Fibrosis: Sirtuins at the checkpoints of myofibroblast differentiation and profibrotic activity.

Fibrotic diseases are still a serious concern for public health, due to their high prevalence, complex etiology and lack of successful treatments. Fibrosis consists of excessive accumulation of extracellular matrix components. As a result, the structure and function of tissues are impaired, thus potentially leading to organ failure and death in several chronic diseases. Myofibroblasts represent the principal cellular mediators of fibrosis, due to their extracellular matrix producing activity, and originate from different types of precursor cells, such as mesenchymal cells, epithelial cells and fibroblasts. Profibrotic activation of myofibroblasts can be triggered by a variety of mechanisms, including the transforming growth factor-ß signalling pathway, which is a major factor driving fibrosis. Interestingly, preclinical and clinical studies showed that fibrotic degeneration can stop and even reverse by using specific antifibrotic treatments. Increasing scientific evidence is being accumulated about the role of sirtuins in modulating the molecular pathways responsible for the onset and development of fibrotic diseases. Sirtuins are NAD+ -dependent protein deacetylases that play a crucial role in several molecular pathways within the cells, many of which at the crossroad between health and disease. In this context, we will report the current knowledge supporting the role of sirtuins in the balance between healthy and diseased myofibroblast activity. In particular, we will address the signalling pathways and the molecular targets that trigger the differentiation and profibrotic activation of myofibroblasts and can be modulated by sirtuins.

Sweeteners modulate bioactivity of endothelial progenitor cells but not induce detrimental effects both on inflammation and behavioural changes.

This study sought to determine the possible detrimental effects of several low- or non-caloric sweeteners on endothelial progenitor cells (EPCs), inflammation and behavioural changes in mice. C57BL/6 male mice received low and high dose of natural and artificial sweeteners for 4 weeks. EPCs, physical and biochemical variables, inflammation and behavioural changes were evaluated. A significant reduction of about 25% of EPCs was found when mice received a moderate amount of all sweeteners (p < .05). This reduction was more strongly significant when a double dose of glucose, aspartame, rebaudioside A and cyclamate (p < .005) in comparison to fructose and sucrose (p < .05) was administered. During inflammation carrageenan-induced, all sweeteners produced a significant increase of EPCs compared to the control group (p < .05). Consumption of glucose and sugar substitutes affect mouse EPC number according to the absence or presence of an inflammatory status, but does not induce detrimental effects on inflammation and behavioural changes.

Sirtuins as Mediator of the Anti-Ageing Effects of Calorie Restriction in Skeletal and Cardiac Muscle.

Fighting diseases and controlling the signs of ageing are the major goals of biomedicine. Sirtuins, enzymes with mainly deacetylating activity, could be pivotal targets of novel preventive and therapeutic strategies to reach such aims. Scientific proofs are accumulating in experimental models, but, to a minor extent, also in humans, that the ancient practice of calorie restriction could prove an effective way to prevent several degenerative diseases and to postpone the detrimental signs of ageing. In the present review, we summarize the evidence about the central role of sirtuins in mediating the beneficial effects of calorie restriction in skeletal and cardiac muscle since these tissues are greatly damaged by diseases and advancing years. Moreover, we entertain the possibility that the identification of sirtuin activators that mimic calorie restriction could provide the benefits without the inconvenience of this dietary style.

Genetic and Epigenetic Control of CDKN1C Expression: Importance in Cell Commitment and Differentiation, Tissue Homeostasis and Human Diseases.

The CDKN1C gene encodes the p57Kip2 protein which has been identified as the third member of the CIP/Kip family, also including p27Kip1 and p21Cip1. In analogy with these proteins, p57Kip2 is able to bind tightly and inhibit cyclin/cyclin-dependent kinase complexes and, in turn, modulate cell division cycle progression. For a long time, the main function of p57Kip2 has been associated only to correct embryogenesis, since CDKN1C-ablated mice are not vital. Accordingly, it has been demonstrated that CDKN1C alterations cause three human hereditary syndromes, characterized by altered growth rate. Subsequently, the p57Kip2 role in several cell phenotypes has been clearly assessed as well as its down-regulation in human cancers. CDKN1C lies in a genetic locus, 11p15.5, characterized by a remarkable regional imprinting that results in the transcription of only the maternal allele. The control of CDKN1C transcription is also linked to additional mechanisms, including DNA methylation and specific histone methylation/acetylation. Finally, long non-coding RNAs and miRNAs appear to play important roles in controlling p57Kip2 levels. This review mostly represents an appraisal of the available data regarding the control of CDKN1C gene expression. In addition, the structure and function of p57Kip2 protein are briefly described and correlated to human physiology and diseases.

