Temporal miRNA remodeling defines a 24-hour regulatory threshold for immune-inflammatory activation in intermittent hypoxia-induced cardiomyocyte injury.

BACKGROUND: Intermittent hypoxia (IH), a hallmark of obstructive sleep apnea (OSA), contributes to cardiac injury through mechanisms including oxidative stress, inflammation, and immune dysregulation. However, the temporal threshold at which miRNA-mediated transcriptional responses are activated, as well as their immunoregulatory roles in this process, remain unclear. METHODS: An in vitro IH model was established in H9C2 cardiomyocytes exposed to IH for 8 h and 24 h. Cellular injury, mitochondrial dysfunction, inflammatory responses, and apoptosis were assessed. Small RNA sequencing and mRNA transcriptome profiling were performed to identify IH-responsive miRNAs and their regulatory networks. Integrated miRNA-mRNA analysis was conducted to reveal potential immune-inflammatory pathways, and miR-146b-5p was further validated using inhibitor-mediated functional assays. RESULTS: IH induced severity-dependent cardiomyocyte injury, characterized by mitochondrial membrane depolarization, increased expression of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β), and activation of caspase-3-mediated apoptosis. No DEmiRs were detected at 8 h, whereas 18 DEmiRs were identified at 24 h, indicating a critical 24-hour threshold for IH-induced miRNA activation. Integrated miRNA-mRNA analysis revealed significant enrichment in immune-related pathways, including: Cytokine-cytokine receptor interaction, IL-17 signaling, Toll-like receptor signaling, PD-L1/PD-1 checkpoint, MAPK, FoxO, Wnt, and mTOR pathways. Key hub genes such as Fos, Gsk3b, Adam17, Cd200, Socs6, and Smad4 were identified as central regulators of inflammation and immune responses. Among the DEmiRs, miR-146b-5p, miR-26a-5p, and miR-23a-3p were highlighted as potential modulators of immune-inflammatory signaling under IH conditions. Taken together, our findings identify miR-146b-5p as a critical IH-responsive miRNA and reveal that its upregulation contributes to the progression of IH-induced cardiomyocyte injury through activation of inflammatory and apoptotic pathways. CONCLUSION: This study identifies a 24-hour temporal threshold for miRNA-mediated immune regulation in IH-induced cardiomyocyte injury. Integrated miRNA-mRNA analysis reveals that IH-responsive miRNAs are involved in immune-inflammatory pathways and identifies miR-146b-5p as a key candidate regulator. Functional validation demonstrates that IH exposure induces miR-146b-5p upregulation, which contributes to cardiomyocyte injury by promoting inflammatory responses and apoptosis. Importantly, inhibition of miR-146b-5p alleviates IH-induced cellular damage by reducing TNF-α expression, suppressing cleaved caspase-3 activation, and restoring Bcl-2 levels. These findings provide mechanistic insights into the role of miRNAs in early immune dysregulation and suggest that targeting specific miRNAs, particularly miR-146b-5p, may offer novel therapeutic strategies for preventing IH-related cardiomyopathy.