Caveolin-3 and Caveolin-1 Interaction Decreases Channel Dysfunction Due to Caveolin-3 Mutations.

Caveolae constitute membrane microdomains where receptors and ion channels functionally interact. Caveolin-3 (cav-3) is the key structural component of muscular caveolae. Mutations in CAV3 lead to caveolinopathies, which result in both muscular dystrophies and cardiac diseases. In cardiomyocytes, cav-1 participates with cav-3 to form caveolae; skeletal myotubes and adult skeletal fibers do not express cav-1. In the heart, the absence of cardiac alterations in the majority of cases may depend on a conserved organization of caveolae thanks to the expression of cav-1. We decided to focus on three specific cav-3 mutations (Δ62-64YTT; T78K and W101C) found in heterozygosis in patients suffering from skeletal muscle disorders. We overexpressed both the WT and mutated cav-3 together with ion channels interacting with and modulated by cav-3. Patch-clamp analysis conducted in caveolin-free cells (MEF-KO), revealed that the T78K mutant is dominant negative, causing its intracellular retention together with cav-3 WT, and inducing a significant reduction in current densities of all three ion channels tested. The other cav-3 mutations did not cause significant alterations. Mathematical modelling of the effects of cav-3 T78K would impair repolarization to levels incompatible with life. For this reason, we decided to compare the effects of this mutation in other cell lines that endogenously express cav-1 (MEF-STO and CHO cells) and to modulate cav-1 expression with an shRNA approach. In these systems, the membrane localization of cav-3 T78K was rescued in the presence of cav-1, and the current densities of hHCN4, hKv1.5 and hKir2.1 were also rescued. These results constitute the first evidence of a compensatory role of cav-1 in the heart, justifying the reduced susceptibility of this organ to caveolinopathies.

Omics Analyses of Stromal Cells from ACM Patients Reveal Alterations in Chromatin Organization and Mitochondrial Homeostasis.

Arrhythmogenic cardiomyopathy (ACM) is a genetic disorder characterized by ventricular arrhythmias, contractile dysfunctions and fibro-adipose replacement of myocardium. Cardiac mesenchymal stromal cells (CMSCs) participate in disease pathogenesis by differentiating towards adipocytes and myofibroblasts. Some altered pathways in ACM are known, but many are yet to be discovered. We aimed to enrich the understanding of ACM pathogenesis by comparing epigenetic and gene expression profiles of ACM-CMSCs with healthy control (HC)-CMSCs. Methylome analysis identified 74 differentially methylated nucleotides, most of them located on the mitochondrial genome. Transcriptome analysis revealed 327 genes that were more expressed and 202 genes that were less expressed in ACM- vs. HC-CMSCs. Among these, genes implicated in mitochondrial respiration and in epithelial-to-mesenchymal transition were more expressed, and cell cycle genes were less expressed in ACM- vs. HC-CMSCs. Through enrichment and gene network analyses, we identified differentially regulated pathways, some of which never associated with ACM, including mitochondrial functioning and chromatin organization, both in line with methylome results. Functional validations confirmed that ACM-CMSCs exhibited higher amounts of active mitochondria and ROS production, a lower proliferation rate and a more pronounced epicardial-to-mesenchymal transition compared to the controls. In conclusion, ACM-CMSC-omics revealed some additional altered molecular pathways, relevant in disease pathogenesis, which may constitute novel targets for specific therapies.

Extracellular Matrix-Based Approaches in Cardiac Regeneration: Challenges and Opportunities.

Cardiac development is characterized by the active proliferation of different cardiac cell types, in particular cardiomyocytes and endothelial cells, that eventually build the beating heart. In mammals, these cells lose their regenerative potential early after birth, representing a major obstacle to our current capacity to restore the myocardial structure and function after an injury. Increasing evidence indicates that the cardiac extracellular matrix (ECM) actively regulates and orchestrates the proliferation, differentiation, and migration of cardiac cells within the heart, and that any change in either the composition of the ECM or its mechanical properties ultimately affect the behavior of these cells throughout one's life. Thus, understanding the role of ECMs' proteins and related signaling pathways on cardiac cell proliferation is essential to develop effective strategies fostering the regeneration of a damaged heart. This review provides an overview of the components of the ECM and its mechanical properties, whose function in cardiac regeneration has been elucidated, with a major focus on the strengths and weaknesses of the experimental models so far exploited to demonstrate the actual pro-regenerative capacity of the components of the ECM and to translate this knowledge into new therapies.

