Downregulation in Connexin43 expression is associated with reduced ferroptosis and enhanced tolerance to myocardial ischemia-reperfusion injury in type 1 diabetic mice

Abstract ID 22723 Poster Board 308 Patients with diabetes are more vulnerable to myocardial ischemia/reperfusion injury (MIRI). However, animal studies showed that short-term diabetes may have a protective effect on MIRI, but the mechanism is unclear. Studies showed that abnormal cardiac Connexin43 (Cx43) protein expression is associated with a variety of pathological conditions, such as myocardial ischemia and diabetic cardiomyopathy, while down-regulating Cx43 can alleviate acute kidney injury by inhibiting ferroptosis. However, the role of Cx43 in myocardial IRI in diabetic conditions and in particular its potential interplay with cardiac ferroptosis in this pathology is unknown. Thus, the present study aimed to explore the role and mechanism of Cx43 and ferroptosis in relation to myocardial susceptibility to IRI in diabetes. Streptozotocin (STZ)-induced diabetes mice and age-matched control mice were subjected to myocardial IRI by occluding the left coronary artery for 30 minutes followed by 2 hours of reperfusion respectively at 1,2 and 5 weeks of diabetes induction. In vitro, rat origin H9C2 cardiomyocytes (H9C2) were exposed to 35 mmol/L high glucose(HG) for 48h in the absence or presence of Cx43 gene knockdown with siRNA, followed by hypoxia for 6 hours and reoxygenation for 12 hours. Myocardial infarct size was assessed by TTC/ Evan blue staining. The heart function of mice was measured by echocardiography. Myocardial mitochondrial damage was measured by transmission electron microscopy. Serum CK-MB and malondialdehyde (MDA) levels were measured by ELISA. Cell viability was detected by the CCK-8 assay, apoptosis by the TUNEL assay. BODIPY staining for ROS production. Western blot analysis was performed to assess cardiac Cx43 protein and ferroptosis-related protein expression. Compared to controls, infarct size was reduced in 1-2 weeks DM mice after I/R, but significantly increased at DM 5 weeks (p<0.05). Post-ischemic cardiac function was improved in 1 week mice and worsened in 5 weeks DM mice as compared to that in the non-diabetic control mice, which was associated with reduced ferroptosis in DM 1-2 weeks and significantly enhanced ferroptosis in DM 5 weeks mice (all p< 0.05 vs. control). Also, cardiac Cx43 expression was significantly lower after I/R in DM 1-2 weeks mice compared to controls and significantly higher in DM 5 weeks mice. Cx43 inhibitor GAP19 significantly attenuated ferroptosis and reduced post-ischemic myocardial infarct size in 5 weeks DM mice. Application of Erastin (ferroptosis activator) reversed the cardioprotective effect of GAP19. In vitro, H9C2 cells exposed to hypoxia and reoxygenation (H/R) under high glucose environment showed a significant increase in cellular injury, as evidenced by increased LDH release, reduced cell viability, significantly increased lipid peroxidation product (MDA) and ferroptosis, which was concomitant with significantly elevated Cx43 expression. Cx43 gene knockdown in H9C2 resulted in a significant increase in glutathione peroxidase 4 (Gpx4) protein expression, reduction in MDA production and ferroptosis, and subsequently reduced post-hypoxic cell viability under high glucose (all p< 0.05 vs. control). However, these beneficial effects of Cx43 gene knock-down were significantly attenuated or eliminated by Erastin. It is concluded that down-regulating the expression of Cx43 in the diabetic myocardium may subsequently inhibit ferroptosis, thereby increasing myocardial tolerance to ischemia-reperfusion injury.