Expression Of Mitochondrial Fusion Proteins Research Articles

ADT-OH exhibits anti-metastatic activity on triple-negative breast cancer by combinatorial targeting of autophagy and mitochondrial fission

High basal autophagy and enhanced mitochondrial fission in triple-negative breast cancer (TNBC) cells support cell migration and promote plasticity of cancer cell metabolism. Here, we suggest a novel combination therapy approach for the treatment of TNBC that targets Drp1-mediated mitochondrial fission and autophagy pathways. Hydrogen sulfide (H2S) mediates a myriad of biological processes, including autophagy and mitochondrial function. In this study, we demonstrated that 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione (ADT-OH), one of the most widely utilized sustained-release H2S donors, effectively suppresses metastasis of TNBC cells in the absence of proliferation inhibition in vitro and in vivo. ADT-OH treatment ameliorated autophagy flux by suppressing autophagosome formation and induced mitochondrial elongation through decreasing expression of dynamin-related protein 1 (Drp1) and increasing expression of mitochondrial fusion protein (Mfn2). At the same time, ADT-OH downregulated mitophagy flux and inhibited mitochondrial function, eventually leading to the inhibition of migration and invasion in TNBC cells. In vivo, intraperitoneal administration of ADT-OH revealed a potent anti-metastatic activity in three different animal models, the MDA-MB-231 orthotopic xenograft model, the 4T1-Luci orthotopic model and the 4T1-Luci tail vein metastasis model. However, ADT-OH has an extremely low water solubility, which is a significant barrier to its effectiveness. Thus, we demonstrated that the solubility of ADT-OH in water can be improved significantly by absorption with hydroxypropyl-β-cyclodextrin (CD). Remarkably, the obtained CD-ADT-OH demonstrated superior anti-cancer effect to ADT-OH in vivo. Altogether, this study describes a novel regulator of mammalian mitochondrial fission and autophagy, with potential utility as an experimental therapeutic agent for metastatic TNBC.

Nicotinamide mononucleotide alleviates seizures via modulating SIRT1-PGC-1α mediated mitochondrial fusion and fission.

Both human and animal experiments have demonstrated that energy metabolism dysfunction in neurons after seizures is associated with an imbalance in mitochondrial fusion/fission dynamics. Effective neuronal mitochondrial dynamics regulation strategies remain elusive. Nicotinamide mononucleotide (NMN) can ameliorate mitochondrial functional and oxidative stress in age-related diseases. But whether NMN improves mitochondrial energy metabolism to exert anti-epileptic effects is unclear. This study aims to clarify if NMN can protect neurons from pentylenetetrazole (PTZ) or Mg2+ -free-induced mitochondrial disorder and apoptosis via animal and cell models. We established a continuous 30-day PTZ (37 mg/kg) intraperitoneal injection-induced epileptic mouse model and a cell model induced by Mg2+ -free solution incubation to explore the neuroprotective effects of NMN. We found that NMN treatment significantly reduced the seizure intensity of PTZ-induced epileptic mice, improved their learning and memory ability, and enhanced their motor activity and exploration desire. At the same time, invitro and invivo experiments showed that NMN can inhibit neuronal apoptosis and improve the mitochondrial energy metabolism function of neurons. In addition, NMN down-regulated the expression of mitochondrial fission proteins (Drp1 and Fis1) and promoted the expression of mitochondrial fusion proteins (Mfn1 and Mfn2) by activating the SIRT1-PGC-1α pathway, thereby inhibiting PTZ or Mg2+ -free extracellular solution-induced mitochondrial dysfunction, cell apoptosis, and oxidative stress. However, combined intervention of SIRT1 inhibitor, Selisistat, and PGC-1α inhibitor, SR-18292, eliminated the regulatory effect of NMN pre-treatment on mitochondrial fusion and fission proteins and apoptosis-related proteins. Therefore, NMN intervention may be a new potential treatment for cognitive impairment and behavioral disorders induced by epilepsy, and targeting the SIRT1-PGC-1α pathway may be a promising therapeutic strategy for seizures.

Mitochondrial fusion induced by transforming growth factor-β1 serves as a switch that governs the metabolic reprogramming during differentiation of regulatory T cells

Although metabolic reprogramming during the differentiation of regulatory T cells (Treg cells) has been extensively studied, the molecular switch to alter energy metabolism remains undefined. The present study explores the critical role of mitochondrial dynamics in the reprogramming and consequent generation of Treg cells. The results showed that during Treg cell differentiation, mitochondrial fusion but not fission led to elevation of oxygen consumption rate values, facilitation of metabolic reprogramming, and increase of number of Treg cells and expression of Foxp3 in vitro and in vivo. Mechanistically, mitochondrial fusion favored fatty acid oxidation but restricted glycolysis in Treg cells through down-regulating the expression of HIF-1α. Transforming growth factor-β1 (TGF-β1) played a crucial role in the induction of mitochondrial fusion, which activated Smad2/3, promoted the expression of PGC-1α and therefore facilitated the expression of mitochondrial fusion proteins. In conclusion, during Treg cell differentiation, TGF-β1 promotes PGC-1α-mediated mitochondrial fusion, which drives metabolic reprogramming from glycolysis to fatty acid oxidation via suppressing HIF-1α expression, and therefore favors the generation of Treg cells. The signals and proteins involved in mitochondrial fusion are potential therapeutic targets for Treg cell-related diseases.

