Myocardial Fibrosis in Hypertrophic Cardiomyopathy: A Perspective from Fibroblasts.

Hypertrophic cardiomyopathy (HCM) is the most common inherited heart disease and the leading cause of sudden cardiac death in young people. Mutations in genes that encode structural proteins of the cardiac sarcomere are the more frequent genetic cause of HCM. The disease is characterized by cardiomyocyte hypertrophy and myocardial fibrosis, which is defined as the excessive deposition of extracellular matrix proteins, mainly collagen I and III, in the myocardium. The development of fibrotic tissue in the heart adversely affects cardiac function. In this review, we discuss the latest evidence on how cardiac fibrosis is promoted, the role of cardiac fibroblasts, their interaction with cardiomyocytes, and their activation via the TGF-ß pathway, the primary intracellular signalling pathway regulating extracellular matrix turnover. Finally, we summarize new findings on profibrotic genes as well as genetic and non-genetic factors involved in the pathophysiology of HCM.

GCN5 contributes to intracellular lipid accumulation in human primary cardiac stromal cells from patients affected by Arrhythmogenic cardiomyopathy.

Arrhythmogenic cardiomyopathy (ACM) is a genetic disease associated with sudden cardiac death and cardiac fibro-fatty replacement. Over the last years, several works have demonstrated that different epigenetic enzymes can affect not only gene expression changes in cardiac diseases but also cellular metabolism. Specifically, the histone acetyltransferase GCN5 is known to facilitate adipogenesis and modulate cardiac metabolism in heart failure. Our group previously demonstrated that human primary cardiac stromal cells (CStCs) contribute to adipogenesis in the ACM pathology. Thus, this study aims to evaluate the role of GCN5 in ACM intracellular lipid accumulation. To do so, CStCs were obtained from right ventricle biopsies of ACM patients and from samples of healthy cadaveric donors (CTR). GCN5 expression was increased both in ex vivo and in vitro ACM samples compared to CTR. When GCN5 expression was silenced or pharmacologically inhibited by the administration of MB-3, we observed a reduction in lipid accumulation and a mitigation of reactive oxygen species (ROS) production in ACM CStCs. In agreement, transcriptome analysis revealed that the presence of MB-3 modified the expression of pathways related to cellular redox balance. Altogether, our findings suggest that GCN5 inhibition reduces fat accumulation in ACM CStCs, partially by modulating intracellular redox balance pathways.

A detailed characterization of the hyperpolarization-activated "funny" current (If) in human-induced pluripotent stem cell (iPSC)-derived cardiomyocytes with pacemaker activity.

Properties of the funny current (If) have been studied in several animal and cellular models, but so far little is known concerning its properties in human pacemaker cells. This work provides a detailed characterization of If in human-induced pluripotent stem cell (iPSC)-derived pacemaker cardiomyocytes (pCMs), at different time points. Patch-clamp analysis showed that If density did not change during differentiation; however, after day 30, it activates at more negative potential and with slower time constants. These changes are accompanied by a slowing in beating rate. If displayed the voltage-dependent block by caesium and reversed (Erev) at - 22 mV, compatibly with the 3:1 K+/Na+ permeability ratio. Lowering [Na+]o (30 mM) shifted the Erev to - 39 mV without affecting conductance. Increasing [K+]o (30 mM) shifted the Erev to - 15 mV with a fourfold increase in conductance. pCMs express mainly HCN4 and HCN1 together with the accessory subunits CAV3, KCR1, MiRP1, and SAP97 that contribute to the context-dependence of If. Autonomic agonists modulated the diastolic depolarization, and thus rate, of pCMs. The adrenergic agonist isoproterenol induced rate acceleration and a positive shift of If voltage-dependence (EC50 73.4 nM). The muscarinic agonists had opposite effects (Carbachol EC50, 11,6 nM). Carbachol effect was however small but it could be increased by pre-stimulation with isoproterenol, indicating low cAMP levels in pCMs. In conclusion, we demonstrated that pCMs display an If with the physiological properties expected by pacemaker cells and may thus represent a suitable model for studying human If-related sinus arrhythmias.

