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.

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.

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.

SCN4A as modifier gene in patients with myotonic dystrophy type 2.

A patient with an early severe myotonia diagnosed for Myotonic Dystrophy type 2 (DM2) was found bearing the combined effects of DM2 mutation and Nav1.4 S906T substitution. To investigate the mechanism underlying his atypical phenotype,whole-cell patch-clamp in voltage- and current-clamp mode was performed in myoblasts and myotubes obtained from his muscle biopsy. Results characterizing the properties of the sodium current and of the action potentials have been compared to those obtained in muscle cells derived from his mother, also affected by DM2, but without the S906T polymorphism. A faster inactivation kinetics and a +5 mV shift in the availability curve were found in the sodium current recorded in patient's myoblasts compared to his mother. 27% of his myotubes displayed spontaneous activity. Patient's myotubes showing a stable resting membrane potential had a lower rheobase current respect to the mother's while the overshoot and the maximum slope of the depolarizing phase of action potential were higher. These findings suggest that SCN4A polymorphisms may be responsible for a higher excitability of DM2 patients sarcolemma, supporting the severe myotonic phenotype observed. We suggest SCN4A as a modifier factor and that its screening should be performed in DM2 patients with uncommon clinical features.

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.

Biomolecular diagnosis of myotonic dystrophy type 2: a challenging approach.

Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are the most common adult form of muscular dystrophy, characterized by autosomal dominant progressive myopathy, myotonia, and multiorgan involvement. The onset and symptoms of the myotonic dystrophies are diverse, complicating their diagnoses and limiting a comprehensive approach to their clinical care. Diagnostic delay in DM2 is due not only to the heterogeneous phenotype and the aspecific onset but also to the unfamiliarity with the disorder by most clinicians. Moreover, the DM2 diagnostic odyssey is complicated by the difficulties to develop an accurate, robust, and cost-effective method for a routine molecular assay. The aim of this review is to underline by challenging approach the diagnostic limits and pitfalls that could results in failure to recognize the presence of DM2 disease. Understanding and preventing delays in DM2 diagnosis may facilitate family planning, improve symptom management in the short term, and facilitate more specific treatment in the long term.

Activation of the Pro-Oxidant PKCßII-p66Shc Signaling Pathway Contributes to Pericyte Dysfunction in Skeletal Muscles of Patients With Diabetes With Critical Limb Ischemia.

Critical limb ischemia (CLI), foot ulcers, former amputation, and impaired regeneration are independent risk factors for limb amputation in subjects with diabetes. The present work investigates whether and by which mechanism diabetes negatively impacts on functional properties of muscular pericytes (MPs), which are resident stem cells committed to reparative angiomyogenesis. We obtained muscle biopsy samples from patients with diabetes who were undergoing major limb amputation and control subjects. Diabetic muscles collected at the rim of normal tissue surrounding the plane of dissection showed myofiber degeneration, fat deposition, and reduction of MP vascular coverage. Diabetic MPs (D-MPs) display ultrastructural alterations, a differentiation bias toward adipogenesis at the detriment of myogenesis and an inhibitory activity on angiogenesis. Furthermore, they have an imbalanced redox state, with downregulation of the antioxidant enzymes superoxide dismutase 1 and catalase, and activation of the pro-oxidant protein kinase C isoform ß-II (PKCßII)-dependent p66Shc signaling pathway. A reactive oxygen species scavenger or, even more effectively, clinically approved PKCßII inhibitors restore D-MP angiomyogenic activity. Inhibition of the PKCßII-dependent p66Shc signaling pathway could represent a novel therapeutic approach for the promotion of muscle repair in individuals with diabetes.

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.

Myotonic Dystrophy Type 2: An Update on Clinical Aspects, Genetic and Pathomolecular Mechanism.

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 CNBP. Despite clinical and genetic similarities, DM1 and DM2 are distinct disorders. The pathogenesis of DM is explained by a common RNA gain-of-function mechanism in which the CUG and CCUG repeats alter cellular function, including alternative splicing of various genes. However additional pathogenic mechanism like changes in gene expression, modifier genes, protein translation and micro-RNA metabolism may also contribute to disease pathology and to clarify the phenotypic differences between these two types of myotonic dystrophies.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.

Progression of muscle histopathology but not of spliceopathy in myotonic dystrophy type 2.

