Moderated Poster session - Genetic, Epigenetic & Integrative480Inhibiting mitochondrial fission with Mdivi-1 directs cardiac differentiation of human induced pluripotent stem cells via protein kinase CK2481A novel role of tristetraprolin in preventing mitochondrial dysfunction in the heart against iron deficiency by optimizing expression of Rieske iron-sulfur protein482Different therapeutic approaches to downregulate the activation of the hepatic interleukin-6/stat3/complement pathway in two models of autoimmune myocarditis483In vitro and in vivo genome engineering of Dilated Cardiomyopathy caused by phospholamban R14 deletion.484Contractile dysfunction of induced pluripotent stem cell-derived cardiomyocytes from a duchenne muscular dystrophy patient485Cigarette smoking increases expression of the G protein-coupled receptor 15 mRNA by change in CpG methylation486Cardiogenic potential of iPSC from cardiac progenitor cells

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Introduction: Mitochondria are morphologically dynamic organelles which are constantly undergoing fusion and fission, processes essential to maintain the organelle fidelity and these also determine cell fate. Human induced pluripotent stem cells (iPSCs) showed fragmented mitochondria, and we set out to explore whether inhibiting mitochondrial fission affects differentiation of iPSCs, using Mdivi-1, a known inhibitor of mitochondrial fission protein DRP1. Aims: To examine the potential of Mdivi-1 to promote cardiac differentiation of human iPSCs. Methods: Cardiac differentiation of human iPSCs (iPS(foreskin)-2 and CERA007c6 cell lines) was induced by forming embryoid bodies (EB) for 6 days, which were then transferred to gelatin-coated plates. iPSCs were treated with either DMSO or 5 μM Mdivi-1 with or without 5 μM TBCA (a selective inhibitor of protein kinase CK2) during EB formation. End point analyses include mitochondrial morphology using MitoTracker red staining, the percentage of beating EBs, expression of cardiac markers by RT-qPCR, calcium imaging by fluorescence Fluo-4 dye, electrophysiological behaviour by microelectrode arrays and protein kinase profiling by KINOMEscan assays. Results: Mitochondria in undifferentiated iPSCs were mainly fragmented with perinuclear localisation, while mitochondria in differentiated cardiomyocytes appeared elongated and distributed throughout the cytoplasm. Treatment of iPSCs with Mdivi-1 during EB formation resulted in a 3-fold increase in beating EBs. The beating cardiomyocytes expressed several key cardiac markers, cycled calcium ion and were responsive to isoprenaline and carbachol. When screened against a panel of 468 protein kinases, which covers over 80% of the human kinome, Mdivi-1 showed only one candidate kinase target – CSNK2A2 (protein kinase CK2α’). Western blotting analysis showed a significant increase in CK2α’ protein expression in EBs treated with Mdivi-1, and treatment with TBCA attenuated the cardiogenic effect of Mdivi-1. Conclusion & significance: Mdivi-1 can significantly enhance differentiation of human iPSCs into functional cardiomyocytes. These cardiomyocytes can be used for drug screening, disease modelling or for treatment of heart disease. Mdivi-1 is a highly selective inhibitor that shows a clinically safe prolife. This inhibitor allows timely and short-term manipulation of mitochondrial morphology for benefit in cell differentiation.

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