Activities Of Mitochondrial Respiratory Chain Complexes Research Articles

Mitochondrial OXPHOS is involved in the protective effects of L-arginine against heat-induced low sperm motility of boar.

The present study aimed to analyze the time- and temperature-responses of boar sperm and clarify the mechanism underlying the protective effects of L-arginine on heat-induced low sperm motility. Mature boar sperm was used to evaluate the effects of temperature, exposure time, L-arginine level and their interactions on sperm motility, respectively. Results showed increasing exposure time resulted in the decreased total motility and rate of rapid progressive sperm, and the increased rates of the immotile sperm and the sperm shaking in place at 38 and 39 °C, respectively (P < 0.05). L-arginine supplementation at the dose of 1.0 mM increased total motility and decreased rate of immotile sperm (P < 0.05). Heat at 39 °C decreased total motile and rate of rapid progressive sperm (P < 0.05), increased the level of sperm reactive oxygen species (ROS) (P < 0.05), reduced mitochondrial membrane potential (ΔΨm), ATP content and the activities of mitochondrial respiratory chain complexes (MRCC) ΙΙΙ and V (P < 0.05), which were attenuated by L-arginine supplementation. There were significant increases in the relative mRNA expression of nuclear respiratory factor 1 and peroxisome proliferator-activated receptor gamma coactivator-1 alpha in heat-exposed group without L-arginine supplementation. In conclusion, the rising temperatures impacted boar sperm motility in a time-dependent manner. In vitro addition of L-arginine to boar semen had a dose-dependent effect on sperm motility and sperm incubated with 1.0 mM L-arginine showed elevated motility. L-arginine supplementation can ameliorate heat-induced increase in ROS level and decreases in MRCC activities, which further maintain mitochondrial oxidative phosphorylation function, ATP synthesis and boar sperm motility.

AKT-GSK3β Signaling Pathway Regulates Mitochondrial Dysfunction-Associated OPA1 Cleavage Contributing to Osteoblast Apoptosis: Preventative Effects of Hydroxytyrosol.

Oxidative stress (OS) induces osteoblast apoptosis, which plays a crucial role in the initiation and progression of osteoporosis. Although OS is closely associated with mitochondrial dysfunction, detailed mitochondrial mechanisms underlying OS-induced osteoblast apoptosis have not been thoroughly elucidated to date. In the present study, we found that mitochondrial abnormalities largely contributed to OS-induced osteoblast apoptosis, as evidenced by enhanced production of mitochondrial reactive oxygen species; considerable reduction in mitochondrial respiratory chain complex activity, mitochondrial membrane potential, and adenosine triphosphate production; abnormality in mitochondrial morphology; and alteration of mitochondrial dynamics. These mitochondrial abnormalities were primarily mediated by an imbalance in mitochondrial fusion and fission through a protein kinase B- (AKT-) glycogen synthase kinase 3β- (GSK3β-) optic atrophy 1- (OPA1-) dependent mechanism. Hydroxytyrosol (3,4-dihydroxyphenylethanol (HT)), an important compound in virgin olive oil, significantly prevented OS-induced osteoblast apoptosis. Specifically, HT inhibited OS-induced mitochondrial dysfunction by decreasing OPA1 cleavage and by increasing AKT and GSK3β phosphorylation. Together, our results indicate that the AKT-GSK3β signaling pathway regulates mitochondrial dysfunction-associated OPA1 cleavage, which may contribute to OS-induced osteoblast apoptosis. Moreover, our results suggest that HT could be an effective nutrient for preventing osteoporosis development.

Total Salvianolic Acid Injection Prevents Ischemia/Reperfusion-Induced Myocardial Injury Via Antioxidant Mechanism Involving Mitochondrial Respiratory Chain Through the Upregulation of Sirtuin1 and Sirtuin3.

