Neuropeptide Y promotes adipogenesis of human cardiac mesenchymal stromal cells in arrhythmogenic cardiomyopathy.

BACKGROUND: Arrhythmogenic Cardiomyopathy (AC) is a familial cardiac disease, mainly caused by mutations in desmosomal genes. AC hearts show fibro-fatty myocardial replacement, which favors stress-related life-threatening arrhythmias, predominantly in the young and athletes. AC lacks effective therapies, as its pathogenesis is poorly understood. Recently, we showed that cardiac Mesenchymal Stromal Cells (cMSCs) contribute to adipose tissue in human AC hearts, although the underlying mechanisms are still unclear. PURPOSE: We hypothesize that the sympathetic neurotransmitter, Neuropeptide Y (NPY), participates to cMSC adipogenesis in human AC. METHODS: For translation of our findings, we combined in vitro cytochemical, molecular and pharmacologic assays on human cMSCs, from myocardial biopsies of healthy controls and AC patients, with the use of existing drugs to interfere with the predicted AC mechanisms. Sympathetic innervation was inspected in human autoptic heart samples, and NPY plasma levels measured in healthy and AC subjects. RESULTS: AC cMSCs expressed higher levels of pro-adipogenic isotypes of NPY-receptors (i.e. Y1-R, Y5-R). Consistently, NPY enhanced adipogenesis in AC cMSCs, which was blocked by FDA-approved Y1-R and Y5-R antagonists. AC-associated PKP2 reduction directly caused NPY-dependent adipogenesis in cMSCs. In support of the involvement of sympathetic neurons (SNs) and NPY in AC myocardial remodeling, patients had elevated NPY plasma levels and, in human AC hearts, SNs accumulated in fatty areas and were close to cMSCs. CONCLUSIONS: Independently from the disease origin, AC causes in cMSCs a targetable gain of responsiveness to NPY, which leads to increased adipogenesis, thus playing a role in AC myocardial remodeling.

Excess TGF-ß1 Drives Cardiac Mesenchymal Stromal Cells to a Pro-Fibrotic Commitment in Arrhythmogenic Cardiomyopathy.

Arrhythmogenic Cardiomyopathy (ACM) is characterized by the replacement of the myocardium with fibrotic or fibro-fatty tissue and inflammatory infiltrates in the heart. To date, while ACM adipogenesis is a well-investigated differentiation program, ACM-related fibrosis remains a scientific gap of knowledge. In this study, we analyze the fibrotic process occurring during ACM pathogenesis focusing on the role of cardiac mesenchymal stromal cells (C-MSC) as a source of myofibroblasts. We performed the ex vivo studies on plasma and right ventricular endomyocardial bioptic samples collected from ACM patients and healthy control donors (HC). In vitro studies were performed on C-MSC isolated from endomyocardial biopsies of both groups. Our results revealed that circulating TGF-ß1 levels are significantly higher in the ACM cohort than in HC. Accordingly, fibrotic markers are increased in ACM patient-derived cardiac biopsies compared to HC ones. This difference is not evident in isolated C-MSC. Nevertheless, ACM C-MSC are more responsive than HC ones to TGF-ß1 treatment, in terms of pro-fibrotic differentiation and higher activation of the SMAD2/3 signaling pathway. These results provide the novel evidence that C-MSC are a source of myofibroblasts and participate in ACM fibrotic remodeling, being highly responsive to ACM-characteristic excess TGF-ß1.

Clinical and Molecular Data Define a Diagnosis of Arrhythmogenic Cardiomyopathy in a Carrier of a Brugada-Syndrome-Associated PKP2 Mutation.

