Abstract P1133: Bmal1 Drives Postnatal Cardiac Hypertrophy Via Circadian Chromatin Remodeling Of The Pro-hypertrophic Gene Sik1

Abstract

Background: Neonatal cardiomyocyte physiological hypertrophy is temporally encoded, yet the chromatin remodeling mechanisms driving this behavior are unclear. In other tissues, genes regulated by the master circadian rhythm factor BMAL1 display circadian oscillations in histone acetylation and nucleosome and RNAPII occupancy. Hypothesis: BMAL1 regulates temporal chromatin remodeling and expression of genes critical to neonatal cardiac hypertrophy. Methods: RNAPII and BMAL1 ChIP-seq and ATAC-seq data were analyzed to identify genes at which BMAL1 may drive chromatin remodeling. Bmal1 was knocked down in neonatal rat ventricular myocytes via siRNA and hypertrophy was assessed by measuring cell size and L-azidohomoalanine incorporation into total protein, and Nppa/Nppb RT-qPCR. Bmal1 target gene promoter accessibility was assessed via MNase-qPCR and levels of chromatin-associated nucleosome core histones H2A, H2B, H3 and H4 were measured by immunoblotting of acid soluble chromatin fractions. Results: RNAPII occupancy at Bmal1 target genes was increased in adult as compared to E12.5 hearts. BMAL1 ChIP-seq and ATAC-seq revealed cardiac-specific BMAL1 co-localization (other tissues served as controls) with accessibility at the pro-hypertrophic gene salt-inducible kinase 1 (Sik1). BMAL1 knockdown impaired serum-stimulated hypertrophy, indicated by 30% decrease in cell size, 50% decrease in L-azidohomoalanine incorporation into total protein, and 80% decrease in Nppa/Nppb mRNA. BMAL1 knockdown decreased mRNA and promoter accessibility of BMAL1 targets Per2 and Sik1 and decreased chromatin-associated histones by 50%. Finally, Sik1 knockdown decreased myocyte size and Nppb mRNA. Conclusion: These findings indicate that BMAL1 controls Sik1 expression—and possibly that of other growth inducing genes—in part by modulating nucleosome assembly at specific loci, thereby regulating neonatal myocyte physiological hypertrophy.