Sirtuin1 (Sirt1) and Sirtuin3 (Sirt3) are known to participate in regulating mitochondrial function. However, whether Total Salvianolic Acid Injection (TSI) protects against myocardial ischemia/reperfusion (I/R) injury through regulating Sirt1, Sirt3, and mitochondrial respiratory chain complexes is unclear. The aim of this study was to explore the effects of TSI on I/R-induced myocardial injury and the underlying mechanism. Male Sprague-Dawley rats were subjected to 30 min occlusion of the left anterior descending coronary artery followed by 90 min reperfusion with or without TSI treatment (8 mg/kg/h). The results demonstrated that TSI attenuated I/R-induced myocardial injury by the reduced infarct size, recovery of myocardial blood flow, and decreased cardiac apoptosis. Moreover, TSI protected heart from oxidative insults, such as elevation of myeloperoxidase, malondialdehyde, hydrogen peroxide, ROS, as well as attenuated I/R-elicited downregulation of Sirt1, Sirt3, NADH dehydrogenase [ubiquinone] 1 alpha subcomplex 10 (NDUFA10), succinate dehydrogenase complex, subunit A, flavoprotein variant (SDHA), and restoring mitochondrial respiratory chain complexes activity. The in vitro study in H9c2 cells using siRNA transfection further confirmed the critical role of Sirt1 and Sirt3 in the effect of TSI on the expression of NDUFA10 and SDHA. These results demonstrated that TSI attenuated I/R-induced myocardial injury via inhibition of oxidative stress, which was related to the activation of NDUFA10 and SDHA through the upregulation of Sirt1 and Sirt3.

Inhibition of Mitochondrial Complex Function-The Hepatotoxicity Mechanism of Emodin Based on Quantitative Proteomic Analyses.

Emodin is the main component of traditional Chinese medicines including rhubarb, Polygonum multiflorum, and Polygonum cuspidatum. It has confirmed hepatotoxicity and may be the main causative agent of liver damage associated with the above-mentioned traditional Chinese medicines. However, current research does not explain the mechanism of emodin in hepatotoxicity. In this study, L02 cells were used as a model to study the mechanism of emodin-induced hepatocyte apoptosis using quantitative proteomics, and the results were verified by Western blot. A total of 662 differentially expressed proteins were discovered and analyzed using Gene Ontology (GO) and pathway enrichment analysis. The results show that the oxidative phosphorylation pathway is highly represented. Abnormalities in this pathway result in impaired mitochondrial function and represent mitochondrial damage. This result is consistent with mitochondria membrane potential measurements. Analysis of differentially expressed proteins revealed that emodin mainly affects oxidative phosphorylation pathways by inhibiting the function of the mitochondrial respiratory chain complexes; the mitochondrial respiratory chain complex activity assay result also confirmed that emodin could inhibit the activity of all mitochondrial complexes. This results in an increase in caspase-3, a decrease in mitochondrial membrane potential (MMP,) an increase in reactive oxygen species (ROS), and disorders in ATP synthesis, etc., eventually leading to mitochondrial damage and hepatocyte apoptosis in vitro.

Novel role of mitochondrial GTPases 1 in pathological cardiac hypertrophy

While most mitochondrial proteins are encoded in the nucleus and translated on cytosolic/endoplasmic reticulum ribosomes, proteins encoded by mitochondrial DNA are translated on mitochondrial ribosomes. Mitochondrial GTPases 1 (MTG1) regulates mitochondrial ribosome assembly and translation, but its impact on cardiac adaptation to stress is unknown. Here, we found that MTG1 is dramatically elevated in hearts of dilated cardiomyopathy patients and in mice exposed to left ventricular pressure overload (AB). To examine the role of MTG1 in cardiac hypertrophy and heart failure, MTG1 loss/gain of function studies were performed in cultured cardiomyocytes and mice exposed to hypertrophic stress. MTG1 shRNA and adenoviral overexpression studies indicated that MTG1 expression attenuates angiotensin II-induced hypertrophy in cultured cardiomyocytes, while MTG1 KO mice exhibited no observable cardiac phenotype under basal conditions. MTG1 deficiency significantly exacerbated AB-induced cardiac hypertrophy, expression of hypertrophic stress markers, fibrosis, and LV dysfunction in comparison to WT mice. Conversely, transgenic cardiac MTG1 expression attenuated AB-induced hypertrophy and LV dysfunction. Mechanistically, MTG1 preserved mitochondrial respiratory chain complex activity during pressure overload, which further attenuated ROS generation. Moreover, we demonstrated that TAK1, P38 and JNK1/2 activity is downregulated in the MTG1 overexpression group. Importantly, dampening oxidative stress with N-acetylcysteine (NAC) lowered hypertrophy in MTG1 KO to WT levels. Collectively, our data indicate that MTG1 protects against pressure overload-induced cardiac hypertrophy and dysfunction by preserving mitochondrial function and reducing oxidative stress and downstream TAK1 stress signaling.

