miR-221-3p-mediated CPT2 suppression links cardiomyocyte metabolic stress to NET-associated inflammation in myocardial ischemia-reperfusion injury: Multi-omics evidence and experimental validation.

BACKGROUND: Impaired fatty acid oxidation (FAO) and neutrophil extracellular trap (NET) formation (NETosis) contribute to myocardial ischemia-reperfusion injury (MIRI). Whether cardiomyocyte metabolic dysfunction contributes to neutrophil activation remains unclear. METHODS: We performed two-sample Mendelian randomization (MR) of 1400 plasma metabolites with ischemic heart disease (IHD) as the outcome, followed by summary-data-based MR (SMR) to prioritize candidate genes for the selected metabolite. Five mouse MIRI microarray datasets and a published cardiac single-cell RNA-sequencing dataset were analyzed to identify the relevant cell type and predict intercellular signaling. Direct binding of miR-221-3p to the Cpt2 3' untranslated region (3'UTR) was tested by dual-luciferase reporter assay. The proposed pathway was then examined in vivo using miR-221-3p antagomir, AAV9-shCpt2, and DNase I. Untargeted myocardial metabolomics was performed in Sham, MIRI, and MIRI + anti-miR-221-3p mice. RESULTS: MR identified 18 metabolites associated with IHD after false-discovery-rate (FDR) correction. Nervonoylcarnitine (C24:1) was the only risk-direction acylcarnitine (OR = 1.065, q = 0.030), whereas octadecanedioylcarnitine (C18-DC) showed the strongest protective association (OR = 0.924, q = 2.13 × 10-6). SMR using C24:1 as the exposure prioritized CPT2 as a candidate gene (b_SMR = -0.52). Across five MIRI microarray datasets, Cpt2 was shared by downregulated FAO and upregulated NET-related gene sets (area under the curve [AUC] = 0.869) and was negatively correlated with Ncf4 (r = -0.83) and Cyba (r = -0.80). Single-cell analysis localized Cpt2 downregulation to cardiomyocytes at day 1 after reperfusion. The cardiomyocyte FAO score was inversely correlated with the neutrophil NETosis score across animals (ρ = -0.49, P = 0.036), and CellChat predicted increased damage-associated molecular pattern (DAMP) and chemokine signaling from Cpt2-low cardiomyocytes to myeloid cells. Mmu-miR-221-3p was the only upregulated MIRI miRNA predicted by both miRWalk and TargetScan to target Cpt2, and the interaction was confirmed by reporter assay. In vivo, miR-221-3p antagomir pre-treatment restored CPT2, reduced reactive oxygen species (ROS) and NET-associated markers, lowered apparent infarct burden (percentage of total left ventricular area, %LV), and improved acute cardiac function. Concurrent AAV9-shCpt2 largely reversed these effects. Annotated C24:1 increased 68.9-fold in MIRI myocardium and decreased 11.4-fold after antagomir treatment (P = 9.2 × 10-4). CONCLUSION: MR and SMR prioritized C24:1 and CPT2, and subsequent transcriptomic, single-cell, in vivo, and metabolomic analyses supported the involvement of miR-221-3p-mediated CPT2 suppression in cardiomyocyte FAO impairment and NET-associated inflammation during acute MIRI. In this pre-treatment model, miR-221-3p inhibition restored CPT2, reduced cardiac injury and NET-associated changes, and lowered myocardial C24:1.