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Direct conversion of quiescent cardiomyocytes to pacemaker cells by expression of Tbx18

Nature Biotechnology volume 31, pages 54–62 (2013)Cite this article

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Abstract

The heartbeat originates within the sinoatrial node (SAN), a small structure containing <10,000 genuine pacemaker cells. If the SAN fails, the ∼5 billion working cardiomyocytes downstream of it become quiescent, leading to circulatory collapse in the absence of electronic pacemaker therapy. Here we demonstrate conversion of rodent cardiomyocytes to SAN cells in vitro and in vivo by expression of Tbx18, a gene critical for early SAN specification. Within days of in vivo Tbx18 transduction, 9.2% of transduced, ventricular cardiomyocytes develop spontaneous electrical firing physiologically indistinguishable from that of SAN cells, along with morphological and epigenetic features characteristic of SAN cells. In vivo, focal Tbx18 gene transfer in the guinea-pig ventricle yields ectopic pacemaker activity, correcting a bradycardic disease phenotype. Myocytes transduced in vivo acquire the cardinal tapering morphology and physiological automaticity of native SAN pacemaker cells. The creation of induced SAN pacemaker (iSAN) cells opens new prospects for bioengineered pacemakers.

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Figure 1: Tbx18-transduced NRVMs become spontaneously beating cardiomyocytes.
Figure 2: Tbx18-transduced myocytes recapitulate major calcium clock characteristics of genuine SAN pacemakers.
Figure 3: Morphological, epigenetic and functional features of Tbx18-transduced myocytes.
Figure 4: Tbx18 converts adult guinea pig ventricular myocytes into pacemaker cells in vivo.
Figure 5: De novo automaticity responds to autonomic regulation.
Figure 6: Single-cell, qRT-PCR of long-term Tbx18-VMs indicates persistent automaticity even after Tbx18 expression had waned.

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References

  1. Chien, K.R., Domian, I.J. & Parker, K.K. Cardiogenesis and the complex biology of regenerative cardiovascular medicine. Science 322, 1494–1497 (2008).

    Article  CAS  Google Scholar 

  2. Bleeker, W.K., Mackaay, A.J., Masson-Pevet, M., Bouman, L.N. & Becker, A.E. Functional and morphological organization of the rabbit sinus node. Circ. Res. 46, 11–22 (1980).

    Article  CAS  Google Scholar 

  3. Cho, H.C. & Marbán, E. Biological therapies for cardiac arrhythmias: can genes and cells replace drugs and devices? Circ. Res. 106, 674–685 (2010).

    Article  CAS  Google Scholar 

  4. Miake, J., Marban, E. & Nuss, H.B. Biological pacemaker created by gene transfer. Nature 419, 132–133 (2002).

    Article  CAS  Google Scholar 

  5. Qu, J. et al. Expression and function of a biological pacemaker in canine heart. Circulation 107, 1106–1109 (2003).

    Article  Google Scholar 

  6. Kehat, I. et al. Electromechanical integration of cardiomyocytes derived from human embryonic stem cells. Nat. Biotechnol. 22, 1282–1289 (2004).

    Article  CAS  Google Scholar 

  7. Xue, T. et al. Functional integration of electrically active cardiac derivatives from genetically engineered human embryonic stem cells with quiescent recipient ventricular cardiomyocytes: insights into the development of cell-based pacemakers. Circulation 111, 11–20 (2005).

    Article  Google Scholar 

  8. Ripplinger, C.M. & Bers, D.M. Human biological pacemakers: intrinsic variability and stability. Circulation 125, 856–858 (2012).

    Article  Google Scholar 

  9. Christoffels, V.M., Smits, G.J., Kispert, A. & Moorman, A.F. Development of the pacemaker tissues of the heart. Circ. Res. 106, 240–254 (2010).

    Article  CAS  Google Scholar 

  10. Blaschke, R.J. et al. Targeted mutation reveals essential functions of the homeodomain transcription factor Shox2 in sinoatrial and pacemaking development. Circulation 115, 1830–1838 (2007).

