormal splicing. A subset of the abnormally regulated genes was further analyzed at the protein level. TNNT3 and LDB3 showed abnormal splicing with significant differences in proportions between DM2 and DM1. The differential abnormal splicing patterns for TNNT3 and LDB3 appeared more pronounced in DM2 relative to DM1 and are among the first molecular differences reported between the two diseases. In addition to these specific differences, the majority of the analyzed genes showed an overall increased expression at the mRNA level. In particular, there was a more global abnormality of all different myosin isoforms in both DM1 and DM2 with increased transcript levels and a differential pattern of protein expression. Atrophic fibers in DM2 patients expressed only the fast myosin isoform, while in DM1 patients they co-expressed fast and slow isoforms. However, there was no increase of total myosin protein levels, suggesting that aberrant protein translation and/or turnover may also be involved.

Common micro-RNA signature in skeletal muscle damage and regeneration induced by Duchenne muscular dystrophy and acute ischemia.

The aim of this work was to identify micro-RNAs (miRNAs) involved in the pathological pathways activated in skeletal muscle damage and regeneration by both dystrophin absence and acute ischemia. Eleven miRNAs were deregulated both in MDX mice and in Duchenne muscular dystrophy patients (DMD signature). Therapeutic interventions ameliorating the mdx-phenotype rescued DMD-signature alterations. The significance of DMD-signature changes was characterized using a damage/regeneration mouse model of hind-limb ischemia and newborn mice. According to their expression, DMD-signature miRNAs were divided into 3 classes. 1) Regeneration miRNAs, miR-31, miR-34c, miR-206, miR-335, miR-449, and miR-494, which were induced in MDX mice and in DMD patients, but also in newborn mice and in newly formed myofibers during postischemic regeneration. Notably, miR-206, miR-34c, and miR-335 were up-regulated following myoblast differentiation in vitro. 2) Degenerative-miRNAs, miR-1, miR-29c, and miR-135a, that were down-modulated in MDX mice, in DMD patients, in the degenerative phase of the ischemia response, and in newborn mice. Their down-modulation was linked to myofiber loss and fibrosis. 3) Inflammatory miRNAs, miR-222 and miR-223, which were expressed in damaged muscle areas, and their expression correlated with the presence of infiltrating inflammatory cells. These findings show an important role of miRNAs in physiopathological pathways regulating muscle response to damage and regeneration.