Skeletal Muscle Mitochondria Research Articles

Human Cachexia Induces Changes in Mitochondria, Autophagy and Apoptosis in the Skeletal Muscle.

Cachexia is a wasting syndrome characterized by the continuous loss of skeletal muscle mass due to imbalance between protein synthesis and degradation, which is related with poor prognosis and compromised quality of life. Dysfunctional mitochondria are associated with lower muscle strength and muscle atrophy in cancer patients, yet poorly described in human cachexia. We herein investigated mitochondrial morphology, autophagy and apoptosis in the skeletal muscle of patients with gastrointestinal cancer-associated cachexia (CC), as compared with a weight-stable cancer group (WSC). CC showed prominent weight loss and increased circulating levels of serum C-reactive protein, lower body mass index and decreased circulating hemoglobin, when compared to WSC. Electron microscopy analysis revealed an increase in intermyofibrillar mitochondrial area in CC, as compared to WSC. Relative gene expression of Fission 1, a protein related to mitochondrial fission, was increased in CC, as compared to WSC. LC3 II, autophagy-related (ATG) 5 and 7 essential proteins for autophagosome formation, presented higher content in the cachectic group. Protein levels of phosphorylated p53 (Ser46), activated caspase 8 (Asp384) and 9 (Asp315) were also increased in the skeletal muscle of CC. Overall, our results demonstrate that human cancer-associated cachexia leads to exacerbated muscle-stress response that may culminate in muscle loss, which is in part due to disruption of mitochondrial morphology, dysfunctional autophagy and increased apoptosis. To the best of our knowledge, this is the first report showing quantitative morphological alterations in skeletal muscle mitochondria in cachectic patients.

Mechanisms of Energy Metabolism in Skeletal Muscle Mitochondria Following Radiation Exposure.

An understanding of cellular processes that determine the response to ionizing radiation exposure is essential for improving radiotherapy and assessing risks to human health after accidental radiation exposure. Radiation exposure leads to many biological effects, but the mechanisms underlying the metabolic effects of radiation are not well known. Here, we investigated the effects of radiation exposure on the metabolic rate and mitochondrial bioenergetics in skeletal muscle. We show that ionizing radiation increased mitochondrial protein and mass and enhanced proton leak and mitochondrial maximal respiratory capacity, causing an increase in the fraction of mitochondrial respiration devoted to uncoupling reactions. Thus, mice and cells treated with radiation became energetically efficient and displayed increased fatty acid and amino acid oxidation metabolism through the citric acid cycle. Finally, we demonstrate that radiation-induced alterations in mitochondrial energy metabolism involved adenosine monophosphate-activated kinase signaling in skeletal muscle. Together, these results demonstrate that alterations in mitochondrial mass and function are important adaptive responses of skeletal muscle to radiation.

Regulation of mitochondrial metabolism in murine skeletal muscle by the medium-chain fatty acid receptor Gpr84.

Fatty acid receptors have been recognized as important players in glycaemic control. This study is the first to describe a role for the medium-chain fatty acid (MCFA) receptor G-protein-coupled receptor (Gpr) 84 in skeletal muscle mitochondrial function and insulin secretion. We are able to show that Gpr84 is highly expressed in skeletal muscle and adipose tissue. Mice with global deletion of Gpr84 [Gpr84 knockout (KO)] exhibit a mild impairment in glucose tolerance when fed a MCFA-enriched diet. Studies in mice and pancreatic islets suggest that glucose intolerance is accompanied by a defect in insulin secretion. MCFA-fed KO mice also exhibit a significant impairment in the intrinsic respiratory capacity of their skeletal muscle mitochondria, but at the same time also exhibit a substantial increase in mitochondrial content. Changes in canonical pathways of mitochondrial biogenesis and turnover are unable to explain these mitochondrial differences. Our results show that Gpr84 plays a crucial role in regulating mitochondrial function and quality control.-Montgomery, M. K., Osborne, B., Brandon, A. E., O'Reilly, L., Fiveash, C. E., Brown, S. H. J., Wilkins, B. P., Samsudeen, A., Yu, J., Devanapalli, B., Hertzog, A., Tolun, A. A., Kavanagh, T., Cooper, A. A., Mitchell, T. W., Biden, T. J., Smith, N. J., Cooney, G. J., Turner, N. Regulation of mitochondrial metabolism in murine skeletal muscle by the medium-chain fatty acid receptor Gpr84.

Estrogens, Estrogen Receptors Effects on Cardiac and Skeletal Muscle Mitochondria.

Mitochondria are unique organelles present in almost all cell types. They are involved not only in the supply of energy to the host cell, but also in multiple biochemical and biological processes like calcium homeostasis, production, and regulation of reactive oxygen species (ROS), pH control, or cell death. The importance of mitochondria in cell biology and pathology is increasingly recognized. Being maternally inherited, mitochondria exhibit a tissue-specificity, because most of the mitochondrial proteins are encoded by the nuclear genome. This renders them exquisitely well-adapted to the physiology of the host cell. It is thus not surprising that mitochondria show a sexual dimorphism and that they are also prone to the influence of sex chromosomes and sex hormones. Estrogens affect mitochondria through multiple processes involving membrane and nuclear estrogen receptors (ERs) as well as more direct effects. Moreover, estrogen receptors have been identified within mitochondria. The effects of estrogens on mitochondria comprise protein content and specific activity of mitochondrial proteins, phospholipid content of membranes, oxidant and anti-oxidant capacities, oxidative phosphorylation, and calcium retention capacities. Herein we will briefly review the life cycle and functions of mitochondria, the importance of estrogen receptors and the effects of estrogens on heart and skeletal muscle mitochondria.

