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Human ES-cell-derived cardiomyocytes electrically couple and suppress arrhythmias in injured hearts

Abstract

Transplantation studies in mice and rats have shown that human embryonic-stem-cell-derived cardiomyocytes (hESC-CMs) can improve the function of infarcted hearts1,2,3, but two critical issues related to their electrophysiological behaviour in vivo remain unresolved. First, the risk of arrhythmias following hESC-CM transplantation in injured hearts has not been determined. Second, the electromechanical integration of hESC-CMs in injured hearts has not been demonstrated, so it is unclear whether these cells improve contractile function directly through addition of new force-generating units. Here we use a guinea-pig model to show that hESC-CM grafts in injured hearts protect against arrhythmias and can contract synchronously with host muscle. Injured hearts with hESC-CM grafts show improved mechanical function and a significantly reduced incidence of both spontaneous and induced ventricular tachycardia. To assess the activity of hESC-CM grafts in vivo, we transplanted hESC-CMs expressing the genetically encoded calcium sensor, GCaMP3 (refs 4, 5). By correlating the GCaMP3 fluorescent signal with the host ECG, we found that grafts in uninjured hearts have consistent 1:1 host–graft coupling. Grafts in injured hearts are more heterogeneous and typically include both coupled and uncoupled regions. Thus, human myocardial grafts meet physiological criteria for true heart regeneration, providing support for the continued development of hESC-based cardiac therapies for both mechanical and electrical repair.

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Figure 1: Transplanted hESC-CMs partially remuscularize injured guinea-pig hearts, preserve mechanical function and reduce arrhythmia susceptibility.
Figure 2: hESC-CM grafts in uninjured hearts show 1:1 coupling with host myocardium.
Figure 3: hESC-CM grafts show 1:1 host–graft coupling in the majority of injured recipient hearts, but the extent of coupling and pattern of activation is variable.

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Acknowledgements

We thank Y. Tallini, L. Linares, B. Johnson and S. Dupras for advice and technical assistance. This work was partly supported by a grant from Geron Corporation (M.A.L.), as well as by US National Institutes of Health grants K08-HL80431 (M.A.L.), R01-HL064387 (M.A.L. and C.E.M.), P01-HL094374 (M.A.L. and C.E.M.), R01-HL084642 (C.E.M.), P01-GM81619 (C.E.M.), U01-HL100405 (M.A.L. and C.E.M.) and R01-HL095828 (N.S. and M.W.K.). Animal experiments were supported in part by the University of Washington’s Mouse Metabolic Phenotyping Center, U24-DK076126.

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Authors

Contributions

Author Contributions S.F., Y.S., W.-Z.Z., D.F., M.I.K., J.D.G., C.E.M. and M.A.L. designed the study. Y.S. and S.F. led the arrhythmia and calcium imaging experiments, respectively. Y.S. developed and performed the telemetry and programmed electrical stimulation. T.D., J.L.M., A.I., R.H. and M.V. performed telemetric ECG interpretation. The GCaMP3-expressing hESC line was generated by J.G., N.J.P. and B.V.B. V.M., J.K., K.W.M., S.F. and Y.S. carried out and analysed immunohistochemistry experiments. S.F., H.R. and M.A.L. developed and performed guinea-pig-specific in situ hybridization. S.F., W.-Z.Z. and D.F. carried out and analysed the GCaMP3 imaging experiments. D.F., M.W.K. and N.S. developed and analysed the voltage mapping experiments. All authors contributed to data analysis and interpretation. Y.S. created the figures with the assistance of S.F., W.-Z.Z., K.W.M. and D.F. C.E.M. and M.A.L. wrote the manuscript.

Corresponding authors

Correspondence to Charles E. Murry or Michael A. Laflamme.

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Competing interests

J.D.G. was an employee of Geron Corporation when many of these experiments were performed. M.A.L. and C.E.M. are founders and equity holders in BEAT Bio.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-14, Supplementary Tables 1-4, additional references and legends for Supplementary Movies 1-6. (PDF 2175 kb)

Supplementary Movie 1

This movie shows that GCaMP3-positive hESC-CMs exhibit robust fluorescent transients in vitro (see Supplementary Information file for full legend). (MOV 6990 kb)

Supplementary Movie 2

This movie shows intravital imaging of GCaMP3-positive hESC-CM grafts in the uninjured heart (see Supplementary Information file for full legend). (MOV 8809 kb)

Supplementary Movie 3

This movie shows confocal Z-stack of a GCaMP3-positive hESC-CM graft that has been dual-labeled with anti-GFP and guinea pig-specific in situ probe(see Supplementary Information file for full legend). (MOV 1917 kb)

Supplementary Movie 4

This movie shows intravital imaging of a cryoinjured heart with GCaMP3-positive hESC-CM graft that exhibited uniform 1:1 host-graft coupling (see Supplementary Information file for full legend). (MOV 1493 kb)

Supplementary Movie 5

This movie shows intravital imaging of a cryoinjured heart with GCaMP3-positive hESC-CM graft that was not coupled with host myocardium (see Supplementary Information file for full legend). (MOV 1562 kb)

Supplementary Movie 6

This movie shows intravital imaging of a cryoinjured heart with coupled and uncoupled GCaMP3-positive hESC-CM graft, examined under spontaneous and paced conditions (see Supplementary Information file for full legend). (MOV 8483 kb)

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Shiba, Y., Fernandes, S., Zhu, WZ. et al. Human ES-cell-derived cardiomyocytes electrically couple and suppress arrhythmias in injured hearts. Nature 489, 322–325 (2012). https://doi.org/10.1038/nature11317

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