Eicosapentaenoic Acid Preserves Mitochondrial Quality and Attenuates Cardiac Remodeling After Myocardial Infarction in Rats.

Eicosapentaenoic acid (EPA) reduces the risk of ischemic heart diseases and is a component of mitochondria. We herein investigated whether dietary EPA mediated mitochondrial fatty acid compositions, dynamics, and functions, resulting in the attenuation of cardiac remodeling after myocardial infarction (MI). The coronary artery of male rats was ligated to induce MI, and they were then treated with or without EPA (1000mg/kg/day) for 12weeks. The EPA treatment improved left ventricular systolic function and increased the mitochondrial content of EPA in the non-infarct region 12weeks after MI. The content of ATP and mitochondrial complex II, III, and IV activities decreased after MI but were maintained by the EPA treatment in association with the preservation of optic atrophy 1, a mitochondrial fusion protein. The present results suggest that dietary EPA increased the mitochondrial content of EPA and preserved the expression of mitochondrial fusion proteins and energy metabolism, which attenuated left ventricular remodeling after MI.

Cadmium-induced apoptosis of Leydig cells is mediated by excessive mitochondrial fission and inhibition of mitophagy

Cadmium is one of the environmental and occupational pollutants and its potential adverse effects on human health have given rise to substantial concern. Cadmium causes damage to the male reproductive system via induction of germ-cell apoptosis; however, the underlying mechanism of cadmium-induced reproductive toxicity in Leydig cells remains unclear. In this study, twenty mice were divided randomly into four groups and exposed to CdCl2 at concentrations of 0, 0.5, 1.0 and 2.0 mg/kg/day for four consecutive weeks. Testicular injury, abnormal spermatogenesis and apoptosis of Leydig cells were observed in mice. In order to investigate the mechanism of cadmium-induced apoptosis of Leydig cells, a model of mouse Leydig cell line (i.e. TM3 cells) was subjected to treatment with various concentrations of CdCl2. It was found that mitochondrial function was disrupted by cadmium, which also caused a significant elevation in levels of mitochondrial superoxide and cellular ROS. Furthermore, while cadmium increased the expression of mitochondrial fission proteins (DRP1 and FIS1), it reduced the expression of mitochondrial fusion proteins (OPA1 and MFN1). This led to excessive mitochondrial fission, the release of cytochrome c and apoptosis. Conversely, cadmium-induced accumulation of mitochondrial superoxide was decreased by the inhibition of mitochondrial fission through the use of Mdivi-1 (an inhibitor of DRP1). Mdivi-1 also partially prevented the release of cytochrome c from mitochondria to cytosol and attenuated cell apoptosis. Finally, given the accumulation of LC3II and SQSTM1/p62 and the obstruction of Parkin recruitment into damaged mitochondria in TM3 cells, the autophagosome-lysosome fusion was probably inhibited by cadmium. Overall, these findings suggest that cadmium induces apoptosis of mouse Leydig cells via the induction of excessive mitochondrial fission and inhibition of mitophagy.

A small molecule M1 promotes optic nerve regeneration to restore target-specific neural activity and visual function

Axon regeneration is an energy-demanding process that requires active mitochondrial transport. In contrast to the central nervous system (CNS), axonal mitochondrial transport in regenerating axons of the peripheral nervous system (PNS) increases within hours and sustains for weeks after injury. Yet, little is known about targeting mitochondria in nervous system repair. Here, we report the induction of sustained axon regeneration, neural activities in the superior colliculus (SC), and visual function recovery after optic nerve crush (ONC) by M1, a small molecule that promotes mitochondrial fusion and transport. We demonstrated that M1 enhanced mitochondrial dynamics in cultured neurons and accelerated invivo axon regeneration in the PNS. Ex vivo time-lapse imaging and kymograph analysis showed that M1 greatly increased mitochondrial length, axonal mitochondrial motility, and transport velocity in peripheral axons of the sciatic nerves. Following ONC, M1 increased the number of axons regenerating through the optic chiasm into multiple subcortical areas and promoted the recovery of local field potentials in the SC after optogenetic stimulation of retinal ganglion cells, resulting in complete recovery of the pupillary light reflex, and restoration of the response to looming visual stimuli was detected. M1 increased the gene expression of mitochondrial fusion proteins and major axonal transport machinery in both the PNS and CNS neurons without inducing inflammatory responses. The knockdown of two key mitochondrial genes, Opa1 or Mfn2, abolished the growth-promoting effects of M1 after ONC, suggesting that maintaining a highly dynamic mitochondrial population in axons is required for successful CNS axon regeneration.

