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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 signaling 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) Test if targeting mitochondria can 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 are being used, namely iPSC derived cardiomyocytes (iPSC-CMs) and primary cardiac stromal cells (i.e. Cardiac Fibroblasts or 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.In detail, right ventricular CStCs from ACM patients were obtained thanks to the ongoing collaboration with the Centro Cardiologico Monzino (Milano). Non-patient cells (Controls) were purchased. Cells were cultivated and amplified in Basal Medium (BM) consisting of IMDM (Lonza), supplemented with 20% Fetal Bovine Serum (FBS, Hyclone, Italy), 10.000U/ml Penicillin, 10,000μg/ml Streptomycin (Pen/strep, Thermo Fisher Scientific) and 20mM L-Glutamine (L-Glu, Thermo Fisher Scientific). Adipogenic transdifferentiation of CStCs (one feature of ACM) was induced in BM half depleted in FBS and supplemented with IBMX, Indomethacin and Hydrocortisone (also called here adipogenic medium, or AM). Experiments and sample collection were performed on cells in BM, or after 3, 7 or 14 days of AM.Human iPSCs were generated at the Institute for Biomedicine, EURAC from Peripheral Blood Mononuclear Cells (PBMCs) of one patient sample (ACM) and one familial healthy individual (CTR) as described in Meraviglia et al., Stem Cell Res, 2021. In addition, iPSC coming from an ACM patient carrying a PKP2 mutation and its isogenic control were generated (Meraviglia et al., Stem Cell Res, 2021).All these lines were used for CM-related experiments. CM were derived from human iPSC as described by De Bortoli et al., 2023. Briefly, cardiac differentiation was induced in cells using the PSC Cardiomyocyte Differentiation Kit (Thermo Fisher Scientific). After 25 days from the start of the differentiation, the beating monolayers were enriched from non-CMs cells using the PSC-Derived Cardiomyocyte Isolation Kit (Miltenyi Biotec). Purified hiPSC-CMs were maintained in culture for 3, 7 or 14 days in BM and AM. Basal medium, also named EB2%, was composed of High Glucose DMEM (Gibco), 2% Hyclone Fetal Bovine Defined (GE Healthcare Life Sciences), 1% non-essential Amino Acids (Gibco), 1% Penicillin/Streptomycin (Gibco) and 0,09% β-mercapto-ethanol (Gibco). Adipogenic Medium was composed of basal medium supplemented with 50μg/ml Insulin (Sigma-Aldrich), 0.5 μM Dexamethasone (Sigma-Aldrich), 0.25 mM 3-isobutyl-1 methylxanthine (IBMX) (Sigma-Aldrich), 200 μM Indomethacin (Sigma Aldrich) and 5μM Rosiglitazone (Vinci- Biochem) and is used to induce adipogenic differentiation. Experiments and sample collection were performed on cells in BM, and AM after 3, 7 or 14 days. Results and future workWP1 ACTION 1.1 Mitochondrial MetabolismAs previously demonstrated by our group, ACM CStCs exposed to adipogenic medium accumulate intracellular lipid faster and more abundantly than control CStCs (Volani, Pagliaro et al., 2022). To assess whether mitochondrial activity could contribute to this increased lipid accumulation, mitochondrial metabolic activity was evaluated by High-Resolution Respirometry (HRR). Expression in CTR and ACM cells of subunits of the five Electron Transport Chain (ETC) complexes (in order, NDUFB8, SDHB, UQCR2, COX2, ATP5A) have also been investigated by immunoblotting to unravel eventual metabolic differences. Proximity of mitochondria with lipid droplets was assessed by confocal imaging to determine if mitochondria could have a direct access to lipid droplets as a source of energy while mitochondrial membrane potential (MMP), closely related to mitochondrial metabolic activity, was also determined by fluorescence microscopy of the MMP sensible dye TMRM.ACM-CStCs in basal medium displayed a lower mitochondrial-linked respiration compared to CTR CStCs, but no difference in MMP or ETC complex expression were observed. On the contrary, after 7 days of adipogenic medium, mitochondrial respiratory capacity was higher in ACM CStCs compared to CTR CStCs, while MMP was similar between ACM and CTR CStCs. Preliminary observations (n=3) indicate that UQCR2 (Complex III) expression was lower in ACM CStCs after 7 and 15 days of adipogenic medium, while COX2 (Complex IV) expression was higher in ACM CStCs after 3 days of adipogenic medium. Of note, the interpretation of all these results is tightly related to mitochondrial load of cells. Results obtained