There is an urgent clinical need to develop new therapeutic approaches to treat heart failure, but the biology of cardiovascular regeneration is complex. A new generation of reprogramming technology involves trans-differentiating one adult somatic cell type directly into another. We reported previously that administration of gene transfer vectors encoding Gata4 (G), Mef 2c (M) and Tbx5 (T), reprograms rat cardiac fibroblasts into induced cardiomyocytes (iCMs) in vitro and improves cardiac function in myocardial infarction models. Previous cardiac reprogramming studies were restricted to rodent and human fibroblasts. However studies in large animal models relevant for pre-clinical studies is lacking.
The Aim of the present study was to determine the optimal combination of factors necessary for direct reprogramming of porcine fibroblasts towards a cardiomyocyte lineage for the first time. Here we have used human lentiviral system to express various cardiomyocyte enriched transcriptional regulators such as GMT, Hand2, myocardin, and two microRNAs, miR-590 and mir-199, in porcine cardiac fibroblasts. Reprogramming of fibroblasts into iCMs was determined 4 weeks post-virus transduction by FACS analysis for the activation of endogenous cardiac troponin T (cTnT).
GMT alone was not sufficient to reprogram porcine fibroblasts although this combination was shown previously to be sufficient to convert rodent fibroblasts into iCMs. Addition of mir-199 to GMT was also not sufficient to promote reprogramming. However, adding miR-590 or Hand2, and myocardin (HM) to GMT resulted in reprogramming, although the efficiency remained low. Together, our results show that combination of 5 distinct transcription regulators (GMTHM) or GMT plus mir-590 is necessary for reprogramming porcine fibroblast.
These findings demonstrate that pig fibroblasts can be directly reprogrammed toward the cardiomyocyte lineage, and represent a step toward possible therapeutic application of this reprogramming approach in a pre-clinical setting.