Macrophage-enriched HMOX1 is an ischemia-reperfusion-responsive marker in the heart

Myocardial ischemia-reperfusion (I/R) injury triggers rapid innate immune activation, yet cell-type-resolved, I/R-responsive markers that bridge macrophage biology and translational relevance remain insufficiently defined. This study aimed to identify cell-type-resolved I/R-responsive markers in the heart and to clarify the translational relevance of macrophage-enriched HMOX1 in myocardial ischemia-reperfusion injury.

A murine bulk myocardial transcriptomic dataset (GSE160516, spanning 6, 24, and 72 h post-reperfusion time points) was analyzed using differential expression and weighted gene co-expression network analysis (WGCNA), followed by functional enrichment and protein-protein interaction analyses. Candidate genes were prioritized through interpretable feature-selection models [least absolute shrinkage and selection operator (LASSO) regression and shapley additive explanations (SHAP)-based random forest analysis]. Cell-type specificity was resolved in an independent single-cell RNA-sequencing dataset (GSE288103). The top-ranked candidate was subsequently validated in a rat myocardial I/R model using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Complementary human genetic colocalization and in silico docking analyses were conducted as exploratory, hypothesis-generating exercises.

An I/R-associated co-expression module was enriched for immune and inflammatory pathways. Across multiple prioritization strategies, HMOX1 consistently ranked among the leading candidates. In bulk data, HMOX1 correlated with macrophage-related signals, and single-cell analysis confirmed predominant expression in macrophages. RT-qPCR demonstrated elevated myocardial HMOX1 transcript levels in rats following I/R. An independent external bulk RNA-seq cohort (GSE225105) showed a directionally concordant increase in HMOX1 expression, although this did not reach statistical significance in the limited sample. Colocalization and docking analyses yielded preliminary, hypothesis-generating signals.

Integrated bulk and single-cell transcriptomic analyses, together with cross-species RT-qPCR validation, identify HMOX1 as a macrophage-enriched, I/R-responsive marker. These findings delineate a tractable candidate for future mechanistic studies investigating macrophage-associated responses in myocardial I/R injury.