by TOM20 Western blot, Citrate Synthase (CS) activity assay and MitoTracker measurement by Flow cytometry experiments showed an increase in mitochondrial density after adipogenic treatment but no significant difference between CTR and ACM CStCs. Therefore, to obtain a more conclusive indication digital droplet PCR was performed to assess mtDNA copies. First results (N= 5 each group) revealed no major difference between CTR and ACM CStCs.Lipid accumulation was investigated in iPSC-CMs cultured in BM and adipogenic medium for 3, 7 and 14 days. After 14 days of culture (around days 40-45 of age) ACM-derived cells did not manifest large lipid deposition in EB2% (BM) compared to CTR cells (CTR, N=4; ACM, N=4) in line with the recently published manuscript by De Bortoli et al., 2023. Instead, the exposition to the adipogenic medium for 14 days is not sufficient to manifest a large lipid deposition in neither ACM nor CTR cells (CTR, N=4; ACM, N=4). Accordingly, mitochondrial respiratory capacity measured by means of high-Resolution Respirometry (HRR) showed no significant difference in iPSC-CMs cultured both in basal medium (EB2%) and adipogenic medium (CTR, N=4; ACM, N=4).  The expression profile of the different mitochondrial complexes is under investigation. Preliminary experiments on MMP revealed no significant difference between ACM and CTR-derived CMs.WP1 ACTION 1.2 Mitochondrial dynamicsIn cells, mitochondria are organized in network. This network integrity is allowed by events of fusion and fission, also called mitochondrial dynamics, which are tightly related to mitochondrial activity. Mitochondrial dynamics, indicated as a ratio of connectivity versus fragmentation, was measured on fixed cells from ACM and CTR origin by confocal imaging of mitochondrial network (Grp75 staining). In addition, gene and protein expression of players involved in mitochondrial fusion (MFN1, MFN2, OPA1), fission (FIS1, DRP1) and mitophagy (PINK, PRKN) were assessed by RT-qPCR and Western blot analysis.No difference in mitochondrial branching was observed between ACM and CTR CStCs after adipogenic treatment. This result was included in the manuscript published last year (Volani, Pagliaro et al., 2022 J Cell Mol Med). Interestingly, results obtained on CStCs in basal conditions showed less connected mitochondrial network in ACM cells compared to CTR; this is in agreement with the decreased mitochondrial respiratory capacity observed in ACM CStCs.The expression analysis of the genes and proteins involved in mitochondrial dynamics and mitophagy was performed in BM and AM. In BM no major changes were observed, however, a slightly higher expression of genes involved in mitochondrial fusion MFN1 and OPA1 were observed in ACM CStCs after 3 days of adipogenic treatment, as well increased levels of the protein involved in mitochondrial fission FIS1 after 3 and 15 days of adipogenic treatment . In conclusion, in CStCs mitochondrial dynamics show some changes at the beginning of the AM treatment (day3), suggesting an active remodeling of the mitochondrial needs after the induction of the adipogenic process.We also started investigating mitochondrial dynamics in iPSC-CMs. Preliminary experiments on the expression levels of proteins involved in mitochondrial fusion and fission did not reveal major differences between CTR and ACM. In addition, as for CStCs, iPSC-CMs specimens were fixed after 3,7 and 14 days of BM (EB2%) and AM. Next, cells staining was performed using a marker of sarcomeric structures (a-actinin) together with the usual mitochondrial staining (Grp75). Unfortunately, Grp75 mitochondrial staining resulted in some technical challenges, probably because of cell density and/or type of fixation. To overcome this issue, preliminary experiments were performed on iPSC-CMs living cells using the mitochondrial dye, MitoTraker, showing nice mitochondrial branching. This approach will be used to monitor mitochondrial fragmentation. Additional experiments are planned after having received the results from the transmission electron microscopy analysis (see next section action 1.3).WP 1 ACTION 1.3 Mitochondrial ultrastructureMitochondrial ultrastructure was determined by Transmission Electron Microscopy (TEM), in collaboration with Dr. Michael Blumer at the University of Innsbruck. Results obtained on ACM and CTR CStCs from three independent individuals exposed to adipogenic medium for 7 days showed no evident mitochondrial ultrastructure alteration, with