Roles of TRPA1 and TRPV1 in cigarette smoke -induced airway epithelial cell injury model

Transient receptor potential protein (TRP) ion channels TRPA1 and TRPV1 may be important in mediating airway tissue injury and inflammation. This study was designed to clarify the role of TRPA1 and TRPV1 channels in cigarette smoke extract (CSE)-induced damage to bronchial and alveolar epithelial cells. Alveolar epithelial (A549) cells and bronchial epithelial (Beas-2B) cells were treated with CSE in the presence and absence of a TRPA1 inhibitor (100 μM, A967079), a TRPV1 inhibitor (100 μM, AMG9810) or both. DCFH-DA and MitoSOX Red probes were used to assay intracellular and mitochondrial oxidative stress, respectively. The mRNA levels of inflammatory mediators (IL-1β, IL-8, IL-18, IL-33) and antioxidants (HO-1, NQO1, MnSOD, catalase) and the protein expression levels of mitochondrial and inflammasome factors (MFN2, OPA1, DRP1, MFF, NLRP3,caspase-1) were respectively detected by RT-PCR and Western Blot. The results were validated in TRPA1 shRNA and TRPV1 shRNA cells. In both cell types, 10% CSE increased intracellular and mitochondrial oxidative stress, induced Ca2+ influx, increased inflammatory gene expression, reduced antioxidant gene expression and inhibited the activities of mitochondrial respiratory chain (MRC) complexes. 10% CSE increased the expression of mitochondrial fission proteins (MFF and DRP1), Caspase-1 and NLRP3 protein expression and decreased that of mitochondrial fusion proteins (MFN2 and OPA1). Both inhibitors and gene-knockout of TRPA1 and TRPV1 reduced oxidative stress, blocked Ca2+ influx, and inhibited inflammatory and increased antioxidant gene expression. They also prevented the changes in mitochondrial fission and fusion proteins and in MRC complexes activities induced by CSE. Both TRPA1 and TRPV1 mediate CSE-induced damage of bronchial and alveolar epithelial cells via modulation of oxidative stress, inflammation and mitochondrial damage and their inhibition should be considered as potential therapy for COPD.

Effects of Xingpi Kaiyu Fang on ATP, Na/K-ATPase, and Respiratory Chain Complexes of Hippocampus and Gastrocnemius Muscle in Depressed Rats.

Objective To clarify the effectiveness and mechanism of the Chinese herbal formula Xingpi Kaiyu Fang (XPKYF) which is composed of American ginseng (Xi-Yang-shen), Radix curcumae (Yu-Jin), Acori tatarinowii rhizoma (Shi-Chang-pu), and Hypericum perforatum (Guan-Ye-lian-qiao) in depressed rats. Methods The rat model of depression was established by chronic unpredictable mild stress (CUMS) method for 6 weeks. Rats were randomly divided into six groups: control group, CUMS group, CUMS+XPKYF (3.6g/kg/d, 7.2g/kg/d, 14.4g/kg/d) groups, and CUMS+sertraline (4.5mg/kg/d) group. The sucrose preference test and the forced swimming test were performed to assess the rats' depression behavior. Mitochondrial ultrastructure was observed by transmission electron microscope and adenosine triphosphate (ATP) content, sodium potassium ATPase (Na/K-ATPase) activity, and mitochondrial respiratory chain complexes activities in hippocampus and gastrocnemius muscle were measured at the 14th and 42nd day. Results Rats subjected to six weeks of CUMS exhibited decreased sucrose preference ratio and prolonged immobility time. CUMS reduced ATP content in hippocampus, decreased Na/K-ATPase activity and respiratory chain complex I, III, and IV activities in hippocampus and gastrocnemius muscle, and damaged mitochondrial ultrastructure of hippocampus and gastrocnemius muscle. XPKYF at 14.4g/kg, the efficacy trend of which was better than the other drug groups, could prevent the stress-induced depressed behavior changes, inhibit the decrease of Na/K-ATPase activity in hippocampus, inhibit the decrease of respiratory chain complex III activities in hippocampus and gastrocnemius muscle, and protect mitochondria from ultrastructural damage. Conclusions Energy deficiency and damaged mitochondrial ultrastructure were found in hippocampus and gastrocnemius muscle of depressed rats established by CUMS. XPKYF could partly reverse alterations in ATP, Na/K-ATPase, and respiratory chain complexes of hippocampus and gastrocnemius muscle and protect mitochondria from ultrastructural damage. This provides another experimental evidence for the clinical application of XPKYF in the treatment of depression.

