Tamer M A Mohamed1, Nicole R Stone1, Emily C Berry1, Ethan Radzinsky1, Yu Huang1, Karishma Pratt1, Yen-Sin Ang1, Pengzhi Yu1, Haixia Wang1, Shibing Tang1, Sergey Magnitsky1, Sheng Ding1, Kathryn N Ivey1, Deepak Srivastava2. 1. From Gladstone Institute of Cardiovascular Disease and Roddenberry Center for Stem Cell Biology and Medicine, San Francisco, CA (T.M.A.M., N.R.S., E.C.B., E.R., Y.H., K.P., Y.-S.A., P.Y., H.W., S.T., S.D., K.N.I., D.S.); Institute of Cardiovascular Sciences, University of Manchester, UK (T.M.A.M.); Faculty of Pharmacy, Zagazig University, Egypt (T.M.A.M.); and Departments of Radiology (S.M.), Pharmaceutical Chemistry (S.D.), Pediatrics (K.N.I., D.S.), and Biochemistry and Biophysics (D.S.), University of California San Francisco. 2. From Gladstone Institute of Cardiovascular Disease and Roddenberry Center for Stem Cell Biology and Medicine, San Francisco, CA (T.M.A.M., N.R.S., E.C.B., E.R., Y.H., K.P., Y.-S.A., P.Y., H.W., S.T., S.D., K.N.I., D.S.); Institute of Cardiovascular Sciences, University of Manchester, UK (T.M.A.M.); Faculty of Pharmacy, Zagazig University, Egypt (T.M.A.M.); and Departments of Radiology (S.M.), Pharmaceutical Chemistry (S.D.), Pediatrics (K.N.I., D.S.), and Biochemistry and Biophysics (D.S.), University of California San Francisco. deepak.srivastava@gladstone.ucsf.edu.
Abstract
BACKGROUND: Reprogramming of cardiac fibroblasts into induced cardiomyocyte-like cells in situ represents a promising strategy for cardiac regeneration. A combination of 3 cardiac transcription factors, Gata4, Mef2c, and Tbx5 (GMT), can convert fibroblasts into induced cardiomyocyte-like cells, albeit with low efficiency in vitro. METHODS: We screened 5500 compounds in primary cardiac fibroblasts to identify the pathways that can be modulated to enhance cardiomyocyte reprogramming. RESULTS: We found that a combination of the transforming growth factor-β inhibitor SB431542 and the WNT inhibitor XAV939 increased reprogramming efficiency 8-fold when added to GMT-overexpressing cardiac fibroblasts. The small molecules also enhanced the speed and quality of cell conversion; we observed beating cells as early as 1 week after reprogramming compared with 6 to 8 weeks with GMT alone. In vivo, mice exposed to GMT, SB431542, and XAV939 for 2 weeks after myocardial infarction showed significantly improved reprogramming and cardiac function compared with those exposed to only GMT. Human cardiac reprogramming was similarly enhanced on transforming growth factor-β and WNT inhibition and was achieved most efficiently with GMT plus myocardin. CONCLUSIONS: Transforming growth factor-β and WNT inhibitors jointly enhance GMT-induced direct cardiac reprogramming from cardiac fibroblasts in vitro and in vivo and provide a more robust platform for cardiac regeneration.
BACKGROUND: Reprogramming of cardiac fibroblasts into induced cardiomyocyte-like cells in situ represents a promising strategy for cardiac regeneration. A combination of 3 cardiac transcription factors, Gata4, Mef2c, and Tbx5 (GMT), can convert fibroblasts into induced cardiomyocyte-like cells, albeit with low efficiency in vitro. METHODS: We screened 5500 compounds in primary cardiac fibroblasts to identify the pathways that can be modulated to enhance cardiomyocyte reprogramming. RESULTS: We found that a combination of the transforming growth factor-β inhibitor SB431542 and the WNT inhibitor XAV939 increased reprogramming efficiency 8-fold when added to GMT-overexpressing cardiac fibroblasts. The small molecules also enhanced the speed and quality of cell conversion; we observed beating cells as early as 1 week after reprogramming compared with 6 to 8 weeks with GMT alone. In vivo, mice exposed to GMT, SB431542, and XAV939 for 2 weeks after myocardial infarction showed significantly improved reprogramming and cardiac function compared with those exposed to only GMT. Human cardiac reprogramming was similarly enhanced on transforming growth factor-β and WNT inhibition and was achieved most efficiently with GMT plus myocardin. CONCLUSIONS: Transforming growth factor-β and WNT inhibitors jointly enhance GMT-induced direct cardiac reprogramming from cardiac fibroblasts in vitro and in vivo and provide a more robust platform for cardiac regeneration.
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