Multilayer Timing of Cardiac Circadian Regulation Informs Prevention and Treatment of Cardiovascular Disease.

Background: Cardiovascular diseases remain the leading cause of death and economic burden worldwide. Increasing evidence indicates that sleep disturbance and circadian rhythm disruption are major risk drivers for hypertension, coronary artery disease, heart failure, and arrhythmia. Although the classical transcription-translation feedback loop (TTFL) model explains the basic mechanism of rhythm generation, increasing evidence suggests that the heart-an organ with high metabolic demand-maintains circadian stability through coordinated transcriptional, translational, and post-translational regulation. Methods: We developed an integrative, time-resolved, multilayer in silico framework to systematically analyze cardiac circadian regulation by combining mouse heart time-series RNA-seq (GSE54650), proteomics (PXD002870), phosphoproteomics (PXD036824), BMAL1 and Rev-erbα ChIP-seq, and enhancer RNA (eRNA) datasets. Rhythmicity was assessed using MetaCycle, with cross-layer comparisons evaluating concordance and divergence between transcriptomic and proteomic rhythms, and translation efficiency (TE) estimated from protein-to-mRNA ratios. Enhancer-gene coupling, transcription factor binding, and phosphorylation motif analyses were integrated to investigate multilayer regulatory coordination. Results: We identified 2552 rhythmic transcripts and 139 rhythmic proteins, with only 31 genes rhythmic at both layers, indicating substantial RNA-protein phase decoupling in the heart. Temporal stability of TE correlated positively with protein amplitude, suggesting that stable translation supports robust protein rhythmicity. Phosphoproteomic analyses revealed enrichment of SP motifs mediated by proline-directed kinases in rhythmic proteins. BMAL1 binding was associated with enhanced transcriptional amplitude, whereas REV-ERBα binding was associated with delayed target gene expression, forming complementary enhancer-level regulatory dynamics. Conclusion: This study supports a multilayered integrative model of cardiac circadian regulation in which rhythmic gene expression is jointly shaped by transcriptional activation, translational precision, and post-translational modification. By extending the classical "clock-transcription-protein" paradigm, our findings highlight enhancer-level control mediated by BMAL1 and Rev-erbα as an important mechanism contributing to the stabilization of cardiac circadian timing.