    Article  CAS  Google Scholar 

  11. Espinoza-Lewis, R.A. et al. Shox2 is essential for the differentiation of cardiac pacemaker cells by repressing Nkx2–5. Dev. Biol. 327, 376–385 (2009).

    Article  CAS  Google Scholar 

  12. Hoogaars, W.M. et al. Tbx3 controls the sinoatrial node gene program and imposes pacemaker function on the atria. Genes Dev. 21, 1098–1112 (2007).

    Article  CAS  Google Scholar 

  13. Mori, A.D. et al. Tbx5-dependent rheostatic control of cardiac gene expression and morphogenesis. Dev. Biol. 297, 566–586 (2006).

    Article  CAS  Google Scholar 

  14. Wiese, C. et al. Formation of the sinus node head and differentiation of sinus node myocardium are independently regulated by Tbx18 and Tbx3. Circ. Res. 104, 388–397 (2009).

    Article  CAS  Google Scholar 

  15. Kapoor, N., Galang, G., Marbán, E. & Cho, H.C. Transcriptional suppression of Connexin43 by Tbx18 undermines cell-cell electrical coupling in postnatal cardiomyocytes. J. Biol. Chem. 286, 14073–14079 (2011).

    Article  CAS  Google Scholar 

  16. Mangoni, M.E. & Nargeot, J. Genesis and regulation of the heart automaticity. Physiol. Rev. 88, 919–982 (2008).

    Article  CAS  Google Scholar 

  17. DiFrancesco, D. The role of the funny current in pacemaker activity. Circ. Res. 106, 434–446 (2010).

    Article  CAS  Google Scholar 

  18. Nakayama, T., Kurachi, Y., Noma, A. & Irisawa, H. Action potential and membrane currents of single pacemaker cells of the rabbit heart. Pflugers Archiv: Eur. J. Physiol. 402, 248–257 (1984).

    Article  CAS  Google Scholar 

  19. van Ginneken, A.C. & Giles, W. Voltage clamp measurements of the hyperpolarization-activated inward current I(f) in single cells from rabbit sino-atrial node. J. Physiol. (Lond.) 434, 57–83 (1991).

    Article  CAS  Google Scholar 

  20. Lakatta, E.G., Maltsev, V.A. & Vinogradova, T.M. A coupled SYSTEM of intracellular Ca2+ clocks and surface membrane voltage clocks controls the timekeeping mechanism of the heart's pacemaker. Circ. Res. 106, 659–673 (2010).

    Article  CAS  Google Scholar 

  21. Bogdanov, K.Y., Vinogradova, T.M. & Lakatta, E.G. Sinoatrial nodal cell ryanodine receptor and Na+-Ca2+ exchanger: molecular partners in pacemaker regulation. Circ. Res. 88, 1254–1258 (2001).

    Article  CAS  Google Scholar 

  22. Vinogradova, T.M. et al. High basal protein kinase A-dependent phosphorylation drives rhythmic internal Ca2+ store oscillations and spontaneous beating of cardiac pacemaker cells. Circ. Res. 98, 505–514 (2006).

    Article  CAS  Google Scholar 

  23. Lyashkov, A.E. et al. Calcium cycling protein density and functional importance to automaticity of isolated sinoatrial nodal cells are independent of cell size. Circ. Res. 100, 1723–1731 (2007).

    Article  CAS  Google Scholar 

  24. Honjo, H., Boyett, M.R., Kodama, I. & Toyama, J. Correlation between electrical activity and the size of rabbit sino-atrial node cells. J. Physiol. (Lond.) 496, 795–808 (1996).

    Article  CAS  Google Scholar 

  25. Trautwein, W. & Uchizono, K. Electron microscopic and electrophysiologic study of the pacemaker in the sino-atrial node of the rabbit heart. Z. Zellforsch. Mikrosk. Anat. 61, 96–109 (1963).

