Fig 2: 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.

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: Norepinephrine induces clock genes and suppresses cell cycle genes in NMCMs.(A) Norepinephrine concentration in heart with disrupted sympathetic innervation (SNi) is profoundly suppressed. (B) Norepinephrine induces the expression of circadian genes in wild-type NMCMs after 48 hours of treatment. (C) Norepinephrine decreased the expression of mitosis-regulating genes in wild-type NMCMs. Norepinephrine failed to suppress mitosis-regulating genes apart from Aurkb in cardiomyocytes isolated from Per1/Per2 DKO neonatal mouse hearts. 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.