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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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References

  1. Murry, C. E. & Keller, G. Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development. Cell 132, 661–680 (2008)

    Article  CAS  Google Scholar 

  2. Passier, R., van Laake, L. W. & Mummery, C. L. Stem-cell-based therapy and lessons from the heart. Nature 453, 322–329 (2008)

    Article  ADS  CAS  Google Scholar 

  3. Srivastava, D. & Ivey, K. N. Potential of stem-cell-based therapies for heart disease. Nature 441, 1097–1099 (2006)

    Article  ADS  CAS  Google Scholar 

  4. Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676 (2006)

    Article  CAS  Google Scholar 

  5. Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007)

    Article  CAS  Google Scholar 

  6. Yamanaka, S. & Blau, H. M. Nuclear reprogramming to a pluripotent state by three approaches. Nature 465, 704–712 (2010)

    Article  ADS  CAS  Google Scholar 

  7. Ieda, M. et al. Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors. Cell 142, 375–386 (2010)

    Article  CAS  Google Scholar 

  8. Szabo, E. et al. Direct conversion of human fibroblasts to multilineage blood progenitors. Nature 468, 521–526 (2010)

    Article  ADS  CAS  Google Scholar 

  9. Vierbuchen, T. et al. Direct conversion of fibroblasts to functional neurons by defined factors. Nature 463, 1035–1041 (2010)

    Article  ADS  CAS  Google Scholar 

  10. Zhou, Q., Brown, J., Kanarek, A., Rajagopal, J. & Melton, D. A. In vivo reprogramming of adult pancreatic exocrine cells to β-cells. Nature 455, 627–632 (2008)

    Article  ADS  CAS  Google Scholar 

  11. Huang, P. et al. Induction of functional hepatocyte-like cells from mouse fibroblasts by defined factors. Nature 475, 386–389 (2011)

    Article  CAS  Google Scholar 

  12. Silva, J. et al. Promotion of reprogramming to ground state pluripotency by signal inhibition. PLoS Biol. 6, e253 (2008)

    Article  Google Scholar 

  13. Ieda, M. et al. Cardiac fibroblasts regulate myocardial proliferation through β1 integrin signaling. Dev. Cell 16, 233–244 (2009)

    Article  CAS  Google Scholar 

  14. Byun, J. et al. Myocardial injury-induced fibroblast proliferation facilitates retroviral-mediated gene transfer to the rat heart in vivo . J. Gene Med. 2, 2–10 (2000)

    Article  CAS  Google Scholar 

  15. Snider, P. et al. Origin of cardiac fibroblasts and the role of periostin. Circ. Res. 105, 934–947 (2009)

    Article  ADS  CAS  Google Scholar 

  16. Baudino, T. A., Carver, W., Giles, W. & Borg, T. K. Cardiac fibroblasts: friend or foe? Am. J. Physiol. Heart Circ. Physiol. 291, H1015–H1026 (2006)

    Article  CAS  Google Scholar 

  17. Camelliti, P., Borg, T. K. & Kohl, P. Structural and functional characterisation of cardiac fibroblasts. Cardiovasc. Res. 65, 40–51 (2005)

    Article  CAS  Google Scholar 

  18. Snider, P. et al. Periostin is required for maturation and extracellular matrix stabilization of noncardiomyocyte lineages of the heart. Circ. Res. 102, 752–760 (2008)

    Article  CAS  Google Scholar 

  19. Takeda, N. et al. Cardiac fibroblasts are essential for the adaptive response of the murine heart to pressure overload. J. Clin. Invest. 120, 254–265 (2010)

    Article  CAS  Google Scholar 

  20. Soriano, P. Generalized lacZ expression with the ROSA26 Cre reporter strain. Nature Genet. 21, 70–71 (1999)

    Article  CAS  Google Scholar 

  21. Bhowmick, N. A. et al. TGF-β signaling in fibroblasts modulates the oncogenic potential of adjacent epithelia. Science 303, 848–851 (2004)

    Article  ADS  CAS  Google Scholar 

  22. Murry, C. E., Kay, M. A., Bartosek, T., Hauschka, S. D. & Schwartz, S. M. Muscle differentiation during repair of myocardial necrosis in rats via gene transfer with MyoD. J. Clin. Invest. 98, 2209–2217 (1996)