Long-term oral administration of naringenin counteracts aging-related retinal degeneration via regulation of mitochondrial dynamics and autophagy.

Aging-related retinal degeneration can manifest as decreased visual function due to damage to retinal structures and dysfunction in retinal homeostasis. Naringenin, a flavonoid, has beneficial effects in preventing cellular aging, preserving the functionality of photoreceptors, and slowing down visual function loss. However, the role and potential mechanism of naringenin in the aging mouse retina require further investigation. In this study, we evaluated the effects of naringenin on the aging eye using electroretinogram (ERG) and hematoxylin and eosin staining and explored its potential mechanism by western blotting. ERG showed that naringenin increased the amplitude of the a- and b-waves of scotopic 3.0, 10.0, and the a-wave amplitude of photopic 3.0 in the aging mouse retina. Furthermore, administration of naringenin prevented aging-induced retinal degeneration in the total retina, ganglion cell, inner plexiform layer, inner nuclear layer, and outer nuclear layer. The expression of mitochondrial fusion protein two was increased, OPA1 protein expression and the ratio of L-OPA1/S-OPA1 were unchanged, and dynamin-related protein one was decreased in the 12-month-old mice treated with naringenin compared with the 12-month-old mice treated with vehicle. Furthermore, the downregulation of age-related alterations in autophagy was significantly rescued in the aging mice by treatment with naringenin. Taken together, these results suggest that the oral administration of naringenin improves visual function, retinal structure, mitochondrial dynamics, and autophagy in the aging mouse retinas. Naringenin may be a potential dietary supplement for the prevention or treatment of aging-related retinal degeneration.

Guanxinning Injection Combined With Ischemic Postconditioning Attenuate Myocardial Ischemic Reperfusion Injury in Chronic Renal Failure Rats by Modulating Mitochondrial Dynamics.

PurposeSalvia miltiorrhiza Bge. (Danshen, DS) and Ligusticum chuanxiong Hort. (Chuanxiong, CX) have been widely used in traditional Chinese medicine to prevent and treat myocardial ischemia and renal insufficiency, and their extracts (Guanxinning injection, GXN) have been reported to exhibit antioxidant, anti-inflammatory, and anti-ischemia-reperfusion injury properties. It is well-established that ischemic postconditioning (IPOC) can protect against myocardial ischemia-reperfusion (I/R) injury in rats with chronic renal failure (CRF). However, little is known on whether GXN combined with IPOC may affect myocardial I/R injury in CRF rats. We sought to observe the effect of GXN combined with IPOC on myocardial I/R injury in CRF rats by quantifying changes in the expression of proteins related to mitochondrial dynamics.Materials and MethodsIn a survey, 90 Wistar rats were randomly divided into 6 groups (15 rats per group): CRF group, I/R group, comorbid group (CRF + I/R), IPOC group, IPOC + GXN group and the sham group. Changes in blood myocardial injury markers, urea, and creatinine were analyzed. Heart tissues were harvested for histomorphometry and western blotting when rats were sacrificed. Myocardial infarction area was measured by Evans blue and Triphenyltetrazolium chloride solution staining. The expressions of mitochondrial fission relative proteins (DRP1 and FIS1) and mitochondrial fusion relative proteins (OPA1 and MFN1) were detected by western blotting.ResultsIPOC could significantly decrease myocardial injury markers and myocardial area of necrosis (AN)/area at risk (AAR) of the comorbid model rats. Further results showed that GXN combined with IPOC could significantly reduce CK-MB levels and myocardial AN/AAR in comorbid model rats compared with the IPOC group. Meanwhile, both IPOC and IPOC + GXN significantly reduced DRP1 levels and increased the MFN1 and OPA1 protein levels in the comorbid model rats. However, compared with the IPOC group, MFN1 and OPA1 protein levels increased significantly in the IPOC + GXN group.ConclusionExtracts of DS and CX combined with IPOC exert a protective effect against myocardial I/R injury in rats with CRF, mediated by increased expression of mitochondrial fusion proteins (MFN1 and OPA1).

The effect of gestational diabetes on the expression of mitochondrial fusion proteins in placental tissue.

