OBJECTIVE: Mitofusin-2 (MFN2) is a mitochondrial membrane protein that plays a critical role in regulating mitochondrial fusion and cellular metabolism. To further elucidate the impact of MFN2, this study aimed to investigate its significance on hepatocellular carcinoma (HCC) cell function and its potential role in mediating chemosensitivity.
METHODS: This study investigated the effects of silencing and overexpressing MFN2 on the survival, proliferation, invasion and migration abilities, and sorafenib resistance of MHCC97-L HCC cells. Additional experiments were conducted using XAV939 (a β-catenin inhibitor) and HLY78 (a β-catenin activator) to further validate these findings.
RESULTS: Silencing MFN2 significantly promoted the survival and proliferation of MHCC97-L cells, enhanced their invasion and migration capacities, increased the IC50 of sorafenib, reduced the percentage of TUNEL-positive cells, and decreased the expression of proapoptotic proteins. Additionally, silencing MFN2 markedly induced the nuclear translocation of β-catenin, increased β-catenin acetylation levels and enhanced the expression of the downstream regulatory proteins Snail1 and Vimentin while inhibiting E-cadherin expression. Conversely, overexpressing MFN2 reversed the effects observed in MHCC97-L cells mentioned above. The results confirmed that silencing MFN2 activated the β-catenin/epithelial-mesenchymal transition (EMT) pathway and reduced the sensitivity of cells to sorafenib, which could be reversed by XAV939 treatment. Conversely, overexpression of MFN2 inhibited the β-catenin/EMT pathway and increased the sensitivity of cells to sorafenib, which could be altered by HLY78.
CONCLUSIONS: Low expression of MFN2 in HCC cells promotes the nuclear translocation of β-catenin, thereby activating the EMT pathway and mediating resistance to sorafenib.

Androgen excess and metabolic abnormality largely contribute to the pathogenesis of polycystic ovarian syndrome (PCOS), which primarily precipitates ovarian dysfunction and infertility in reproductive-age women. Impaired mitochondrial function and epigenetic alteration have been linked to the development of PCOS. However, it is unknown whether acetate would exert a therapeutic effect on ovarian mitochondrial dysfunction in PCOS. Herein, the study hypothesized that acetate reverses ovarian mitochondrial dysfunction in experimental PCOS rat model, possibly through modulation of mitofusin-2 (MFn2). Eight-week-old female Wistar rats were randomized into four groups (n = 5). Induction of PCOS was performed by 1 mg/kg letrozole (p.o.), administered for 21 days. Thereafter, the rats were treated with acetate (200 mg/kg; p.o.) for 6 weeks. The PCOS rats demonstrated androgen excess, multiple ovarian cysts, elevated anti-mullerian hormone and leptin and decreased SHBG, adiponectin and 17-β estradiol with corresponding increase in ovarian transforming growth factor-β1. Additionally, inflammation (tumor growth factor and nuclear factor-kB), elevated caspase-6, decreased hypoxia-inducible factor-1α and elevated histone deacetylase-2 (HDAC2) were observed in the ovaries of PCOS rats, while mitochondrial abnormality with evidence of decreased adenosine triphosphate synthase and MFn2 was observed in rats with PCOS. Treatment with acetate reversed the alterations. The present results collectively suggest that acetate ameliorates ovarian mitochondrial abnormality, a beneficial effect that is accompanied by MFn2 with consequent normalization of reproductive-endocrine profile and ovarian function. Perhaps, the present data provide hope for PCOS individuals that suffer infertility.

The experimental myocardial infarction (MI) model originating from isoproterenol (ISO) is frequently preferred in research due to its similarity to MI-induced damage in humans. Beneficial effects of L-arginine (L-Arg), a semi-essential amino acid, in cardiovascular diseases have been shown in many studies. This study was carried out to determine whether L-Arg pre-intervention has protective effects on heart tissue in the experimental MI model. The 28 rats used in the study were randomly divided into 4 equal groups: control, L-Arg, ISO, and L-Arg+ISO. Upon completion of all applications, cardiac markers in serum and biochemical, histopathological, and immunohistochemical examinations in cardiac tissues were performed. Cardiac markers, histopathological changes, oxidative stress, inflammation, and apoptosis were increased in the experimental MI model. In addition, administration of ISO deregulated OTULIN levels and mitochondrial dynamics in heart tissue. However, L-Arg pre-intervention showed a significant protective effect against changes in ISO-induced MI. L-Arg supplementation with cardioprotective effect may reduce the risks of possible pathophysiological processes in MI.