    Article  CAS  Google Scholar 

  26. Cao, R. et al. Role of histone H3 lysine 27 methylation in Polycomb-group silencing. Science 298, 1039–1043 (2002).

    Article  CAS  Google Scholar 

  27. Santos-Rosa, H. et al. Active genes are tri-methylated at K4 of histone H3. Nature 419, 407–411 (2002).

    Article  CAS  Google Scholar 

  28. Cho, H.C., Kashiwakura, Y. & Marbán, E. Creation of a biological pacemaker by cell fusion. Circ. Res. 100, 1112–1115 (2007).

    Article  CAS  Google Scholar 

  29. Kashiwakura, Y., Cho, H.C., Barth, A.S., Azene, E. & Marbán, E. Gene transfer of a synthetic pacemaker channel into the heart: a novel strategy for biological pacing. Circulation 114, 1682–1686 (2006).

    Article  Google Scholar 

  30. Goldberger, E. How to Interpret Electrocardiograms in Terms of Vectors: a Practical Manual (Charles C Thomas, Springfield, Ill, 1968).

  31. 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 

  32. Yu, J. et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917–1920 (2007).

    Article  CAS  Google Scholar 

  33. Cai, C.L. et al. A myocardial lineage derives from Tbx18 epicardial cells. Nature 454, 104–108 (2008).

    Article  CAS  Google Scholar 

  34. Christoffels, V.M. et al. Tbx18 and the fate of epicardial progenitors. Nature 458, E8–9. E discussion 458, 9–10 (2009).

    Article  CAS  Google Scholar 

  35. Rosemann, M. et al. Allelic imbalance at intragenic markers of Tbx18 is a hallmark of murine osteosarcoma. Carcinogenesis 24, 371–376 (2003).

    Article  CAS  Google Scholar 

  36. Kojima, M., Sada, H. & Sperelakis, N. Developmental changes in beta-adrenergic and cholinergic interactions on calcium-dependent slow action potentials in rat ventricular muscles. Br. J. Pharmacol. 99, 327–333 (1990).

    Article  CAS  Google Scholar 

  37. Nagashima, M. et al. Alternation of inwardly rectifying background K+ channel during development of rat fetal cardiomyocytes. J. Mol. Cell. Cardiol. 33, 533–543 (2001).

    Article  CAS  Google Scholar 

  38. Zhang, Y. et al. Dedifferentiation and proliferation of mammalian cardiomyocytes. PLoS ONE 5, e12559 (2010).

    Article  Google Scholar 

  39. Izumo, S., Nadal-Ginard, B. & Mahdavi, V. Protooncogene induction and reprogramming of cardiac gene expression produced by pressure overload. Proc. Natl. Acad. Sci. USA 85, 339–343 (1988).

    Article  CAS  Google Scholar 

  40. Dobrzynski, H. et al. Computer three-dimensional reconstruction of the sinoatrial node. Circulation 111, 846–854 (2005).

    Article  CAS  Google Scholar 

  41. Sola, C. et al. Atrial natriuretic factor in the vena cava and sinus node. J. Histochem. Cytochem. 38, 1123–1135 (1990).

    Article  CAS  Google Scholar 

  42. Komuro, I. & Izumo, S. Csx: a murine homeobox-containing gene specifically expressed in the developing heart. Proc. Natl. Acad. Sci. USA 90, 8145–8149 (1993).

    Article  CAS  Google Scholar 

  43. Lints, T.J., Parsons, L.M., Hartley, L., Lyons, I. & Harvey, R.P. Nkx-2.5: a novel murine homeobox gene expressed in early heart progenitor cells and their myogenic descendants. Development 119, 419–431 (1993).

    CAS  PubMed  Google Scholar 

  44. Espinoza-Lewis, R.A. et al. Ectopic expression of Nkx2.5 suppresses the formation of the sinoatrial node in mice. Dev. Biol. 356, 359–369 (2011).