Gestational diabetes mellitus (GDM) poses a risk factor for fetal mortality and morbidity by directly affecting the placenta and fetus. Mitochondria are dynamic organelles that play a key role in energy production and conversion in placental tissue. Mitochondrial fusion and fission proteins are important in terms of providing mitochondrial dynamics, the adaptation of the cell to different conditions, and maintaining the metabolic stability of the cells. Although GDM shares many features with Type 2 diabetes mellitus (T2DM), different effects of these conditions on the mother and the child suggest that GDM may have specific pathological effects on placental cells. The aim of this study is to investigate the expression of mitochondrial dynamics, and mitochondrial protein folding markers in placentas from GDM patients and women with pre-existing diabetes mellitus. Placentas were properly collected from women, who had pre-existing diabetes (Pre-DM), from women with gestational diabetes mellitus (GDM) and from healthy (non-diabetic) pregnant women. Levels of mitochondrial fusion markers were determined in these placentas by real time quantitative PCR and Western blot experiments. mRNA expressions and protein levels of mitochondrial fusion markers, mitofusin 1, mitofusin 2 (MFN1 and MFN2) and optical atrophy 1 (OPA1) proteins were found to be significantly lower in both Pre-DM placentas and those with GDM compared to healthy (non-diabetic) control group. Likewise, proteins involved in mitochondrial protein folding were also found to be significantly reduced compared to control group. Diabetes during pregnancy leads to processes that correlate with mitochondria dysfunction in placenta. Our results showed that mitochondrial fusion markers significantly decrease in placental tissue of women with GDM, compared to the healthy non-diabetic women. The decrease in mitochondrial fusion markers was more severe during GDM compared to the Pre-DM. Our results suggest that there may be differences in the pathophysiology of these conditions.

Secreted Frizzled-Related Protein 5 Protects Against Cardiac Rupture and Improves Cardiac Function Through Inhibiting Mitochondrial Dysfunction.

Background: Secreted frizzled-related protein 5 (Sfrp5) has been suggested to be a protective regulatory protein in coronary heart disease. However, the role of Sfrp5 in regulating ischemic injury and its consequences is not known. The aim of our study was to explore the effects of Sfrp5 on hearts after myocardial infarction (MI) and to investigate the underlying mechanisms.Methods and Results: We found that Sfrp5 was downregulated over time in the heart tissue of MI mice. To further elucidate the role of Sfrp5 during MI, we established a cardiac overexpression of an Sfrp5 mouse model using the cardiotropic adeno-associated virus serotype 9 (AAV9). Overexpression of Sfrp5 significantly reduced infarct size as demonstrated by a decrease in mortality owing to cardiac rupture. Moreover, cardiac overexpression of Sfrp5 increased left ventricular function and mitochondrial biogenesis, decreased cardiomyocyte apoptosis, suppressed inflammation reaction, inhibited oxidative stress, and ameliorated cardiac remodeling as demonstrated by left ventricular ejection fraction, mitochondrial morphology, heart weight, NADH oxidase activity levels, and myocardial fibrosis at 2 weeks post-MI. At the molecular level, overexpression of Sfrp5 significantly increased the expression of p-AMPKThr172 protein with higher expression of mitochondrial fusion protein (MFN1 and MFN2) and lower expression of mitochondrial fission protein (p-Drp1Ser616/Mid49/MFF/Fis-1). In isolated neonatal rat cardiac myocytes, Sfrp5 treatment attenuated hypoxia-induced mitochondrial dysfunction. Inhibition of AMPK activity with compound C abrogated this benefit.Conclusions: Sfrp5 overexpression inhibits ischemic injury, reduces risk of cardiac rupture, ameliorates post-MI remodeling, and decreases the progression to heart failure via disrupting mitochondrial dysfunction and partly through normalizing the AMPK activity.

Dexmedetomidine ameliorates endotoxin-induced acute lung injury in vivo and in vitro by preserving mitochondrial dynamic equilibrium through the HIF-1a/HO-1 signaling pathway

Increasing lines of evidence identified that dexmedetomidine (DEX) exerted protective effects against sepsis-stimulated acute lung injury via anti-inflammation, anti-oxidation and anti-apoptosis. However, the mechanisms remain unclear. Herein, we investigated whether DEX afforded lung protection by regulating the process of mitochondrial dynamics through the HIF-1a/HO-1 pathway in vivo and in vitro. Using C57BL/6J mice exposed to lipopolysaccharide, it was initially observed that preemptive administration of DEX (50μg/kg) alleviated lung pathologic injury, reduced oxidative stress indices (OSI), improved mitochondrial dysfunction, upregulated the expression of HIF-1α and HO-1, accompanied by shifting the dynamic course of mitochondria into fusion. Moreover, HO-1-knockout mice or HO-1 siRNA transfected NR8383 cells were pretreated with HIF-1α stabilizer DMOG and DEX to validate the effect of HIF-1a/HO-1 pathway on DEX-mediated mitochondrial dynamics in a model of endotoxin-induced lung injury. We found that pretreatment with DEX and DMOG distinctly relieved lung injury, decreased the levels of mitochondrial ROS and mtDNA, reduced OSI, increased nuclear accumulation of HIF-1a and HO-1 protein in wild type mice but not HO-1 KO mice. Similar observations were recapitulated in NC siRNA transfected NR8383 cells after LPS stimulation but not HO-1 siRNA transfected cells. Concertedly, DEX reversed the impaired mitochondrial morphology in LPS stimulated-wild type mice or NC siRNA transfected NR8383 cells, upregulated the expression of mitochondrial fusion protein, while downregulated the expression of fission protein in HIF-1a/HO-1 dependent pathway. Altogether, our data both in vivo and in vitro certified that DEX treatment ameliorated endotoxin-induced acute lung injury by preserving the dynamic equilibrium of mitochondrial fusion/fission through the regulation of HIF-1a/HO-1 signaling pathway.

