Autophagy is involved in the protective effect of endophilin A2 on H₂O₂-induced apoptosis in H9C2 cardiomyocytes

Highlights

• EndoA2 promotes the association between Bif-1 and beclin-1 to strengthen autophagy.

Abstract

Apoptosis plays a critical role in normal embryonic development and tissue homeostasis regulation. EndophilinA2 (EndoA2) is widely reported to regulate endocytosis. Additionally, EndoA2 has been demonstrated to be involved in tumor metastasis, neuroregulation and vascular function. In this study, we used siRNA and Ad-EndoA2 transfection strategy to investigate whether EndoA2 provides a protective effect against apoptosis induced by H₂O₂ in H9C2 cardiomyocytes and the underlying mechanisms. We found that EndoA2 siRNA knockdown promoted H₂O₂-induced apoptosis in H9C2 cardiomyocytes, evidenced by decreased cell number, increased apoptotic cells, and activation of caspase-3. In contrast, EndoA2 overexpression showed the opposite effects and inhibited H₂O₂-induced apoptosis in H9C2 cardiomyocytes. Further studies revealed that EndoA2 overexpression strengthened autophagy, evidenced by the increased LC3 II/I ratio and P62 degradation, whereas EndoA2 siRNA knockdown produced the opposite effects. Furthermore, we revealed that there was an interaction between Bif-1 and Beclin-1. Upon H₂O₂ treatment, the association of Bif-1 and Beclin-1 remarkably increased. EndoA2 overexpression further promoted the binding of Bif-1 with Beclin-1, whereas EndoA2 siRNA knockdown reduced this association. These data strongly suggested that EndoA2 inhibited H₂O₂-induced apoptosis in H9C2 cardiomyocytes, possibly by promoting Bif-1 to form a complex with Beclin-1 and strengthening autophagy. This study provides a novel target for heart diseases.

Introduction

Although oxidative stress caused by reactive oxygen species (ROS) is usually an endogenous antioxidant defense mechanism in cardiomyocytes, excessive ROS causes adverse left ventricular remodeling and ultimately heart failure [1,2]. ROS, including H₂O₂, peroxynitrite, superoxide and hydroxyl radicals, are likely involved in the pathogenesis of myocardial injury, through the induction of damage to DNA, protein and lipids [3]. There are multiple signaling pathways regulating the level of ROS, including autophagy [4,5]. Autophagy is an evolutionarily conserved process for bulk degradation and recycling of cytoplasmic components. To date, autophagy has been implicated in a number of pathological and biological processes, such asaging [6], tumorigenesis [7], cell death [8] and differentiation [9]. Recent studies on autophagy in cardiac injury or disease have suggested both protective and deleterious roles of autophagy, depending on the model system [10,11]. During the early stage of disease, autophagy represents a compensatory mechanism to maintain homeostasis [7]. On the other hand, excessive activation of autophagy can exert a significant contributory role in disease pathogenesis [12,13].

EndoA2 is widely expressed in eukaryotic cells, such as tumor cells, neurocytes and smooth muscle cells. It's reported to participate in regulating various pathological and biological progressions, including tumor metastasis [14], atherosclerosis [15] and vascular remodeling [16]. Our previous study showed that EndoA2 inhibited H₂O₂-induced apoptosis in rat basilar artery smooth muscle cells by blockade of Bax translocation from the cytosol to mitochondria, suggesting a protective role of EndoA2 in vascular remodeling [17]. However, the function of EndoA2 in the heart system, are largely unknown. In this study, we investigated whether EndoA2 influenced H₂O₂-induced apoptosis in H9C2 cardiomyocytes and the underlying mechanisms. Our results showed that EndoA2 protects against H₂O₂-induced apoptosis through strengthening autophagy in H9C2 cardiomyocytes.