N-Acetyl Cysteine Restores Limb Function, Improves Mitochondrial Respiration, and Reduces Oxidative Stress in a Murine Model of Critical Limb Ischaemia

The aim of this study was to investigate whether antioxidant therapy might decrease oxidative stress related deleterious effects in the setting of critical limb ischaemia (CLI). Twenty Swiss mice were submitted to sequential right femoral and iliac ligatures; the left limb served as control. The mice were assigned to two groups: in the first group (no-treatment group, n=10) no treatment was administered; in the second group (N-acetyl cysteine [NAC] group, n=10) NAC was administered by dissolution in drinking water for 4 weeks, starting on day 7, when CLI was effective. Clinical and functional scores were assessed by two blinded investigators. Mice were killed on day 40 and mitochondrial respiratory chain complex activities, calcium retention capacity, oxidative stress, and histological analysis were analysed. Ischaemic muscles in the no-treatment group showed significantly impaired mitochondrial respiration and calcium retention capacity, with increased production of reactive oxygen species; but no statistical difference was noticed when comparing ischaemic muscles in the NAC group (n=10) to contralateral muscles (n=10) and to control muscles in the no-treatment group (n=10). Ischaemic muscles in the no-treatment group exhibited myopathic features such as wider range in fibre size, rounded shape, centrally located nuclei, and smaller cross sectional areas, but none of these features were observed in contralateral muscles or in NAC-group muscles (ischaemic or controls). Targeting inhibition of oxidative stress may be a potential therapeutic strategy for muscle protection in CLI and might be considered as potential adjunctive therapy to revascularisation procedures.

The Contribution of Different Components in QiShenYiQi Pills® to Its Potential to Modulate Energy Metabolism in Protection of Ischemic Myocardial Injury.

Ischemic heart diseases remain a challenge for clinicians. QiShenYiQi pills® (QSYQ) has been reported to be curative during coronary heart diseases with modulation of energy metabolism as one of the underlying mechanisms. In this study, we detected the effect of QSYQ and its components on rat myocardial structure, mitochondrial respiratory chain complexes activity and energy metabolism, and heart function after 30 min of cardiac ischemia, with focusing on the contribution of each component to its potential to regulate energy metabolism. Results showed that treatment with QSYQ and all its five components protected myocardial structure from damage by ischemia. QSYQ also attenuated release of myocardial cTnI, and restored the production of ATP after cardiac ischemia. AS-IV and Rb1, but not Rg1, R1, and DLA, had similar effect as QSYQ in regulation of energy metabolism. These results indicate that QSYQ may prevent ischemia-induced cardiac injury via regulation of energy metabolism, to which each of its components contributes differently.

Formoterol attenuates increased oxidative stress and myosin protein loss in respiratory and limb muscles of cancer cachectic rats.

Muscle mass loss and wasting are characteristic features of patients with chronic conditions including cancer. Therapeutic options are still scarce. We hypothesized that cachexia-induced muscle oxidative stress may be attenuated in response to treatment with beta2-adrenoceptor-selective agonist formoterol in rats. In diaphragm and gastrocnemius of tumor-bearing rats (108 AH-130 Yoshida ascites hepatoma cells inoculated intraperitoneally) with and without treatment with formoterol (0.3 mg/kg body weight/day for seven days, daily subcutaneous injection), redox balance (protein oxidation and nitration and antioxidants) and muscle proteins (1-dimensional immunoblots), carbonylated proteins (2-dimensional immunoblots), inflammatory cells (immunohistochemistry), and mitochondrial respiratory chain (MRC) complex activities were explored. In the gastrocnemius, but not the diaphragm, of cancer cachectic rats compared to the controls, protein oxidation and nitration levels were increased, several functional and structural proteins were carbonylated, and in both study muscles, myosin content was reduced, inflammatory cell counts were greater, while no significant differences were seen in MRC complex activities (I, II, and IV). Treatment of cachectic rats with formoterol attenuated all the events in both respiratory and limb muscles. In this in vivo model of cancer-cachectic rats, the diaphragm is more resistant to oxidative stress. Formoterol treatment attenuated the rise in oxidative stress in the limb muscles, inflammatory cell infiltration, and the loss of myosin content seen in both study muscles, whereas no effects were observed in the MRC complex activities. These findings have therapeutic implications as they demonstrate beneficial effects of the beta2 agonist through decreased protein oxidation and inflammation in cachectic muscles, especially the gastrocnemius.