Allelic Complexity in Long QT Syndrome: A Family-Case Study.

Congenital long QT syndrome (LQTS) is associated with high genetic and allelic heterogeneity. In some cases, more than one genetic variant is identified in the same (compound heterozygosity) or different (digenic heterozygosity) genes, and subjects with multiple pathogenic mutations may have a more severe disease. Standard-of-care clinical genetic testing for this and other arrhythmia susceptibility syndromes improves the identification of complex genotypes. Therefore, it is important to distinguish between pathogenic mutations and benign rare variants. We identified four genetic variants (KCNQ1-p.R583H, KCNH2-p.C108Y, KCNH2-p.K897T, and KCNE1-p.G38S) in an LQTS family. On the basis of in silico analysis, clinical data from our family, and the evidence from previous studies, we analyzed two mutated channels, KCNQ1-p.R583H and KCNH2-p.C108Y, using the whole-cell patch clamp technique. We found that KCNQ1-p.R583H was not associated with a severe functional impairment, whereas KCNH2-p.C108Y, a novel variant, encoded a non-functional channel that exerts dominant-negative effects on the wild-type. Notably, the common variants KCNH2-p.K897T and KCNE1-p.G38S were previously reported to produce more severe phenotypes when combined with disease-causing alleles. Our results indicate that the novel KCNH2-C108Y variant can be a pathogenic LQTS mutation, whereas KCNQ1-p.R583H, KCNH2-p.K897T, and KCNE1-p.G38S could be LQTS modifiers.

Osteosarcoma cells induce endothelial cell proliferation during neo-angiogenesis.

Understanding the mechanisms inducing endothelial cell (EC) proliferation following tumor microenvironment stimuli may be important for the development of antiangiogenic therapies. Here, we show that cyclin-dependent kinase 2 and 5 (Cdk2, Cdk5) are important mediators of neoangiogenesis in in vitro and in vivo systems. Furthermore, we demonstrate that a specific Yin Yang 1 (YY1) protein-dependent signal from osteosarcoma (SaOS) cells determines proliferation of human aortic endothelial cells (HAECs). Following tumor cell stimuli, HAECs overexpress Cdk2 and Cdk5, display increased Cdk2 activity, undergo enhanced proliferation, and form capillary-like structures. Moreover, Roscovitine, an inhibitor of Cdks, blunted overexpression of Cdk2 and Cdk5 and Cdk2 activity induced by the YY1-dependent signal secreted by SaOS cells. Furthermore, Roscovitine decreased HAEC proliferation and angiogenesis (the latter by 70% in in vitro and 50% in in vivo systems; P < 0.01 vs. control). Finally, the finding that Roscovitine triggers apoptosis in SaOS cells as well as in HAECs by activating caspase-3/7 indicates multiple mechanisms for the potential antitumoral effect of Roscovitine. Present work suggests that Cdk2 and Cdk5 might be pharmacologically accessible targets for both antiangiogenic and antitumor therapy.

Recent advances in proteomic technologies applied to cardiovascular disease.

In recent years, the diagnosis of cardiovascular disease (CVD) has increased its potential, also thanks to mass spectrometry (MS) proteomics. Modern MS proteomics tools permit analyzing a variety of biological samples, ranging from single cells to tissues and body fluids, like plasma and urine. This approach enhances the search for informative biomarkers in biological samples from apparently healthy individuals or patients, thus allowing an earlier and more precise diagnosis and a deeper comprehension of pathogenesis, development and outcome of CVD to further reduce the enormous burden of this disease on public health. In fact, many differences in protein expression between CVD-affected and healthy subjects have been detected, but only a few of them have been useful to establish clinical biomarkers because they did not pass the verification and validation tests. For a concrete clinical support of MS proteomics to CVD, it is, therefore, necessary to: ameliorate the resolution, sensitivity, specificity, throughput, precision, and accuracy of MS platform components; standardize procedures for sample collection, preparation, and analysis; lower the costs of the analyses; reduce the time of biomarker verification and validation. At the same time, it will be fundamental, for the future perspectives of proteomics in clinical trials, to define the normal protein maps and the global patterns of normal protein levels, as well as those specific for the different expressions of CVD.

