Development of FRDA iPSCs-derived Cardiomyocytes as a tool to evaluate efficacy of potential therapeutic compounds

Friedreich ataxia is a disease mainly due to the low expression of frataxin protein with consequent production of ROS and free iron accumulation. Since one of the major causes of death is associated with cardiac hypertrophy, it is of crucial importance to develop a cellular cardiac model that specifically recapitulates cardiac phatophysiology of FA. Therefore, our aim concerns the generation of a robust method of differentiation and characterization of cardiomyocytes derived from patient iPSCs, a tissue specific cells able to recapitulate the main phenotypic hallmarks of the disease. The final aim of this project is the validation of etravirine effects on FRDA iPSCsderived cardiomyocytes, by promotion of frataxin accumulation and restoration of any evident functional defect. We developed and verified a reproducible procedure of differentiation based on temporal modulation of Wnt/βcatenin pathway; named GiWi protocol (Xiaojun L. et al 2012). In detail, temporal application of GSK3 inhibitor combinate with chemical Wnt pathway inhibitor are able to differentiat iPSCs into functional cardiomyocytes. After 24h from the induction of the Wnt pathway, mediated by the inhibition of GSK3, the cells result positive for the mesodermal induction marker Brachyury. The subsequent total inhibition of a family of proteins known as porcupine (PORCN), drives the mesoderm cells towards a cardiogenic fate by activating some key genes such as Nkx2.5, Isl1 and GATA4. After 9-10 days of culture, the cardiogenic cells organize themselves to form recognizable beating areas due to the expression of terminal differentiation markers such as MLC2a, cTnT and alpha-actinin that identify the terminal differentiation in cardiomyocytes. Our first goal was to prove the differences in frataxin expression between healthy control and FRDA cells during differentiation. We observed that low levels of frataxin protein expression is maintained during all steps of differentiation in FRDA cardiomyocytes as compared to healthy control. Moreover, we evaluated and observed that specific hypertrophic markers are differentially expressed between healthy control and FRDA iPSCs-derived cardiomyocyte. We then identified the minimal etravirine concentration that could significantly increase frataxin expression in FRDA iPSCs-derived cardiomyocytes. Our results show that etravirine is able to significantly increase frataxin level up to 40% at 1uM in this cell type. Following these results, our future study will be to analyze if etravirine is able to rescue the observed hypertrophic phenotype in cardiomyocytes and the evaluation of oxidative stress-mediated cell death with and without etravirine.