Enhancement of ANGPTL4 exacerbates post-ischemic myocardial ferroptosis and cellular injury in diabetic conditions via impairing AKT/AMPK signaling

Abstract ID 97790 Poster Board 070 Background: Myocardial infarction as a result of ischemic heart disease is the primary cause of death in patients with type 2 diabetes mellitus (T2DM). Reperfusion therapy restores blood flow, but paradoxically exacerbates myocardial injury, known as ischemia/reperfusion injury (I/RI). Ferroptosis is an important type of cardiomyocyte death caused by infarction-reperfusion, especially in the later phase of reperfusion. Research has shown that abnormal expression of the Angiopoietin-like protein 4(ANGPTL4) is associated with various pathological conditions, such as myocardial ischemia and diabetic cardiomyopathy. The role of ANGPTL4 in diabetic myocardial IRI and its potential interaction with myocardial cell ferroptosis in this pathology is unknown. Methods: Male C57BL/6 mice were fed with a high-fat diet (HFD) for 6 weeks and received intraperitoneal injection of low dose streptozotocin to induce T2DM. In vivo diabetic myocardial I/R model was induced by occluding the left anterior descending (LAD) artery for 30 mins, followed by 2h reperfusion. Sham operations were performed by passing a silk thread under the LAD without occlusion. Infarct size was determined by using Evans blue/TTC staining, and cardiac function was determined by echocardiography. In vitro, The cardiac origin HL-1 cells were exposed to high glucose (HG) and palmitic acid (PAL) for 24 hours, followed by H/R (6 hours hypoxia followed by 12 hours reoxygenation) in the absence or presence of ANGPTL4 gene knockdown or AMPK gene overexpression. Results: The result showed that ANGPTL4 increased significantly in the diabetic mouse myocardium after I/RI and in H/R-stimulated HL-1 cells, but cardiac levels of p-AMPK and p-AKT reduced as compared to non-diabetic control that was accompanied with reduced GPX4 protein expression and increased oxidative stress and ferroptosis. Knockdown of ANGPTL4 in HL-1 cells with ANGPTL4 siRNA significantly enhanced GPX4 protein expression, reduced ferroptosis as evidenced by reduced production of reactive substances, ferrous ion content and lipid peroxidation and attenuated H/R-induced cell injury that was concomitant enhanced protein levels of p-AMPK and p-AKT. Activation of Akt is known to protect against myocardial I/RI via inhibition of cell apoptosis, and activation of AMPK may be related to the inhibition of cell ferroptosis. Of note, overexpression of AMPK in HL-1 cells cultured under diabetic conditions with high glucose and palmitate reversed H/R induced reductions in p-Akt and GPX4, and significantly reduced post-hypoxic ferroptosis and cardiomyocyte injuries without significant impact on the increased post-hypoxic ANGPTL4 protein expression. Conclusion: Findings of the current study are indicative that excessive enhancement of ANGPTL4 after myocardial I/RI in diabetic conditions may exacerbate post-ischemic myocardial injury via impairing P-AMPK/P-AKT signaling and that AMPK works downstream of ANGPTL4. Funding: This study was supported by National Natural Science Foundation of China (NSFC, 81970427,82270306), and The Hong Kong Polytechnic University Translational Research Grant HK from Faculty of Health and Social Sciences (P0048507).