Microbiota, type 2 diabetes and non-alcoholic fatty liver disease: protocol of an observational study.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is characterized by triglyceride accumulation in the hepatocytes in the absence of alcohol overconsumption, commonly associated with insulin resistance and obesity. Both NAFLD and type 2 diabetes (T2D) are characterized by an altered microbiota composition, however the role of the microbiota in NAFLD and T2D is not well understood. To assess the relationship between alteration in the microbiota and NAFLD while dissecting the role of T2D, we established a nested study on T2D and non-T2D individuals within the Cooperative Health Research In South Tyrol (CHRIS) study, called the CHRIS-NAFLD study. Here, we present the study protocol along with baseline and follow-up characteristics of study participants. METHODS: Among the first 4979 CHRIS study participants, 227 individuals with T2D were identified and recalled, along with 227 age- and sex-matched non-T2D individuals. Participants underwent ultrasound and transient elastography examination to evaluate the presence of hepatic steatosis and liver stiffness. Additionally, sampling of saliva and faeces, biochemical measurements and clinical interviews were carried out. RESULTS: We recruited 173 T2D and 183 non-T2D participants (78% overall response rate). Hepatic steatosis was more common in T2D (63.7%) than non-T2D (36.3%) participants. T2D participants also had higher levels of liver stiffness (median 4.8 kPa, interquartile range (IQR) 3.7, 5.9) than non-T2D participants (median 3.9 kPa, IQR 3.3, 5.1). The non-invasive scoring systems like the NAFLD fibrosis score (NFS) suggests an increased liver fibrosis in T2D (mean - 0.55, standard deviation, SD, 1.30) than non-T2D participants (mean - 1.30, SD, 1.17). DISCUSSION: Given the comprehensive biochemical and clinical characterization of study participants, once the bioinformatics classification of the microbiota will be completed, the CHRIS-NAFLD study will become a useful resource to further our understanding of the relationship between microbiota, T2D and NAFLD.

Subchronic exposure to titanium dioxide nanoparticles modifies cardiac structure and performance in spontaneously hypertensive rats.

BACKGROUND: Non-communicable diseases, intended as the results of a combination of inherited, environmental and biological factors, kill 40 million people each year, equivalent to roughly 70% of all premature deaths globally. The possibility that manufactured nanoparticles (NPs) may affect cardiac performance, has led to recognize NPs-exposure not only as a major Public Health concern, but also as an occupational hazard. In volunteers, NPs-exposure is problematic to quantify. We recently found that inhaled titanium dioxide NPs, one of the most produced engineered nanomaterials, acutely increased cardiac excitability and promoted arrhythmogenesis in normotensive rats by a direct interaction with cardiac cells. We hypothesized that such scenario can be exacerbated by latent cardiovascular disorders such as hypertension. RESULTS: We monitored cardiac electromechanical performance in spontaneously hypertensive rats (SHRs) exposed to titanium dioxide NPs for 6 weeks using a combination of cardiac functional measurements associated with toxicological, immunological, physical and genetic assays. Longitudinal radio-telemetry ECG recordings and multiple-lead epicardial potential mapping revealed that atrial activation times significantly increased as well as proneness to arrhythmia. At the third week of nanoparticles administration, the lung and cardiac tissue encountered a maladaptive irreversible structural remodelling starting with increased pro-inflammatory cytokines levels and lipid peroxidation, resulting in upregulation of the main pro-fibrotic cardiac genes. At the end of the exposure, the majority of spontaneous arrhythmic events terminated, while cardiac hemodynamic deteriorated and a significant accumulation of fibrotic tissue occurred as compared to control untreated SHRs. Titanium dioxide nanoparticles were quantified in the heart tissue although without definite accumulation as revealed by particle-induced X-ray emission and ultrastructural analysis. CONCLUSIONS: The co-morbidity of hypertension and inhaled nanoparticles induces irreversible hemodynamic impairment associated with cardiac structural damage potentially leading to heart failure. The time-dependence of exposure indicates a non-return point that needs to be taken into account in hypertensive subjects daily exposed to nanoparticles.

