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In vivo cardiac reprogramming contributes to zebrafish heart regeneration

Abstract

Despite current treatment regimens, heart failure remains the leading cause of morbidity and mortality in the developed world due to the limited capacity of adult mammalian ventricular cardiomyocytes to divide and replace ventricular myocardium lost from ischaemia-induced infarct1,2. Hence there is great interest to identify potential cellular sources and strategies to generate new ventricular myocardium3. Past studies have shown that fish and amphibians and early postnatal mammalian ventricular cardiomyocytes can proliferate to help regenerate injured ventricles4,5,6; however, recent studies have suggested that additional endogenous cellular sources may contribute to this overall ventricular regeneration3. Here we have developed, in the zebrafish (Danio rerio), a combination of fluorescent reporter transgenes, genetic fate-mapping strategies and a ventricle-specific genetic ablation system to discover that differentiated atrial cardiomyocytes can transdifferentiate into ventricular cardiomyocytes to contribute to zebrafish cardiac ventricular regeneration. Using in vivo time-lapse and confocal imaging, we monitored the dynamic cellular events during atrial-to-ventricular cardiomyocyte transdifferentiation to define intermediate cardiac reprogramming stages. We observed that Notch signalling becomes activated in the atrial endocardium following ventricular ablation, and discovered that inhibiting Notch signalling blocked the atrial-to-ventricular transdifferentiation and cardiac regeneration. Overall, these studies not only provide evidence for the plasticity of cardiac lineages during myocardial injury, but more importantly reveal an abundant new potential cardiac resident cellular source for cardiac ventricular regeneration.

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Figure 1: Genetic ventricular-specific cardiomyocyte ablation results in ventricular cardiomyocyte death and subsequent regeneration.
Figure 2: In vivo cardiac reprogramming contributes to zebrafish ventricular regeneration.
Figure 3: Dynamic cellular remodelling occurs during zebrafish cardiac regeneration.
Figure 4: Notch signalling is required for zebrafish cardiac regeneration.

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Acknowledgements

We thank N. Tedeschi, L. Pandolfo and A. Ayala for fish care; O. Huang, J. Kim, T. Kuo and J. Sun for experimental assistance; S. Tu and other laboratory members for comments on the manuscript; Q. Liu for anti-N-cadherin antibody; M. Lardelli, J. Lenis and W. Clements for plasmids; and N. Lawson for the Notch reporter line. This work was supported in part by grants from the American Heart Association to D.Y. (0940041N), R.Z. (11POST7090024) and H.Y. (12POST12050080); the Packard Foundation and the National Institutes of Health (NIH) (HL54737) to D.Y.R.S.; NIH/NHLBI (NIH Heart, Lung, and Blood Institute) to J.C.; and the NIH (OD007464, HL104239) to N.C.C.

Author information

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Authors

Contributions

N.C.C. initiated the project when he was in the laboratory of D.Y.R.S. by generating and validating some of the cardiac chamber specific transgenic lines. Additional experimental design was done with the help of R.Z. and D.Y. R.Z., P.H., Ku.Ou., D.L., G.K. and N.C.C. conducted experiments. R.Z., H.Y. and N.C.C. generated and characterized transgenic lines for lineage tracing. Ka.Oc. and J.C. helped with cardiac function analysis. Y.-F.L. and D.Y. provided key reagents. R.Z., P.H., D.L., D.Y. and N.C.C. prepared the manuscript. All authors commented on the manuscript.

Corresponding author

Correspondence to Neil C. Chi.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Figures

This file contains Supplementary Figures 1-19. (PDF 2195 kb)

Cardiac function is normal in DMSO-treated control zebrafish

Tg(vmhc:mCherry-NTR) larvae were treated with DMSO at 3 dpf. Both ventricle and atrium of 24 hpt control hearts contract similarly to the age-matched wild type heart at 4 dpf. Ventral view, anterior to the top. Ventricle on the left and atrium on the right. (MOV 6677 kb)

Ventricular function is significantly reduced in 24 hpt ablated larvae

Tg(vmhc:mCherry-NTR) zebrafish were treated with 5 mM MTZ at 3 dpf. The atrium of 24 hpt ablated hearts contracts strongly while the ventricle contracts weakly at 4 dpf. Ventral view, anterior to the top. Ventricle on the left and atrium on the right. (MOV 7891 kb)

GFP genetically labeled atrial cardiomyocytes migrate into the ventricle during zebrafish ventricular regeneration

Tg(vmhc:mCherry-NTR;amhc:CreERT2;β-act2:RSG) zebrafish were treated with 4-hydroxytamoxifen from 72-78 hpf to genetically label atrial cardiomyocytes with GFP. Ventricular cardiomyocytes were then ablated at 5 dpf using 5mM MTZ. GFP fluorescence time-lapse imaging shows migration of GFP genetically labeled atrial cardiomyocytes into the regenerating ventricle from 24-84 hpt. Time interval is every 15 minutes. Ventral view, anterior to the top. Ventricle on the left and atrium on the right. (MOV 8279 kb)

GFP genetically labeled atrial cardiomyocytes gradually express vmhc:cherry fluorescence as they migrate into the injured ventricle

Tg(vmhc:mCherry-NTR;amhc:CreERT2;β-act2:RSG) zebrafish were treated with 4-hydroxytamoxifen from 72-78 hpf to genetically label atrial cardiomyocytes with GFP. Ventricular cardiomyocytes were then ablated at 5 dpf using 5mM MTZ. Red fluorescence time-lapse imaging shows that GFP genetically labeled atrial cardiomyocytes (from Supplementary video 3) gradually express vmhc:cherry fluorescence as they migrate into the injured ventricle from 24-84 hpt. Time interval is every 15 minutes. Ventral view, anterior to the top. Ventricle on the left and atrium on the right. (MOV 8405 kb)

Ventricular function gradually recovers during migration of GFP genetically labeled atrial cardiomyocytes into the ventricle

Tg(vmhc:mCherry-NTR;amhc:CreERT2;β-act2:RSG) zebrafish were treated with 4-hydroxytamoxifen from 72-78 hpf to genetically label atrial cardiomyocytes with GFP. Ventricular cardiomyocytes were then ablated at 5 dpf using 5mM MTZ. Bright field time-lapse imaging shows ventricular function gradually recovers during migration of GFP genetically labeled atrial cardiomyocytes (from Supplementary video 3) into the regenerating ventricle from 24-84 hpt. Time interval is every 15 minutes. Ventral view, anterior to the top. Ventricle on the left and atrium on the right. (MOV 8718 kb)

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Zhang, R., Han, P., Yang, H. et al. In vivo cardiac reprogramming contributes to zebrafish heart regeneration. Nature 498, 497–501 (2013). https://doi.org/10.1038/nature12322

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