Effects of endurance training on skeletal muscle mitochondrial function in Huntington disease patients

BackgroundMitochondrial dysfunction may represent a pathogenic factor in Huntington disease (HD). Physical exercise leads to enhanced mitochondrial function in healthy participants. However, data on effects of physical exercise on HD skeletal muscle remains scarce. We aimed at investigating adaptations of the skeletal muscle mitochondria to endurance training in HD patients.MethodsThirteen HD patients and 11 healthy controls completed 26 weeks of endurance training. Before and after the training phase muscle biopsies were obtained from M. vastus lateralis. Mitochondrial respiratory chain complex activities, mitochondrial respiratory capacity, capillarization, and muscle fiber type distribution were determined from muscle samples.ResultsCitrate synthase activity increased during the training intervention in the whole cohort (P = 0.006). There was no group x time interaction for citrate synthase activity during the training intervention (P = 0.522). Complex III (P = 0.008), Complex V (P = 0.043), and succinate cytochrome c reductase (P = 0.008) activities increased in HD patients and controls by endurance training. An increase in mass-specific mitochondrial respiratory capacity was present in HD patients during the endurance training intervention. Overall capillary-to-fiber ratio increased in HD patients by 8.4% and in healthy controls by 6.4% during the endurance training intervention.ConclusionsSkeletal muscle mitochondria of HD patients are equally responsive to an endurance-training stimulus as in healthy controls. Endurance training is a safe and feasible option to enhance indices of energy metabolism in skeletal muscle of HD patients and may represent a potential therapeutic approach to delay the onset and/or progression of muscular dysfunction.Trial registrationClinicalTrials.gov NCT01879267. Registered May 24, 2012.

The low affinity neurotensin receptor antagonist levocabastine impairs brain nitric oxide synthesis and mitochondrial function by independent mechanisms.

Neurotensin is known to inhibit neuronal Na+ , K+ -ATPase, an effect that is rescued by nitric oxide (NO) synthase inhibition. However, whether the neurotensinergic and the nitrergic systems are independent pathways, or are mechanistically linked, remains unknown. Here, we addressed this issue and found that the administration of low affinity neurotensin receptor (NTS2) antagonist, levocabastine (50μg/kg, i.p.) inhibited NO synthase (NOS) activity by 74 and 42% after 18h in synaptosomal and mitochondrial fractions isolated from the Wistar rat cerebral cortex, respectively; these effects disappeared 36h after levocabastine treatment. Intriguingly, whereas neuronal NOS protein abundance decreased (by 56%) in synaptosomes membranes, it was enhanced (by 86%) in mitochondria 18h after levocabastine administration. Levocabastine enhanced the respiratory rate of synaptosomes in the presence of oligomycin, but it failed to alter the spare respiratory capacity; furthermore, the mitochondrial respiratory chain (MRC) complexes I-IV activities were severely diminished by levocabastine administration. The inhibition of NOS and MRC complexes activities were also observed after incubation of synaptosomes and mitochondria with levocabastine (1μM) invitro. These data indicate that the NTS2 antagonist levocabastine regulates NOS expression and activity at the synapse, suggesting an interrelationship between the neurotensinergic and the nitrergic systems. However, the bioenergetics effects of NTS2 activity inhibition are likely to be independent from the regulation of NO synthesis.

Clinical validity of biochemical and molecular analysis in diagnosing Leigh syndrome: a study of 106 Japanese patients

