MOLECULAR MEDICINE REPORTS 24: 549, 2021 HIGD‑1B inhibits hypoxia‑induced mitochondrial fragmentation by regulating OPA1 cleavage in cardiomyocytes YAN PANG1, ZHIDE ZHU2, ZHIHAO WEN1, JUNSHEN LU3, HAO LIN4, MEILING TANG1, ZHILIANG XU2 and JIANQI LU1 1Department of Cardiology, The First Affiliated Hospital, Guangxi University of Chinese Medicine, Nanning, Guangxi 530023; 2Academic Affairs Section, Guangxi University of Chinese Medicine, Nanning, Guangxi 530000; 3Academic Affairs Section, Guangxi University of Traditional Chinese Medicine Attached Chinese Medicine School, Nanning, Guangxi 530001; 4Department of Geriatrics, Danzhou Traditional Chinese Medicine Hospital, Danzhou, Hainan 571700, P.R. China Received August 17, 2020; Accepted March 9, 2021 DOI: 10.3892/mmr.2021.12188 Abstract. The dynamic regulation of mitochondrial Introduction morphology is key for eukaryotic cells to manage physiological challenges. Therefore, it is important to understand the molec‑ Mitochondria are essential subcellular organelles that are ular basis of mitochondrial dynamic regulation. The aim of the involved in respiration, oxidative phosphorylation and apop‑ present study was to explore the role of HIG1 hypoxia inducible tosis. Balanced cycles of fusion and fission are essential for domain family member 1B (HIGD‑1B) in hypoxia‑induced maintaining mitochondrial morphology and activity (1). An mitochondrial fragmentation. Protein expression was deter‑ aberrant balance between fission and fusion is associated mined via western blotting. Immunofluorescence assays were with a number of neurodegenerative disorders and cardiac performed to detect the subcellular location of HIGD‑1B. Cell diseases (2,3). For example, peroxisome proliferator‑activated Counting Kit‑8 assays and flow cytometry were carried out receptor‑γ coactivator‑1α (PGC‑1α) expression, which is to measure cell viability and apoptosis, respectively. Protein decreased in postmortem brains of patients with Parkinson's interactions were evaluated by co‑immunoprecipitation. In the disease, protected against neurotoxicity induced by present study, it was found that HIGD‑1B serves a role in cell 1‑methyl‑4‑phenyl‑1,2,3,6‑tetrahydrodropyridine (4). Fission survival by maintaining the integrity of the mitochondria under is regulated by dynamin‑related protein 1 (DRP1), which hypoxic conditions. Knockdown of HIGD‑1B promoted mito‑ can assemble into multimeric ring‑like structures and wrap chondrial fragmentation, while overexpression of HIGD‑1B around the constriction sites of dividing mitochondria (5). increased survival by preventing activation of caspase‑3 and ‑9. Mitochondrial outer membrane dynamin‑like guanosine HIGD‑1B expression was associated with cell viability and triphosphatases (GTPases) mitofusin (MFN)‑1 and ‑2 apoptosis in cardiomyocytes. Furthermore, HIGD‑1B delayed orchestrate outer mitochondrial membrane fusion (6), while the cleavage process of optic atrophy 1 (OPA1) and stabi‑ optic atrophy 1 (OPA1) is required for inner mitochondrial lized mitochondrial morphology by interacting with OPA1. membrane fusion (7). OPA1 is encoded by eight mRNA splice Collectively, the results from the present study identified a role forms, which are produced by differential splicing. Cleavage for HIGD‑1B as an inhibitor of the mitochondrial fission in of OPA1 is a key regulatory step for coordinating fusion and cardiomyocytes. fission of mitochondria (8). There are two mitochondrial inner membrane‑embedded AAA proteases (i‑AAA), metalloendo‑ peptidase OMA1, mitochondrial (OMA1) and ATP‑dependent zinc metalloprotease YME1L1 (YME1L), which can convert long forms (L‑OPA1) into short forms (S‑OPA1). The i‑AAA protease YME1L regulates OPA1 cleavage at site S2, whereas OMA1 mediates mitochondrial fragmentation by transforma‑ tion of L‑OPA1 into S‑OPA1 at site S1 (9,10). The balance Correspondence to: Professor Jianqi Lu, Department of between the two forms of OPA1 maintains normal mitochon‑ Cardiology, The First Affiliated Hospital, Guangxi University of Chinese Medicine, 89‑9 Dongge Road, Qingxiu, Nanning, Guangxi drial morphology; fusion involves L‑OPA1, whereas S‑OPA1 530023, P.R. China is associated with fission (11,12). Furthermore, OPA1 serves an E‑mail: [email protected] important role in maintaining mitochondrial inner structure and crista remodeling (13). Key words: HIG1 hypoxia inducible domain family member 1B, Hypoxia is associated with heart disease, stroke and optic atrophy 1, hypoxia, mitochondria, cardiomyocyte tumor microenvironment (14). It has also been demonstrated that hypoxia induces mitochondrial fragmentation (15). The HIG1 hypoxia inducible domain (HIGD) family of genes, 2 PANG et al: HIGD‑1B INHIBITS OPA1 CLEAVAGE whose expression is induced during