The Histone Deacetylase Inhibitor Suberoylanilide Hydroxamic Acid (SAHA) Restores Cardiomyocyte Contractility in a Rat Model of Early Diabetes.

In early diabetes, hyperglycemia and the associated metabolic dysregulation promote early changes in the functional properties of cardiomyocytes, progressively leading to the appearance of the diabetic cardiomyopathy phenotype. Recently, the interplay between histone acetyltransferases (HAT) and histone deacetylases (HDAC) has emerged as a crucial factor in the development of cardiac disorders. The present study evaluates whether HDAC inhibition can prevent the development of cardiomyocyte contractile dysfunction induced by a short period of hyperglycemia, with focus on the potential underlying mechanisms. Cell contractility and calcium dynamics were measured in unloaded ventricular myocytes isolated from the heart of control and diabetic rats. Cardiomyocytes were either untreated or exposed to the pan-HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) for 90 min. Then, a fraction of each group of cells was used to evaluate the expression levels of proteins involved in the excitation-contraction coupling, and the cardiomyocyte metabolic activity, ATP content, and reactive oxygen species levels. SAHA treatment was able to counteract the initial functional derangement in cardiomyocytes by reducing cell oxidative damage. These findings suggest that early HDAC inhibition could be a promising adjuvant approach for preventing diabetes-induced cardiomyocyte oxidative damage, which triggers the pro-inflammatory signal cascade, mitochondrial damage, and ventricular dysfunction.

The arrhythmogenic cardiomyopathy-specific coding and non-coding transcriptome in human cardiac stromal cells.

BACKGROUND: Arrhythmogenic cardiomyopathy (ACM) is a genetic autosomal disease characterized by abnormal cell-cell adhesion, cardiomyocyte death, progressive fibro-adipose replacement of the myocardium, arrhythmias and sudden death. Several different cell types contribute to the pathogenesis of ACM, including, as recently described, cardiac stromal cells (CStCs). In the present study, we aim to identify ACM-specific expression profiles of human CStCs derived from endomyocardial biopsies of ACM patients and healthy individuals employing TaqMan Low Density Arrays for miRNA expression profiling, and high throughput sequencing for gene expression quantification. RESULTS: We identified 3 miRNAs and 272 genes as significantly differentially expressed at a 5% false discovery rate. Both the differentially expressed genes as well as the target genes of the ACM-specific miRNAs were found to be enriched in cell adhesion-related biological processes. Functional similarity and protein interaction-based network analyses performed on the identified deregulated genes, miRNA targets and known ACM-causative genes revealed clusters of highly related genes involved in cell adhesion, extracellular matrix organization, lipid transport and ephrin receptor signaling. CONCLUSIONS: We determined for the first time the coding and non-coding transcriptome characteristic of ACM cardiac stromal cells, finding evidence for a potential contribution of miRNAs, specifically miR-29b-3p, to ACM pathogenesis or phenotype maintenance.

HDAC Inhibition Improves the Sarcoendoplasmic Reticulum Ca2+-ATPase Activity in Cardiac Myocytes.

