Hydroxysafflor yellow A ameliorates myocardial ischemia/reperfusion injury by promoting MDH1-mediated mitochondrial metabolic homeostasis.

BACKGROUND

Myocardial ischemia/reperfusion injury (MIRI) is a critical complication in the management of acute myocardial infarction, substantially compromising clinical outcomes. Hydroxysafflor yellow A (HSYA), a natural compound derived from Carthamus tinctorius L., has been approved by the National Medical Products Administration for the management of cardiovascular and cerebrovascular diseases. While empirical evidence supports its therapeutic efficacy against MIRI, the underlying mechanism remains incompletely characterized.

PURPOSE: This study aimed to elucidate the mechanism of action and direct targets of HSYA treatment for MIRI.

METHODS

Pharmacological evaluations were conducted via a left anterior descending-ligated MIRI model in male C57BL/6 mice and a hypoxia/reoxygenation (H/R)-injured H9c2 cell model. Reducing dimethyl labeling with tandem orthogonal proteolysis-activity-based protein profiling (rdTOP-ABPP) in combination with liquid chromatography‒tandem mass spectrometry was employed to investigate the mechanism of action and identify direct targets and binding sites. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were conducted to elucidate the therapeutic mechanisms of HSYA. A cellular thermal shift assay was used to detect the drug-target protein binding efficiency. Enzymatic activity profiling was systematically conducted to evaluate how HYSA modulates core metabolic enzymes. Furthermore, small-interfering RNA transfection was implemented to establish a causal linkage between the candidate molecular target and its core biological functions.

RESULTS

We found that HSYA administration in MIRI-induced mice significantly reduced the myocardial infarct size, improved cardiac function, inhibited apoptosis, and restored myocardial bioenergetics by increasing ATP production and balancing the NAD+/NADH level. In H/R-injured H9c2 cells, HSYA dose-dependently reduced mitochondrial damage, decreased reactive oxygen species production, inhibited mitochondrial permeability transition pore opening, and promoted mitochondrial energy metabolism, including the levels of glycolysis and oxidative phosphorylation. Cysteine (Cys) activity-based protein profiling revealed that malate dehydrogenase 1 (MDH1), an essential metabolic enzyme involved in maintaining the mitochondrial energy metabolism homeostasis, was identified as a core target of HSYA. HSYA covalently activated MDH1 through Cys residue 137, thereby promoting MDH1's bioenergetic enzymatic activity and restoring mitochondrial energy metabolism homeostasis. Importantly, disruption of MDH1 or the predicted binding site significantly abolished the cardioprotective benefits of HSYA.

CONCLUSIONS

HSYA, an allosteric MDH1 activator, is a potential therapeutic strategy for regulating mitochondrial energy metabolism homeostasis and improving mitochondrial dysfunction in MIRI. Our study provides a scientific rationale for the clinical application of HSYA.