Myotonic dystrophy type 2 (DM2) is an autosomal dominant progressive disease involving skeletal and cardiac muscle and brain. It is caused by a tetranucleotide repeat within the first intron of the CNBP gene that leads to an alteration of the alternative splicing of several genes. To understand the molecular mechanisms that play a role in DM2 progression, the evolution of skeletal muscle histopathology and biomolecular findings in successive biopsies have been studied. Biceps brachii biopsies from 5 DM2 patients who underwent two successive biopsies at different years of age have been used. Muscle histopathology has been assessed on sections immunostained with fast or slow myosin. FISH in combination with MBNL1-immunofluorescence has been performed to evaluate ribonuclear inclusion and MBNL1 foci dimensions in myonuclei. Gene and protein expression and alteration of alternative splicing of several genes have been evaluated over time. All DM2 patients examined show a worsening of muscle histopathology and an increase of foci dimensions over time. The progressive worsening of myotonia in DM2 patients may be due to the decrease of CLCN1 mRNA observed in all patients examined. However, a worsening of alternative splicing alterations has not been evidenced over time. The data obtained in this study confirm that DM2 is a slow progression disease since histological and biomolecular alterations observed in skeletal muscle are minimal even after 10-year interval. The data indicate that muscle morphological alterations evolve more rapidly over time than the molecular changes thus indicating that muscle biopsy is a more sensitive tool than biomolecular markers to assess disease progression at muscle level.

Genome wide identification of aberrant alternative splicing events in myotonic dystrophy type 2.

Myotonic dystrophy type 2 (DM2) is a genetic, autosomal dominant disease due to expansion of tetraplet (CCTG) repetitions in the first intron of the ZNF9/CNBP gene. DM2 is a multisystemic disorder affecting the skeletal muscle, the heart, the eye and the endocrine system. According to the proposed pathological mechanism, the expanded tetraplets have an RNA toxic effect, disrupting the splicing of many mRNAs. Thus, the identification of aberrantly spliced transcripts is instrumental for our understanding of the molecular mechanisms underpinning the disease. The aim of this study was the identification of new aberrant alternative splicing events in DM2 patients. By genome wide analysis of 10 DM2 patients and 10 controls (CTR), we identified 273 alternative spliced exons in 218 genes. While many aberrant splicing events were already identified in the past, most were new. A subset of these events was validated by qPCR assays in 19 DM2 and 15 CTR subjects. To gain insight into the molecular pathways involving the identified aberrantly spliced genes, we performed a bioinformatics analysis with Ingenuity system. This analysis indicated a deregulation of development, cell survival, metabolism, calcium signaling and contractility. In conclusion, our genome wide analysis provided a database of aberrant splicing events in the skeletal muscle of DM2 patients. The affected genes are involved in numerous pathways and networks important for muscle physio-pathology, suggesting that the identified variants may contribute to DM2 pathogenesis.

Dysregulation and cellular mislocalization of specific miRNAs in myotonic dystrophy type 1.

Myotonic Dystrophy Type-1 (DM1) is caused by the expansion of a CTG repeat with a peculiar pattern of multisystemic involvement affecting skeletal muscles, the heart, the eye, the central nervous system and the endocrine system. Since microRNA expression is disrupted in several myopathies, the expression of 24 candidate microRNAs was analyzed in skeletal muscle biopsies of 15 DM1 patients. Controls were constituted by biopsies without overt pathological features derived from 14 subjects with suspected neuromuscular disorder of undetermined nature. We found that miR-1 and miR-335 were up-regulated, whereas miR-29b and c, and miR-33 were down-regulated in DM1 biopsies compared to controls. We also found that the cellular distribution of muscle specific miR-1, miR-133b and miR-206 was severely altered in DM1 skeletal muscles. MicroRNA dysregulation was likely functionally relevant, since it impacted on the expression of the predicted miR-1, and miR-29 targets. The observed miRNA dysregulations and myslocalizations may contribute to DM1 pathogenetic mechanisms.

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.

Effect of glucose stress conditions in BL6T murine melanoma cells.

Elevated expression of stress proteins can be a characteristic of human cancer and may be involved in the development of resistance to some types of chemotherapeutic agent. In this paper, the effect of physiological stress conditions, such as glucose deprivation, was investigated in overexpressing nPKCdelta murine melanoma BL6 (BL6T) cells. Glucose stress conditions decreased the proliferative capacity, increasing the percentage of BL6T cells in the G0/G1 phase of the cell cycle. Furthermore, under such conditions, nPKCdelta, whose subcellular localization is cell cycle dependent, showed a cytoplasmic and perinuclear localization by immunohistochemistry, this being typical for cells in G0/G1 phase. Moreover, these cells expressed GRP-78, a known stress protein. On the other hand, glucose depletion enhanced intracellular melanin as well as tyrosinase activity and expression. In summary, these data demonstrate that stress conditions can modify the biological characteristics of BL6T cells, and therefore can select a quiescent cellular population.