Abstract
The development of therapeutic approaches based on direct cardiac reprogramming of fibroblasts into induced-cardiomyocytes (iCM) has emerged as an attractive strategy to repair the injured myocardium. The identification of the mechanisms driving lineage conversion represents a crucial step toward the development of new and more efficient regenerative strategies. To this aim, here we show that pre-treatment with the Bmi1 inhibitor PTC-209 is sufficient to increase the efficiency of Chemical-induced Direct Cardiac Reprogramming both in mouse embryonic fibroblasts and adult cardiac fibroblasts. PTC-209 induces an overall increase of spontaneously beating iCM at end-stage of reprogramming, expressing high levels of late cardiac markers Troponin T and myosin muscle light chain-2v. The inhibition of Bmi1 expression occurring upon PTC-209 pre-treatment was maintained throughout the reprogramming protocol, contributing to a significant gene expression de-regulation. RNA profiling revealed that, upon Bmi1 inhibition a significant down-regulation of genes associated with immune and inflammatory signalling pathways occurred, with repression of different genes involved in interleukin, cytokine and chemokine pathways. Accordingly, we observed the down-regulation of both JAK/STAT3 and MAPK/ERK1-2 pathway activation, highlighting the crucial role of these pathways as a barrier for cardiac reprogramming. These findings have significant implications for the development of new cardiac regenerative therapies.
Highlights
The adult mammalian heart is unable to fully restore cardiac function after injury, due to the lack of endogenous repair mechanisms[1,2]
We found that 24 hours pre-treatment with PTC-209 is sufficient to increase the efficiency of Chemical-induced Direct Cardiac Reprogramming (CiDCR) both in mouse embryonic fibroblasts (MEFs) and adult (5 weeks old) cardiac fibroblasts (CFs)
At day 16 for MEFs or 20 for CFs, the medium was changed into Cardiomyocyte Maintaining Medium (CMM)
Summary
The adult mammalian heart is unable to fully restore cardiac function after injury, due to the lack of endogenous repair mechanisms[1,2]. Since the first attempt based on retroviral delivery of the pivotal cardiac transcription factors (TFs) Gata[4], Mef2c, and Tbx[5] (G-M-T)[5], alternative sets of reprogramming factors based on different TFs combinations[6,7] or microRNAs8,9, alone or with small chemical compounds capable to inhibit specific signalling pathways or enzymes involved in epigenetic modifications, have been reported[4,10]. Recent work[11] demonstrated that a solo chemical compound cocktail was able to functionally replace ectopic expression of TFs. The cocktail, comprising six molecules including CHIR99021 (C- a GSK3 inhibitor), RepSox (R- a TGFβR1 inhibitor), Forskolin (F- which sustains cAMP synthesis), Valproic Acid (VPA - a HDAC inhibitor), Parnate (P- an inhibitor of lysine-specific demethylase 1) and TTNPB (T- a highly selective retinoic acid analogue) and named CRFVPT, could induce beating clusters of cardiac cells from mouse fibroblasts in vitro[11] and in vivo[12], with low efficacy. The increase of the efficiency of the reprogramming progress due to the interventions on the epigenetic profile has prompted new strategies to improve the whole process
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