A K184S/K185S mutant full-length murine ds-HMGB1 preserved native conformation and the disulfide bond while enhancing binding to RAGE and TLR4-MD2 assembly, and it worsened ischemic myocardial injury in vitro and in vivo, supporting HMGB1 as a therapeutic target for acute myocardial infarction and providing a standardized reagent.
Key Findings
Methods
The authors generated a K184S/K185S mutant full-length murine disulfide-oxidized HMGB1 (ds-HMGB1) as a standardized reagent intended to overcome limitations of commercial HMGB1 preparations.
Commercial HMGB1 reagents were described as having mixed proteoforms, unstable biological activity and low receptor affinity.
A K184S/K185S mutant HMGB1 plasmid was constructed and the protein was purified by three-step chromatography.
Structural modeling, mass spectrometry, SPR and PLA were used to analyze conformation, modification and receptor binding.
Results
The K184S/K185S mutation preserved the native overall conformation and functional disulfide bond of HMGB1.
Conformation and modification were assessed by structural modeling and mass spectrometry.
The preserved disulfide bond is the feature defining the ds-HMGB1 proteoform.
Results
The mutation markedly enhanced HMGB1 binding to RAGE and promoted TLR4-MD2 complex assembly.
Receptor binding kinetics were analyzed by SPR, and receptor interactions/complex assembly by PLA.
The authors present this as addressing the low receptor affinity of commercial HMGB1.
Results
In hypoxic cardiomyocyte models, mutant ds-HMGB1 induced robust intracellular ROS accumulation, mitochondrial dysfunction and cardiomyocyte apoptosis.
Mutant ds-HMGB1 acted either directly on cardiomyocytes or synergized with hypoxia.
Outcomes were measured using flow cytometry, qPCR and Western blot among other methods.
Results
Mutant ds-HMGB1 acted via endothelial-cardiomyocyte paracrine signaling to synergize with hypoxia in causing cardiomyocyte injury.
A hypoxic endothelial-cardiomyocyte co-culture system was used.
This paracrine route produced ROS accumulation, mitochondrial dysfunction and apoptosis in cardiomyocytes.
Results
In a mouse myocardial infarction model, mutant ds-HMGB1 administration expanded infarct size, impaired cardiac systolic function and exacerbated inflammatory infiltration in cardiac tissue.
Assessment used echocardiography and histological staining.
The findings support a role for ds-HMGB1 in aggravating ischemic myocardial injury in vivo.
Results
RNA-seq identified 53 core myocardial injury genes specifically upregulated by ds-HMGB1 under ischemic stress.
Transcriptomic profiling was applied to examine signaling pathways and molecular phenotypes.
The authors describe these as key myocardial injury genes modulated by ds-HMGB1.
Conclusions
The authors conclude that the mutant ds-HMGB1 supplies a standardized experimental reagent and multi-layered mechanistic evidence supporting HMGB1 as a therapeutic target for acute myocardial infarction.
The reagent addresses intrinsic drawbacks of commercial HMGB1 preparations.
The work is framed as a theoretical framework for HMGB1-targeted therapies against myocardial damage.
What This Means
Heart attacks damage heart muscle partly through inflammation. One molecule released from injured cells, HMGB1, acts as a danger signal, particularly in its disulfide-oxidized form. Studying it has been hard because commercial HMGB1 products are mixtures of different forms, vary in activity, and bind poorly to their receptors. This study built an engineered mouse version of the protein, with two amino acid changes (K184S/K185S), that keeps its normal shape and key disulfide bond but binds more strongly to the immune receptors RAGE and TLR4-MD2.
In lab experiments, this engineered protein caused heart muscle cells to build up reactive oxygen species, develop mitochondrial problems and undergo programmed cell death, especially under low-oxygen conditions. It did this by acting directly on the heart cells and also through signals from neighboring blood vessel lining cells. In mice with induced heart attacks, giving the protein produced larger damaged areas, weaker pumping function and more inflammatory cell infiltration. Gene activity analysis pinpointed 53 genes that rose specifically with this protein under ischemic stress.
This research suggests that HMGB1 in its disulfide form worsens heart attack injury and could be a target for future treatments. The engineered protein may also help researchers study HMGB1 more consistently. The work was done in cells and mice, so it does not show whether HMGB1-targeted treatments would help people.
Sun G, Xu X, Jiang S, Tang J, Zhao L, Yan H, et al.. (2026). Disulfide-oxidized HMGB1 mutant construction and its mechanism in AMI-induced myocardial injury.. Apoptosis : an international journal on programmed cell death. https://doi.org/10.1007/s10495-026-02448-2