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In vivo reprogramming of murine cardiac fibroblasts into induced cardiomyocytes

Abstract

The reprogramming of adult cells into pluripotent cells or directly into alternative adult cell types holds great promise for regenerative medicine. We reported previously that cardiac fibroblasts, which represent 50% of the cells in the mammalian heart, can be directly reprogrammed to adult cardiomyocyte-like cells in vitro by the addition of Gata4, Mef2c and Tbx5 (GMT). Here we use genetic lineage tracing to show that resident non-myocytes in the murine heart can be reprogrammed into cardiomyocyte-like cells in vivo by local delivery of GMT after coronary ligation. Induced cardiomyocytes became binucleate, assembled sarcomeres and had cardiomyocyte-like gene expression. Analysis of single cells revealed ventricular cardiomyocyte-like action potentials, beating upon electrical stimulation, and evidence of electrical coupling. In vivo delivery of GMT decreased infarct size and modestly attenuated cardiac dysfunction up to 3 months after coronary ligation. Delivery of the pro-angiogenic and fibroblast-activating peptide, thymosin β4, along with GMT, resulted in further improvements in scar area and cardiac function. These findings demonstrate that cardiac fibroblasts can be reprogrammed into cardiomyocyte-like cells in their native environment for potential regenerative purposes.

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Figure 1: Genetic lineage tracing demonstrates in vivo reprogramming of cardiac fibroblasts to CM-like cells.
Figure 2: Cellular analysis of the degree of in vivo cardiac reprogramming.
Figure 3: Electrophysiological properties of iCMs.
Figure 4: In vivo delivery of cardiac reprogramming factors improves cardiac function after myocardial infarction.

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Acknowledgements

We are grateful for technical assistance from the Gladstone Histology Core (C. Miller), Gladstone Genomics Core (L. Ta, Y. Hao, B. Chadwick), UCSF MRI Core (M. Wendland, J. Hawkins) and Laboratory for Cell Analysis at UCSF (S. Elmes). We thank all the members of the Srivastava laboratory for helpful discussions; G. Howard and B. Taylor for editorial help; and B. Bruneau and B. Conklin for helpful discussions and critical reviews of the manuscript. We also thank J. Nerbonne, N. Foeger, and members of the Nerbonne laboratory for assistance with the adult myocyte isolation protocol. L.Q. is a postdoctoral scholar of the California Institute for Regenerative Medicine (CIRM). V.V. is supported by grants from the GlaxoSmithKline Research and Education Foundation and the NIH/NHLBI (K08HL101989). J.-d.F. is supported by a postdoctoral fellowship from American Heart Association. S.J.C. was supported by R01 HL060714 from NHLBI/NIH. D.S. was supported by grants from NHLBI/NIH, CIRM, the Younger Family Foundation, Roddenberry Foundation and the L.K. Whittier Foundation. This work was supported by NIH/NCRR grant (C06 RR018928) to the Gladstone Institutes.

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Authors

Contributions

L.Q. designed, supervised and performed the experiments. Y.H. performed all surgeries, echoes and ECGs, and contributed to tissue sectioning and sample preparation. C.I.S. performed all cellular electrophysiology experiments. A.F. quantified scar size and induced CMs and helped with mouse colony maintenance. V.V. helped with isolation of adult CMs and implantation of transmitters. S.J.C. provided periostin-Cre:Rosa26-lacZ mice and supplemental data. J.-d.F. provided initial reagents and technical knowledge and helpful discussion. D.S. designed and supervised the work. L.Q. and D.S. wrote the manuscript.

Corresponding author

Correspondence to Deepak Srivastava.

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Competing interests

D.S. is a member of the Scientific Advisory Board of iPierian Inc., and RegeneRx Pharmaceuticals.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-14 and legends for Supplementary Movies 1-2. (PDF 7082 kb)

Supplementary Movie 1

This file contains four videomicroscopy segments, edited together, showing a pair of Fluo-4-loaded myocytes – see Supplementary Information file for full legend. (MOV 8570 kb)

Supplementary Movie 2

This file contains a high-speed camera recording shows contraction of an induced cardiomyocyte (iCM) and an endogenous cardiomyocyte (CM) upon electrical stimulation – see Supplementary Information file for full legend. (MOV 5025 kb)

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Qian, L., Huang, Y., Spencer, C. et al. In vivo reprogramming of murine cardiac fibroblasts into induced cardiomyocytes. Nature 485, 593–598 (2012). https://doi.org/10.1038/nature11044

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