BACKGROUND: Myocardial ischemia (MI) threatens the health of middle-aged and older adults by reducing cardiac oxygen supply and function. Current therapies, including vasodilation, thrombolysis, and interventions, focus on relieving symptoms and improving blood flow but do not adequately address underlying energy metabolism issues. Ligustilide exerts a protective effect on the cardiovascular system and holds the potential for ameliorating MI
however, there is currently no systematic elucidation of ligustilide's target and action mechanism for MI. PURPOSE: This study aimed to comprehensively assess ligustilide's potential targets for improving acute MI and elucidate its underlying mechanism. METHODS: The therapeutic effects of ligustilide were evaluated at doses of 30, 15, and 7.5 mg/kg over 7 days in a murine model of acute MI induced by isoproterenol hydrochloride. The putative target protein was identified through target fishing, in-gel imaging, and thermal shift assay (TSA), followed by tissue and cell localization studies via a ligustilide probe. The interaction sites between ligustilide and the target protein were elucidated using protein profiling, molecular docking, and TSA at the protein level. Subsequently, knockdown and reconstruction tests were employed at the cellular level to identify the functionally active sites where ligustilide binds to the target protein. Finally, molecular docking and molecular dynamics simulations were conducted to elucidate the underlying mechanism by which ligustilide enhances creatine kinase, M-type (CKMM) protein activity. RESULTS: The covalent bonding of ligustilide in cardiac tissue enhances the therapeutic effect on acute MI in mice. For the first time, we found ligustilide specifically targets Cys254 of the CKMM protein following epoxidation. This irreversible binding effectively reduces the proximity between creatine and ATP, promoting creatine phosphorylation and ultimately increasing the creatine phosphate (CP) level by 9.50 % to 19.31 %. The accumulation of CP alleviates MI by enhancing energy metabolism, mitigating oxidative stress, and suppressing inflammatory responses. CONCLUSIONS: Our study unveiled ligustilide as a CKMM activator, which effectively enhances the content of CP and mitigates acute MI. The findings significantly contribute to advancing our understanding of ligustilide's function for myocardial protection while proposing a novel activation mechanism of CKMM to improve MI. And the insight into the covalent regulation of the active pocket on CKMM may lead to an alternative therapeutic strategy against acute MI.