Direct differentiation of rat skin fibroblasts into cardiomyocytes

Abstract

Myocardial infarction (MI) is one of the major cardiovascular diseases caused by diminished supply of nutrients and oxygen to the heart due to obstruction of the coronary artery. Different treatment options are available for cardiac diseases, however, they do not completely repair the damage. Therefore, reprogramming terminally differentiated fibroblasts using transcription factors is a promising strategy to differentiate them into cardiac like cells in vitro and to increase functional cardiomyocytes and reduce fibrotic scar in vivo. In this study, skin fibroblasts were selected for reprogramming because they serve as a convenient source for the autologous cell therapy. Fibroblasts were isolated from skin of rat pups, propagated, and directly reprogrammed towards cardiac lineage. For reprogramming, two different approaches were adopted, i.e., cells were transfected with: (1) combination of cardiac transcription factors; GATA4, MEF2c, Nkx2.5 (GMN), and (2) combination of cardiac transcription factors; GATA4, MEF2c, Nkx2.5, and iPSC factors; Oct4, Klf4, Sox2 and cMyc (GMNO). After 72 h of transfection, cells were analyzed for the expression of cardiac markers at the mRNA and protein levels. For in vivo study, rat MI models were developed by ligating the left anterior descending coronary artery and the reprogrammed cells were transplanted in the infarcted heart. qPCR results showed that the reprogrammed cells exhibited significant upregulation of cardiac genes. Immunocytochemistry analysis further confirmed cardiomyogenic differentiation of the reprogrammed cells. For the assessment of cardiac function, animals were analyzed via echocardiography after 2 and 4 weeks of cell transplantation. Echocardiographic results showed that the hearts transplanted with the reprogrammed cells improved ejection fraction, fractional shortening, left ventricular internal systolic and diastolic dimensions, and end systolic and diastolic volumes. After 4 weeks of cell transplantation, heart tissues were harvested and processed for histology. The histological analysis showed that the reprogrammed cells improved wall thickness of left ventricle and reduced fibrosis significantly as compared to the control. It is concluded from the study that novel combination of cardiac transcription factors directly reprogrammed skin fibroblasts and differentiated them into cardiomyocytes. These differentiated cells showed cardiomyogenic characters in vitro, and reduced fibrosis and improved cardiac function in vivo. Furthermore, direct reprogramming of fibroblasts transfected with cardiac transcription factors showed better regeneration of the injured myocardium and improved cardiac function as compared to the indirect approach in which combination of cardiac and iPSC factors were used. The study after further optimization could be used as a better strategy for cell-based therapeutic approaches for cardiovascular diseases.

Keywords: Cardiac regeneration; Cell reprogramming; Myocardial infarction; Transcription factors.