Enhanced Generation of Induced Cardiomyocytes Using a Small‐Molecule Cocktail to Overcome Barriers to Cardiac Cellular Reprogramming

Abstract

Background Given known inefficiencies in reprogramming of fibroblasts into mature induced cardiomyocytes ( iCM s), we sought to identify small molecules that would overcome these barriers to cardiac cell transdifferentiation. Methods and Results We screened alternative combinations of compounds known to impact cell reprogramming using morphologic and functional cell differentiation assays in vitro. After screening 6 putative reprogramming factors, we found that a combination of the histone deacetylase inhibitor sodium butyrate, the WNT inhibitor ICG ‐001, and the cardiac growth regulator retinoic acid (RA) maximally enhanced iCM generation from primary rat cardiac fibroblasts when combined with administration of the cardiodifferentiating transcription factors Gata4, Mef2C, and Tbx5 ( GMT ) compared with GMT administration alone (23±1.5% versus 3.3±0.2%; P <0.0001). Expression of the cardiac markers cardiac troponin T, Myh6, and Nkx2.5 was upregulated as early as 10 days after GMT –sodium butyrate, ICG‐001, and RA treatment. Human iCM generation was likewise enhanced when administration of the human cardiac reprogramming factors GMT , Hand2, and Myocardin plus miR‐590 was combined with sodium butyrate, ICG‐001, and RA compared with GMT , Hand2, and Myocardin plus miR‐590 treatment alone (25±1.3% versus 5.7±0.4%; P <0.0001). Rat and human iCM s also more frequently demonstrated spontaneous beating in coculture with neonatal cardiomyocytes with the addition of sodium butyrate, ICG‐001, and RA to transcription factor cocktails compared with transcription factor treatment alone. Conclusions The combined administration of histone deacetylase and WNT inhibitors with RA enhances rat and human iCM generation induced by transcription factor administration alone. These findings suggest opportunities for improved translational approaches for cardiac regeneration.

Cardiac Regeneration: New Insights Into the Frontier of Ischemic Heart Failure Therapy

“…Dual inhibition of TGF-β and WNT signaling increased the number of contracting cells generated by polycistronic GMT overexpression in neonatal CFs by ~8-fold vs. GMT alone, resulting in ~40% contracting cells after 7 weeks of culture (Mohamed et al, 2017 ). Dual inhibition of histone deacetylases (HDACs) and WNT signaling alongside administration of RA increased expression of cTnT by ~7-fold compared to GMT alone in rat CFs, which coincided with increased expression of cardiac genes including Gata4, Mef2c, Tbx5, Tnnt2, Nkx2-5 , and Myh6 (Singh et al, 2020 ). Notably, though neither compound treated reprogrammed cells nor untreated cells spontaneously contracted in culture, GMT-reprogrammed cells treated with HDAC/WNT inhibitors and RA synchronously contracted following 4 week coculture with neonatal rat CMs.…”

“…Targeted depletion of additional epigenetic regulators including lysine-specific methyltransferases Kmt2a, Kmt2b , and Kmt2e via shRNA delivery impaired the reprogramming process whereas depletion of chromatin remodeler complex and cohesion complex subunits Ruvbl1/Bcor and Stag2 , respectively, enhanced reprogramming by polycistronic M-G-T (Zhou et al, 2018 ). HDAC inhibition alone or in conjunction with WNT inhibition and RA stimulation improved reprogramming by GMT, which coincided with elevated global H3K27ac levels in rat CFs (Singh et al, 2020 ). Further, overexpression of HDAC1 counteracted the effects of HDAC inhibition, indicating that HDACs antagonize reprogramming by GMT ( Figure 3 ).…”

“…GHMT + MYOCD or GMT + miR-590 both induced expression of cTnT in ~5% of HCFs (Singh et al, 2016 ). Dual inhibition of HDACs and WNT alongside administration of RA further improved reprogramming by GHMT, MYOCD, and miR-590 compared to vehicle control (25% vs. 5.7% cTnT positive cells) (Singh et al, 2020 ). Though neither reprogramming condition generated spontaneously beating cells, ~5% of reprogrammed cells treated with these compounds synchronously contracted after 4 weeks of coculture with neonatal rat CMs.…”

Clinical potential of angiogenic therapy and cellular reprogramming

“…6,[42][43][44][45] Small molecules are particularly useful for suppressing profibrotic and inflammatory pathways during cardiac reprogramming, and significantly improved in vitro reprogramming efficiency is achieved with inhibitors of transforming growth factor-b, Rho-associated kinase, prostaglandin, and interleukin-1b signaling. 42,[44][45][46] Importantly, Mohamed and colleagues 44 demonstrated small molecules also improve the in vivo efficacy of cardiac reprogramming. Anti-inflammatory strategies could be particularly important for cardiac reprogramming in chronic HF, given the persistent myocardial inflammation in this condition.…”

Small Molecule Epigenetic Modulators in Pure Chemical Cell Fate Conversion

“…Furthermore, Ding's group reported that a small molecule combination (CHIR99021, SB431542, Parnate, and Forskolin) was sufficient to complete the conversion of cardiomyocytes from mouse fibroblasts with Oct4 alone [ 59 ]. It was also reported that small molecules (NaB, RA, and ICG-001) were able to improve rat and human cardiac cell generation induced by transcription factors (Gata4, Mef2C, and Tbx5) [ 60 ]. In 2015, Xie's team transdifferentiated mouse fibroblasts into cardiomyocytes by passing a cardiac progenitor stage with six small molecules (CHIR99021, RepSox, Forskolin, VPA, Parnate, and TTNPB), while the induced cardiomyocytes were cultured in cardiomyocyte maintenance medium containing CHIR99021, PD0325901, LIF, and insulin [ 61 ].…”