MiR-199a-5p aggravates hypoxia/reoxygenation-induced cardiomyocyte ferroptosis by blocking HSPB1-Keap1/Nrf2/ARE signaling.

Ferroptosis plays a crucial role in hypoxia/reoxygenation (H/R)-induced cardiomyocyte injury and acute myocardial infarction (AMI), yet the involvement of microRNA-199a-5p (miR-199a-5p) in this process remains insufficiently understood. In this study, serum miR-199a-5p levels were markedly elevated in AMI patients and positively correlated with myocardial injury markers cardiac troponin I and creatine kinase-MB, while H/R stimulation similarly upregulated miR-199a-5p expression in AC16 cardiomyocytes. Functional experiments demonstrated that miR-199a-5p overexpression exacerbated oxidative stress and ferroptosis, as evidenced by increased lactate dehydrogenase release, malondialdehyde production, Fe²+ accumulation, lipid peroxidation, and glutathione depletion, whereas miR-199a-5p inhibition conferred significant protection against H/R-induced injury. Mechanistically, heat shock protein β‑1 (HSPB1) was identified as a direct downstream target of miR-199a-5p, with HSPB1 overexpression alleviating and its silencing aggravating ferroptotic responses under H/R conditions. Rescue assays further confirmed that HSPB1 mediates the pro-ferroptotic effects of miR-199a-5p. At the molecular level, the miR-199a-5p/HSPB1 axis regulated key ferroptosis-related proteins, including ACSL4, SLC7A11, and GPX4, and disrupted Keap1/Nrf2/ARE antioxidant signaling during H/R injury. Collectively, these findings indicate that miR-199a-5p aggravates H/R-induced cardiomyocyte ferroptosis by suppressing HSPB1 and impairing Nrf2-dependent antioxidant defense, suggesting that circulating miR-199a-5p may serve as a biomarker of myocardial injury and a potential therapeutic target in ischemic heart disease.