OBJECTIVE: The excessive accumulation of lipid droplets (LDs) is a defining characteristic of nonalcoholic fatty liver disease (NAFLD). The interaction between LDs and mitochondria is functionally important for lipid metabolism homeostasis. Exercise improves NAFLD, but it is not known if it has an effect on hepatic LD-mitochondria interactions. Here, we investigated the influence of exercise on LD-mitochondria interactions and its significance in the context of NAFLD.
RESULTS: Mice were fed high-fat diet (HFD) or HFD-0.1 % methionine and choline-deficient diet (MCD) to emulate simple hepatic steatosis or non-alcoholic steatohepatitis, respectively. In both models, aerobic exercise decreased the size of LDs bound to mitochondria and the number of LD-mitochondria contacts. Analysis showed that the effects of exercise on HOMA-IR and liver triglyceride levels were independent of changes in body weight, and a positive correlation was observed between the number of LD-mitochondria contacts and NAFLD severity and with the lipid droplet size bound to mitochondria. Cellular fractionation studies revealed that ATP-coupled respiration and fatty acid oxidation (FAO) were greater in hepatic peridroplet mitochondria (PDM) from HFD-fed exercised mice than from equivalent sedentary mice. Finally, exercise increased FAO and mitofusin-2 abundance exclusively in PDM through a mechanism involving the curvature of mitochondrial membranes and the abundance of saturated lipids. Accordingly, hepatic mitofusin-2 ablation prevented exercise-induced FAO in PDM.
CONCLUSIONS: This study demonstrates that aerobic exercise has beneficial effects in murine NAFLD models by lessening the interactions between hepatic LDs and mitochondria, and by decreasing LD size, correlating with a reduced severity of NAFLD. Additionally, aerobic exercise increases FAO in PDM and this process is reliant on Mfn-2 enrichment, which modifies LD-mitochondria communication.

Zinc homeostasis is essential for maintaining redox balance, cell proliferation, and apoptosis. However, excessive zinc exposure is toxic and leads to mitochondrial dysfunction. In this study, we established a zinc overload model by treating rat cardiomyocyte H9c2 cells with Zn2+ at different concentrations. Our results showed that zinc overload increased LDH and reactive oxygen species (ROS) levels, leading to cell death, mitochondrial membrane potential decrease and impaired mitochondrial function and dynamics. Furthermore, zinc overload activated the PINK1/Parkin signaling pathway and induced mitochondrial autophagy via ROS, while NAC inhibited mitophagy and weakened the activation of PINK1/Parkin pathway, thereby preserving mitochondrial biogenesis. In addition, our data also showed that Mfn2 deletion increased ROS production and exacerbated cytotoxicity induced by zinc overload. Our results therefore suggest that Zn2+-induced ROS generation causes mitochondrial autophagy and mitochondrial dysfunction, damaging H9c2 cardiomyocytes. Additionally, Mfn2 may play a key role in zinc ion-mediated endoplasmic reticulum and mitochondrial interactions. Our results provide a new perspective on zinc-induced toxicology.

