Machine learning identifies autophagy biomarkers driving microvascular injury in cardiac ischemia/reperfusion

Ischemic heart disease (IHD) remains a leading global cause of mortality. While reperfusion is essential for treating ischemic cardiomyopathy, it paradoxically induces myocardial ischemia/reperfusion (I/R) injury targeting the microvascular endothelium. The link between autophagy and microvascular damage in myocardial I/R requires clarification.

This study aimed to identify autophagy-related biomarkers of myocardial I/R injury using integrated bioinformatics and experimental validation, characterize the associated molecular and immune features, and evaluate the role of Myc in cardiac microvascular endothelial injury.

Bioinformatics analysis of Gene Expression Omnibus (GEO) multi-chip datasets intersected autophagy-related genes (HADb) with differentially expressed genes (DEGs) to identify signature biomarkers using machine-learning algorithms. Diagnostic efficacy was validated using a nomogram. Mechanistic exploration through gene set enrichment analysis (GSEA) and immune profiling revealed I/R subtypes. Biomarker expression was externally validated in the GSE168610 dataset. Myocardial I/R models were assessed using echocardiography (cardiac function), Evans blue/TTC staining (infarction area), and ink perfusion (microvascular density). Hub gene expression was quantified using quantitative polymerase chain reaction (qPCR), western blot (WB), and immunohistochemistry (IHC). In vitro, c-Myc-knockout cardiac endothelial cells underwent hypoxia/reoxygenation (H/R), followed by analysis of autophagy (WB), apoptosis (TUNEL), and autophagic vacuoles (transmission electron microscopy (TEM)).

Seven autophagy-related DEGs were identified and linked to muscle proliferation, protease activation, and oncogenesis. Four signature genes (Casp4, Cdkn1a, Myc, and Rgs19) were identified by 3 machine-learning algorithms, with diagnostic validation. In the development cohort, the AUCs ranged from 0.756 to 0.876. In the external-validation cohort, the AUC was 1.000 for Casp4, Myc, and Rgs19 and 0.875 for Cdkn1a. GSEA linked these genes to tricarboxylic acid cycle (TCA) dysregulation and immune infiltration. Unsupervised clustering identified 2 molecular subtypes. C1 exhibited higher expression of 6 of the 7 autophagy-related DEGs, whereas C2 showed higher proportions of mast cells and activated dendritic cells and enrichment of mitochondrial and energy-metabolism pathways. c-Myc knockout reduced endothelial apoptosis and autophagic injury.

c-Myc emerged as a critical driver linking excessive autophagy to microvascular damage, providing a promising diagnostic and therapeutic target for reperfusion injury management.