a Department of Physiology and Pathophysiology , Tianjin Medical University , Tianjin , China.
b Department of Cardiology , General Hospital, Tianjin Medical University , Tianjin , China.
Free Radic Res. 2018 Jan;52(1):80-91. doi: 10.1080/10715762.2017.1414949. Epub 2017 Dec 19.
Zinc plays a role in autophagy and protects cardiac cells from ischemia/reperfusion injury. This study aimed to test if zinc can induce mitophagy leading to attenuation of mitochondrial superoxide generation in the setting of hypoxia/reoxygenation (H/R) in cardiac cells. H9c2 cells were subjected to 4 h hypoxia followed by 2 h reoxygenation. Under normoxic conditions, treatments of cells with ZnCl increased both the LC3-II/LC3-I ratio and GFP-LC3 puncta, implying that zinc induces autophagy. Further experiments showed that endogenous zinc is required for the autophagy induced by starvation and rapamycin. Zinc down-regulated TOM20, TIM23, and COX4 both in normoxic cells and the cells subjected to H/R, indicating that zinc can trigger mitophagy. Zinc increased ERK activity and Beclin1 expression, and zinc-induced mitophagy was inhibited by PD98059 and Beclin1 siRNA during reoxygenation. Zinc-induced Beclin1 expression was reversed by PD98059, implying that zinc promotes Beclin1 expression via ERK. In addition, zinc failed to induce mitophagy in cells transfected with PINK1 siRNA and stabilized PINK1 in mitochondria. Moreover, zinc-induced PINK1 stabilization was inhibited by PD98059. Finally, zinc prevented mitochondrial superoxide generation and dissipation of mitochondrial membrane potential (ΔΨ) at reoxygenation, which was blocked by both the Beclin1 and PINK1 siRNAs, suggesting that zinc prevents mitochondrial oxidative stress through mitophagy. In summary, zinc induces mitophagy through PINK1 and Beclin1 via ERK leading to the prevention of mitochondrial superoxide generation in the setting of H/R. Clearance of damaged mitochondria may account for the cardioprotective effect of zinc on H/R injury.
锌在自噬中发挥作用，并保护心脏细胞免受缺血/再灌注损伤。本研究旨在测试锌是否可以诱导细胞自噬，从而减轻心脏细胞缺氧/复氧（H/R）期间线粒体超氧化物的产生。将 H9c2 细胞置于 4 小时缺氧后再复氧 2 小时。在正常氧条件下，用 ZnCl2 处理细胞可增加 LC3-II/LC3-I 比值和 GFP-LC3 斑点，表明锌诱导自噬。进一步的实验表明，饥饿和 rapamycin 诱导的自噬需要内源性锌。锌下调正常氧细胞和 H/R 细胞中的 TOM20、TIM23 和 COX4，表明锌可触发细胞自噬。锌增加 ERK 活性和 Beclin1 表达，并且在复氧期间 PD98059 和 Beclin1 siRNA 抑制锌诱导的自噬。锌诱导的 Beclin1 表达被 PD98059 逆转，表明锌通过 ERK 促进 Beclin1 表达。此外，锌未能在转染 PINK1 siRNA 的细胞中诱导自噬，并稳定线粒体内的 PINK1。此外，PD98059 抑制锌诱导的 PINK1 稳定。最后，锌可防止复氧时线粒体中超氧阴离子的产生和线粒体膜电位（ΔΨ）的耗散，Beclin1 和 PINK1 siRNA 均阻断了这一过程，表明锌通过自噬防止线粒体氧化应激。总之，锌通过 ERK 诱导 PINK1 和 Beclin1 诱导自噬，从而防止 H/R 期间线粒体中超氧阴离子的产生。清除受损线粒体可能是锌对 H/R 损伤的心脏保护作用的原因。
