Abstract 33: Unlocking Reprogramming Capability: Silencing Antiplasticity Gene P63 Enhances the Reprogramming of Fibroblasts into Induced Cardiomyocytes

Objective: In situ cellular reprogramming of cardiac fibroblasts into (induced) cardiomyocytes (iCMs) represents a promising new potential intervention for the treatment of heart failure. Despite encouraging in vivo data in rodent myocardial infarction models, the relative resistance of human cells to reprogramming may be a significant barrier to the clinical application of this new therapy. We hypothesized that knockdown of the anti-plasticity gene p63 could therefore be used to enhance cellular reprogramming efficiency. Methods: p63 knockout (KO) murine embryonic fibroblasts (MEFs) and MEFs treated with p63 silencing shRNA were assessed for expression of the cardiomyocyte marker Cardiac Troponin T (cTnT) and pro-cardiogenic genes, with or without the treatment with known cardiac transcription factors Hand2 and Myocardin (HM). Results: After 3 wks in culture, expression of the cardiomyocyte marker cTnT (FACS) was significantly greater in p63 KO MEFs than in wild-type (WT) MEFs or WT MEFs treated with transcription factors Hand2 and Myocardin (39% ± 8%, 2.0% ± 1% and 2.7 ± 0.3%, respectively, p < 0.05). Treatment of p63 KO MEFs with Hand2 and Myocardin further increased cTnT expression up to 74% ± 3%. Treatment of WT MEFs with p63 shRNA likewise yielded a 20-fold increase in cTnT expression (qPCR) without HM and a 600-fold increase with HM when compared to non-silencing shRNA treated MEFs. Consistent with these findings, p63 KO or p63 shRNA-treated MEFs demonstrated increased expression (qPCR) of pro-cardiogenic genes Gata4, Mef2c and Tbx5 compared to naïve or non-silencing shRNA treated MEFs. After treatment with p63 shRNA, adult human epidermal cells also demonstrated increased expression of cTnT, myosin heavy chain and pro-cardiogenic genes when analyzed by qPCR. Conclusions: Downregulation of the anti-plasticity gene p63 enhances cellular reprogramming efficiency and iCM generation, as reflected in the increased expression of the cardiomyocyte marker cTnT and pro-cardiogenic genes Gata4, Mef2c and Tbx5. Use of such cellular plasticity enhancing strategies may be a useful strategy to overcome barriers to cellular reprogramming in the clinical arena.