The experimental myocardial infarction (MI) model originating from isoproterenol (ISO) is frequently preferred in research due to its similarity to MI-induced damage in humans. Beneficial effects of L-arginine (L-Arg), a semi-essential amino acid, in cardiovascular diseases have been shown in many studies. This study was carried out to determine whether L-Arg pre-intervention has protective effects on heart tissue in the experimental MI model. The 28 rats used in the study were randomly divided into 4 equal groups: control, L-Arg, ISO, and L-Arg+ISO. Upon completion of all applications, cardiac markers in serum and biochemical, histopathological, and immunohistochemical examinations in cardiac tissues were performed. Cardiac markers, histopathological changes, oxidative stress, inflammation, and apoptosis were increased in the experimental MI model. In addition, administration of ISO deregulated OTULIN levels and mitochondrial dynamics in heart tissue. However, L-Arg pre-intervention showed a significant protective effect against changes in ISO-induced MI. L-Arg supplementation with cardioprotective effect may reduce the risks of possible pathophysiological processes in MI.

High-fat consumption promotes the development of obesity, which is associated with various chronic illnesses. Mitochondria are the energy factories of eukaryotic cells, maintaining self-stability through a fine-tuned quality-control network. In the present study, we evaluated high-fat diet (HFD)-induced changes in mitochondrial ultrastructure and dynamics protein expression in multiple organs. C57BL/6J male mice were fed HFD or normal diet (ND) for 24 weeks. Compared with ND-fed mice, HFD-fed mice exhibited increased body weight, cardiomyocyte enlargement, pulmonary fibrosis, hepatic steatosis, renal and splenic structural abnormalities. The cellular apoptosis of the heart, liver, and kidney increased. Cellular lipid droplet deposition and mitochondrial deformations were observed. The proteins related to mitochondrial biogenesis (TFAM), fission (DRP1), autophagy (LC3 and LC3-II: LC3-I ratio), and mitophagy (PINK1) presented different changes in different organs. The mitochondrial fusion regulators mitofusin-2 (MFN2) and optic atrophy-1 (OPA1) were consistently downregulated in multiple organs, even the spleen. TOMM20 and ATP5A protein were enhanced in the heart, skeletal muscle, and spleen, and attenuated in the kidney. These results indicated that high-fat feeding caused pathological changes in multiple organs, accompanied by mitochondrial ultrastructural damage, and MFN2 and OPA1 downregulation. The mitochondrial fusion proteins may become promising targets and/or markers for treating metabolic disease.

During myocardial ischemia and reperfusion (IR) injury matrix metalloproteinase-2 (MMP-2) is rapidly activated in response to oxidative stress. MMP-2 is a multifunctional protease that cleaves both extracellular and intracellular proteins. Oxidative stress also impairs mitochondrial function which is regulated by different proteins, including mitofusin-2 (Mfn-2), which is lost in IR injury. Oxidative stress and mitochondrial dysfunction trigger the NLRP3 inflammasome and the innate immune response which invokes the de novo expression of an N-terminal truncated isoform of MMP-2 (NTT-MMP-2) at or near mitochondria. We hypothesized that MMP-2 proteolyzes Mfn-2 during myocardial IR injury, impairing mitochondrial function and enhancing the inflammasome response. Isolated hearts from mice subjected to IR injury (30 min ischemia/40 min reperfusion) showed a significant reduction in left ventricular developed pressure (LVDP) compared to aerobically perfused hearts. IR injury increased MMP-2 activity as observed by gelatin zymography and increased degradation of troponin I, an intracellular MMP-2 target. MMP-2 preferring inhibitors, ARP-100 or ONO-4817, improved post-ischemic recovery of LVDP compared to vehicle perfused IR hearts. In muscle fibers isolated from IR hearts the rates of mitochondrial oxygen consumption and ATP production were impaired compared to those from aerobic hearts, whereas ARP-100 or ONO-4817 attenuated these reductions. IR hearts showed higher levels of NLRP3, cleaved caspase-1 and interleukin-1β in the cytosolic fraction, while the mitochondria-enriched fraction showed reduced levels of Mfn-2, compared to aerobic hearts. ARP-100 or ONO-4817 attenuated these changes. Co-immunoprecipitation showed that MMP-2 is associated with Mfn-2 in aerobic and IR hearts. ARP-100 or ONO-4817 also reduced infarct size and cell death in hearts subjected to 45 min ischemia/120 min reperfusion. Following myocardial IR injury, impaired contractile function and mitochondrial respiration and elevated inflammasome response could be attributed, at least in part, to MMP-2 activation, which targets and cleaves mitochondrial Mfn-2. Inhibition of MMP-2 activity protects against cardiac contractile dysfunction in IR injury in part by preserving Mfn-2 and suppressing inflammation.

