Effects of hypoxia/reoxygenation on apoptosis, autophagy and pyroptosis in H9C2 cardiomyocytes

Abstract

Objective To investigate the effects of hypoxia/reoxygenation (HR) on apoptosis, autophagy and pyroptosis in H9C2 cardiomyocytes. Methods The cultured H9C2 cardiomyocytes were divided into two groups according to the random number table method: a normal control (Ctrl) group and an HR group. The H9C2 cardiomyocytes in the HR group were deprived of oxygen and glucose for 8 h in a hypoxic incubator, followed by reoxygenation for 12 h. Cell damage was assessed through detection of lactate dehy-drogenase (LDH) content in culture medium and determination of cell viability by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) method (n=6). Apoptosis related proteins [cysteinyl aspartate-specific proteinase (caspase)-3, B-cell lymphoma/leuke-mia 2 (Bcl-2) and Bcl-associate x protein (Bax)], autophagy related proteins [(light chain 3 (LC3) Ⅱ/Ⅰ , p62, Beclin-1, and phosphory-lated mammalian target of rapamycin (p-mTOR)]and pyroptosis related proteins [(nod-like receptor pyrin domain 3 (NLRP3), apoptosis associated speck-like protein (ASC), caspase-1p20, interleukin (IL)-1β, and IL-18]were detected by Western blot (n=9). Cell apopto-sis, autophagy and pyroptosis were further assessed by immunofluorescence staining (n=6) Results Compared with the Ctrl group, the HR group presented reduced viability of H9C2 cardiomyocytes (P<0.05), increased LDH release (P<0.05), and up-regulated expres-sion of activated caspase-3 and Bax (P<0.05), as well as decreased expression of Bcl-2 (P<0.05). TUNEL staining showed that apoptosis significantly was enhanced in the cells after HR (P<0.05). These results indicated that apoptosis and cell damage were enhanced after HR. In contrast, the expression of p-mTOR and p62 increased (P<0.05), but the expression of LC3 Ⅱ/Ⅰ and Beclin-1 protein decreased(P<0.05). The immunofluorescence staining of LC3 Ⅱ/Ⅰ showed that the number of autophagosome within the cells increased after HR(P<0.05), indicating that autophagy in H9C2 myocardial cells was significantly inhibited. Meanwhile, the expression of NLRP3 inflamma-some, ASC and caspase-1p20 protein was remarkably up-regulated (P<0.05), and the expression of activated inflammatory cellular factors IL-1β and IL-18 increased (P<0.05), suggesting that NLRP3 inflammasomes were activated and pyroptosis enhanced after HR. Conclusions Autophagy is inhibited in H9C2 cardiomyocytes during HR injury, but pyroptosis and apoptosis are enhanced. Key words: Autophagy; Apoptosis; Pyroptosis; Cardiomyocytes; Hypoxia-reoxygenation injury