Fig 2: In hearts with disrupted sympathetic cardiac innervation and in Per1/Per2 DKO hearts, suppression of Wee1 kinase activates the Cdk1/cyclin B1 mitosis entry complex.(A) Schematic representation of the different proteins linking the cell cycle with the circadian cycle. Wee1 is a kinase that phosphorylates and inactivates Cdk1, not allowing the Cdk1/cyclin B1 complex to induce entry into mitosis. Cdc25 is a phosphatase with the opposite effect. (B) Gene expression analysis of factors known to link Per1/Per2 and cell cycle showed that Wee1 is consistently decreased in both hearts with inhibited sympathetic innervation and Per1/Per2 DKO. Cdc25 is significantly increased in SNi hearts. (C and D) Western blot analysis confirmed decreased expression of Wee1 protein kinase and increased expression of Cdk1 and cyclin B1 proteins in hearts with disrupted sympathetic innervation and Per1/Per2 DKO. Phosphorylated Tyr15 Cdk1 was decreased in both mouse models suggestive of increased Cdk1 activation. (E) Norepinephrine treatment of wild-type NMCMs induced Wee1 gene expression; however, this effect was not observed in Per1/Per2 DKO cardiomyocytes. Student’s t test was used for two-group analysis. ANOVA test was used for multiple group comparisons. Data are presented as means ± SEM. Only P values <0.1 are reported.

Fig 3: Inhibition of cardiac SNs results in up-regulation of cell cycle genes and down-regulation of Per1 and Per2 genes.(A) GO analysis of RNA-sequencing data of P7 ventricles with disrupted sympathetic innervation compared to controls. Cell cycle, mitosis, and nuclear division genes were up-regulated (red), while circadian rhythm genes (green), calcium handling, cell size, muscle contraction, muscle action potential, heart rate, and axonogenesis genes were down-regulated. (All hearts were isolated at the same time, ~2 p.m.) (B) Quantitative PCR analysis of cell cycle regulators showing increased expression of genes regulating S phase and mitosis (M). (C) Gene expression analysis of circadian genes, showing decreased expression of Per1 and Per2 circadian cycle regulators. (D) Per1 and Per2 proteins were significantly reduced in hearts with suppressed sympathetic innervation. (E) Calcium handling genes were decreased in hearts with disrupted sympathetic innervation. Student’s t test was used for two-group analysis. Data are presented as means ± SEM. Only P values <0.1 are reported.

Fig 4: Per1/Per2 DKO hearts have more proliferative neonatal cardiomyocytes.(A) Per1/Per2 DKO mice developed increased heart size, whereas total body weight remained unchanged at P14. (B) The size of individual Per1/Per2 DKO cardiomyocytes was decreased. (C) The total number of cardiomyocytes in Per1/Per2 DKO hearts was increased. (D) Per1/Per2 DKO myocytes were more mononucleated at P14. Scale bars, 20 µm. (E) Immunofluorescence staining of P7 cardiac sections for pH3 showed more Per1/Per2 DKO cardiomyocytes entering mitosis. Scale bars, 25 µm. (F) Immunofluorescence staining of P7 cardiac sections for EdU showed more Per1/Per2 DKO cardiomyocytes in the S phase. Scale bars, 25 µm. (G) Gene expression analysis of the major cell cycle regulators showed increased expression of genes regulating mitosis. Student’s t test was used for two-group analysis. Data are presented as means ± SEM. Only P values <0.1 are reported.