Molecular Mechanisms Underlying Sex-Related Differences In Mitochondrial Response To Myocardial Ischemia/Reperfusion

Introduction: Cardiac dysfunction adversely impacts patient outcomes following surgically induced myocardial ischemia/reperfusion (I/R). Maintaining mitochondrial activity is crucial to reducing myocardial I/R. Inflammation and oxidative stress triggered by I/R impair mitochondria. Our group has shown sex differences in myocardial functional recovery after I/R. Here, we determined whether sex disparity in mitochondrial response to inflammatory mediator - TNFα and to reactive oxygen species - H 2 O 2 is responsible for sex differences in cardiac dysfunction. Given the key role of mitochondrial connexin43 (Cx43) in cardiac protection initiated by ischemic preconditioning, we also investigated the implication of Cx43 in sex-related mitochondrial response. Methods: Cardiomyocytes (male & female; C57BL/6 & doxycycline inducible Tnnt2-controlled Cx43 knockout) were subjected to simulated I/R (20’ I/60’ R), TNFα (10ng/ml), or H 2 O 2 (50 μM), +/- 17β-estradiol (E2).Mitochondrial membrane potential (ΔΨm) analyzed by JC1 red to JC1 green fluorescence intensity ratio (Fig. A) and mitochondrial respiration capacity by Seahorse XF Cell Mito Stress Test were determined.Cx43 was assessed in mitochondria by Western blots and Immunoelectron microscopy. Results: Improved ΔΨm (Fig. B) and preserved mitochondrial respiratory capacity were observed in female cardiomyocytes than in male ones following simulated I/R, TNFα, or H 2 O 2 .E2 restored mitochondrial activity in cardiomyocytes (Fig. C, D). In addition, E2 increased Cx43 content and phosphorylation in cardiac mitochondria. Ablation of cardiac Cx43 abolished E2-mediated mitochondrial protection during TNFα or H 2 O 2 challenge (Fig. E). Conclusions: Female cardiomyocytes were more resistant to I/R, TNFα, or H 2 O 2 stimulation than age-matched males, with greater protective role of mitochondrial Cx43 in female hearts. E2 usage regulated mitochondrial Cx43 and preserved cardiac mitochondrial performance.