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.

Authors

Malike Mutailipu, Abudousaimi Aini, Duolikun Mutailifu, Subi Maimaitirexiti, Maiwurulila Maiwulanjiang, Diliyaer Paerhati, Abudunaibi Maimaitiaili

Published in

International immunopharmacology. Volume 187. Pages 117265. Aug 10, 2026. Epub Aug 10, 2026.

Abstract

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.
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.
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).
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.

PMID:
42574811
Bibliographic data and abstract were imported from PubMed on 11 Aug 2026.

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