The lysosomal membrane protein Sidt2 is a vital regulator of mitochondrial quality control in skeletal muscle.

The role of Sidt2 in the process of glucose and lipid metabolism has been recently reported. However, whether Sidt2 is involved in the metabolic regulation in skeletal muscle remains unknown. In this study, for the first time, using skeletal muscle-selective Sidt2 knockout mice, we found that Sidt2 was vital for the quality control of mitochondria in mouse skeletal muscle. These mice showed significantly reduced muscle tolerance and structurally abnormal mitochondria. Deletion of the Sidt2 gene resulted in decreased expression of mitochondrial fusion protein 2 (Mfn2) and Dynamin-related protein 1 (Drp1), as well as peroxisome proliferator-activated receptor γ coactivator-1 (PGC1-α). In addition, the clearance of damaged mitochondria in skeletal muscle was inhibited upon Sidt2 deletion, which was caused by blockade of autophagy flow. Mechanistically, the fusion of autophagosomes and lysosomes was compromised in Sidt2 knockout skeletal muscle cells. In summary, the deletion of the Sidt2 gene not only interfered with the quality control of mitochondria, but also inhibited the clearance of mitochondria and caused the accumulation of a large number of damaged mitochondria, ultimately leading to the abnormal structure and function of skeletal muscle.

Propofol alleviates hypoxic neuronal injury by inhibiting high levels of mitochondrial fusion and fission.

This study aimed to explore the mechanism of propofol in alleviating neuronal oxidative damage. The neuron cells were randomly assigned to normal group (NOR), model group (MOD), and propofol administration group (MED). A 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was carried out to detect the viability of neuron cells, reverse transcription PCR (RT-PCR) assay to determine the gene expression of Fis and Mfn1, and Western blot assay to determine the protein expression of Caspase-3, Caspase-9, Bax, Bcl-2, and COX-2. According to the results of cell proliferation rate, under normal circumstances, neuron cells would have some programmed death and weak apoptosis, while after hypoxia-reoxygenation, the apoptosis rate of neuron cells gradually increased with the increase of culture time, which was significantly higher than that of the NOR. After the addition of propofol, the overall apoptosis rate of neuron cells slowly increased, significantly lower than that in the MOD and close to that in the NOR. Compared with the NOR, the ROS content in the MOD was significantly reduced, and compared with the MOD, the ROS content in the MED significantly recovered. Furthermore, the RT-PCR results showed that compared with the NOR, the expression of mitochondrial fusion protein (Mfnl) in the MOD group declined significantly, and the expression of mitochondrial fission protein 1 (Fis1) increased significantly, while after the addition of propofol, the expression of Mfnl and Fis1 was closed to that in the NOR. WB results showed that compared with the NOR, the expression of apoptosis proteins (Caspase-3, Caspase-9, Bax, and COX-2) in the MOD increased significantly, and the expression of Bcl-2 reduced significantly (all p<0.05), and the addition of propofol improved the expression of corresponding proteins. Propofol could alleviate hypoxic neuronal injury by inhibiting high levels of mitochondrial fusion and fission.

Fluoride-induced renal dysfunction via respiratory chain complex abnormal expression and fusion elevation in mice

A fluoride exposure mouse model is established to evaluate the relationship between mitochondrial respiratory chain complexes and renal dysfunction. Morphological changes in kidney tissues were observed. Renal function and cell proliferation in the kidneys were evaluated. The expression of mitochondrial fusion protein including mitofusin-1 (Mfn1) and optic atrophy 1 (OPA1), and mitochondrial respiratory chain complex subunits, including NDUFV2, SDHA, CYC1 and COX Ⅳ, were detected via real-time polymerase chain reaction, immunohistochemistry staining and Western blot, respectively. Results showed that the structures of renal tubule, renal glomerulus and renal papilla were seriously damaged. Renal function was impaired, and cell proliferation was remarkably inhibited by excessive fluoride in kidney. The mRNA and protein expression levels of Mfn1, OPA1, NDUFV2, CYC1 and COX Ⅳ were significantly increased after excessive fluoride exposure. However, the mRNA and protein expression of SDHA significantly decreased. Overall, our findings revealed that excessive fluoride can damage kidney structure, inhibit renal cell proliferation, interfere with the expression of mitochondrial respiratory chain complexes and elevate mitochondrial fusion. Consequently, renal function disorder occurred.