SERCA2a is the Ca2+ ATPase playing the major contribution in cardiomyocyte (CM) calcium removal. Its activity can be regulated by both modulatory proteins and several post-translational modifications. The aim of the present work was to investigate whether the function of SERCA2 can be modulated by treating CMs with the histone deacetylase (HDAC) inhibitor suberanilohydroxamic acid (SAHA). The incubation with SAHA (2.5 µM, 90 min) of CMs isolated from rat adult hearts resulted in an increase of SERCA2 acetylation level and improved ATPase activity. This was associated with a significant improvement of calcium transient recovery time and cell contractility. Previous reports have identified K464 as an acetylation site in human SERCA2. Mutants were generated where K464 was substituted with glutamine (Q) or arginine (R), mimicking constitutive acetylation or deacetylation, respectively. The K464Q mutation ameliorated ATPase activity and calcium transient recovery time, thus indicating that constitutive K464 acetylation has a positive impact on human SERCA2a (hSERCA2a) function. In conclusion, SAHA induced deacetylation inhibition had a positive impact on CM calcium handling, that, at least in part, was due to improved SERCA2 activity. This observation can provide the basis for the development of novel pharmacological approaches to ameliorate SERCA2 efficiency.

Exploring digenic inheritance in arrhythmogenic cardiomyopathy.

BACKGROUND: Arrhythmogenic cardiomyopathy (ACM) is an inherited genetic disorder, characterized by the substitution of heart muscle with fibro-fatty tissue and severe ventricular arrhythmias, often leading to heart failure and sudden cardiac death. ACM is considered a monogenic disorder, but the low penetrance of mutations identified in patients suggests the involvement of additional genetic or environmental factors. METHODS: We used whole exome sequencing to investigate digenic inheritance in two ACM families where previous diagnostic tests have revealed a PKP2 mutation in all affected and some healthy individuals. In family members with PKP2 mutations we determined all genes that harbor variants in affected but not in healthy carriers or vice versa. We computationally prioritized the most likely candidates, focusing on known ACM genes and genes related to PKP2 through protein interactions, functional relationships, or shared biological processes. RESULTS: We identified four candidate genes in family 1, namely DAG1, DAB2IP, CTBP2 and TCF25, and eleven candidate genes in family 2. The most promising gene in the second family is TTN, a gene previously associated with ACM, in which the affected individual harbors two rare deleterious-predicted missense variants, one of which is located in the protein's only serine kinase domain. CONCLUSIONS: In this study we report genes that might act as digenic players in ACM pathogenesis, on the basis of co-segregation with PKP2 mutations. Validation in larger cohorts is still required to prove the utility of this model.

Acetylation mediates Cx43 reduction caused by electrical stimulation.

Communication between cardiomyocytes depends upon gap junctions (GJ). Previous studies have demonstrated that electrical stimulation induces GJ remodeling and modifies histone acetylase (HAT) and deacetylase (HDAC) activities, although these two results have not been linked. The aim of this work was to establish whether electrical stimulation modulates GJ-mediated cardiac cell-cell communication by acetylation-dependent mechanisms. Field stimulation of HL-1 cardiomyocytes at 0.5 Hz for 24 h significantly reduced connexin43 (Cx43) expression and cell-cell communication. HDAC activity was down-regulated whereas HAT activity was not modified resulting in increased acetylation of Cx43. Consistent with a post-translational mechanism, we did not observe a reduction in Cx43 mRNA in electrically stimulated cells, while the proteasomal inhibitor MG132 maintained Cx43 expression. Further, the treatment of paced cells with the HAT inhibitor Anacardic Acid maintained both the levels of Cx43 and cell-cell communication. Finally, we observed increased acetylation of Cx43 in the left ventricles of dogs subjected to chronic tachypacing as a model of abnormal ventricular activation. In conclusion, our findings suggest that altered electrical activity can regulate cardiomyocyte communication by influencing the acetylation status of Cx43.

The Cooperative Health Research in South Tyrol (CHRIS) study: rationale, objectives, and preliminary results.

