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In situ reprogramming of cardiac fibroblasts into cardiomyocytes in mouse heart with chemicals

Abstract

Cardiomyocytes are terminal differentiated cells and have limited ability to proliferate or regenerate. Condition like myocardial infarction causes massive death of cardiomyocytes and is the leading cause of death. Previous studies have demonstrated that cardiac fibroblasts can be induced to transdifferentiate into cardiomyocytes in vitro and in vivo by forced expression of cardiac transcription factors and microRNAs. Our previous study have demonstrated that full chemical cocktails could also induce fibroblast to cardiomyocyte transdifferentiation both in vitro and in vivo. With the development of tissue clearing techniques, it is possible to visualize the reprogramming at the whole-organ level. In this study, we investigated the effect of the chemical cocktail CRFVPTM in inducing in situ fibroblast to cardiomyocyte transdifferentiation with two strains of genetic tracing mice, and the reprogramming was observed at whole-heart level with CUBIC tissue clearing technique and 3D imaging. In addition, single-cell RNA sequencing (scRNA-seq) confirmed the generation of cardiomyocytes from cardiac fibroblasts which carries the tracing marker. Our study confirms the use of small molecule cocktails in inducing in situ fibroblast to cardiomyocyte reprogramming at the whole-heart level and proof-of-conceptly providing a new source of naturally incorporated cardiomyocytes to help heart regeneration.

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Fig. 1: CRFVPTM induce in situ fibroblast-to-cardiomyocyte transdifferentiation in Fsp1-Cre:R26R-loxp-tdTomato mice.
Fig. 2: Whole-heart imaging of hearts from Fsp1-Cre:R26R-loxp-tdTomato mice.
Fig. 3: Single-cell transcriptomics analysis reveals the in situ generation of CiCM.
Fig. 4: CRFVPTM induce in situ fibroblast-to-cardiomyocyte transdifferentiation in PDGFRα-DreER:R26R-rox-tdTomato mice.
Fig. 5: Whole-heart imaging of hearts from PDGFRα-DreER:R26R-rox-tdTomato mice.
Fig. 6: Refining of the chemical cocktail in vivo with Fsp1-Cre:R26R-loxp-tdTomato mice.

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Acknowledgements

This work was supported by grants from the Ministry of Science and Technology of China (2022YFA1104700 and 2022ZD0204700 to XX), the National Natural Science Foundation of China (82121005 and 82330113 to XX, 31900518 to CWH), Youth Innovation Promotion Association of the Chinese Academy of Sciences (2022280 to RG), Fundamental Research Projects of Science & Technology Innovation and Development Plan in Yantai City (2023JCYJ063 to RG), and Taishan Scholars Program to XX. We thank Shanghai Genenergy Biotechnology Co. Ltd. for their support in single-cell sequencing. Also, we appreciate Shanghai Yifu Technologies Co. Ltd. and Light Innovation Technology Ltd. for their assistance in heart CUBIC-clearing and 3D-imaging.

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ZYC and SJJ designed and conducted most of the experiments, analyzed the results; ZYC wrote the paper; CWH, WZT and XYR provided technical assistance in animal study; RG provided some critical suggestions for the study; XX conceived the idea and supervised the study, analyzed the results, and wrote the paper. All authors reviewed the results and approved the final version of the manuscript.

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Correspondence to Xin Xie.

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Chen, Zy., Ji, Sj., Huang, Cw. et al. In situ reprogramming of cardiac fibroblasts into cardiomyocytes in mouse heart with chemicals. Acta Pharmacol Sin (2024). https://doi.org/10.1038/s41401-024-01308-6

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