hypoxia, is conserved instructions in 96‑well plates. Briefly, AC16 cells were seeded throughout evolution (16). There are five human HIGD genes, (5x103 cells/well) in a 96‑well plate and cultured at 37˚C for HIGD‑1A, ‑1B, ‑1C, ‑2A and ‑2B. HIGD‑1A is regulated by 48 h. Cells were incubated with the CCK‑8 reagent for 1 h. hypoxia‑inducible factor‑1 (HIF1) under hypoxic conditions. Relative viability of cells was assessed at 450 nm absorbance. HIGD‑1A is a survival factor that contains two transmem‑ For crystal violet (CV) staining assay, cells were fixed with brane domains oriented in a ‘N‑terminal outside‑C‑terminal 4% formaldehyde at room temperature for 15 min, followed outside and loop inside’ conformation (17). As a mitochondrial by staining with a crystal violet solution (crystal violet 0.2%, inner membrane protein, HIGD‑1A knockdown decreases ethanol 2%) at room temperature for 10 min, and then the cytochrome c oxidase activity, leading to increased mito‑ colonies were photographed using a dissection microscope. chondrial fission and cell death in response to hypoxia (18). For trypan blue exclusion assay, 0.1 ml trypan blue stock solu‑ Ameri et al (19) reported that HIGD‑1A promotes tumor cell tion (cat. no. 15250061; Thermo Fisher Scientific, Inc.) was survival by regulating AMPK activity and levels of cellular added to 1 ml cells at room temperature for 5 min, and then reactive oxygen species (ROS) in vivo. In addition, HIGD‑1A the numbers of blue stained‑cells and the number of total cells prevents OPA1 cleavage and is required for the functional were counted under a light microscope at x10 magnification. integrity of mitochondria (20). Both HIGD‑1A and HIGD‑2A promote cell survival in a number of cell lines in response Immunofluorescence. AC16 cells were divided into the to hypoxia, suggesting that the HIGD gene family may be a following four groups: i) control + normoxia group, cells were potential anti‑apoptosis factor (21). HIGD‑1B and HIGD‑1A cultured under normoxic conditions without any treatment; share 42.4% homology and their similarity is highest in the ii) control + hypoxia group, cells were cultured under hypoxic transmembrane domain. However, the role of HIGD‑1B conditions without any treatment; iii) Vector + hypoxia group, in hypoxia‑induced mitochondrial fragmentation and cell cells were cultured under hypoxic conditions and transfected apoptosis is not clearly understood. In the present study, the with empty vector; and iv) HIGD‑1B‑Flag + hypoxia group, biological function of HIGD‑1B in cardiomyocytes was char‑ cells were cultured under hypoxic conditions and transfected acterized and the underlying mechanisms were investigated. with HIGD‑1B‑Flag vector. Subsequently, AC16 cells (5x103) Knockdown or overexpression experiments were performed were cultured on glass coverslips at 37˚C for 48 h, and stained to examine the effects of HIGD‑1B on OPA1 expression. with 100 nM MitoTracker red CMXRos at 37˚C for 30 min, then The interaction between HIGD‑1B and OPA1 was measured fixed with 4% formaldehyde at 4˚C for 30 min. After washing via co‑immunoprecipitation (Co‑IP) assays. The effect of twice with ice‑cold PBS, the samples were blocked with HIGD‑1B on cell viability was determined via Cell Counting 1% BSA in PBS with Tween‑20 (0.05%) at 4˚C for 1 h. Then, Kit‑8 (CCK‑8) and apoptosis assays. the cells were incubated overnight at 4˚C with anti‑HIGD‑1B antibody (1:500; cat. no. ab238867; Abcam). Next, the samples Materials and methods were washed with PBS and incubated at room temperature for 1 h with goat anti‑rabbit IgG H&L (1:1,000; cat. no. ab96899; Cell culture. AC16 human cardiomyocyte and 293T embryonic DyLight 488; Abcam). In order to determine the morphology kidney cell lines were purchased from American Type Culture of the mitochondria, the cells were classified into three types Collection and cultured in DMEM supplemented with 10% fetal under a fluorescence microscope (magnification, x400): bovine serum (both Gibco; Thermo Fisher Scientific, Inc.) and Tubular, intermediate and fragmented. ‘Tubular’ referred to 1% penicillin‑streptomycin at 37˚C in a humidified incubator the cells that exhibited the most interconnected mitochondria, with 5% CO2. Hypoxia experiments were performed using a while ‘fragmented’ referred to cells that primarily contained hypoxia chamber (Billups‑Rothenberg, Inc.). AC16 cells were spherical mitochondrial segments. cultured under hypoxic conditions of 1% oxygen (5% CO2 and 94% N2). Small interfering (si)RNA‑mediated knockdown of Western blot analysis. Total protein was extracted from cells HIGD‑1B was performed using specific siRNAs targeting using RIPA buffer (Beijing Solarbio Science & Technology HIGD‑1B (cat. no. SR324184; OriGene Technologies, Inc.) and