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

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.