Sall4 and Myocd Empower Direct Cardiac Reprogramming From Adult Cardiac Fibroblasts After Injury

Abstract

Direct conversion of fibroblasts into induced cardiomyocytes (iCMs) holds promising potential to generate functional cardiomyocytes for drug development and clinical applications, especially for direct in situ heart regeneration by delivery of reprogramming genes into adult cardiac fibroblasts in injured hearts. For a decade, many cocktails of transcription factors have been developed to generate iCMs from fibroblasts of different tissues in vitro and some were applied in vivo. Here, we aimed to develop genetic cocktails that induce cardiac reprogramming directly in cultured cardiac fibroblasts isolated from adult mice with myocardial infarction (MICFs), which could be more relevant to heart diseases. We found that the widely used genetic cocktail, Gata4, Mef2c, and Tbx5 (GMT) were inefficient in reprogramming cardiomyocytes from MICFs. In a whole well of a 12-well plate, less than 10 mCherry+ cells (<0.1%) were observed after 2 weeks of GMT infection with Myh6-reporter transgenic MICFs. By screening 22 candidate transcription factors predicted through analyzing the gene regulatory network of cardiac development, we found that five factors, GMTMS (GMT plus Myocd and Sall4), induced more iCMs expressing the cardiac structural proteins cTnT and cTnI at a frequency of about 22.5 ± 2.7% of the transduced MICFs at day 21 post infection. What is more, GMTMS induced abundant beating cardiomyocytes at day 28 post infection. Specifically, Myocd contributed mainly to inducing the expression of cardiac proteins, while Sall4 accounted for the induction of functional properties, such as contractility. RNA-seq analysis of the iCMs at day 28 post infection revealed that they were reprogrammed to adopt a cardiomyocyte-like gene expression profile. Overall, we show here that Sall4 and Myocd play important roles in cardiac reprogramming from MICFs, providing a cocktail of genetic factors that have potential for further applications in in vivo cardiac reprogramming.

Defining cardiac fibrosis complexity and regulation towards therapeutic development

“…Further applications of single‐cell omics at protein and lipid levels may provide new pathology‐specific targets. Additionally, reprogramming rather than removal of fibroblasts may prove beneficial, methods of which are emerging at an in vitro level 173 . Combining fibroblast targeting and reprogramming technologies could allow not only for the removal of pathogenic subtypes but replacement with reparative or functional cells.…”

“…Additionally, reprogramming rather than removal of fibroblasts may prove beneficial, methods of which are emerging at an in vitro level. 173 Combining fibroblast targeting and reprogramming technologies could allow not only for the removal of pathogenic subtypes but replacement with reparative or functional cells. Furthermore, insights gained from therapeutic endeavours for cancer-associated fibroblast (CAF) modulation should be considered as their heterogeneity and phenotypic signatures mirror those of cardiac fibroblasts.…”

Epigenetic Repression of Chloride Channel Accessory 2 Transcription in Cardiac Fibroblast: Implication in Cardiac Fibrosis

“…Cell Culture, Plasmids, Transient Transfection, and Reporter Assay Primary cardiac fibroblasts were isolated and maintained in DMEM supplemented with 10% FBS as previously described (Gao et al, 2020;Liu et al, 2020;He et al, 2021;Zhao et al, 2021). Mouse embryonic fibroblasts (MEFs) were isolated and maintained in DMEM supplemented with 10% FBS as previously described (Angrisani et al, 2021).…”

Improving Cardiac Reprogramming for Heart Regeneration in Translational Medicine

“…In this study, the combination of Hand2, Nkx2.5, Gata4, Mef2c, and Tbx5 (HNGMT) is >50-fold more efficient than GMT alone, with higher cardiac related gene expression and more cells exhibiting calcium transient and spontaneous beating. Focusing on cardiac fibroblasts isolated from adult mice with myocardial infarction (MICFs), a five factor combination, GMTMS (GMT plus Myocd and Sall4), induces more iCMs expressing the cardiac structural and functional proteins [ 8 ].…”