mitochondria having normal shape, dimension, and regular cristae. The same observation was true for ACM and CTR CStCs grown in basal conditions. Data were included in the manuscript by Volani, Pagliaro et al (Volani, Pagliaro et al., 2022 J Cell Mol Med).A similar analysis of mitochondrial ultrastructure of iPSC-CMs is also ongoing. Samples were collected at day 7 and 14 of BM and AM. Unfortunately, samples collected so far, using the same protocol as for CStCs, did not allow sample preservation for TEM analysis and results had to be discarded. To overcome this issue, different strategies of sample preparation, including fixation and collection, were tested and iPSC-CMs are now in Innsbruck at the facility of Dr. Blumer for TEM analysis. These samples include a set of iPSC-CMs from CTR and ACM specimens . This analysis will give us important information on the mitochondrial ultrastructure fitness of iPSC-CMs in BM and could be used to decide how and if to go on with additional experiments on mitochondrial dynamics.WP2 ACTION 2.1 ROS source definitionAim of the WP2 is to investigate in detail the molecular pathways related to ROS homeostasis. This part of the project follows previous effort of our laboratory showing a crosstalk between lipid accumulation and ROS levels (Volani et al, Journal of Cellular and Molecular Medicine, 2022). In particular, we showed higher ROS levels and lipid peroxidation in ACM specimens. In addition, we investigated the effects of the drug MB-3 that was able to efficiently reduce intracellular lipid accumulation, while boosting cellular ROS detoxification systems by acting on glutathione homeostasis.In the context of the present project, our previous results were further developed by the determination of ROS origin (especially mitochondrial ROS origin) and the assessment of the antioxidant defense capacity of CTR and ACM cell. As first, we measure the total antioxidant capacity (TAC) of CStCs in BM and after 3 and 7 days of AM (N=4 samples each time and treatment). No difference was detected in BM, but a lower TAC was present after 3 days of AM in ACM CStCs compared to CTR. Similarly, the indirect impact of oxidative stress on nuclear (pH2AX) and lipid peroxidation (4HNE) as well as expression measurements of oxidative stress response genes (HIF1A, HIF1B, NFE2L2) showed a potential difference between ACM and CTR after 3 days of AM. Moreover, the analysis of one of the proteins involved in cellular detoxification, namely superoxide dismutase 2 (SOD2), revealed an increased expression overtime due to the AM but no significant change between ACM and CTR.Measurements of ROS levels have proven to be a challenging task, and major effort are now place into this working package. Specifically, direct ROS levels are investigated in CStCs using fluorescent dyes measuring by confocal imaging of both ROS levels by confocal imaging (MitoSOX, MitoTracker Red CM-H2XRos) and mitochondrial network (MitoTracker Green, MitoTracker DeepRed). In addition, the simultaneous measurement of mitochondrial respiratory capacity and ROS production (HRR-AmpRed) in CStCs in BM and AM (day 3 and day7) was performed in the previous months in Innsbruck. Analysis of the data is currently ongoing and should give us soon information on the possibly susceptibility of ACM to ROS.WP2 ACTION 2.2 Analysis of ROS contribution to ACM pathologyImmunoblotting, FACS and microscopy imaging experiments were originally planned to discriminate different forms of cell deaths. However, our data on cell viability (% living cells) in CStCs showed no significant difference between ACM and CTR [CTR (BM, N=4): mean±SD 95.8±1.8; ACM (BM, N=6): 93.6±2.0; CTR (AM 7d, N=6): 84.9±3.1; ACM (AM 7d, N=5): 84.7±4.3], therefore a further check cell death mechanisms characterization is not planned for the moment. To additionally investigate the ROS contribution to the ACM pathology, we are currently performing experiments using inducers of ROS production, namely MitoPQ and PEITC, as well as the ROS scavenger mitoTempo. In these experiments, we focus on the lipid accumulation, on one side, and the oxidative environment, on the other side. Since the ROS inducers are quite tricky to handle, we are currently working on the establishment of the proper experimental protocol to avoid too severe side effects. Our partners in Innsbruck are responsible for the execution of this action.WP3 ACTION 3.1The compound library screening strategy takes advantage of the capacity of CStCs