The Cooperative Health Research In South Tyrol (CHRIS) study is a population-based study with a longitudinal lookout to investigate the genetic and molecular basis of age-related common chronic conditions and their interaction with life style and environment in the general population. All adults of the middle and upper Vinschgau/Val Venosta are invited, while 10,000 participants are anticipated by mid-2017. Family participation is encouraged for complete pedigree reconstruction and disease inheritance mapping. After a pilot study on the compliance with a paperless assessment mode, computer-assisted interviews have been implemented to screen for conditions of the cardiovascular, endocrine, metabolic, genitourinary, nervous, behavioral, and cognitive system. Fat intake, cardiac health, and tremor are assessed instrumentally. Nutrient intake, physical activity, and life-course smoking are measured semi-quantitatively. Participants are phenotyped for 73 blood and urine parameters and 60 aliquots per participant are biobanked (cryo-preserved urine, DNA, and whole and fractionated blood). Through liquid-chromatography mass-spectrometry analysis, metabolite profiling of the mitochondrial function is assessed. Samples are genotyped on 1 million variants with the Illumina HumanOmniExpressExome array and the first data release including 4570 fully phenotyped and genotyped samples is now available for analysis. Participants' follow-up is foreseen 6 years after the first visit. The target population is characterized by long-term social stability and homogeneous environment which should both favor the identification of enriched genetic variants. The CHRIS cohort is a valuable resource to assess the contribution of genomics, metabolomics, and environmental factors to human health and disease. It is awaited that this will result in the identification of novel molecular targets for disease prevention and treatment.

Endoscopic findings in uninvestigated dyspepsia.

BACKGROUND: It is important to know the causes of dyspepsia to establish the therapeutic approach. Dyspepsia is a frequent syndrome in our country, where there are restrictions to endoscopy and high prevalence of Helicobacter pylori (H. pylori) infection. This study aimed to assess the endoscopic findings of the syndrome, in an outpatient screening clinic of a tertiary hospital in São Paulo. METHODS: Outpatients with uninvestigated dyspepsia, according to Rome III criteria, answered a dyspepsia questionnaire and underwent esophagogastroduodenoscopy. The Rapid Urease Test was applied to fragments of the antral mucosa and epidemiological data were collected from the studied population. Organic dyspepsia findings were analyzed with different variables to verify statistically significant associations. RESULTS: Three hundred and six patients were included and 282 were analyzed in the study. The mean age was 44 years and women comprised 65% of the sample. Forty-five percent of the patients reported alarm symptoms. Functional dyspepsia was found in 66% of the patients (20% with normal endoscopy results and 46% with gastritis), 18% had GERD and 13% had ulcers (duodenal in 9% and gastric in 4%). Four cases of gastric adenocarcinoma were identified (1.4%), one without alarm characteristics, 1 case of adenocarcinoma of the distal esophagus and 1 case of gastric lymphoma. The prevalence of H. pylori was 54% and infection, age and smoking status were associated with organic dyspepsia. The age of 48 years was indicative of alarm signs. CONCLUSIONS: The endoscopic diagnosis of uninvestigated dyspepsia in our setting showed a predominance of functional disease, whereas cancer was an uncommon finding, despite the high prevalence of H. pylori. Organic dyspepsia was associated with infection, age and smoking status.

Human chorionic villus mesenchymal stromal cells reveal strong endothelial conversion properties.

Chorion, amnion and villi are reservoirs of mesenchymal stromal cells (StC) and the hypothesis that StC from fetal tissues retain higher plasticity compared to adult StC has been suggested. Aimed at investigating this aspect, a series of in vitro experiments were performed with StC isolated from first trimester human chorionic villi (CVStC). CVStC were cultured in: (i) standard mesenchymal medium (MM) and (ii) AmniomaxII® (AM), specifically designed to grow amnion-derived cells in prenatal diagnostic procedures. Cells were then exposed to distinct differentiation treatments and distinguished according to morphology, immunophenotype and molecular markers. Human StC obtained from adult bone marrow (BMStC) were used as control. CVStC cultured either in MM or AM presented stromal morphology and immunophenotype, were negative for pluripotency factors (Nanog, Oct-4 and Sox-2), lacked detectable telomerase activity and retained high genomic stability. In AM, however, CVStC exhibited a faster proliferation rate compared to BMStC or CVStC kept in MM. During differentiation, CVStC were less efficient than BMStC in acquiring adipocytes and osteocytes features; the cardiomyogenic conversion occurred at low efficiency in both cell types. Remarkably, in the presence of pro-angiogenic factors, CVStC reprogrammed toward an endothelial-like phenotype at significantly higher efficiency than BMStC. This effect was particularly evident in CVStC expanded in AM. Mechanistically, the reduced CVStC expression of anti-angiogenic microRNA could support this process. The present study demonstrates that, despite of fetal origin, CVStC exhibit restricted plasticity, distinct from that of BMStC and predominantly directed toward the endothelial lineage.

