Abstract Body (Do not enter title and authors here): [Background]
Since mammalian cardiomyocytes (CMs) are terminally differentiated cells that have largely lost their proliferative capacity, the heart has limited regenerative capacity. Therefore, elucidating the mechanisms that regulate proliferative activities of CMs may provide novel insights into the development of heart regenerative therapy. However, the regulatory mechanisms of CM proliferation remain to be fully elucidated.

[Objective]
This study aims to address the regulatory mechanisms of CM proliferation using multiple comprehensive analyses.

[Methods and Results]
Three comprehensive analyses were performed to explore regulators of CM proliferation.
(1) RNA sequencing (RNA-seq) analysis using the neonatal rat cardiomyocytes (NRCMs) overexpressing Runx1, a proliferative gene. In Runx1 overexpressing NRCMs, 613 genes were upregulated and 553 genes were downregulated. (2) RNA-seq analysis using proliferating CMs. Fluorescent Ubiquitination-based Cell Cycle Indicator was used for fluorescent labeling of proliferating cells, and labeled cells were collected by flow cytometry. In proliferating NRCMs, 213 genes were upregulated and 336 genes were downregulated. (3) Single-cell RNA-seq data set of 1- and 7-day-old rat hearts obtained from a public database. Eleven CM clusters were detected, with cluster 5 composed of S phase cells. Pseudotime trajectory analysis revealed a trajectory from G1 phase to S and G2/M phases in CMs on postnatal day1. However, on postnatal day 7, the trajectory of CMs was interrupted at cluster 5, suggesting cell cycle arrest. Comparison of gene expression in cluster 5 showed that 335 genes were upregulated and 389 genes were downregulated in postnatal 1-day-old CMs. These three analyses were integrated, and we found that the expression of Myomesin 2 (Myom2) was commonly decreased in the proliferating CMs in each analysis, suggesting that increased expression of Myom2 might suppress the proliferation. In fact, immunofluorescent staining revealed that overexpression of Myom2 in NRCMs by adenoviral vector reduced the ratio of Ki-67⁺ proliferating CMs.

[Conclusion]
Multiple comprehensive analyses identified Myom2 as a candidate inhibitor of CM proliferation. The suppression of Myom2 expression could be a promising therapeutic strategy for heart regeneration.