Here we screened eight cardiogenic compounds and found that
Here, we screened eight cardiogenic compounds and found that a combination of fibroblast growth factor (FGF) 2, FGF10, and vascular endothelial growth factor (VEGF), termed FFV, greatly improved the quality of cardiac reprogramming in mouse fibroblasts under serum-free conditions. FFV treatment activated multiple cardiac transcriptional regulators and converted partially reprogrammed iCMs into functional iCMs through the p38 mitogen-activated protein kinase (MAPK) and phosphoinositol 3-kinase (PI3K)/AKT pathways. It also eliminated the need of Gata4 in cardiac reprogramming.
Discussion We revealed that the cardiac reprogramming process can be divided into early and late stages with distinct molecular features, and that the factors enhancing cardiac reprogramming may function at different stages. Consistent with previous reports, we found that the addition of Hand2 to GMT increased the generation of spontaneously contracting iCMs, at least in part, by promoting the generation of ARQ 621 expressing sarcomeric genes during early reprogramming (Addis et al., 2013; Song et al., 2012). In contrast, FFV promoted cardiac reprogramming by converting partially reprogrammed iCMs into functional iCMs at the late stage of reprogramming, without increasing the numbers of partially reprogrammed cells or cell proliferation. Thus, the addition of Hand2 and FFV to GMT functioned at both the early and late stages of reprogramming and synergistically increased the cardiac reprogramming efficiency in this study. For directed cardiac differentiation from PSCs, Activin-Nodal, BMP, and Wnt activation cause PSCs to commit to the CPC fate, and subsequent inhibition of Wnt signaling induces cardiac differentiation (Burridge et al., 2014). Kattman et al. (2011) used FGF and VEGF for cardiac differentiation, but the effects of these growth factors on cardiac differentiation and the underlying mechanisms remain undetermined. We found that Activin, BMP, and Wnt signaling activators did not promote cardiac reprogramming, while FFV activated multiple cardiac transcriptional regulators and increased the generation of functional iCMs through the p38MAPK and PI3K/AKT pathways. FFV-induced Gata6, Hand2, and Ppargc1a expression was inhibited by pretreatment with either p38MAPK or PI3K inhibitor, while the expression of Nkx2.5 was largely mediated through the PI3K/AKT pathway. In agreement with this, the expression of Nkx2.5 was regulated by PI3K/AKT activities in the cardiac differentiation of PSCs, suggesting a link between the mechanisms of iCM maturation and cardiac differentiation of PSCs (Naito et al., 2003; Roggia et al., 2007). Consistent with our results, Zhou et al. (2015) recently reported that addition of AKT1 to GHMT greatly enhanced cardiac reprogramming through pathways involving the mitochondrial target of rapamycin complex1 (mTORC1) and forkhead box o3 (Foxo3). While we found that the p38MAPK and PI3K/AKT pathways were critical for cardiac reprogramming, there are many other pathways involved in FFV signaling. Identification of such pathways could enhance our understanding of the molecular mechanism underlying cardiac reprogramming and promote reprogramming efficiency. We also found that FFV enabled cardiac reprogramming with only Mef2c and Tbx5 through the induction of Gata4 and other reprogramming factors. Recently, Baeyens et al. (2014) reported that the transient administration of two growth factors, epidermal growth factor and ciliary neurotrophic factor, into adult diabetic mice efficiently converted pancreatic exocrine cells into functional β-cells, and ameliorated hyperglycemia without genetic manipulation in vivo. This effect was mediated through the induction of the expression of Neurogenin 3, which is one of the reprogramming factors for β-cells. Given that the in vivo environment might be more permissive than culture dishes for reprogramming, FFV with fewer reprogramming factors might be sufficient to repair damaged hearts by in vivo reprogramming. In agreement with this, in vivo cardiac reprogramming by GMT with VEGF administration enhanced the efficacy of GMT-mediated functional recovery in mice after myocardial infarction (Mathison et al., 2012). Further in vitro studies in human cells and in vivo experiments in larger animals are needed to apply this strategy to regenerative therapies. Nevertheless, we believe that our defined culture conditions could serve as a powerful platform that can be used to identify new compounds that enhance cardiac reprogramming and reduce genetic manipulation, which would be highly desirable for the development of pharmacological cardiac regeneration.