Transient Reprogramming of Neonatal Cardiomyocytes to a Proliferative Dedifferentiated State

Abstract

Zebrafish and urodele amphibians are capable of extraordinary myocardial regeneration thanks to the ability of their cardiomyocytes to undergo transient dedifferentiation and proliferation. Somatic cells can be temporarily reprogrammed to a proliferative, dedifferentiated state through transient expression of Oct3/4, Sox2, Klf4 and c-Myc (OSKM) transcription factors. Here, we utilized an OSKM-encoding non-integrating vector to induce transient reprogramming of mammalian cardiomyocytes in vitro. Reprogramming factor expression in neonatal rat cardiomyocytes triggered rapid cell dedifferentiation characterized by downregulation of cardiomyocyte specific gene and protein expression, sarcomere dis-assembly and loss of autorhythmic contractile activity. Concomitantly, a significant increase in cell cycle related gene expression and Ki67 positive cells was observed, indicating that dedifferentiated cardiomyocytes possess an enhanced proliferative capacity. A small proportion of cardiomyocytes progressed through mesenchymal to epithelial transition, further indicating the initiation of cell reprogramming. However, complete reprogramming to a pluripotent-like state was not achieved for the duration of the study (20 days), both in standard and embryonic stem cell culture media conditions. The transient nature of this partial reprogramming response was confirmed as cardiomyocyte-specific cell morphology, gene expression and contractile activity were recovered by day 15 after viral transduction. Further investigations into the complete downstream biological effects of ectopic OSKM expression in cardiomyocytes and the fate of these reprogrammed cells are warranted. Our results to date suggest that transient reprogramming could be a feasible strategy to recapitulate regenerative mechanisms of lower vertebrates and inform direct gene therapy approaches to cardiac regenerative medicine.