Plakophilin-2 (PKP2) is the most frequently mutated desmosomal gene in arrhythmogenic cardiomyopathy (ACM), a disease characterized by structural and electrical alterations predominantly affecting the right ventricular myocardium. Notably, ACM cases without overt structural alterations are frequently reported, mainly in the early phases of the disease. Recently, the PKP2 p.S183N mutation was found in a patient affected by Brugada syndrome (BS), an inherited arrhythmic channelopathy most commonly caused by sodium channel gene mutations. We here describe a case of a patient carrier of the same BS-related PKP2 p.S183N mutation but with a clear diagnosis of ACM. Specifically, we report how clinical and molecular investigations can be integrated for diagnostic purposes, distinguishing between ACM and BS, which are increasingly recognized as syndromes with clinical and genetic overlaps. This observation is fundamentally relevant in redefining the role of genetics in the approach to the arrhythmic patient, progressing beyond the concept of "one mutation, one disease", and raising concerns about the most appropriate approach to patients affected by structural/electrical cardiomyopathy. The merging of genetics, electroanatomical mapping, and tissue and cell characterization summarized in our patient seems to be the most complete diagnostic algorithm, favoring a reliable diagnosis.

Fibrosis in Arrhythmogenic Cardiomyopathy: The Phantom Thread in the Fibro-Adipose Tissue.

Arrhythmogenic cardiomyopathy (ACM) is an inherited heart disorder, predisposing to malignant ventricular arrhythmias leading to sudden cardiac death, particularly in young and athletic patients. Pathological features include a progressive loss of myocardium with fibrous or fibro-fatty substitution. During the last few decades, different clinical aspects of ACM have been well investigated but still little is known about the molecular mechanisms that underlie ACM pathogenesis, leading to these phenotypes. In about 50% of ACM patients, a genetic mutation, predominantly in genes that encode for desmosomal proteins, has been identified. However, the mutation-associated mechanisms, causing the observed cardiac phenotype are not always clear. Until now, the attention has been principally focused on the study of molecular mechanisms that lead to a prominent myocardium adipose substitution, an uncommon marker for a cardiac disease, thus often recognized as hallmark of ACM. Nonetheless, based on Task Force Criteria for the diagnosis of ACM, cardiomyocytes death associated with fibrous replacement of the ventricular free wall must be considered the main tissue feature in ACM patients. For this reason, it urges to investigate ACM cardiac fibrosis. In this review, we give an overview on the cellular effectors, possible triggers, and molecular mechanisms that could be responsible for the ventricular fibrotic remodeling in ACM patients.

Calcium as a Key Player in Arrhythmogenic Cardiomyopathy: Adhesion Disorder or Intracellular Alteration?

Arrhythmogenic cardiomyopathy (ACM) is an inherited heart disease characterized by sudden death in young people and featured by fibro-adipose myocardium replacement, malignant arrhythmias, and heart failure. To date, no etiological therapies are available. Mutations in desmosomal genes cause abnormal mechanical coupling, trigger pro-apoptotic signaling pathways, and induce fibro-adipose replacement. Here, we discuss the hypothesis that the ACM causative mechanism involves a defect in the expression and/or activity of the cardiac Ca2+ handling machinery, focusing on the available data supporting this hypothesis. The Ca2+ toolkit is heavily remodeled in cardiomyocytes derived from a mouse model of ACM defective of the desmosomal protein plakophilin-2. Furthermore, ACM-related mutations were found in genes encoding for proteins involved in excitation‒contraction coupling, e.g., type 2 ryanodine receptor and phospholamban. As a consequence, the sarcoplasmic reticulum becomes more eager to release Ca2+, thereby inducing delayed afterdepolarizations and impairing cardiac contractility. These data are supported by preliminary observations from patient induced pluripotent stem-cell-derived cardiomyocytes. Assessing the involvement of Ca2+ signaling in the pathogenesis of ACM could be beneficial in the treatment of this life-threatening disease.

Cyclophilin A in Arrhythmogenic Cardiomyopathy Cardiac Remodeling.

