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.