    Article  CAS  Google Scholar 

  23. Kisanuki, Y. Y. et al. Tie2-Cre transgenic mice: A new model for endothelial cell-lineage analysis in vivo . Dev. Biol. 230, 230–242 (2001)

    Article  CAS  Google Scholar 

  24. Sohal, D. S. et al. Temporally regulated and tissue-specific gene manipulations in the adult and embryonic heart using a tamoxifen-inducible Cre protein. Circ. Res. 89, 20–25 (2001)

    Article  CAS  Google Scholar 

  25. Hsieh, P. C. et al. Evidence from a genetic fate-mapping study that stem cells refresh adult mammalian cardiomyocytes after injury. Nature Med. 13, 970–974 (2007)

    Article  CAS  Google Scholar 

  26. Li, J. et al. Cardiac-specific loss of N-cadherin leads to alteration in connexins with conduction slowing and arrhythmogenesis. Circ. Res. 97, 474–481 (2005)

    Article  CAS  Google Scholar 

  27. Saffitz, J. E., Laing, J. G. & Yamada, K. A. Connexin expression and turnover: implications for cardiac excitability. Circ. Res. 86, 723–728 (2000)

    Article  CAS  Google Scholar 

  28. Bock-Marquette, I., Saxena, A., White, M. D., Dimaio, J. M. & Srivastava, D. Thymosin β4 activates integrin-linked kinase and promotes cardiac cell migration, survival and cardiac repair. Nature 432, 466–472 (2004)

    Article  ADS  CAS  Google Scholar 

  29. Goldstein, A. L., Hannappel, E. & Kleinman, H. K. Thymosin β4: actin-sequestering protein moonlights to repair injured tissues. Trends Mol. Med. 11, 421–429 (2005)

    Article  CAS  Google Scholar 

  30. Hinkel, R. et al. Thymosin β4 is an essential paracrine factor of embryonic endothelial progenitor cell-mediated cardioprotection. Circulation 117, 2232–2240 (2008)

    Article  CAS  Google Scholar 

  31. Smart, N. et al. Thymosin β4 induces adult epicardial progenitor mobilization and neovascularization. Nature 445, 177–182 (2007)

    Article  ADS  CAS  Google Scholar 

  32. Bock-Marquette, I. et al. Thymosin β4 mediated PKC activation is essential to initiate the embryonic coronary developmental program and epicardial progenitor cell activation in adult mice in vivo . J. Mol. Cell. Cardiol. 46, 728–738 (2009)

    Article  CAS  Google Scholar 

  33. Smart, N. et al. De novo cardiomyocytes from within the activated adult heart after injury. Nature 474, 640–644 (2011)

    Article  CAS  Google Scholar 

  34. Qian, L. et al. miR-24 inhibits apoptosis and represses Bim in mouse cardiomyocytes. J. Exp. Med. 208, 549 (2011)

    Article  CAS  Google Scholar 

  35. Larsen, T. H., Saetersdal, T. & Grong, K. The ultrastructure of the myocyte in different regions of experimental infarcts in the cat heart. Res. Exp. Med. (Berl.) 186, 295–306 (1986)

    Article  CAS  Google Scholar 

  36. Xu, H., Guo, W. & Nerbonne, J. M. Four kinetically distinct depolarization-activated K+ currents in adult mouse ventricular myocytes. J. Gen. Physiol. 113, 661–678 (1999)

    Article  CAS  Google Scholar 

  37. Fenske, S. et al. HCN3 contributes to the ventricular action potential waveform in the murine heart. Circ. Res. 109, 1015–1023 (2011)

    Article  CAS  Google Scholar 

  38. Kurrelmeyer, K. M. et al. Endogenous tumor necrosis factor protects the adult cardiac myocyte against ischemic-induced apoptosis in a murine model of acute myocardial infarction. Proc. Natl Acad. Sci. USA 97, 5456–5461 (2000)

    Article  ADS  CAS  Google Scholar 

  39. Cheng, H., Lederer, W. J. & Cannell, M. B. Calcium sparks: elementary events underlying excitation–contraction coupling in heart muscle. Science 262, 740–744 (1993)

    Article  ADS  CAS  Google Scholar 

  40. Ljubojević, S. et al. In situ calibration of nucleoplasmic versus cytoplasmic Ca2+ concentration in adult cardiomyocytes. Biophys. J. 100, 2356–2366 (2011)

    Article  ADS  Google Scholar 

Download references

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 and Affiliations

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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