FOXO1 Overactivation via Enhancing CD36 Exacerbates Myocardial Ischemia Reperfusion Injury in Diabetic Rats

Abstract

Diabetes are more vulnerable to myocardial ischemia/reperfusion injury (MIRI), and the mechanism is unclear. Fatty acid (FA) uptake and oxidation is increased in diabetic heart, leading to cardiac metabolic inefficiency and increased ROS production, which renders the hearts more vulnerable to MIRI. Forkhead box protein O1(FOXO1), a transcriptional factor, is overactivated in diabetic heart that is concomitant with the increase of lipid transporter CD36 and exacerbated MIRI. Yet, whether FOXO1-mediated cardiac metabolic changes contributes to increased susceptibility of diabetic hearts to MIRI and its potential interplay with CD36 in this pathology is unknown. Thus, we hypothesized that FOXO1 overactivation increases diabetic hearts susceptibility to MIRI by increasing CD36, and that inhibition of FOXO1 may suppress CD36, thereby to inhibit FA utilization and reduce ROS production, which in turn attenuates MIRI.Non-diabetic control (C) and Streptozotocin (STZ)-induced Type 1 diabetic rats (D) at 4 weeks of diabetes induction were subjected to 30 minutes coronary ligation and followed by 2 hours reperfusion. The left ventricular (LV) functions were assessed by Pressure-volume (PV) loop analysis. Results showed that nuclear FOXO1 was progressively increased in the heart of diabetic rats with the progress of the diseases (0, 1, 4 weeks of D rats), and became significantly higher than that in the control at 4 weeks of diabetes (P<0.05). Consequently, D rats were administered 50 mg/kg AS1842856(AS, FOXO1 specific inhibitor) by gavage twice a day for one week starting at the third week after inducing diabetes before being subjected to MIRI at 4 weeks of the diabetes. PV loop analysis indicated that heart rate(bpm), LV ejection fraction (%), dp/dt max(mm Hg/s), -dp/dt min((mm Hg/s) were all significantly reduced while post-ischemic infarct size and plasma lactate dehydrogenase (LDH) increased in diabetic rats relative to control (all P <0.05). AS slightly improved LV functions and significantly reduced infarct size and LDH after MIRI, indicating cardio-protective effects of FOXO1 inhibition.Primary cardiomyocytes cultured in high glucose/high lipid (HG/HL, 30mM/L glucose with 1mM/L palmitic acid) for 48 hours were subjected to 45 minutes hypoxia and 2 hours reoxygenaration (H/R), demonstrated significant increases in LDH release, cleaved-caspase3 expression and decrease in cell viability (all P<0.05, vs. normal culture). Meanwhile, HG/HL culture increased FOXO1 expression and proportionally increased FOXO1 nuclear retention, accompanied with significant increases in CD36 and cytochrome C protein expression. H/R can increase FOXO1 expression and CD36 expression in normal cultured cardiomyocytes, while H/R didn’t further increase CD36 and cytochrome C expression under HG/HL. Pretreatment with 1 mM AS for 1 hour before subjecting the cells to H/R significantly reduced LDH, cleaved-caspase3, CD36 and cytochrome C and increase cell viability in HG/HL cultured cardiomyocytes.In conclusion, FOXO1 overactivation exacerbates diabetic hearts MIRI by enhancing CD36 expression, which potentially increase FA uptake and oxidation to enhance myocardial susptibility to MIRI under diabetic/high glucose conditions.