GSK3β-mediated phosphorylation of IF1 at Ser27 destabilizes mitochondrial ATP synthase inhibitory factor 1 to aggravate cardiac ischemia/reperfusion injury.

OBJECTIVES: Myocardial ischemia-reperfusion (I/R) injury is a major cause of ischemic cardiomyopathy and chronic heart failure, underscoring the need for new cardioprotective strategies. ATP synthase inhibitory factor 1 (IF1) maintains mitochondrial function and limits oxidative damage during I/R injury. Our previous work showed that IF1 protects the heart through AMPK activation; however, IF1 protein levels decline sharply during I/R despite stable mRNA expression, suggesting post-translational regulation. This study aimed to elucidate the molecular mechanism underlying IF1 instability and to characterize a newly identified phosphorylation site at serine 27 (S27). METHODS: Mass spectrometric analysis of HEK293 cells overexpressing human IF1 identified a novel phosphorylation site at serine 27 (S27), and subsequent experiments validated that phosphorylation at this specific site was markedly induced during the I/R process. The functional role of IF1 was further investigated in an IF1-KO mouse model of myocardial I/R, comparing the effects of wild-type IF1 (WT) and a non-phosphorylatable IF1 mutant (S27A). RESULTS: Mass spectrometric analysis revealed S27 as a novel phosphorylation site of IF1 that is markedly induced during I/R injury. This phosphorylation promoted the interaction between IF1 and the E3 ubiquitin ligase NEDD4, enhancing IF1 ubiquitination and proteasomal degradation. In vivo, restoration of IF1-either WT or S27A-attenuated acute and chronic myocardial injury, preserved mitochondrial integrity, and improved cardiac function. Notably, the S27A mutant, resistant to phosphorylation-dependent degradation, provided superior cardioprotection compared with WT IF1. CONCLUSIONS: Glycogen Synthase Kinase-3 beta (GSK3β) mediated phosphorylation of IF1 at the newly identified S27 site destabilizes IF1 via the ubiquitin-proteasome pathway, contributing to myocardial I/R injury. Blocking this modification stabilizes IF1 and enhances cardioprotection. These findings reveal a previously unrecognized mechanism of IF1 regulation and identify S27 phosphorylation as a promising therapeutic target against reperfusion-induced cardiac damage.