Single-cell RNA-sequencing reveals the effects of right ventricular volume overload on cardiomyocyte proliferation via immune responses
Background: Right ventricular volume overload (RVVO) is one of the most important hemodynamic characteristics in children with congenital heart diseases (CHDs) and heart failure, and cardiomyocyte (CM) proliferation is a vital factor for improving cardiac performance. However, whether and how RVVO reboots CM proliferation remains elusive.  Methods and Results: We first created a prepubertal RVVO mouse model by an abdominal- aorta- and inferior- vena- cava- fistula (ACF) surgery on postnatal day 7(P7). Then we performed bulk RNA-seq on RVs of RVVO mice on P14, which showed that the top 30 enriched gene ontology(GO) terms of upregulated genes were associated with cell proliferation and that cell cycle was the fourth top enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway terms. Immunofluorescence staining confirmed the results, showing significantly increased numbers of phosphorylated histone 3/Aurora B-positive CMs in RVs with RVVO when compared to those in control RVs. Single RNA-seq performed on P21 further confirmed the results, showing increased percentage of CMs, with five sub-functional CMs, in which immature CMs increased while mature CMs decreased. In addition, RNA-seq/flow cytometry demonstrated that RVVO induced an immune response. Inhibiting the immune response with cyclosporin A(CsA) caused the gene expression profile of RVVO mice to shift towards that of sham mice, with a significant decrease in cell cycle associated genes' enrichment. Conclusions: RVVO temporarily reboots prepubertal CM proliferation via immune responses. This study may provide an opportunity to create a novel paradigm to treat pediatric CHDs or heart failure in considering the importance of RVVO and CM proliferation in CHDs or heart failure. Overall design: We used C57/BL6J mice in this study and performed abdominal- aorta- and inferior- vena- cava- fistula (ACF) surgery on prepubertal mice on postnatal day 7 (P7), which is the edge of the CM proliferation window (P1-P7). Then we collected the right ventricular tissue of mouse pups on postnatal day 21 for single-cell RNA-seq analysis to investigate whether RVVO reactivates CM proliferation.