Leigh syndrome (LS) is a progressive neurodegenerative disorder of infancy and early childhood. It is clinically diagnosed by typical manifestations and characteristic computed tomography (CT) or magnetic resonance imaging (MRI) studies. Unravelling mitochondrial respiratory chain (MRC) dysfunction behind LS is essential for deeper understanding of the disease, which may lead to the development of new therapies and cure. The aim of this study was to evaluate the clinical validity of various diagnostic tools in confirming MRC disorder in LS and Leigh-like syndrome (LL). The results of enzyme assays, molecular analysis, and cellular oxygen consumption rate (OCR) measurements were examined. Of 106 patients, 41 were biochemically and genetically verified, and 34 had reduced MRC activity but no causative mutations. Seven patients with normal MRC complex activities had mutations in the MT-ATP6 gene. Five further patients with normal activity in MRC were identified with causative mutations. Conversely, 12 out of 60 enzyme assays performed for genetically verified patients returned normal results. No biochemical or genetic background was confirmed for 19 patients. OCR was reduced in ten out of 19 patients with negative enzyme assay results. Inconsistent enzyme assay results between fibroblast and skeletal muscle biopsy samples were observed in 33% of 37 simultaneously analyzed cases. These data suggest that highest diagnostic rate is reached using a combined enzymatic and genetic approach, analyzing more than one type of biological materials where suitable. Microscale oxygraphy detected MRC impairment in 50% cases with no defect in MRC complex activities.

Protein profiling and functional analysis of liver mitochondria from rats with nonalcoholic steatohepatitis

Mitochondrial dysfunction is closely associated with the pathogenesis of nonalcoholic steatohepatitis (NASH). The aim of the present study was to comprehensively determine mitochondrial abnormalities in NASH by detecting the proteomics in liver mitochondria in a NASH rat model, which was induced for 16 weeks by the provision of a high fat and high cholesterol diet (HFD). Serum parameters, including triglycerides, total cholesterol, low-density lipoprotein cholesterol and high-density lipoprotein cholesterol were determined, and hematoxylin and eosin staining of liver tissues was examined to evaluate the NASH rat model. Various parameters associated with mitochondrial function were examined, including mitochondrial DNA (mtDNA) copy number, mitochondrial membrane potential (MMP) and mitochondrial respiratory chain complex (MRC) activity. The mitochondrial proteomics were analyzed and identified using isobaric tags for relative and absolute quantitation labeling coupled with two-dimensional liquid chromatography-tandem mass spectrometry. The identified proteins were classified and grouped using the Blast2GO program against the non-redundant protein database, the Kyoto Encyclopedia of Genes and Genomes database and the Cluster of Orthologous Groups of proteins database. Compared with the control, mtDNA copy number, MMP, and activities of MRC I and III were decreased markedly in the HFD group. A total of 18 upregulated and 13 downregulated proteins were identified, with a significant 1.2-fold difference between the control and NASH groups. The dysregulated proteins were closely involved in mitochondrial oxidative phosphorylation, the lipid metabolic process and fatty acid β-oxidation. The results of the present study provide important proteomic information regarding liver mitochondria in NASH and serve as a basis for further detailed investigations of the pathogenesis of NASH.

Placental respiratory chain complex activities in high risk pregnancies

Objectives: Mitochondrial oxidative phosphorylation is the key energy source for placental functions and fetal growth. The purpose of this study was to investigate the function of placenta in high risk pregnancies by measuring mitochondrial respiratory chain complex (RCC) activities, and to evaluate the correlation between double test risk ratio and RCC activities.Methods: The placenta samples were collected from 50 pregnant women. The controls consisted of 20 normal uncomplicated pregnancies and the study group (n = 30) consisted of preeclampsia (PE), intrauterin growth restriction (IUGR), advanced maternal age (AMA), twins and preterm deliveries. Complexes I, II–III, IV and citrate synthase (CS) activities were measured by spectrophotometric assays.Results: Complexes I, II–III and IV activities were significantly lower in the study group than the controls (p < 0.05). Complexes I and II–III activities were significantly reduced in placenta of preterm deliveries compared with the controls (p < 0.003). Double test risk ratio was above the cut-off limit (1:300) in 43% of the study group in which decreased complexes I and II–III activities were observed.Conclusions: Impaired placental mitochondria RCC functions can lead to adverse pregnancy outcomes. Pregnant women with high risk in double test should be monitored carefully in terms of PE, IUGR and preterm delivery.