A Novel Algorithmic Approach to the Analysis of Multi-Electrode Array Signals of hiPSC-Derived Cardiomyocytes.

In order to perform in vitro cardiotoxicity screening of pharmacological substances, multi-electrode array systems are increasingly used to measure the extracellular field potentials of cell layers of human induced pluripotent stem cell cardiomyocytes. The analysis of the field potentials is usually performed using complex analysis software provided by the hardware manufacturers. In the case of the Cardiac Analysis Tool software from Axion Biosystems, inconsistencies were found in the results, which can significantly influence the cardiotoxicity screening results. In order to obtain more reliable results, a new algorithm was developed and implemented in an easy-to-use software tool, the INCardio Data Analysis Tool, which, due to its high degree of automation, can also be used by inexperienced users. The validation reveals differences in the results of the two tools both in depolarization spike amplitudes and in the time course of the field potential durations. The manual analysis of all signals affected by deviations shows that the results of the newly developed Data Analysis Tool are correct in all cases and can therefore be classified as more accurate and reliable than the reference analysis software.

Generation of human induced pluripotent stem cell line EURACi015-A from a patient affected by dilated cardiomyopathy carrying the Lamin A/C p.Glu161Lys mutation.

Dilated cardiomyopathy (DCM) is a common heart disorder caused by genetic and non-genetic etiologies, characterized by left ventricular dilatation and contractile dysfunction. Here, we created a human induced pluripotent stem cell line from peripheral blood mononuclear cells using non-integrating vectors from a patient carrying a heterozygous LMNA variant (c.481G > A, p.Glu161Lys, NM_170707.4). The obtained EURACi015-A line, showed the typical morphology of pluripotent cells, normal karyotype and exhibited pluripotency markers and a trilineage differentiation potential. This cell line can be successfully differentiated into cardiomyocytes and endothelial cells. This line represents a human in vitro model to study the genetic basis of DCM.

Modeling incomplete penetrance in arrhythmogenic cardiomyopathy by human induced pluripotent stem cell derived cardiomyocytes.

Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) are commonly used to model arrhythmogenic cardiomyopathy (ACM), a heritable cardiac disease characterized by severe ventricular arrhythmias, fibrofatty myocardial replacement and progressive ventricular dysfunction. Although ACM is inherited as an autosomal dominant disease, incomplete penetrance and variable expressivity are extremely common, resulting in different clinical manifestations. Here, we propose hiPSC-CMs as a powerful in vitro model to study incomplete penetrance in ACM. Six hiPSC lines were generated from blood samples of three ACM patients carrying a heterozygous deletion of exon 4 in the PKP2 gene, two asymptomatic (ASY) carriers of the same mutation and one healthy control (CTR), all belonging to the same family. Whole exome sequencing was performed in all family members and hiPSC-CMs were examined by ddPCR, western blot, Wes™ immunoassay system, patch clamp, immunofluorescence and RNASeq. Our results show molecular and functional differences between ACM and ASY hiPSC-CMs, including a higher amount of mutated PKP2 mRNA, a lower expression of the connexin-43 protein, a lower overall density of sodium current, a higher intracellular lipid accumulation and sarcomere disorganization in ACM compared to ASY hiPSC-CMs. Differentially expressed genes were also found, supporting a predisposition for a fatty phenotype in ACM hiPSC-CMs. These data indicate that hiPSC-CMs are a suitable model to study incomplete penetrance in ACM.

