Hypoxia-induced mitochondrial dysfunction and mitophagy in the small yellow croaker (Larimichthys polyactis)

Mitophagy serves as a pivotal mechanism for regulating the quantity and quality of mitochondria within cells, exerting significant influence on various processes such as cell differentiation, oxidative stress, inflammatory responses, and apoptosis. Currently, research on whether and how fish activate mitophagy under hypoxic stress conditions is still insufficient. In this study, to determine the mechanisms whereby marine fish adapt to hypoxic environments from the perspective of mitophagy, we used the small yellow croaker (Larimichthys polyactis) as the research subject and combined in vivo (liver) and in vitro (small yellow croaker fry [SYCF] cell line) hypoxic stress experiments. Fish exposed to hypoxic conditions were found to be characterized by liver tissue damage, and we detected significant elevations in the levels of hydrogen peroxide in liver tissues and reactive oxygen species (ROS) in SYCF cells, along with significant reductions in mitochondrial membrane potential. These findings thus indicate that hypoxic stress leads to tissue damage, excessive ROS production, and mitochondrial damage. In further experiments, we pre-treated SYCF cells with the antioxidant N-acetylcysteine, which was found to effectively reduce ROS levels and prevented the loss of mitochondrial membrane potential, thereby indicating that ROS play a crucial role in hypoxic stress-induced mitochondrial damage. Subsequently, to investigate whether hypoxic stress activates mitophagy to remove damaged mitochondria, we examined changes in the mRNA expression of mitophagy-related genes (bnip3, lc3b, bnip3l, beclin1, fundc1, and ulk1) in the liver and SYCF cells of L. polyactis exposed to hypoxic stress, and detected a significant upregulation of the mRNA expression of these genes. Furthermore, examination of liver ultrastructure and changes in the co-localization of mitochondria and lysosomes in SYCF cells revealed that hypoxic stress induces the formation of autophagosomes and autolysosomes in the liver, with an enhanced co-localization of mitochondria and lysosomes being observed after 6 h of hypoxia, which gradually increased with a prolongation of hypoxic exposure. We have, for the first time, exhibited the formation process of autophagosomes and the subsequent formation of autolysosomes in fish under hypoxic stress. These findings reveal the induction of mitophagy in L. polyactis in response to hypoxic stress, and indicate that these fish may initiate a mitophagic response to remove damaged mitochondria, reduce excessive ROS accumulation, and maintain cellular homeostasis. Our findings will not only lay a biological foundation for the breeding of hypoxia-tolerant strains of L. polyactis but also provide new insights into the mechanisms underlying the adaptation of marine fish to hypoxic environments.