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

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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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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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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