Inhibition of TGFβ Signaling Increases Direct Conversion of Fibroblasts to Induced Cardiomyocytes

Abstract

Recent studies have been successful at utilizing ectopic expression of transcription factors to generate induced cardiomyocytes (iCMs) from fibroblasts, albeit at a low frequency in vitro. This work investigates the influence of small molecules that have been previously reported to improve differentiation to cardiomyocytes as well as reprogramming to iPSCs in conjunction with ectopic expression of the transcription factors Hand2, Nkx2.5, Gata4, Mef2C, and Tbx5 on the conversion to functional iCMs. We utilized a reporter system in which the calcium indicator GCaMP is driven by the cardiac Troponin T promoter to quantify iCM yield. The TGFβ inhibitor, SB431542 (SB), was identified as a small molecule capable of increasing the conversion of both mouse embryonic fibroblasts and adult cardiac fibroblasts to iCMs up to ∼5 fold. Further characterization revealed that inhibition of TGFβ by SB early in the reprogramming process led to the greatest increase in conversion of fibroblasts to iCMs in a dose-responsive manner. Global transcriptional analysis at Day 3 post-induction of the transcription factors revealed an increased expression of genes associated with the development of cardiac muscle in the presence of SB compared to the vehicle control. Incorporation of SB in the reprogramming process increases the efficiency of iCM generation, one of the major goals necessary to enable the use of iCMs for discovery-based applications and for the clinic.

Direct Cardiac Reprogramming

“…More recently, the same group used this reporter system to identify small molecules sufficient to enhance cardiac reprogramming and demonstrated that the transforming growth factor β (TGFβ) inhibitor, SB431542, was capable of increasing the conversion of both mouse embryonic fibroblasts and adult CFs into calcium transient + iCMs up to 5-fold. 89 Inhibition of TGFβ signaling at early in the reprogramming process led to the greatest increase in the conversion of fibroblasts to iCMs.…”

“…MiR-133 promoted cardiac induction by Snai1 suppression and silencing the fibroblast signature at the early stage. Given that TGFβ is an activator of Snai1, it is conceivable that TGFβ inhibitor-mediated cardiac reprogramming in mouse fibroblasts, reported by Ifkovits et al, 89 might be partly mediated through suppression of Snai1 and silencing fibroblast signatures. Intriguingly, this process is similar to the mesenchymal-to-epithelial transition process mediated through Snai1 suppression in iPSC generation.…”

Heart Development, Diseases, and Regeneration – New Approaches From Innervation, Fibroblasts, and Reprogramming –

“…8 Recently, it was reported that chemical compounds, including transforming growth factor β inhibitors, also promote cardiac reprogramming by suppressing fibroblast signatures. 35, 36 A major issue in direct reprogramming is that the efficiency of generating functional iCMs from fibroblasts was low. miRNAs, to GMT could result in reprogramming of human fibroblasts into iCMs.…”

Direct Reprogramming of Fibroblasts into Cardiomyocytes for Cardiac Regenerative Medicine

“…They also found that GCaMP5 helps track the location of rare beating iCMs that represent fully reprogrammed cells. With the same method, Ifkovits et al 58 found that a small-molecule inhibitor of TGFβ, SB432542, increased reprogramming efficiency via GMTHN up to nearly 5-fold and generated more beating iCMs in mouse embryonic fibroblasts.…”