Rictor regulates mitochondrial calcium signaling in mouse embryo stem cell-derived cardiomyocytes

To explore the expression, localization and regulatory effect on mitochondrial calcium signaling of Rictor in embryonic stem cell-derived cardiomyocytes (ESC-CMs). Classical embryonic stem cell cardiomyogenesis model was used for differentiation of mouse embryonic stem cells into cardiomyocytes. The location of Rictor in ESC-CMs was investigated by immunofluorescence and Western blot. The expression of Rictor in mouse embryonic stem cells was interfered with lentiviral technology, then the superposition of mitochondria and endoplasmic reticulum (ER) in ESC-CMs was detected with immunofluorescence method; the cellular ultrastructure of ESC-CMs was observed by transmission electron microscope; the mitochondrial calcium transients of ESC-CMs was detected by living cell workstation;immunoprecipitation was used to detect the interaction between 1,5,5-trisphosphate receptor (IP3 receptor, IP3R), glucose-regulated protein 75 (Grp75) and voltage-dependent anion channel 1 (VDAC1) in mitochondrial outer membrane; the expression of mitochondrial fusion protein (mitonusin-2, Mfn2) was detected by Western blot. Rictor was mainly localized in the endoplasmic reticulum and mitochondrial-endoplasmic reticulum membrane (MAM) in ESC-CMs. Immunofluorescence results showed that Rictor was highly overlapped with ER and mitochondria in ESC-CMs. After mitochondrial and ER were labeled with Mito-Tracker Red and ER-Tracker Green, it was demonstrated that the mitochondria of the myocardial cells in the Rictor group were scattered, and the superimposition rate of mitochondria and ER was lower than that of the negative control group (P<0.01). The MAM structures were decreased in ESC-CMs after knockdown of Rictor. The results of the living cell workstation showed that the amplitude of mitochondrial calcium transients by ATP stimulation in ESC-CMs was decreased after knockdown of Rictor (P<0.01). The results of co-immunoprecipitation showed that the interaction between IP3R, Grp75 and VDAC1 in the MAM structure of the cardiomyocytes in the Rictor group was significantly attenuated (P<0.01); the results of Western blot showed that the expression of Mfn2 protein was significantly decreased (P<0.01). Using lentiviral technology to interfere Rictor expression in mouse embryonic stem cells, the release of calcium from the endoplasmic reticulum to mitochondria in ESC-CMs decreases, which may be affected by reducing the interaction of IP3R, Grp75, VDAC1 and decreasing the expression of Mfn2, leading to the damage of MAM structure.

The Effects of TNFα on Mitochondria Morphology are Mediated by Endoplasmic Reticulum Stress in Human Airway Smooth Muscle Cells

Airway inflammation is a key trigger for asthma, and pro‐inflammatory cytokines such as TNFα mediate the airway hyperreactivity and remodeling characteristic of asthma. In various diseases, inflammation leads to an increase in mitochondrial fission, associated with decreased expression of mitochondrial fusion proteins, such as Mfn2. Mitochondrial fission is generally coupled with mitochondrial biogenesis and cell proliferation. Some studies in other cell types suggest that mitochondrial fission may be a result of the accumulation of unfolded or misfolded proteins i.e., endoplasmic reticulum (ER) stress. ER stress activates three intracellular pathways to induce a homeostatic response to restore normal ER function. The IRE1α pathway is activated by autophosphorylation and splices XBP1 mRNA. Previous studies have shown that the XBP1 has downstream effects on mitochondrial proteins. In this study we hypothesized that TNFα induces activation of the IRE1α pathway which leads to a decrease in Mfn2 expression. hASM cells were isolated from lung specimen incidental to patient surgery. The patients were normal as evaluated by the clinical pathologist (no history of asthma or COPD). hASM cells were stimulated with TNFα (20 ng/ml) for 1, 3, 6, 12 or 24 h. Expression of total and phosphorylated IRE1α, Mfn2 and cleaved and uncleaved XBP1 were evaluated by Western blot. RNAscope® was used to measure Mfn2 mRNA expression in hASM cells transfected with non‐phosphorylatable IREα (DeltaP‐IRE1α; GFP tagged) compared to non‐transfected cells. Results were analyzed by 2‐way ANOVA with Bonferroni analysis when justified. We found that exposure of hASM cells to TNFα activated the IRE1α/XBP1 pathway and decreased Mfn2 expression in a time dependent fashion. In addition we found that TNFα exposure in cells expressing the inactive DeltaP‐IRE1α did not lead to a significant decrease in Mfn2 expression. Our results suggest that the IRE1α/XBP1 pathway may be involved in the regulation of mitochondrial fission, mitochondrial biogenesis and cell proliferation.Support or Funding InformationSupported by NIH grant HL126451 (GCS)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cigarette smoke extract may induce lysosomal storage disease-like adverse health effects.