Heart valve engineering: decellularized aortic homograft seeded with human cardiac stromal cells.

BACKGROUND AND AIM OF THE STUDY: The adult human heart contains a cardiac mesenchymal stromal cell (CStC) population with residual cardiovascular plasticity. The study aim was to investigate the ability of CStCs to populate decellularized aortic homograft leaflets, without mechanical stimulation. METHODS: The ability of CStCs to acquire valve endothelial and interstitial cell phenotypes was tested using in vitro assays. First, trypsin-decellularized aortic leaflets were seeded with CStCs under static conditions; tissue section analyses were then performed before and after decellularization, and at 10, 20, and 30 days after reseeding. RESULTS: Following in vitro treatment, the CStCs differentiated along the endothelial lineage, as shown by their capacity to uptake acetylated low-density lipoprotein and to secrete the pro-angiogenic factor, vascular endothelial growth factor. After seeding, CStCs were able to adhere to the leaflet surface, rescuing up to the 90% of the original cell density and expressing the mature endothelial marker, von Willebrandt factor. The CStC supernatants were also positive for matrix metalloprotease-2 (MMP-2), which confirmed the ability of these cells to penetrate within the leaflet structure; this also suggested that CStCs, once engrafted, would contribute to the extracellular matrix turnover. Accordingly, although at a lower efficiency, CStC repopulation was also evident in the inner portions of the leaflet. CONCLUSION: Seeded CStCs were able to reconstitute, without mechanical stimulation, an endothelial-like layer and to partially infiltrate decellularized homograft leaflets. Hence, CStCs appear to be a potentially useful cell type for engineered heart valves.

Generation of human induced pluripotent stem cell line EURACi006-A and its isogenic gene-corrected line EURACi006-A-1 from an arrhythmogenic cardiomyopathy patient carrying the c.1643delG PKP2 mutation.

Arrhythmogenic Cardiomyopathy (ACM) is a rare genetic cardiac disease predominantly associated with mutations in genes of the desmosomes and characterized by arrhythmia and fibro-fatty replacement of the myocardium. We generated human induced pluripotent stem cells (hiPSCs) from one patient affected by ACM carrying the heterozygous c.1643delG (p.G548VfsX15) PKP2 mutation and then corrected the mutation using CRISPR/Cas9 technology. Both original and corrected hiPSC lines showed typical morphology of pluripotent cells, expressed pluripotency markers, displayed a normal karyotype, and differentiated towards the three germ layers. This isogenic hiPSC pair can be used to study the role of the c.1643delG PKP2 mutation in vitro.

Metabolic Signature of Arrhythmogenic Cardiomyopathy.

Arrhythmogenic cardiomyopathy (ACM) is a genetic-based cardiac disease accompanied by severe ventricular arrhythmias and a progressive substitution of the myocardium with fibro-fatty tissue. ACM is often associated with sudden cardiac death. Due to the reduced penetrance and variable expressivity, the presence of a genetic defect is not conclusive, thus complicating the diagnosis of ACM. Recent studies on human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) obtained from ACM individuals showed a dysregulated metabolic status, leading to the hypothesis that ACM pathology is characterized by an impairment in the energy metabolism. However, despite efforts having been made for the identification of ACM specific biomarkers, there is still a substantial lack of information regarding the whole metabolomic profile of ACM patients. The aim of the present study was to investigate the metabolic profiles of ACM patients compared to healthy controls (CTRLs). The targeted Biocrates AbsoluteIDQ® p180 assay was used on plasma samples. Our analysis showed that ACM patients have a different metabolome compared to CTRLs, and that the pathways mainly affected include tryptophan metabolism, arginine and proline metabolism and beta oxidation of fatty acids. Altogether, our data indicated that the plasma metabolomes of arrhythmogenic cardiomyopathy patients show signs of endothelium damage and impaired nitric oxide (NO), fat, and energy metabolism.