from ACM patients to accumulate more neutral lipids than control CStCs. Measurement consists in the measurement of BODIPY fluorescence in CStCs after adipogenic treatment and need to be expressed over cell number (cytotoxicity, proliferation). As results obtained on a classic plate reader (Envision) did not give satisfying results, acquisition strategy was changed for a High-Content Imaging method. High Content Imaging system offered the best compromise to reach the expected output while maintaining a good throughput. Such a system allows the measurement and visualization of lipid droplet accumulation and cell numeration (using DAPI as nucleus staining) by the acquisition of confocal microscopy quality images. A partnership contract was signed with the High-Throughput Screening (HTS) facility of CIBIO (University of Trento), offering an ideal environment in terms of instrumentation, expertise, convenience and close location (important e.g. to assure survival of cells prepared in Bolzano for the planned experiments).Test for automation were performed between February and May 2023. Cardiac fibroblasts from one single origin were selected to improve the reproducibility of the results and was selected on the following parameters: capacity of the cells to accumulate well lipids and in a reproducible manner and capacity to get enough cell material for the test (high number of vials with low passage and capacity to grow and keep good fibroblast features over passages). Tests successfully led to the validation of the staining and measurement strategy, optimization and adaptation of the cell seeding, medium and compounds dispensing and fixation and staining protocols. Last test also included the full automation of all these steps and determination of the z-score of the assay, classifying our assay in the range of “very good assays” and validating the overall strategy.WP3 ACTION 3.2 Library testA library of 407 compounds targeting mitochondria and ROS signaling targets (MedChem Express) was selected and the effect of these compounds on lipid accumulation by CStCs was evaluated. Primary library screening was successfully performed in June 2023. All 407 compounds were tested on the selected primary ACM cardiac fibroblast at two concentrations (1 µM and 10 µM) and in duplicates for each condition for a total of 22 x 96-well plates. Eight DMSO and eight MB-3 control wells were included on each plate to assess the reproducibility of the test and get internal control to each plate.Thanks to the optimization steps performed in WP 3.1 all the plates successfully allowed the imaging of BODIPY and DAPI staining with a high reproducibility as measured on controls z-score. First row data were extracted and information on cell number, number of lipid aggregates and lipid aggregates per cells obtained. Compounds were classified according to two parameters, cell number (Toxic, mild toxic, normal cell number compared to DMSO or increase cell number) and lipid aggregates per cell (increased, similar to DMSO control, small reduction or strong reduction). Around 25% of the compounds showed, at least at one concentration, a decrease in lipid accumulation without affecting cell number proving the importance of mitochondria and ROS signaling in lipid accumulation process. In this active compounds, 24 compounds are showing a strong reduction of lipid accumulation (at least 50% lower than DMSO control) without showing toxicity. This list of compounds will be reduced based on compounds information (pharmacology, family of compounds), targets and in-depth analysis of the images to reduce the list to 4-5 top compounds for further study. Analysis of the targets and pathways related to these compounds is also undergoing to unravel whether specific targets could have a higher impact on lipid accumulation in ACM-CStCs.WP 3 ACTION 3.3 Analysis of selected compound effects on mitochondrial respiration and ROS signalingThe WP3.3 will be defined in detail according to the results of WP1 and especially WP2 ACTION 2.1.To evaluate the compound effects on mitochondrial respiration and ROS signaling, we are currently developing specific tests. Of one them includes the analysis of mitochondrial respiratory capacity in intact cells exposed or not to the compound of interest. In addition the measurement of Dichlorofluorescin (DCF) fluorescence can be assessed to investigate the ability of the compound to act as an antioxidant.

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