Cigarette smoke is known to be associated with the incidence of a variety of pulmonary diseases, and alveolar macrophages are a key player in the defense mechanism against inhalable toxicants. Herein, we have found that a hydrophilic fraction in smoke extracts from 3R4F reference cigarettes (CSE) contains high concentrations of volatile substances compared to cigarette smoke condensate (amphoteric fraction). We also identified the toxic mechanism of CSE using MH-S, a mouse alveolar macrophage cell line. CSE decreased cell viability accompanying increased lactate dehydrogenase release. Additionally, mitochondrial volume and the potential increased along with enhanced expression of mitochondrial fusion proteins and decreased adenosine triphosphate production. Similarly, CSE clearly induced increase of catalase activity and intracellular calcium concentration and decrease of endoplasmic reticulum and lysosome volume at the highest dose. More interestingly, damaged organelles accumulated in the cytosol, and CSE-containing particles specifically penetrated to mitochondria. Meanwhile, any significant change in autophagy related protein expression was not found in CSE-treated cells. Subsequently, we evaluated the effects of CSE on secretion of inflammatory related cytokines and chemokines, considering the relationship between organelle damage and the disturbed immune response. Very importantly, we found that expression of innate and adaptive immunity related mediators is disrupted following CSE exposure. Taken together, we suggest that CSE may cause the accumulation of damaged organelles in the cytoplasm by impairing selective autophagic function. In addition, this accumulation is responsible for the inadequate ability of immune cells to repair the damage of lung tissue following exposure to CSE.

Mitochondrial respiratory chain complex abnormal expressions and fusion disorder are involved in fluoride-induced mitochondrial dysfunction in ovarian granulosa cells

Excessive fluoride intake has a strong female reproductive toxicity, which can result in follicular developmental dysplasia and decrease oocytes developmental potential. The underlying mechanisms of fluoride-induced mitochondrial dysfunction in ovarian granulosa cells remain largely unknown. In this study, the ultrastructure changes of mitochondria and DNA damage in ovarian granulosa cells were observed under transmission electron microscope and TUNEL staining. Then, the ATP content and ROS level in granulosa cells were measured. The expression of mitochondrial fusion proteins and mitochondrial respiratory chain complexes, including OPA1 and Mfn1, and NDUFV2, SDHA and CYC1, in the ovarian tissues were measured by immunohistochemistry, Western blot and Quantitative real-time PCR analyses. The expression of ATP5j and ATP5h in the ovarian tissues was also measured. Results show that fluoride treatment considerably damages mitochondrial ultrastructure and enhances the apoptosis of granulosa cells. The ATP content greatly decreased, whereas the ROS level increased after fluoride treatment. The expression level of Mfn1 in the ovarian tissue was up-regulated, whereas OPA1 expression had no significant change. The expression levels of NDUFV2, SDHA and CYC1 were considerably up-regulated, and the expression of ATP5j and ATP5h were down-regulated after fluoride treatment. In summary, the damage in the mitochondrial ultrastructure, ATP content decrease, ROS level increase and the abnormal expression of OPA1, Mfn1, NDUFV2, SDHA, CYC1, ATP5j and ATP5h in ovary tissue are closely associated with fluoride-induced mitochondrial dysfunction, which might be responsible for the follicular developmental dysplasia and the potential decrease in oocyte development induced by fluoride in female mice.

Role of PI3K/Akt signaling pathway in carbon monoxide-induced up-regulation of mitochondrial fusion proteins in endotoxin-challenged alveolar epithelial cells of rats