Extracellular Cl−-free-induced cardioprotection against hypoxia/reoxygenation is associated with attenuation of mitochondrial permeability transition pore

The isotonic substitution of extracellular chloride by gluconate (extracellular Cl--free) has been demonstrated to elicit cardioprotection by attenuating ischaemia/reperfusion-induced elevation of intracellular chloride ion concentration ([Cl-]i). However, the downstream mechanism underlying the cardioprotective effect of extracellular Cl--free is not fully established. Here, it was investigated whether extracellular Cl--free attenuates mitochondrial dysfunction after hypoxia/reoxygenation (H/R) and whether mitochondrial permeability transition pore (mPTP) plays a key role in the extracellular Cl--free cardioprotection. H9c2 cells were incubated with or without Cl--free solution, in which Cl- was replaced with equimolar gluconate, during H/R. The involvement of mPTP was determined with atractyloside (Atr), a specific mPTP opener. The results showed that extracellular Cl--free attenuated H/R-induced the elevation of [Cl-]i, accompanied by increase of cell viability and reduction of lactate dehydrogenase release. Moreover, extracellular Cl--free inhibited mPTP opening, and improved mitochondria function, as indicated by preserved mitochondrial membrane potential and respiratory chain complex activities, decreased mitochondrial reactive oxygen species generation, and increased ATP content. Intriguingly, pharmacologically opening of the mPTP with Atr attenuated all the protective effects caused by extracellular Cl--free, including suppression of mPTP opening, maintenance of mitochondrial membrane potential, and subsequent improvement of mitochondrial function. These results indicated that extracellular Cl--free protects mitochondria from H/R injury in H9c2 cells and inhibition of mPTP opening is a crucial step in mediating the cardioprotection of extracellular Cl--free.

Sasanquasaponin-induced cardioprotection involves inhibition of mPTP opening via attenuating intracellular chloride accumulation

Sasanquasaponin (SQS) has been reported to elicit cardioprotection by suppressing hypoxia/reoxygenation (H/R)-induced elevation of intracellular chloride ion concentration ([Cl−]i). Given that the increased [Cl−]i is involved to modulate the mitochondrial permeability transition pore (mPTP), we herein sought to further investigate the role of mPTP in the cardioprotective effect of SQS on H/R injury. H9c2 cells were incubated for 24h with or without 10μM SQS followed by H/R. The involvement of mPTP was determined with a specific mPTP agonist atractyloside (ATR). The results showed that SQS attenuated H/R-induced the elevation of [Cl−]i, accompanied by reduction of lactate dehydrogenase release and increase of cell viability. Moreover, SQS suppressed mPTP opening, and protected mitochondria, as indicated by preserved mitochondrial membrane potential and respiratory chain complex activities, decreased mitochondrial reactive oxygen species generation, and increased ATP content. Interestingly, extracellular Cl−-free condition created by replacing Cl− with equimolar gluconate resulted in a decrease in [Cl−]i and induced protective effects similar to SQS preconditioning, whereas pharmacologically opening of the mPTP with ATR abolished all the protective effects induced by SQS or Cl−-free, including suppression of mPTP opening, maintenance of mitochondrial membrane potential, and subsequent improvement of mitochondrial function. The above results allow us to conclude that SQS-induced cardioprotection may be mediated by preserving the mitochondrial function through preventing mPTP opening via inhibition of H/R-induced elevation of [Cl−]i.

Relationships between the activity of respiratory-chain complexes in pre- (biopsy) or post-slaughter muscle samples and feed efficiency in random-bred Ghezel lambs

The present study was conducted to determine the relationship between the activity of mitochondrial respiratory chain complexes in pre- and post-slaughter muscle samples and residual feed intake (RFI) in Ghezel male lambs born as a result of random mating. The study was based on the hypothesis that random-bred lambs with lower feed (or higher) RFI have lower (or higher) respiratory chain-complex activity in muscle samples. Lambs (n = 30) were fed a diet consisting of 70% concentrate and 30% alfalfa hay during a 70-day period. Individual feed intake and average daily gain were recorded to calculate the RFI, feed-conversion ratio (FCR) and adjusted FCR (aFCR). On the basis of these calculations, the lambs were classified into low and high groups for RFI, with FCR and aFCR (n = 22) being one standard deviation above or below the means; this was corroborated by Student’s t-test (P &amp;lt; 0.01). At the end of the experiment, a 10-g biopsy sample was taken from the posterior side of the left femoral biceps. After 24 h, the lambs were slaughtered, and a sample from the posterior side of the right femoral biceps was dissected for determination of mitochondrial protein and respiratory chain-complex activities (Complexes I–V). The RFI was not correlated with the metabolic bodyweight and average daily gain, but was positively correlated (r = 0.56) with the average daily feed intake (P &amp;lt; 0.01); mean daily feed intake in the low-RFI group was 200 g less than that in the high-RFI group. The FCR and aFCR were not significantly (P &amp;gt; 0.05) correlated with average daily feed intake (r = 0.39 and r = 0.36 respectively), but showed a negative correlation (P &amp;lt; 0.01) with average daily gain (r = –0.73 and r = –0.76 respectively). Although very high negative correlations were recorded between the activities of all five respiratory-chain complexes and RFI in muscle samples obtained before (–0.91 to –0.97) and after (–0.92 to –0.97) slaughter, Complexes I and V showed small negative correlations (–0.40) with FCR or aFCR (P &amp;lt; 0.05). Enzyme activities of the respiratory-chain Complexes I, III and V were not significantly different between the pre- and post-slaughter biopsy samples; however, the enzyme activities of respiratory-chain Complexes II and IV were slightly higher in post-slaughter samples (P &amp;lt; 0.01). These results suggested that it may be possible to use the enzymatic activity of respiratory-chain complexes in muscle biopsy samples for screening of lambs for RFI, providing a useful procedure for genetic selection of lambs for this component of feed efficiency. These encouraging results need to be verified in further experiments using other sheep breeds and a larger number of lambs.

