Bmi1 inhibitor PTC-209 promotes Chemically-induced Direct Cardiac Reprogramming of cardiac fibroblasts into cardiomyocytes

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 pretreatment 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.

YAP contributes to DNA methylation remodeling upon mouse embryonic stem cell differentiation

“…Clusters of ChIP-Seq read alignments were identified employing MACS software (version 1.3.7.1). For ChIP-qPCR, samples were prepared as previously described ( 41 ). Supernatant obtained without antibody was used as an input control.…”

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

“…Indeed, pharmacological or genetic inhibition of EGFR signaling increased AGHMT-mediated reprogramming efficiencies in MEFs, adult CFs, and adult TTFs (Hashimoto et al, 2019 ). EGFR signaling also results in the activation of pro-inflammatory signaling via activation of the JAK-STAT pathway, which is known to impair the reprogramming process (Efe et al, 2011 ; Jayawardena et al, 2012 ; Testa et al, 2020 ). Pharmacological inhibition of JAK2 enhanced reprogramming efficiency, similar to EGFR inhibition (Hashimoto et al, 2019 ).…”

“…Knockdown of polycomb repressive complex 1 component Bmi1 facilitated removal of the transcriptionally repressive mark Histone H2A Lysine 119 ubiquitination (H2AK119ub) at cardiac genes and corresponded to increased H3K4me3 levels, increased expression, and increased reprogramming efficiency by polycistronic M-G-T (Zhou et al, 2016 ). Furthermore, 24 h pretreatment of MEFs or CFs with the Bmi1 inhibitor PTC-209 repressed STAT3-, IL-6-, and ERK1/2-mediated inflammatory signaling and resulted in a ~25% increase in the number of αMHC-GFP positive cells generated by the CRFVPT small molecule combination (Testa et al, 2020 ). 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 ).…”

YAP and TAZ Mediators at the Crossroad between Metabolic and Cellular Reprogramming

“…Direct cardiac reprogramming generates cardiomyocytes from fibroblasts without passing through a stem cell state [ 109 ]. This transdifferentiation requires a massive epigenetic remodeling to allow the silencing of fibroblast gene programs and, in parallel, the upregulation of the transcriptional rate of genes in cardiogenic loci [ 110 ]. Very recently, it has been demonstrated that the use of soft matrices resembling the native myocardium improved cardiac reprogramming by suppressing YAP/TAZ signaling [ 111 ].…”