Redox Homeostasis in Cardiovascular Disease: The Role of Mitochondrial Sirtuins.

Cardiovascular disease (CVD) is still the leading cause of death worldwide. Despite successful advances in both pharmacological and lifestyle strategies to fight well-established risk factors, the burden of CVD is still increasing. Therefore, it is necessary to further deepen our knowledge of the pathogenesis of the disease for developing novel therapies to limit even more its related morbidity and mortality. Oxidative stress has been identified as a common trait of several manifestations of CVD and could be a promising target for innovative treatments. Mitochondria are a major source of oxidative stress and sirtuins are a family of enzymes that generate different post-translational protein modifications, thus regulating important cellular processes, including cell cycle, autophagy, gene expression, and others. In particular, three sirtuins, SIRT3, SIRT4, and SIRT5 are located within the mitochondrial matrix where they regulate energy production and antioxidant pathways. Therefore, these sirtuins are strongly involved in the balance between oxidant and antioxidant mechanisms. In this review, we summarize the activities of these sirtuins with a special focus on their role in the control of oxidative stress, in relation to energy metabolism, atherosclerosis, and CVD.

Sample-Pooling Strategy for SARS-CoV-2 Detection among Students and Staff of the University of Sannio.

Since the beginning of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic, it has been clear that testing large groups of the population was the key to stem infection and prevent the effects of the coronavirus disease of 2019, mostly among sensitive patients. On the other hand, time and cost-sustainability of virus detection by molecular analysis such as reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) may be a major issue if testing is extended to large communities, mainly asymptomatic large communities. In this context, sample-pooling and test grouping could offer an effective solution. Here we report the screening on 1195 oral-nasopharyngeal swabs collected from students and staff of the Università degli Studi del Sannio (University of Sannio, Benevento, Campania, Italy) and analyzed by an in-house developed multiplex RT-qPCR for SARS-CoV-2 detection through a simple monodimensional sample pooling strategy. Overall, 400 distinct pools were generated and, within 24 h after swab collection, five positive samples were identified. Out of them, four were confirmed by using a commercially available kit suitable for in vitro diagnostic use (IVD). High accuracy, sensitivity and specificity were also determined by comparing our results with a reference IVD assay for all deconvoluted samples. Overall, we conducted 463 analyses instead of 1195, reducing testing resources by more than 60% without lengthening diagnosis time and without significant losses in sensitivity, suggesting that our strategy was successful in recognizing positive cases in a community of asymptomatic individuals with minor requirements of reagents and time when compared to normal testing procedures.

The Stiffness Comparison Test: A pilot study to determine inter-individual differences in palpatory skill related to gender, age, and occupation-related experience.

BACKGROUND: Manual palpation is a core skill in physical examination. Assessing elastic properties such as tissue stiffness has the potential for being an important diagnostics tool in the detection of cancer and other diseases. OBJECTIVE: The study describes the newly developed Stiffness Comparison Test (SCT). The aim of our study was to test the SCT as a tool to detect interindividual differences in palpation skill related to gender, age and occupational experience. METHODS: We used eight pairs of polyuterhane gel pads with the stiffness difference decreasing from the first to the last pair. Test subjects were asked to palpate each pair and determine stiffness differences. PARTICIPANTS: We recruited 25 osteopaths, 48 other manual therapists and 50 participants from other non-manual professions. RESULTS: As hypothesized there was no significant difference in SCT performance between the sexes (t(121) = 0.288, p = .774). To investigate if an age-related decline would have an effect on palpation skill, we carried out a linear regression. As hypothesized, the model did not predict any significant associations (F(1, 121) = 2.733, b = -0.149, p = .101, R2 = 0.022). To compare the effect of occupational groups on SCT performance a one-way ANOVA was conducted. There were no statistically significant differences between group means (F(2, 120) = 0.598, p = .552). CONCLUSIONS: The SCT can be used as simple and affordable tool for assessment, teaching and training in all disciplines of manual medicine. Further refinements of the tool are suggested to advance its discrimination power.