ADAMTS5 deficiency protects against non-alcoholic steatohepatitis in obesity.

Increased prevalence of obesity is paralleled by an increase in non-alcoholic steatohepatitis (NASH). We previously found that the expression of ADAMTS5 (A Disintegrin And Metalloproteinase with Thrombospondin type 1 motifs; member 5) is enhanced in expanding adipose tissue. However, no information is available on a potential role in liver pathology. We studied the effect of ADAMTS5 deficiency on NASH in mice. Wild-type (Adamts5+/+ ) and deficient (Adamts5-/- ) mice were kept on a standard- or high-fat diet (HFD) for 15 weeks. Alternatively, steatohepatitis was induced with methionine/choline-deficient (MCD) diet. HFD feeding resulted in comparable body weights for both genotypes, but Adamts5-/- mice had approximately 40% lower liver weight (P = 0.0004). In the Adamts5-/- mice, the HFD as well as the MCD diet consistently induced less NASH with less fibrosis. The deteriorating effect of ADAMTS5 on the liver during diet-induced obesity may be due, at least in part, to proteolytic cleavage of the matrix components syndecan-1 and versican, thereby enhancing hepatic triglyceride clearance from the circulation. Plasma lipid levels were elevated in obese Adamts5-/- mice. There was no clear effect of ADAMTS5 deficiency on glycaemia or glucose tolerance, whereas insulin sensitivity was somewhat improved. Furthermore, Adamts5-/- mice were protected from hepatic mitochondrial dysfunction, as indicated by increased mitochondrial respiratory chain complex activity, higher ATP levels and higher expression of antioxidant enzymes. Absence of ADAMTS5 preserves liver integrity in a diet-induced obesity model. Selective targeting of ADAMTS5 could provide a new therapeutic strategy for treatment/prevention of NASH.

Acute and Chronic Treatments with Quetiapine Increase Mitochondrial Respiratory Chain Complex Activity in the Rat Brain.

Several studies have found that the molecular mechanisms of mitochondrial energy metabolism are impaired in major depressive disorder (MDD). Classic antidepressants and atypical antipsychotics can alter the function of enzymes involved in adenosine triphosphate (ATP) metabolism. Quetiapine is an atypical antipsychotic that, in addition to having a therapeutic benefit in treating MDD, appears to exert antioxidant and neuroprotective effects. Therefore, we aimed to evaluate the acute and chronic effects of quetiapine on the activity of enzyme complexes I to IV of the mitochondrial respiratory chain and creatine kinase (CK) in brain regions involved with MDD. After a single dose or serial injections over 14 days of quetiapine (20, 40, and 80 mg) were administered, isolates from the pre- frontal cortex, hippocampus, amygdala and nucleus accumbens were analyzed for enzyme activity levels. The enzyme activity varied according to the dose, brain region, and acute or chronic dosing protocols. In general, complexes I-III activity was increased, especially after acute administration. Acute administration also increased the activity of complex IV and CK in the amygdala while complex I was inhibited in the prefrontal cortex and nucleus accumbens. These results suggest that quetiapine produces an increase in respiratory chain complex activity, which may be underlying its efficacy against psychiatric disorders and neuronal damage.