Spectrum of Rare and Common Genetic Variants in Arrhythmogenic Cardiomyopathy Patients.

Arrhythmogenic cardiomyopathy (ACM) is a rare inherited disorder, whose genetic cause is elusive in about 50-70% of cases. ACM presents a variable disease course which could be influenced by genetics. We performed next-generation sequencing on a panel of 174 genes associated with inherited cardiovascular diseases on 82 ACM probands (i) to describe and classify the pathogenicity of rare variants according to the American College of Medical Genetics and Genomics both for ACM-associated genes and for genes linked to other cardiovascular genetic conditions; (ii) to assess, for the first time, the impact of common variants on the ACM clinical disease severity by genotype-phenotype correlation and survival analysis. We identified 15 (likely) pathogenic variants and 66 variants of uncertain significance in ACM-genes and 4 high-impact variants in genes never associated with ACM (ABCC9, APOB, DPP6, MIB1), which deserve future consideration. In addition, we found 69 significant genotype-phenotype associations between common variants and clinical parameters. Arrhythmia-associated polymorphisms resulted in an increased risk of arrhythmic events during patients' follow-up. The description of the genetic framework of our population and the observed genotype-phenotype correlation constitutes the starting point to address the current lack of knowledge in the genetics of ACM.

Cardiac Biomarkers and Autoantibodies in Endurance Athletes: Potential Similarities with Arrhythmogenic Cardiomyopathy Pathogenic Mechanisms.

The "Extreme Exercise Hypothesis" states that when individuals perform training beyond the ideal exercise dose, a decline in the beneficial effects of physical activity occurs. This is due to significant changes in myocardial structure and function, such as hemodynamic alterations, cardiac chamber enlargement and hypertrophy, myocardial inflammation, oxidative stress, fibrosis, and conduction changes. In addition, an increased amount of circulating biomarkers of exercise-induced damage has been reported. Although these changes are often reversible, long-lasting cardiac damage may develop after years of intense physical exercise. Since several features of the athlete's heart overlap with arrhythmogenic cardiomyopathy (ACM), the syndrome of "exercise-induced ACM" has been postulated. Thus, the distinction between ACM and the athlete's heart may be challenging. Recently, an autoimmune mechanism has been discovered in ACM patients linked to their characteristic junctional impairment. Since cardiac junctions are similarly impaired by intense physical activity due to the strong myocardial stretching, we propose in the present work the novel hypothesis of an autoimmune response in endurance athletes. This investigation may deepen the knowledge about the pathological remodeling and relative activated mechanisms induced by intense endurance exercise, potentially improving the early recognition of whom is actually at risk.

Human Cell Modeling for Cardiovascular Diseases.

The availability of appropriate and reliable in vitro cell models recapitulating human cardiovascular diseases has been the aim of numerous researchers, in order to retrace pathologic phenotypes, elucidate molecular mechanisms, and discover therapies using simple and reproducible techniques. In the past years, several human cell types have been utilized for these goals, including heterologous systems, cardiovascular and non-cardiovascular primary cells, and embryonic stem cells. The introduction of induced pluripotent stem cells and their differentiation potential brought new prospects for large-scale cardiovascular experiments, bypassing ethical concerns of embryonic stem cells and providing an advanced tool for disease modeling, diagnosis, and therapy. Each model has its advantages and disadvantages in terms of accessibility, maintenance, throughput, physiological relevance, recapitulation of the disease. A higher level of complexity in diseases modeling has been achieved with multicellular co-cultures. Furthermore, the important progresses reached by bioengineering during the last years, together with the opportunities given by pluripotent stem cells, have allowed the generation of increasingly advanced in vitro three-dimensional tissue-like constructs mimicking in vivo physiology. This review provides an overview of the main cell models used in cardiovascular research, highlighting the pros and cons of each, and describing examples of practical applications in disease modeling.