Structural and Functional Changes in the Coupling of Fascial Tissue, Skeletal Muscle, and Nerves During Aging.

Aging is a one-way process associated with profound structural and functional changes in the organism. Indeed, the neuromuscular system undergoes a wide remodeling, which involves muscles, fascia, and the central and peripheral nervous systems. As a result, intrinsic features of tissues, as well as their functional and structural coupling, are affected and a decline in overall physical performance occurs. Evidence from the scientific literature demonstrates that senescence is associated with increased stiffness and reduced elasticity of fascia, as well as loss of skeletal muscle mass, strength, and regenerative potential. The interaction between muscular and fascial structures is also weakened. As for the nervous system, aging leads to motor cortex atrophy, reduced motor cortical excitability, and plasticity, thus leading to accumulation of denervated muscle fibers. As a result, the magnitude of force generated by the neuromuscular apparatus, its transmission along the myofascial chain, joint mobility, and movement coordination are impaired. In this review, we summarize the evidence about the deleterious effect of aging on skeletal muscle, fascial tissue, and the nervous system. In particular, we address the structural and functional changes occurring within and between these tissues and discuss the effect of inflammation in aging. From the clinical perspective, this article outlines promising approaches for analyzing the composition and the viscoelastic properties of skeletal muscle, such as ultrasonography and elastography, which could be applied for a better understanding of musculoskeletal modifications occurring with aging. Moreover, we describe the use of tissue manipulation techniques, such as massage, traction, mobilization as well as acupuncture, dry needling, and nerve block, to enhance fascial repair.

The Diversity of Muscles and Their Regenerative Potential across Animals.

Cells with contractile functions are present in almost all metazoans, and so are the related processes of muscle homeostasis and regeneration. Regeneration itself is a complex process unevenly spread across metazoans that ranges from full-body regeneration to partial reconstruction of damaged organs or body tissues, including muscles. The cellular and molecular mechanisms involved in regenerative processes can be homologous, co-opted, and/or evolved independently. By comparing the mechanisms of muscle homeostasis and regeneration throughout the diversity of animal body-plans and life cycles, it is possible to identify conserved and divergent cellular and molecular mechanisms underlying muscle plasticity. In this review we aim at providing an overview of muscle regeneration studies in metazoans, highlighting the major regenerative strategies and molecular pathways involved. By gathering these findings, we wish to advocate a comparative and evolutionary approach to prompt a wider use of "non-canonical" animal models for molecular and even pharmacological studies in the field of muscle regeneration.

Functional characterization of ryanodine receptor (RYR1) sequence variants using a metabolic assay in immortalized B-lymphocytes.

Mutations in the RYR1 gene are linked to malignant hyperthermia (MH), central core disease and multi-minicore disease. We screened by DHPLC the RYR1 gene in 24 subjects for mutations, and characterized functional alterations caused by some RYR1 variants. Three novel sequence variants and twenty novel polymorphisms were identified. Immortalized lymphoblastoid cell lines from patients with RYR1 variants and from controls were stimulated with 4-chloro-m-cresol (4-CmC) and the rate of extracellular acidification was recorded. We demonstrate that the increased acidification rate of lymphoblastoid cells in response to 4-CmC is mainly due to RYR1 activation. Cells expressing RYR1 variants in the N-terminal and in the central region of the protein (p.Arg530His, p.Arg2163Pro, p.Asn2342Ser, p.Glu2371Gly and p.Arg2454His) displayed higher activity compared with controls; this could account for the MH-susceptible phenotype. Cell lines harboring RYR1(Cys4664Arg) were significantly less activated by 4-CmC. This result indicates that the p.Cys4664Arg variant causes a leaky channel and depletion of intracellular stores. The functional changes detected corroborate the variants analyzed as disease-causing alterations and the acidification rate measurements as a means to monitor Ca(2+)-induced metabolic changes in cells harboring mutant RYR1 channels.

RYR1 Sequence Variants in Myopathies: Expression and Functional Studies in Two Families.

