Disulfide-oxidized HMGB1 mutant construction and its mechanism in AMI-induced myocardial injury

Cardiovascular diseases are the leading cause of mortality and disability worldwide. Disulfide-bond oxidized HMGB1 (ds-HMGB1) functions as a core damage-associated molecular pattern that mediates acute myocardial injury. Nevertheless, commercially available HMGB1 reagents exhibit mixed proteoforms, unstable biological activity and low receptor affinity, which greatly hinder mechanistic investigations in this field. This study generated a novel mutant full-length murine ds-HMGB1, systematically dissected its molecular mechanisms underlying aggravated ischemic myocardial injury, and sought to provide a standardized protein reagent and theoretical framework for HMGB1-targeted therapies against myocardial damage. A K184S/K185S mutant HMGB1 plasmid was constructed, and protein purified by three-step chromatography. Structural modeling, mass spectrometry, SPR and PLA were used to analyze conformation, modification and receptor binding kinetics. Hypoxic cardiomyocyte co-culture and mouse myocardial infarction models were established. Flow cytometry, echocardiography, histological staining, qPCR, Western blot and RNA-seq were applied to detect molecular phenotypes and signaling pathways. The K184S/K185S mutation preserved the native overall conformation and functional disulfide bond of HMGB1, while markedly enhancing HMGB1 binding to RAGE and promoting TLR4-MD2 complex assembly. In vitro experiments revealed that mutant ds-HMGB1 either acted directly on cardiomyocytes or synergized hypoxia through endothelial-cardiomyocyte paracrine signaling to induce robust intracellular ROS accumulation, mitochondrial dysfunction and cardiomyocyte apoptosis. In vivo administration expanded myocardial infarct size, impaired cardiac systolic performance and exacerbated inflammatory infiltration in cardiac tissue. Transcriptomic profiling identified 53 core injury genes specifically upregulated by ds-HMGB1 under ischemic stress. The mutant ds-HMGB1 developed in this study addresses the intrinsic drawbacks of commercial HMGB1 preparations. We further identified 53 key myocardial injury genes modulated by ds-HMGB1. This work delivers a standardized experimental reagent and multi-layered mechanistic evidence supporting HMGB1 as a therapeutic target for acute myocardial infarction.