Objective To evaluate the role of 1-phosphatidylinositol 3-kinase/serine threonine kinase(PI3K/Akt)signaling pathway in carbon monoxide(CO)-induced up-regulation of mitochondrial fusion proteins in endotoxin-challenged alveolar epithelial cells of rats. Methods The rat alveolar epithelial cells were cultured in a 5% CO2 cell culture incubator at 37 ℃ with F12K complete medium containing 10% fetal bovine serum and 1% green chain double antibody, and divided into 10 groups (n=5 each) according to the random number table method: control group (C group), endotoxin group (L group), lipopolysaccharide (LPS) plus exogenous CO-releasing agent CO-releasing-molecule-2 (CORM-2) group (L+ CO group), LPS plus PI3K inhibitor LY294002 group (L+ LY group), LPS plus CORM-2 plus LY294002 group (L+ CO+ LY group), LPS plus inactive CO releasing agent iCORM-2 group (L+ iCO group), LPS plus dimethyl sulfoxide (DMSO) group (L+ D group), CORM-2 group (CO group), LY294002 group (LY group) and CORM-2 plus LY294002 group (CO+ LY group). LPS 10 μg/ml was added to the culture medium in group L. CORM-2 100 μmol/L was added to the culture medium and 1 h later 10 μg/ml LPS was added in L+ CO group.LY294002 25 μmol/L was added to the medium, and 1 h later LPS 10 μg/ml was added in L+ LY group.In L+ CO+ LY group, 25 μmol/L LY294002 was added to the culture medium, CORM-2 100 μmol/L was added 1 h later, and then LPS 10 μg/ml was added 1 h later.iCORM 100 μmol/L was added to the culture medium and 1 h later LPS 10 μg/ml was added in L+ iCO group.In L+ D group, the equal concentration of DMSO was added to the culture medium and 1 h later LPS 1 μg/ml was added.CORM-2 100 μmol/L was added to the culture medium in CO group.LY294002 25 μmol/L was added to culture medium in LY group.LY294002 25 μmol/L was added to the culture medium, and 1 h later CORM-2 100 μmol/L was added in CO+ LY group.Cells were harvested after 24 h of incubation for measurement of the malondialdehyde (MDA) content, superoxide dismutase (SOD) activity and expression of heme oxygenase-1 (HO-1), phosphorylated Akt(p-Akt), mitochondrial fusion proteins mitofusion 1(Mfn1), Mfn2 and optic atrophy 1(OPA1) by Western blot. Results Compared with group C, the MDA content was significantly increased and SOD activity was decreased in L, L+ CO, L+ LY, L+ CO+ LY, L+ iCO, L+ D groups, and the expression of HO-1, Mfn1, Mfn2, OPA1 protein and p-Akt was significantly up-regulated in group L (P<0.05). Compared with group L, MDA content was significantly decreased and SOD activity was increased in group L+ CO, MDA content was significantly increased and SOD activity was decreased in group L+ LY , the expression of HO-1, Mfn1, Mfn2, OPA1 protein and p-Akt was significantly up-regulated in group L+ CO, and the expression of HO-1, Mfn1, Mfn2, OPA1 protein and p-Akt was significantly down-regulated in group L+ LY (P<0.05). Compared with group L+ CO, the MDA content was significantly increased, SOD activity was decreased, and the expression of HO-1, Mfn1, Mfn2, OPA1 and p-Akt was down-regulated in group L+ CO+ LY(P<0.05). Conclusion The mechanism by which CO up-regulates the expression of mitochondrial fusion proteins in endotoxin-challenged alveolar epithelial cells is related to activating PI3K/Akt signaling pathway in rats. Key words: 1-phosphoinositide 3-kinase; Protein-serine-threonine kinases; Carbon monoxide; Endotoxins; Pulmonary alveoli; Epithelial cells; Mitochondrial proteins

Chronic cerebral hypoperfusion accelerates Alzheimer’s disease pathology with the change of mitochondrial fission and fusion proteins expression in a novel mouse model

Mitochondrial dynamically undergo massive fusion and fission events to continuously maintain their function in cells. Although an impaired balance of mitochondrial fission and fusion was reported in in-vitro and in-vivo Alzheimer’s disease (AD) model, changes of mitochondrial fission and fusion proteins have not been reported in AD with chronic cerebral hypoperfusion (HP) as an etiological factor related to the development of elder AD. To clarify the impacts of HP on mitochondrial fission and fusion, related oxidative stress in the pathogenesis of AD, and protective effect of galantamine, the novel AD with HP mouse model (APP23 + HP) was applied in this project. Compared with APP23 mice, APP23 + HP mice greatly enhanced the number of Aβ oligomer-positive/phosphorylated tau (pTau) cells, the expression of mitochondrial fission proteins (Drp1 and Fis1), and decreased the expression of mitochondrial fusion proteins (Opa1 and Mfn1) in the cerebral cortex (CTX) and thalamus (TH) at 12 month (M) of age. Moreover, the expression of peroxidation products (4-HNE and 8-OHdG) showed a significant increase in CTX and TH of APP23 + HP mice at 12 M. However, above neuropathological characteristics were retrieved by galantamine (Gal) treatment, detected through immunohistochemical analyses. The present study demonstrates that cerebral HP shifted the balance in mitochondrial morphology from fusion to fission with increasing Aβ oligomer/pTau accumulations in APP23 mice, and such neuropathologic processes were strongly attenuated by Gal treatment.