The skeletal muscle ryanodine receptor (RyR1), i.e., the Ca2+ channel of the sarco/endoplasmic reticulum (S/ER), and the voltage-dependent calcium channel Cav1.1 are the principal channels involved in excitation-contraction coupling in skeletal muscle. RYR1 gene variants are linked to distinct skeletal muscle disorders, including malignant hyperthermia susceptibility and central core disease (CCD), mainly with autosomal dominant inheritance, and autosomal recessive myopathies with a broad phenotypic and histopathological spectrum. The age at onset of RYR1-related myopathies varies from infancy to adulthood. We report the identification of four RYR1 variants in two Italian families: one with myopathy and variants c.4003C>T (p.R1335C) and c.7035C>A (p.S2345R), and another with CCD and variants c.9293G>T (p.S3098I) and c.14771_14772insTAGACAGGGTGTTGCTCTGTTGCCCTTCTT (p.F4924_V4925insRQGVALLPFF). We demonstrate that, in patient-specific lymphoblastoid cells, the c.4003C>T (p.R1335C) variant is not expressed and the in-frame 30-nucleotide insertion variant is expressed at a low level. Moreover, Ca2+ release in response to the RyR1 agonist 4-chloro-m-cresol and to thapsigargin showed that the c.7035C>A (p.S2345R) variant causes depletion of S/ER Ca2+ stores and that the compound heterozygosity for variant c.9293G>T (p.S3098I) and the 30-nucleotide insertion increases RyR1-dependent Ca2+ release without affecting ER Ca2+ stores. In conclusion, we detected and functionally characterized disease-causing variants of the RyR1 channel in patient-specific lymphoblastoid cells. This paper is dedicated to the memory and contribution of Luigi Del Vecchio.

Potential clinical benefits of cell therapy in coronary heart disease: an update.

Cell therapy is a central issue of regenerative medicine and is raising a growing interest in the scientific community, but its full therapeutic potential in coronary heart disease (CHD) has not been reached yet. Several different methods, cell types, delivery routes, and supporting techniques have been attempted and improved to elicit cardiac regeneration in CHD, but only some of them showed a really convincing potential for the use in clinical practice. Here we provide an update on approaches and clinical trials of cell therapy applied to CHD, which are ongoing or that have been realized in the last 5 years. Moreover, we discuss the evidence collected so far in favor or against the validity of stem cell therapy for CHD. In particular, we review and comment the recent advances in cell therapy applied to CHD, the most promising cell types, delivery strategies, biochemical and engineering techniques that have been adopted in this context.

Voltage modulates halothane-triggered Ca2+ release in malignant hyperthermia-susceptible muscle.

Malignant hyperthermia (MH) is a fatal hypermetabolic state that may occur during general anesthesia in susceptible individuals. It is often caused by mutations in the ryanodine receptor RyR1 that favor drug-induced release of Ca2+ from the sarcoplasmic reticulum. Here, knowing that membrane depolarization triggers Ca2+ release in normal muscle function, we study the cross-influence of membrane potential and anesthetic drugs on Ca2+ release. We used short single muscle fibers of knock-in mice heterozygous for the RyR1 mutation Y524S combined with microfluorimetry to measure intracellular Ca2+ signals. Halothane, a volatile anesthetic used in contracture testing for MH susceptibility, was equilibrated with the solution superfusing the cells by means of a vaporizer system. In the range 0.2 to 3%, the drug causes significantly larger elevations of free myoplasmic [Ca2+] in mutant (YS) compared with wild-type (WT) fibers. Action potential-induced Ca2+ signals exhibit a slowing of their time course of relaxation that can be attributed to a component of delayed Ca2+ release turnoff. In further experiments, we applied halothane to single fibers that were voltage-clamped using two intracellular microelectrodes and studied the effect of small (10-mV) deviations from the holding potential (-80 mV). Untreated WT fibers show essentially no changes in [Ca2+], whereas the Ca2+ level of YS fibers increases and decreases on depolarization and hyperpolarization, respectively. The drug causes a significant enhancement of this response. Depolarizing pulses reveal a substantial negative shift in the voltage dependence of activation of Ca2+ release. This behavior likely results from the allosteric coupling between RyR1 and its transverse tubular voltage sensor. We conclude that the binding of halothane to RyR1 alters the voltage dependence of Ca2+ release in MH-susceptible muscle fibers such that the resting membrane potential becomes a decisive factor for the efficiency of the drug to trigger Ca2+ release.

