Single-cell analysis of murine fibroblasts identifies neonatal to adult switching that regulates cardiomyocyte maturation

ARTICLE https://doi.org/10.1038/s41467-020-16204-w OPEN Single-cell analysis of murine fibroblasts identifies neonatal to adult switching that regulates cardiomyocyte maturation 1234567890():,; Yin Wang1,4, Fang Yao1,4, Lipeng Wang2, Zheng Li1, Zongna Ren1, Dandan Li1, Mingzhi Zhang1, Leng Han Shi-qiang Wang2, Bingying Zhou1 & Li Wang 1 ✉ 3, Cardiac maturation lays the foundation for postnatal heart development and disease, yet little is known about the contributions of the microenvironment to cardiomyocyte maturation. By integrating single-cell RNA-sequencing data of mouse hearts at multiple postnatal stages, we construct cellular interactomes and regulatory signaling networks. Here we report switching of fibroblast subtypes from a neonatal to adult state and this drives cardiomyocyte maturation. Molecular and functional maturation of neonatal mouse cardiomyocytes and human embryonic stem cell-derived cardiomyocytes are considerably enhanced upon coculture with corresponding adult cardiac fibroblasts. Further, single-cell analysis of in vivo and in vitro cardiomyocyte maturation trajectories identify highly conserved signaling pathways, pharmacological targeting of which substantially delays cardiomyocyte maturation in postnatal hearts, and markedly enhances cardiomyocyte proliferation and improves cardiac function in infarcted hearts. Together, we identify cardiac fibroblasts as a key constituent in the microenvironment promoting cardiomyocyte maturation, providing insights into how the manipulation of cardiomyocyte maturity may impact on disease development and regeneration. 1 State Key Laboratory of Cardiovascular Disease, Fuwai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100037, China. 2 State Key Laboratory of Membrane Biology, College of Life Sciences, Peking University, Beijing 100871, China. 3 Department of Biochemistry and Molecular Biology, The University of Texas Health Science Center at Houston McGovern Medical School, Houston, TX 77030, USA. 4These authors contributed equally: Yin Wang, Fang Yao. ✉email: NATURE COMMUNICATIONS | (2020)11:2585 | https://doi.org/10.1038/s41467-020-16204-w | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-16204-w U nlike other cell types in the heart, cardiomyocytes (CMs) are terminally differentiated cells that lack proliferative capacity to repair myocardial damage caused by disease or other insults. Therefore, restoring the loss of cardiomyocytes remains a fundamental challenge in the treatment of cardiac injury, such as myocardial infarction. In past decades, enormous efforts were undertaken to reconstitute damaged cardiac tissues, typically using exogenous cell-based or cell-free therapies, which revolutionized our understanding of cardiac regeneration1–9. However, many hurdles still lie ahead on their road into the clinic. Adverse effects, such as arrhythmia and teratoma formation, were frequently observed in studies using pluripotent stem cell-derived cardiomyocytes due to their immature nature1,4. Another strategy commonly attempted was to arouse intrinsic programs to force endogenous mature cardiomyocytes back into the cell cycle, so as to supply the damaged myocardium via proliferation, whose reparative effects were marginal nonetheless2,3,10–14. Hence, development of more promising therapies requires an update on our understanding of cardiomyocyte maturation. Compared to well-established mechanisms of cardiac fate commitment in embryonic development15–21, postnatal cardiomyocyte maturation is much less well molecularly defined, which many recent studies have started to address22,23. A panoply of factors, including transcription factors, microRNAs, as well as endothelial nitric oxide synthase (eNOS), were suggested to play crucial roles in the maturation of the conduction system in the heart, or in controlling other aspects of cardiomyocyte biology, such as metabolism, cell size, contractility, and proliferation20,24,25. These and other studies together suggested multi-layered regulatory networks in cardiomyocyte maturation, urging for more detailed and comprehensive characterization of the underlying molecular events. The cell microenvironment is known to play critical roles in regulating cell fate by producing biophysical and biochemical cues, yet little is known about its roles in cardiomyocyte maturation. A recent study reported enhanced maturation of human embryonic stem cell-derived cardiomyocytes in a 3D tissue-engineered culture environment26. Alternatively, advanced cardiac maturation can be achieved by physical conditioning with increasing intensity over time, further underscoring the importance of the microenvironment in cardiac fate decisions27. In contrast to the biophysical environment, biochemical influences on cardiomyocyte maturation remain to be explored in greater depth. In fact, postnatal heart development offers natural clues to understanding alterations in the cellular microenvironment, as well as their impact on the maturation of cardiomyocytes. Here, we apply single-cell RNA sequencing to analyze the cellular composition and interactions at different postnatal developmental stages, and unveil cardiac fibroblasts as the major constituent in the cellular niche driving cardiomyocyte maturation. Importantly, manipulation of fibroblasts or downstream signaling pathways regulates cardiomyocyte maturity, providing potentially viable strategies to improve outcomes of stem cell-based or cellfree therapies. Results Single-cell analysis of murine postnatal heart development. To understand the molecular underpinnings of cardiomyocyte maturation, we set out to investigate single-cell transcriptomic profiles of mouse cardiac cells at various postnatal developmental stages. To this end, we first acquired a single-cell RNA-Sequencing (scRNA-Seq) dataset of left ventricles from mouse hearts at postnatal days (P) 1, 4, 7, and 14 from a separate study. To 2 further capture the mature cardiac state, we performed scRNASeq on left ventricles of P56-hearts using the same experimental design and platform (Supplementary Fig. 1a). The image-based selection of individual cells used in our system assured the reliability of sample input, precluding dead cells and multiplets (Supplementary Fig. 1a). A total of 2497 cells, including both cardiomyocytes (CMs) and non-cardiomyocytes (NCMs), from two P56-mouse hearts, were sequenced (Supplementary Fig. 1b), of which 2137 qualified for subsequent analysis upon stringent filtering (please see “Methods” for details regarding quality control). All data (P1, P4, P7, P14, and P56) reached a median depth of 233,320 reads/cell, 84% alignment rate/cell, and 2610 genes/cell (Supplementary Fig. 1c–e). Following quality control, we performed t-distributed stochastic neighbor embedding (t-SNE) analysis with Seurat to define major cell clusters (...truncated)