Integrated epigenomic and transcriptomic analyses implicate Car5b in cardiomyocyte hypertrophy

Pathological cardiac hypertrophy is a major risk factor for heart failure and is characterized by transcriptional and metabolic remodeling. Although chromatin accessibility is closely associated with transcriptional regulation, how accessibility changes relate to gene-expression and functional changes during hypertrophic stress remains incompletely understood.

Integrated ATAC-seq and RNA-seq analyses were performed in control and AngII-treated cardiomyocytes, followed by pathway enrichment and mitochondrial gene annotation. Candidate genes were validated in transverse aortic constriction (TAC)-induced hypertrophic mouse hearts and AngII-treated cardiomyocytes, and Car5b was examined using siRNA-mediated knockdown. KEGG pathway enrichment of genes identified by the AngII ATAC-seq/RNA-seq integration was used to nominate pathways for follow-up. PI3K and AKT phosphorylation was evaluated by Western blotting, mitochondrial membrane potential by JC-1 staining, and intracellular reactive oxygen species (ROS) by DCFH-DA/MitoTracker fluorescence staining.

AngII stimulation was associated with widespread chromatin-accessibility remodeling and extensive transcriptional reprogramming. Integrated analysis identified 155 genes with overlapping changes in chromatin accessibility and gene expression; this concordance was interpreted as an association and not as evidence of direct epigenetic regulation. Integration with mitochondrial gene annotation prioritized Car5b, Noct, and Agpat4 as candidate genes. Car5b was consistently upregulated at the mRNA and protein levels in hypertrophic hearts and cardiomyocytes. Car5b knockdown attenuated AngII-induced hypertrophic marker expression and cardiomyocyte enlargement. KEGG pathway enrichment analysis of the 155 genes identified by integrated ATAC-seq and RNA-seq analysis of AngII-treated primary cardiomyocytes revealed the PI3K/AKT signaling pathway as one of the enriched pathways. AngII increased PI3K and AKT phosphorylation, whereas Car5b knockdown was associated with lower phosphorylation levels. Car5b knockdown was also associated with improved mitochondrial membrane potential and reduced intracellular ROS accumulation.

These findings identify Car5b as a mitochondria-related candidate associated with AngII-induced cardiomyocyte hypertrophy. Car5b knockdown attenuated hypertrophic readouts and coincided with reduced PI3K/AKT phosphorylation, improved mitochondrial membrane potential, and lower ROS. The present experiments do not establish the causal sequence among Car5b, PI3K/AKT signaling, and mitochondrial dysfunction, and concordant ATAC-seq/RNA-seq changes do not by themselves demonstrate direct epigenetic regulation. Car5b therefore warrants further mechanistic investigation as a potential modulator of pathological cardiac remodeling.