Repeated mild traumatic brain injuries (rMTBI) affect mitochondrial homeostasis in the brain. However, mechanisms of long-lasting neurobehavioral effects of rMTBI are largely unknown. Mitofusin 2 (Mfn2) is a critical component of tethering complexes in mitochondria-associated membranes (MAMs) and thereby plays a pivotal role in mitochondrial functions. Herein, we investigated the implications of DNA methylation in the Mfn2 gene regulation, and its consequences on mitochondrial dysfunction in the hippocampus after rMTBI. rMTBI dramatically reduced the mitochondrial mass, which was concomitant with decrease in Mfn2 mRNA and protein levels. DNA hypermethylation at the Mfn2 gene promoter was observed post 30 days of rMTBI. The treatment of 5-Azacytidine, a pan DNA methyltransferase inhibitor, normalized DNA methylation levels at Mfn2 promoter, which further resulted into restoration of Mfn2 function. The normalization of Mfn2 function was well correlated with recovery in memory deficits in rMTBI-exposed rats. Since, glutamate excitotoxicity serves as a primary insult after TBI, we employed in vitro model of glutamate excitotoxicity in human neuronal cell line SH-SY5Y to investigate the causal epigenetic mechanisms of Mfn2 gene regulation. The glutamate excitotoxicity reduced Mfn2 levels via DNA hypermethylation at Mfn2 promoter. Loss of Mfn2 caused significant surge in cellular and mitochondrial ROS levels with lowered mitochondrial membrane potential in cultured SH-SY5Y cells. Like rMTBI, these consequences of glutamate excitotoxicity were also prevented by 5-AzaC pre-treatment. Therefore, DNA methylation serves as a vital epigenetic mechanism involved in Mfn2 expression in the brain; and this Mfn2 gene regulation may play a pivotal role in rMTBI-induced persistent cognitive deficits. Closed head weight drop injury method was employed to induce repeated mild traumatic brain (rMTBI) in jury in adult, male Wistar rats. rMTBI causes hyper DNA methylation at the Mfn2 promoter and lowers the Mfn2 expression triggering mitochondrial dysfunction. However, the treatment of 5-azacytidine normalizes DNA methylation at the Mfn2 promoter and restores mitochondrial function.

Pulmonary arterial hypertension (PAH) mainly occurs as a result of abnormal proliferation and apoptosis resistance of pulmonary artery smooth muscle cells (PASMCs). Endothelial progenitor cell (EPC)-derived exosomes (Exos) (EPC-Exos) relieve PAH. However, there is still insufficient knowledge of whether EPC-Exos contribute to the pathological process of PAH, especially for PASMC repair. This study aimed to determine the effects of EPC-Exos on the proliferation, migration, and apoptosis of PASMCs and explore the possible underlying molecular mechanisms through bioinformatics analysis and in vitro testing. Bioinformatics analysis showed that the Ras signaling pathway and Exos were crucial in PAH. The PAH differential microRNAs (miRNAs) and miRNAs identified in EPC-Exos were intersected to obtain miR-21-5p. A target gene prediction program predicted mitofusin-2 (Mfn2) as a potential target of miR-21-5p. Cellular experiments demonstrated that EPC-Exos attenuated the viability, proliferation, migration, and apoptosis resistance of PASMCs under hypoxia. Mechanistically, EPC-Exos significantly upregulated Mfn2 expression and attenuated Ras-Raf-ERK1/2 signaling pathway activity. In conclusion, EPC-Exos suppress cell viability, proliferation, and migration and promote apoptosis in PASMCs under hypoxic conditions. It is possible to transport miR-21-5p to improve the expression of Mfn2 and inhibit the Ras-Raf-ERK1/2 signaling pathway directly or by targeting the expression of Mfn2. EPC-Exos are a potential therapeutic candidate for the treatment of PAH.