Abstract P2007: Bmal1 Drives Postnatal Cardiac Hypertrophy Via Circadian Chromatin Remodeling Of The Pro-hypertrophic Gene Sik1

Abstract

Background: During neonatal cardiac development, myocytes integrate cues such as changes in blood pressure and circulating hormones to execute a temporal genetic response that drives physiological cardiac hypertrophy. Chromatin remodeling precedes the adult heart’s response to growth stimuli, yet the temporal nature of neonatal chromatin remodeling is unclear. In other tissues, genes regulated by the master circadian rhythm factor BMAL1 display circadian oscillations in histone acetylation and nucleosome and RNAP II occupancy. We hypothesized that BMAL1 regulates temporal chromatin remodeling and expression of genes critical for neonatal cardiac hypertrophy. Methods: BMAL1 chromatin immunoprecipitation sequencing (ChIP-Seq) and assay for transposase-accessible chromatin and sequencing (ATAC-Seq) data from murine hearts were analyzed to identify genes at which BMAL1 may drive chromatin remodeling. To interrogate myocyte time-of-day dependent response to growth stimuli, neonatal rat ventricular myocytes (NRVM) were subjected to serum shock synchronization to drive oscillatory BMAL1 expression, followed by treatment with the growth stimulus phenylephrine (PE) at the peak and trough of BMAL1 expression. BMAL1 was knocked down via siRNA and hypertrophy was assessed by measurement of cell size and of RT-PCR of fetal genes (Nppa and Nppb). Results: BMAL1 ChIP-Seq data revealed myocyte-specific BMAL1 localization to regions exhibiting an open chromatin signature based on ATAC-seq, including the pro-hypertrophic gene salt-inducible kinase 1 ( Sik1 ). Serum shock induced oscillations of BMAL1 and histone H3.3: administration of PE at the peak of BMAL1 expression induced a 30% increase in cell size and 2-fold increase in fetal gene expression. PE given at the trough of BMAL1 expression affected neither cell size nor fetal genes, demonstrating a critical role for the circadian clock in myocyte growth. BMAL1 knockdown: decreased expression of its known target and partner clock gene, Per2 , as well as expression of Sik1, Nppa , and Nppb ; decreased myocyte size by 30%; and increased expression of histone H3.3. Conclusions: These data indicate that BMAL1 temporally regulates myocyte growth by modulating histone stoichiometry and expression of Sik1 .