Possible Muscle Repair in the Human Cardiovascular System.

The regenerative potential of tissues and organs could promote survival, extended lifespan and healthy life in multicellular organisms. Niches of adult stemness are widely distributed and lead to the anatomical and functional regeneration of the damaged organ. Conversely, muscular regeneration in mammals, and humans in particular, is very limited and not a single piece of muscle can fully regrow after a severe injury. Therefore, muscle repair after myocardial infarction is still a chimera. Recently, it has been recognized that epigenetics could play a role in tissue regrowth since it guarantees the maintenance of cellular identity in differentiated cells and, therefore, the stability of organs and tissues. The removal of these locks can shift a specific cell identity back to the stem-like one. Given the gradual loss of tissue renewal potential in the course of evolution, in the last few years many different attempts to retrieve such potential by means of cell therapy approaches have been performed in experimental models. Here we review pathways and mechanisms involved in the in vivo repair of cardiovascular muscle tissues in humans. Moreover, we address the ongoing research on mammalian cardiac muscle repair based on adult stem cell transplantation and pro-regenerative factor delivery. This latter issue, involving genetic manipulations of adult cells, paves the way for developing possible therapeutic strategies in the field of cardiovascular muscle repair.

Clinical relevance of epigenetics in the onset and management of type 2 diabetes mellitus.

Epigenetics is involved in the altered expression of gene networks that underlie insulin resistance and insufficiency. Major genes controlling ß-cell differentiation and function, such as PAX4, PDX1, and GLP1 receptor, are epigenetically controlled. Epigenetics can cause insulin resistance through immunomediated pro-inflammatory actions related to several factors, such as NF-kB, osteopontin, and Toll-like receptors. Hereafter, we provide a critical and comprehensive summary on this topic with a particular emphasis on translational and clinical aspects. We discuss the effect of epigenetics on ß-cell regeneration for cell replacement therapy, the emerging bioinformatics approaches for analyzing the epigenetic contribution to type 2 diabetes mellitus (T2DM), the epigenetic core of the transgenerational inheritance hypothesis in T2DM, and the epigenetic clinical trials on T2DM. Therefore, prevention or reversion of the epigenetic changes occurring during T2DM development may reduce the individual and societal burden of the disease.

Epigenetics and type 1 diabetes: mechanisms and translational applications.

Type 1 diabetes (T1D) is an irreversible degenerative disease with severe complications such as heart disease, nephropathy, neuropathy, and retinopathy. Although exogenous insulin administration is a life-saving therapy, it does not cure the disease. This review addresses the epigenetic mechanisms responsible for the development of T1D and discusses epigenetic-based strategies for prevention and treatment of the disease. We describe novel epigenetic biomarkers for the identification of susceptible individuals and the establishment of innovative therapies with epidrugs and cell therapy to regenerate the lost ß-cells. Despite the wealth of promising data regarding the potential benefits of epigenetic tools to reduce the burden of T1D, clinical trials are still very few, and this issue needs to be resolved in the near future.

Evidence of epigenetic tags in cardiac fibrosis.

In cardiac fibrosis, following an injury or a stress, non-functional fibrotic tissue substitutes normal myocardium, thus leading to progressive heart failure. Activated fibroblasts are principal determinants of cardiac fibrosis by producing excessive fibrotic extracellular matrix and causing hypertrophy of cardiomyocytes. Epigenetic changes, such as DNA methylation, histone modifications, and miRNAs have been involved in these mechanisms. Therefore, there is a strong interest in reverting such epigenetic transformations in order to arrest myocardial fibrotic degeneration. Demethylating agents, such as 5-aza-2'-deoxycytidine, 5-azacytidine, some selective histone deacetylase inhibitors, including mocetinostat, trichostatin A, and MPT0E014, have a direct action on important inducers of cardiac fibrosis. Also dietary compounds, such as resveratrol, can suppress the differentiation of fibroblasts to myofibroblasts. Although in vivo and in vitro studies suggest specific epigenetic therapies to treat cardiac fibrosis, the related clinical trials are still lacking. A better understanding of the epigenetic effects of dietary compounds (e.g. curcumin and green tea catechins) on the onset and progression of cardiac fibrosis, will allow the identification of protective dietary patterns and/or the generation of novel potential epidrugs.