ANGPTL4 attenuates hypoxia/reoxygenation-induced cardiomyocyte apoptosis via activation of Akt signaling

Abstract

<b>Abstract ID 15237</b> <b>Poster Board 108</b> Ischemic heart disease (IHD) is a leading cause for morbidity and mortality worldwide. Reperfusion therapy restores blood flow, but paradoxically exacerbates myocardial injury, known as ischemia/reperfusion injury (I/RI). Apoptotic cell death is one of the main forms of post-ischemic cell death. Thus, inhibiting apoptosis may limit the extent of I/RI and lead to better prognosis of post-ischemic myocardial recovery. ANGPTL4, a gene known to regulate lipid metabolism, has been shown to preserve vascular integrity, reduce no-reflow and thus attenuate myocardial I/RI in rabbits. However, whether ANGPTL4 directly impacts cardiomyocytes and alleviates myocardial I/RI through reducing cell apoptosis is unknown . Therefore, we explored whether ANGPTL4 protects against hypoxia/reoxygenation (H/R)-induced cardiomyocyte injury through inhibiting apoptosis. In vivo myocardial I/R model was induced by occluding the left anterior descending (LAD) artery for 30 mins, followed by 2 h 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. The rat cardiomyocyte-derived cell line H9C2 was transfected with scramble or ANGPTL4 siRNA, followed by H/R (6 hours hypoxia followed by 12 h reoxygenation) in the absence or presence of pre-treatment with SC-79 (4 μg/mL, 1h, selective Akt activator) or LW-6 (20 μM, 2h, selective HIF-1α inhibitor). Cell damage was assessed by measuring Lactate dehydrogenase (LDH) release and cell viability by MTT assay. Protein levels of apoptosis markers [Bcl-2, Bax, cleaved caspase 3] and proteins related to pro-survival signaling pathway [Akt and phosphorylated Akt (S473)] were determined by Western blotting. The result showed that ANGPTL4 increased significantly in the mouse myocardium after I/RI and in H/R-stimulated H9C2 cardiomyocytes, indicating that ANGPTL4 in cardiomyocytes may be involved in the pathogenesis of myocardial I/RI. Indeed, knockdown of ANGPTL4 in H9C2 cells with ANGPTL4 siRNA significantly aggravated H/R-induced cell injury (increased LDH level and reduced cell viability) and exacerbated cell apoptosis (greater cleaved caspase 3 expression and Bax/Bcl-2 ratio). Activation of Akt is well known to protect against myocardial I/RI via inhibition of cell apoptosis. In the present study, a significant reduction in p-Akt (S473) was observed in the ANGPTL4-knockdown cells upon H/R stimulation, suggesting that inhibition of ANGPTL4 may exacerbate H/R-induced cell apoptosis via down-regulation of Akt signaling pathway. Indeed, pre-treatment with SC-79 significantly reversed H/R-induced cell apoptosis in ANGPTL4 knockdown H9C2 cardiomyocytes, as evidenced by decreased cleaved caspase 3 expression and Bax/Bcl-2 ratio. Furthermore, HIF-1α is known to be the major transcription factor activated in hypoxia in ischemic conditions. The induction of ANGPTL4 was significantly reduced when H/R-stimulated H9C2 cardiomyocytes were pre-treated with LW-6, indicating that HIF-1α may upregulate ANGPTL4 upon H/R stimulation. It is concluded that upon H/R stimulation, HIF-1α-mediated upregulation of ANGPTL4 may protect against H/R-induced cardiomyocyte injury through activating Akt signaling. This study was supported by Joint Postdoc Scheme (P0038982), Guangdong Basic and Applied Basic Research Foundation (2022A1515011116, 2019A1515110063) and National Natural Science Foundation of China (82002095).