Abstract Body (Do not enter title and authors here): Background: Understanding mechanisms activating the cardiomyocyte (CM) cell-cycle in large mammals is essential to designing molecular therapies for treating injured hearts. Pig CMs are known to exist cell-cycle shortly after birth; however, we have shown that pig CMs actively proliferate when apical resection surgery is performed on postnatal day 1 (AR_P1), and active CM proliferation starts on postnatal day (P)8 following injury. CM single-nucleus RNA-sequencing (snRNAseq) data were collected and processed from uninjured (CTL) pigs and AR_P1 pig hearts on P1, P4, P8, and P28 at the border zone. Before P8, expressions of two heat-shock proteins, HSPA5 and HSP90B1, significantly decreased on P4 and remained low at later timepoints in naïve hearts.
Hypothesis: Stress-responsive heat-shock proteins are upregulated in pig AR_P1 cardiomyocytes before AR_P1P8 and promote CM proliferation.
Approach: Heat-shock protein expressions were evaluated via immunohistological analysis in blindly-selected areas in naïve fetal, CTL-P7, and AR_P1P8 pig hearts. The capability of heatshock proteins in inducing CM cell-cycle was demonstrated in human AC16 CM cell line via lentivirus-mediated overexpression and knockdown of HSPA5 and HSP90B1. Cell-cycle marker PH3 and cell-cycle regulators FOXM1, P53, and antioxidant molecule PRX-V were quantified in AC16 cells via Western blotting.
Results: Following AR_P1, both snRNAseq and immunohistological analysis showed that the expression of heat-shock proteins remained high in AR_P1P8 CMs. In AC16 cells, overexpression (or knockdown) of HSPA5 and HSP90B1 increased (or decreased) the cell cycle. Overexpressing (or knocking down) HSPA5 resulted in an increase (or decrease) of PH3, FOXM1, and PRX-V; while P53 expression decreased (or increased). Overexpressing (or knocking down) HSP90B1 increased (or decreased) PH3 and HSPA5.
Conclusion: HSPA5 and HSP90B1 were upregulated in pig AR_P1 CMs before the active CM proliferation timepoints, and promoted cardiomyocyte cell-cycle activity; the mechanism involved the downstream effectors, including FOXM1, PRX-V, and P53. Designing molecular therapies targeting these genes may promote CM proliferation and protection in translational studies of heart diseases.