Arrhythmogenic cardiomyopathy (ACM) is a genetic disorder characterized by the progressive substitution of functional myocardium with noncontractile fibro-fatty tissue contributing to ventricular arrhythmias and sudden cardiac death. Cyclophilin A (CyPA) is a ubiquitous protein involved in several pathological mechanisms, which also characterize ACM (i.e., fibrosis, inflammation, and adipogenesis). Nevertheless, the involvement of CyPA in ACM cardiac remodeling has not been investigated yet. Thus, we first evaluated CyPA expression levels in the right ventricle (RV) tissue specimens obtained from ACM patients and healthy controls (HC) by immunohistochemistry. Then, we took advantage of ACM- and HC-derived cardiac mesenchymal stromal cells (C-MSC) to assess CyPA modulation during adipogenic differentiation. Interestingly, CyPA was more expressed in the RV sections obtained from ACM vs. HC subjects and positively correlated with the adipose replacement extent. Moreover, CyPA was upregulated at early stages of C-MSC adipogenic differentiation and was secreted at higher level over time in ACM- derived C-MSC. Our study provides novel ex vivo and in vitro information on CyPA expression in ACM remodeling paving the way for future C-MSC-based mechanistic and therapeutic investigations.

Arrhythmogenic cardiomyopathy: what blood can reveal?

Blood, serum and plasma represent accessible sources of data about physiological and pathologic status. In arrhythmogenic cardiomyopathy (ACM), circulating nucleated cells are routinely used for detection of germinal genetic mutations. In addition, different biomarkers have been proposed for diagnostic purposes and for monitoring disease progression, including inflammatory cytokines, markers of myocardial dysfunction and damage, and microRNAs. This review summarizes the current information that can be retrieved from the blood of ACM patients and considers the future prospects. Improvements in current knowledge of circulating factors may provide noninvasive means to simplify and improve the diagnosis, prognosis prediction, and management of ACM patients.

Precise Therapy for Thoracic Aortic Aneurysm in Marfan Syndrome: A Puzzle Nearing Its Solution.

Marfan Syndrome (MFS) is a rare connective tissue disorder, resulting from mutations in the fibrillin-1 gene, characterized by pathologic phenotypes in multiple organs, the most detrimental of which affects the thoracic aorta. Indeed, thoracic aortic aneurysms (TAA), leading to acute dissection and rupture, are today the major cause of morbidity and mortality in adult MFS patients. Therefore, there is a compelling need for novel therapeutic strategies to delay TAA progression and counteract aortic dissection occurrence. Unfortunately, the wide phenotypic variability of MFS patients, together with the lack of a complete genotype-phenotype correlation, have represented until now a barrier hampering the conduction of translational studies aimed to predict disease prognosis and drug discovery. In this review, we will illustrate available therapeutic strategies to improve the health of MFS patients. Starting from gold standard surgical overtures and the description of the main pharmacological approaches, we will comprehensively review the state-of-the-art of in vivo MFS models and discuss recent clinical pharmacogenetic results. Finally, we will focus on induced pluripotent stem cells (iPSC) as a technology that, if integrated with preclinical research and pharmacogenetics, could contribute in determining the best therapeutic approach for each MFS patient on the base of individual differences. Finally, we will suggest the integration of preclinical studies, pharmacogenetics and iPSC technology as the most likely strategy to help solve the composite puzzle of precise medicine in this condition.

Isolation and Characterization of Cardiac Mesenchymal Stromal Cells from Endomyocardial Bioptic Samples of Arrhythmogenic Cardiomyopathy Patients.

A normal adult heart is composed of several different cell types, among which cardiac mesenchymal stromal cells represent an abundant population. The isolation of these cells offers the possibility of studying their involvement in cardiac diseases, and, in addition, provides a useful primary cell model to investigate biological mechanisms. Here, the method for the isolation of C-MSC from arrhythmogenic cardiomyopathy patients' bioptic samples is described. The endomyocardial biopsy sampling is guided in the right ventricular areas adjacent to the scar visualized by electro-anatomical mapping. The digestion of the biopsies in collagenase and their plating on a plastic dish in culture medium to allow C-MSC growth is described. The isolated cells can be expanded in culture for several passages. To confirm their mesenchymal phenotype, the description of immuno-phenotypical characterization is provided. C-MSC are able to differentiate into several cell types like adipocytes, chondrocytes, and osteoblasts: in the context of ACM, characterized by adipocyte deposits in patients' hearts, the protocols for the adipogenic differentiation of C-MSC and the characterization of lipid droplet accumulation are described.