    Article  CAS  Google Scholar 

  45. Li, F., Wang, X., Capasso, J.M. & Gerdes, A.M. Rapid transition of cardiac myocytes from hyperplasia to hypertrophy during postnatal development. J. Mol. Cell. Cardiol. 28, 1737–1746 (1996).

    Article  CAS  Google Scholar 

  46. Walsh, S., Ponten, A., Fleischmann, B.K. & Jovinge, S. Cardiomyocyte cell cycle control and growth estimation in vivo–an analysis based on cardiomyocyte nuclei. Cardiovasc. Res. 86, 365–373 (2010).

    Article  CAS  Google Scholar 

  47. 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 

  48. Weintraub, H. et al. Activation of muscle-specific genes in pigment, nerve, fat, liver, and fibroblast cell lines by forced expression of MyoD. Proc. Natl. Acad. Sci. USA 86, 5434–5438 (1989).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  52. Qian, L. et al. In vivo reprogramming of murine cardiac fibroblasts into induced cardiomyocytes. Nature 485, 593–598 (2012).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  54. Bakker, M.L. et al. T-box transcription factor TBX3 reprograms mature cardiac myocytes into pacemaker-like cells. Cardiovasc. Res. (2012).

  55. Cheng, H. et al. Amplitude distribution of calcium sparks in confocal images: theory and studies with an automatic detection method. Biophys. J. 76, 606–617 (1999).

    Article  CAS  Google Scholar 

  56. Picht, E., Zima, A.V., Blatter, L.A. & Bers, D.M. SparkMaster: automated calcium spark analysis with ImageJ. Am. J. Physiol. Cell Physiol. 293, C1073–C1081 (2007).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank G. Galang, R. Rafi, B. Sun and W. Liu for technical assistance; S. Groenke of J.I. Goldhaber's laboratory for help with SAN tissue dissection; and D. Sareen of Clive Svendsen's laboratory for RNA samples of human iPSCs and the parental fibroblasts. The study was supported by the Cedars-Sinai Board of Governors Heart Stem Cell Center. E.M. holds the Mark S. Siegel Family Professorship. N.K. is supported by the Heart Rhythm Society fellowship. W.L. is supported by the Heart and Stroke Foundation of Canada fellowship. H.C.C. is supported by grants from the American Heart Association (12SDG9020030) and NHLBI (1R01HL111646-01A1).

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  1. Nidhi Kapoor and Wenbin Liang: These authors contributed equally to this work.

Authors and Affiliations

  1. The Cedars-Sinai Heart Institute, Los Angeles, California, USA

    Nidhi Kapoor, Wenbin Liang, Eduardo Marbán & Hee Cheol Cho

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  1. Nidhi Kapoor
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Contributions

All authors contributed to the concepts in this study. N.K. performed Ca2+ signaling experiments, gene array, immunochemistry and in vivo experiments. W.L. performed electrophysiological and morphological characterization of in vivo–transduced cardiomyocytes and Langendorff-perfused whole heart samples. H.C.C. performed electrophysiology experiments, immunostaining, and adenoviral vector production with assistance from N.K. Writing of the paper was done in close collaboration by N.K., W.L., E.M. and H.C.C.

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Correspondence to Eduardo Marbán or Hee Cheol Cho.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–12 (PDF 5492 kb)

Supplementary Movie 1

Spontaneous whole-cell Ca2+ oscillations from GFP-NRVMs loaded with rhod-2AM and recorded in the presence of a gap-junction uncoupler, palmitoleic acid (20μM) (AVI 253030 kb)

Supplementary Movie 2

Spontaneous whole-cell Ca2+ oscillations from Tbx18-NRVMs loaded with rhod-2AM and recorded in the presence of a gap-junction uncoupler, palmitoleic acid (20μM) (AVI 106523 kb)

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Kapoor, N., Liang, W., Marbán, E. et al. Direct conversion of quiescent cardiomyocytes to pacemaker cells by expression of Tbx18. Nat Biotechnol 31, 54–62 (2013). https://doi.org/10.1038/nbt.2465

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