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Mitochondrial dysfunction in ACM pathogenesis. 

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Eurac Research is a private research center​ based in Bozen-Bolzano. Our researchers come from a wide variety of scientific fields and all parts of the world.

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Arrhythmogenic cardiomyopathy (ACM) is characterized by arrhythmia and progressive fibro-fatty substitution of the cardiac tissue leading to ventricular dysfunction. Previously published research provided convincing evidence for the involvement of Reactive Oxygen Species (ROS) in tissue degeneration, with our preliminary data also supporting a broader role for mitochondria in ACM development. The present project, built as a collaboration between EURAC (EURAC, Bolzano/Bozen, South-Tyrol, Italy) and the Medical University of Innsbruck (MUI, Innsbruck, Austria) will: 1) Provide a detailed analysis of structural and functional mitochondrial changes in human ACM cell models;2) Assess the contribution of mitochondrial and non-mitochondrial ROS sources to the ACM pathogenesis;3)  Obtain evidence that targeting mitochondria can reverse ACM-associated alterations (i.e. increased lipid accumulation and higher ROS production).

Theoretical frameworkArrhythmogenic cardiomyopathy (ACM) is one of the major causes of sudden cardiac death, especially in young athletes, characterized by arrhythmia and progressive fibro-fatty substitution leading to ventricular dysfunction. Although symptomatic treatments of the arrhythmic events are routinely performed, no conventional therapies against fibro-fatty degeneration exist except for heart transplantation in patients with end-stage heart failure.Previous research showed the involvement for Reactive Oxygen Species (ROS) in tissue degeneration. Although ROS production and signalling are tightly linked to mitochondrial function, the role of mitochondria in the context of ACM onset and progression has not been studied so far. Our preliminary results with induced pluripotent stem cells derived cardiac myocytes (iPSC-CMs) and human primary cardiac stromal cells (CStCs) that well recapitulate the fatty substitution typical of ACM, indicate that:a) mitochondrial ROS abundance is altered in both cell models of ACMb) mitochondrial metabolism is changed in ACM CStCs.The proposed project sets out to meet the following aims:1) Provide a detailed analysis of structural and functional mitochondrial changes in human ACM cell models;2) Assess the contribution of mitochondrial and non-mitochondrial ROS sources to the ACM pathogenesis;3) Obtain evidence if targeting mitochondria reverse ACM-associated alterations (i.e. increased lipid accumulation and higher ROS production)MethodsTo reach the stated aims two different human cell models of the ACM pathology will used, namely iPS derived cardiomyocytes (iPSC-CMs) and primary stromal cardiac fibroblasts (CStCs). A wide range of techniques including real-time PCR, high resolution respirometry (HRR), Western Blot (WB), flow cytometry, confocal imaging and transmission electron microscopy (TEM) will be performed. Results and future work WP1 ACTION 1.1 Mitochondrial MetabolismMitochondrial metabolism was first assessed measuring the mitochondrial membrane potential using the TMRM dye and flow cytometry analysis. This was performed on control and ARVD patients stromal cells and iPSC-CMs at different time points (3, 7 or 14 days) after adipogenic treatment. First trends seem to show an increase of  mitochondrial membrane potential over time after adipogenic treatment. In cardiac fibroblasts this increase appears more important in ARVD cells than in control cells.In parallel cell pellets and protein lysates were collected and sent to our collaborator in Innsbruck (Jakob Troppmair) for analysis of the expression of the complex I to complex V subunits of the electron transfer system by blue native polyacrylamide gel electrophoresis.A first high-resolution respirometry (HRR) assay was also performed on commercial iPSC-CMs cultivated in basal or adipogenic culture conditions and revealed a strong decrease in mitochondrial respiratory capacity of cardiomyocytes after adipogenic treatment.WP1 ACTION 1.2 Mitochondrial dynamicsPreliminary tests were performed on both iPSC-derived cardiomyocytes and cardiac stromal cells. Staining of mitochondria using GRP-75 antibody was done and mitochondrial network images acquired using confocal microscope.These images will be analyzed by 3D modelling of the mitochondrial network and measurement of its fragmentation. Grp-75 staining will also allow us to assess mitochondrial fusion/fission events.In parallel BODIPY staining of lipid droplets accumulated after adipogenic treatment was performed and will allow the measurement of the distance between droplets and mitochondria.Cell pellets and protein lysates were collected and sent to our collaborator in Innsbruck for protein expression analysis by Western blot of proteins involved in mitochondrial fission/fusion events.WP 1 ACTION 1.3 Mitochondrial ultrastructurePreliminary results were obtained in collaboration with the University of Innsbruck. Cell samples (iPSC-CMs and CStC) were cultivated, treated and pre-fixed at Eurac and sent to our collaborator in Innsbruck. After preparation of these samples by this group, the samples were analyzed by Transmission Electron Microscopy by the group of Michael Blumer. First results using untreated ACM and non-ACM cardiac stromal cells did not revealed structural changes between the two samples.WP2 ACTION 2.1 ROS source definitionCell pellets and protein lysates were collected and sent to our collaborator in Innsbruck for biochemical assays and protein expression analysis by Western blot of proteins involved in hypoxia or oxidative stress response (HIF1/2, NRF2).Moreover in the WP1 ACTION 1.1. the HRR analysis will include the simultaneous measurement of mitochondrial respiratory capacity and reactive oxygen species production.Finally other indirect markers of oxidative stress will be also considered, such as lipid peroxidation markers (malondialdehyde, 4-HNE) and protein nitrosylation (3-nitrotyrosin).WP2 ACTION 2.2 Analysis of ROS contribution to ACM pathologyACM pathology is expected to increase ROS and oxidative stress and thereby cell death. Immunoblotting, FACS and microscopy imagining will be used to discriminate different forms of cell deathsWP 3 Analysis of selected compound effects on mitochondrial respiration and ROS signalingThe WP3 will defined in details according to the results of WP1 and especially WP2 ACTION 2.1

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