Abstract 478: Small Molecule ICG-001, Sodium Butyrate, and Retinoic Acid Enhanced Direct Cardiac Reprogramming of Induced Cardiomyocytes (iCMs)

Abstract

Background: Reprogramming of cardiac fibroblasts into induced cardiomyocyte-like cells (iCMs) in situ represents a promising strategy for cardiac regeneration. A combination of three cardiac transcription factors Gata4, Mef2c and Tbx5 (GMT), and microRNAs can convert fibroblasts into iCMs, albeit with low efficiency in vitro . We aimed to increase the efficiency, maturation and speed of direct reprogramming using a unique combination of small molecules, in combination with previously identified reprogramming transcription factors, that we selected and combined in a cocktail specifically formulated to effect optimal impact on the aspects of reprogramming pathways thought to represent “roadblocks” or points of opportunity in these pathways. Methods: We screened a variety of such candidate compounds in primary rat/human cardiac fibroblast. Cardiac reprogramming was induced by lentivirus encoding Gata4, Mef2c and Tbx5 plus small molecules, and the presence of iCM was confirmed 10 days later by QRT-PCR, IFs and FACS analysis for the activation of endogenous cardiac troponin T (cTnT). Results: After validating and optimizing the dose and conditions for adding small molecules to the reprogramming cocktail, we treated GMT-overexpressing fibroblast with a combination of sodium butyrate (SB, 1mM; HDAC inhibitor), ICG-001(1μM; WNT inhibitor), and retinoic acid (RA, 1μM). We found that that a combination of SB, ICG-001, and RA significantly increased GMT-induced reprogramming efficiency to 22 % cTnT + iCMs from primary rat cardiac fibroblasts compared to 8% in the presence of single compounds and 3.5% without any added small molecule compound alone. QRT-PCR and IFs, analysis shows that a combination of the SB, ICG-001, and RA increased cTnT gene expression 4-6 fold when added to GMT-overexpressing rat cardiac fibroblasts. We also demonstrated that combined addition of SB, ICG-001, and RA increased cardiac fibroblast reprogramming efficiency 4 fold when added to GMTHM plus miR-590-overexpressing human cardiac fibroblasts. Conclusions: Thus, HDAC and WNT inhibitors along with RA, jointly accelerate GMT-induced cardiac reprogramming from rat/human cardiac fibroblasts and pave the way for new translational approaches for cardiac regeneration.