RNA-Binding Protein Signature in Proliferative Cardiomyocytes: A Cross-Species Meta-Analysis from Mouse, Pig, and Human Transcriptomic Profiling Data.

In mammals, because cardiomyocytes withdraw from cell-cycle activities shortly after birth, the heart cannot repair the damage caused by a myocardial injury; thus, understanding how cardiomyocytes proliferate is among the most important topics in cardiovascular sciences. In newborn neonatal mammals, when a left ventricular injury is applied in hearts earlier than postnatal day 7, the cardiomyocytes actively proliferate and regenerate lost myocardium in the following weeks. The regulators promoting cardiomyocyte proliferation were discovered by analyzing transcriptomic data generated from models. Most of these regulators support the mRNA production of cell-cycle machinery, yet the mRNA requires translation into functional proteins under the regulation of RNA-binding proteins (RBPs). In this work, we performed a meta-analysis to study the relationship between RBP expression and cardiomyocyte proliferation. To identify RBPs associated with mouse and pig cardiomyocyte proliferation, the single-nuclei RNA sequencing (snRNA-seq) data from regenerating mouse and pig hearts were reanalyzed via an Autoencoder focusing on RBP expression. We also generated and analyzed new bulk RNA-seq from two human-induced pluripotent stem cell-derived (hiPSC) cardiomyocyte (hiPSC-CM) cell lines; the first cell line was harvested sixteen days after differentiation, when the cells still actively proliferated, and the second cell line was harvested one hundred and forty days after differentiation, when the cells ceased cell cycle activity. Then, the RBP associated with mouse, pig, and hiPSC-CM were compared across species. Twenty-one RBPs were found to be consistently upregulated, and six RBPs were downregulated in proliferating mouse, pig, and hiPSC-derived cardiomyocytes. Among upregulated RBPs across species, an immunofluorescence-based imaging analysis validated the significant increase in the proteins of DHX9, PTBP3, HNRNPUL1, and DDX6 in pig hearts with proliferating CMs. This meta-analysis in all species demonstrated a strong relationship between RBP expression and cardiomyocyte proliferation.