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    • Autophagy as an important cellular process has drawn attenti

    2022-06-21

    Autophagy as an important cellular process has drawn attentions to its role in cardiovascular systems [16]. Autophagy is a self-digestion and degradation process that recycles the contents of the cytosol, including macromolecules and cellular organelles, resulting in self-repair and conservation for survival. Autophagy serves as a programmed cell death pathway when it becomes overactive and prolonged [17]. There are different forms of autophagy including microautophagy, macroautophagy, chaperone-mediated autophagy and etc. Macroautophagy represents a typical process of autophagy (autophagy refers to macroautophagy hereafter) [18]. Autophagy sequestrates damaged organelles and unused long-lived proteins in a double-membrane vesicle known as the autophagosome. The autophagosome further fuses with a lysosome or endosome to form an autolysosome. Lysis of the autophagosome inner membrane and breakdown of the contents take place in the autolysosome, and the resulting useful macromolecules are released back into the cytosol through membrane permeases [17], [18]. Autophagy as an end-effector in ischemic and pharmacologic preconditioning is considered cardioprotective [19], [20], [21]. On the other hand, studies found that autophagy enhanced preconditioning cardiac cell death [22], [23]. HIV-1 gp120 was reported to induce autophagy in vitro in neuronal HAMI3379 australia and T lymphocytes [24], [25], [26]. However, the effects of gp120 on autophagy in cardiovascular cells have not been addressed. This study was designed to investigate the effects of the HIV-1 gp120 on autophagy in an H9c2 cardiomyocyte developed from rat cardiomyoblast [27]. We hypothesized that gp120 could induce autophagy in cardiomyocytes.
    Materials and methods
    Results
    Discussion Despite much research on the pathogenesis associated with HIV-1 infection, the mechanisms associated with the cardiovascular involvement remain unclear. In the findings presented here, we demonstrate that gp120 treated cardiomyocytes show an increase in autophagic activity. We have used a number of different approaches to confirm the increase in autophagy activity in the cardiomyocytes. First, because Beclin 1 and ATG7 are required for autophagy, we examined the levels of these two proteins by Western blot. We found that cardiomyocytes exposed to gp120 consistently expressed higher levels of Beclin 1 and ATG7 when compared to those of vehicle control cells. Autophagosome formation requires Beclin 1–Vps34 core complex for autophagosome nucleation whereas autophagic vesicle elongation is regulated by the covalent binding of ATG5 to ATG12, a process catalyzed by ATG7 and ATG10 [41]. Our findings suggest that autophagy is likely increased. Second, we analyzed the autophagic specific protein LC3 by Western blot. Originally characterized as a microtubule-associated protein light chain 3 (MAP-LC3), LC3 protein is cleaved by the cellular protease ATG4, generating an 18-kDa species termed LC3-I. When autophagy is activated, a series of covalent transfers links LC3 to ATG7, then to ATG3, and finally to phosphatidylethanolamine, generating a 16kDa lipidated species termed LC3-II. This modification allows LC3 to become membrane associated, preferentially associating with the developing and newly formed autophagosomes. LC3-II is associated with mature autophagosomes and is degraded together with its cargo following fusion of the autophagosome with a lysosome [29], [30], [31]. Because conversion of LC3-I to LC3-II is characteristic in autophagy, we compared LC3-II to LC3-I and found the ratio to be increased in gp120 treated cells. This finding combined with the increase in LC3 puncta representing autophagosome in cardiomyocytes further supports an increase in autophagic activity. Third, because an increase in autophagic activity is known to be associated with increased numbers of lysosomes, we also examined the cardiomyocytes for evidence of the expression of LAMP1 by Western blot and formation of lysosome by LAMP 1 intracellular staining. Autophagy is an intracellular degradation system that delivers cytoplasmic contents to the lysosome for degradation and recycling. Similar to the increase in autophagic protein Beclin 1, ATG7 and LC3-II as well as LC3 puncta in gp120 treated cardiomyocytes, we found an increase in LAMP1 protein and LAMP1 puncta in the same cells. In addition, to determine if gp120 can efficiently induce a mature autophagy, we assessed the autophagic flux by analyzing the fusion of the autophagosome and the lysosome in the cardiomyocytes treated with bafilomycin A1, a member of inhibitors of membrane ATPases which is essential in the fusion of the autophagosome and the lysosome [40], [42]. We observed that saturating bafilomycin significantly blocked the formation of lysosome in gp120 treated cardiomyocytes. This data suggests that gp120 can efficiently promote autophagy by induction of mature autolysosomes in cardiomyocytes.