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Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): This research was funded by the Department of Innovation, Research and University of the Autonomous Province of Bolzano-South Tyrol (Italy), and by the Joint Project Südtirol- FWF (Italy-Austria) for A.R. J.T. A.M. R.P.. Background Arrhythmogenic cardiomyopathy (ACM) is a genetic disease associated with sudden cardiac death and fibro-fatty replacement of myocardium. Recently, it has been demonstrated that oxidized lipid contributes to cardiac adipogenesis and that ACM hearts are characterized by an increase in oxidative stress. Purpose As mitochondria are an important source of reactive oxygen species (ROS) within mammalian cells, the present work aims to evaluate if increased oxidative stress observed in ACM hearts is associated with altered mitochondrial function. Methods The oxidative stress marker 4HNE was investigated together with the cardiomyocyte marker cardiac Troponin T on paraffin embedded human ventricular samples and analyzed by confocal microscopy. Human primary cardiac stromal cells (CStCs), obtained from either right ventricle biopsies of ACM patients or healthy cadaveric tissue donor (CTR), were used as cellular model as they are known for their contribution to adipogenesis in the ACM pathology. CStCs were cultured either in basal medium or adipogenic medium (ADIPO) in presence or absence of 500 nM of the ROS scavenger MitoTEMPO. After 7 days of adipogenic differentiation, intracellular lipid droplets accumulation and mitochondrial superoxide levels were measured in CStCs by confocal microscopy using BODIPY 493/503 (0.5 µM) and MitoSOX Red (5 µM) dyes, respectively. The oxidative phosphorylation (OXPHOS) and fatty acid oxidation (FAO) were also evaluated in CStCs by high resolution respirometry. Results 4HNE staining was increased in heart tissues from ACM patients compared to CTRs and particularly evident in non-cardiomyocyte cells. In agreement, a higher MitoSOX fluorescence intensity was found in ACM-CStCs compared to CTR-CStCs in ADIPO medium, confirming higher ROS presence in patient cells. The treatment with MitoTEMPO was efficient in reducing ROS abundance, strongly suggesting mitochondrial origin. Lipid accumulation in ACM-CStCs was also prevented by MitoTEMPO treatment. Preliminary evidence further indicates that mitochondrial respiratory capacity is increased in ACM-CStCs versus CTR-CStCs exposed to adipogenic medium for 7 days. However, transmission electron microscopy analysis and Western blot analysis of Mfn1/2, Opa1, Fis1 and Drp1 did not show an evident alteration neither in mitochondria ultrastructure nor in the expression of proteins regulating mitochondrial dynamics. Conclusions Our data support an alteration of mitochondrial activity in CStCs from ACM patients, apparently not linked to a modified network or morphology of mitochondria but associated with a higher ROS production. [ABSTRACT FROM AUTHOR]

Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): European Regional Development Fund and Interreg V-A Italy-Austria 2014-2020 and Department of Innovation, Research and University of the Autonomous Province of Bolzano-South Tyrol (Italy). Background Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) were previously used to model several inherited heart diseases (1). Among these, arrhythmogenic cardiomyopathy (ACM) is characterized by a fibrofatty myocardial replacement and severe ventricular arrhythmias (2). ACM is mainly caused by mutations affecting proteins of intercalated discs (3) and shows incomplete penetrance and variable expressivity (4). Purpose To evaluate whether hiPSC-CMs provide a good in vitro model to study incomplete penetrance in ACM. Material and methods hiPSCs were generated from buffy coats of 6 close relatives. Three of them were ACM patients carrying a deletion of exon 4 in the PKP2 gene causing a premature stop codon, 2 were asymptomatic (ASY) PKP2 mutation carriers and 1 was a healthy control (CTR). Whole exome sequencing of the 6 individuals was performed. The purified hiPSC-CMs were cultured in basal and adipogenic medium and examined by ddPCR, western blot, Wes™ immunoassay system, patch clamp and immunofluorescence. Results All family members tested negative for additional mutations in ACM genes. As expected, half the amount of wild type (wt) PKP2 mRNA was found in ACM and ASY than in CTR hiPSC-CMs (2.25±0.27vs4.29±0.70 wtPKP2/HPRT1; p=0.028) (2.08±0.38vs4.29±0.70 wtPKP2/HPRT1; p=0.052). Of note, the mutated (mut) PKP2 mRNA was detected and significantly more expressed in ACM than in ASY hiPSC-CMs (0.33±0.04vs0.11±0.06 mutPKP2/HPRT1; p=0.029). While we confirmed a reduced amount of wt plakophillin-2 protein in ACM and ASY compared to CTR hiPSC-CMs (0.19±0.07vs0.81±0.28 wtPKP2/GAPDH; p=0.016), (0.25±0.07vs0.81±0.28 wtPKP2/GAPDH; p=0.11), the truncated protein was not detected. Moreover, a significantly lower expression of the active form of β-catenin (ABC) has been shown in ACM and ASY than in CTR hiPSC-CMs (0.93±0.18vs1.30±0.09 ABC/GAPDH; p=0.057), (0.76±0.10vs1.30±0.09 ABC/GAPDH; p=0.0057). In basal medium, ACM hiPSC-CMs had a significant lower overall sodium current density compared to CTR (-49.82±2.8 vs -69.38±4.8 pA/pF; p<0.0001) and to ASY (-49.82±2.8 vs -68.92±4.3 pA/pF; p<0.0001) hiPSC-CMs. In adipogenic medium, ACM hiPSC-CMs showed a higher lipid accumulation and a higher sarcomere disorganization than CTR (1580±219vs615±199 intensity/nuclei; p=0.0351) (0.019±0.001vs0.028±0.003 myofibril alignment index; p=0.039) and ASY (1580±219vs689±146 intensity/nuclei; p=0.0059) (0.019±0.001vs0.027±0.001 myofibril alignment index; p=0.010) hiPSC-CMs. Conclusions Here we report that ACM express a significantly higher amount of mutated PKP2 mRNA than ASY iPSC-CMs. Even though no differences were detected for plakophilin-2 and active β-catenin proteins between ACM and ASY iPSCM-CMs, the ACM showed a fatty phenotype and altered electrical activity that differed significantly from the CTR as well as from the ASY hiPSC-CMs, demonstrating that these cells provide a valid model to study incomplete penetrance in ACM. [ABSTRACT FROM AUTHOR]