Uremic metabolites impair skeletal muscle mitochondrial energetics through disruption of the electron transport system and matrix dehydrogenase activity.

Chronic kidney disease (CKD) leads to increased skeletal muscle fatigue, weakness, and atrophy. Previous work has implicated mitochondria within the skeletal muscle as a mediator of muscle dysfunction in CKD; however, the mechanisms underlying mitochondrial dysfunction in CKD are not entirely known. The purpose of this study was to define the impact of uremic metabolites on mitochondrial energetics. Skeletal muscle mitochondria were isolated from C57BL/6N mice and exposed to vehicle (DMSO) or varying concentrations of uremic metabolites: indoxyl sulfate, indole-3-acetic-acid, l-kynurenine, and kynurenic acid. A comprehensive mitochondrial phenotyping platform that included assessments of mitochondrial oxidative phosphorylation (OXPHOS) conductance and respiratory capacity, hydrogen peroxide production (JH2O2), matrix dehydrogenase activity, electron transport system enzyme activity, and ATP synthase activity was employed. Uremic metabolite exposure resulted in a ~25-40% decrease in OXPHOS conductance across multiple substrate conditions (P < 0.05, n = 5-6/condition), as well as decreased ADP-stimulated and uncoupled respiratory capacity. ATP synthase activity was not impacted by uremic metabolites; however, a screen of matrix dehydrogenases indicated that malate and glutamate dehydrogenases were impaired by some, but not all, uremic metabolites. Assessments of electron transport system enzymes indicated that uremic metabolites significantly impair complex III and IV. Uremic metabolites resulted in increased JH2O2 under glutamate/malate, pyruvate/malate, and succinate conditions across multiple levels of energy demand (all P < 0.05, n = 4/group). Disruption of mitochondrial OXPHOS was confirmed by decreased respiratory capacity and elevated superoxide production in cultured myotubes. These findings provide direct evidence that uremic metabolites negatively impact skeletal muscle mitochondrial energetics, resulting in decreased energy transfer, impaired complex III and IV enzyme activity, and elevated oxidant production.

Deterioration of mitochondrial bioenergetics and ultrastructure impairment in skeletal muscle of a transgenic minipig model in the early stages of Huntington's disease.

ABSTRACTSkeletal muscle wasting and atrophy is one of the more severe clinical impairments resulting from the progression of Huntington's disease (HD). Mitochondrial dysfunction may play a significant role in the etiology of HD, but the specific condition of mitochondria in muscle has not been widely studied during the development of HD. To determine the role of mitochondria in skeletal muscle during the early stages of HD, we analyzed quadriceps femoris muscle from 24-, 36-, 48- and 66-month-old transgenic minipigs that expressed the N-terminal portion of mutated human huntingtin protein (TgHD) and age-matched wild-type (WT) siblings. We found altered ultrastructure of TgHD muscle tissue and mitochondria. There was also significant reduction of activity of citrate synthase and respiratory chain complexes (RCCs) I, II and IV, decreased quantity of oligomycin-sensitivity conferring protein (OSCP) and the E2 subunit of pyruvate dehydrogenase (PDHE2), and differential expression of optic atrophy 1 protein (OPA1) and dynamin-related protein 1 (DRP1) in the skeletal muscle of TgHD minipigs. Statistical analysis identified several parameters that were dependent only on HD status and could therefore be used as potential biomarkers of disease progression. In particular, the reduction of biomarker RCCII subunit SDH30 quantity suggests that similar pathogenic mechanisms underlie disease progression in TgHD minipigs and HD patients. The perturbed biochemical phenotype was detectable in TgHD minipigs prior to the development of ultrastructural changes and locomotor impairment, which become evident at the age of 48 months. Mitochondrial disturbances may contribute to energetic depression in skeletal muscle in HD, which is in concordance with the mobility problems observed in this model.This article has an associated First Person interview with the first author of the paper.

Linking bioenergetic function of mitochondria to tissue-specific molecular fingerprints.

Mitochondria are dynamic organelles with diverse functions in tissues such as liver and skeletal muscle. To unravel the mitochondrial contribution to tissue-specific physiology, we performed a systematic comparison of the mitochondrial proteome and lipidome of mice and assessed the consequences hereof for respiration. Liver and skeletal muscle mitochondrial protein composition was studied by data-independent ultra-high-performance (UHP)LC-MS/MS-proteomics, and lipid profiles were compared by UHPLC-MS/MS lipidomics. Mitochondrial function was investigated by high-resolution respirometry in samples from mice and humans. Enzymes of pyruvate oxidation as well as several subunits of complex I, III, and ATP synthase were more abundant in muscle mitochondria. Muscle mitochondria were enriched in cardiolipins associated with higher oxidative phosphorylation capacity and flexibility, in particular CL(18:2)4 and 22:6-containing cardiolipins. In contrast, protein equipment of liver mitochondria indicated a shuttling of complex I substrates toward gluconeogenesis and ketogenesis and a higher preference for electron transfer via the flavoprotein quinone oxidoreductase pathway. Concordantly, muscle and liver mitochondria showed distinct respiratory substrate preferences. Muscle respired significantly more on the complex I substrates pyruvate and glutamate, whereas in liver maximal respiration was supported by complex II substrate succinate. This was a consistent finding in mouse liver and skeletal muscle mitochondria and human samples. Muscle mitochondria are tailored to produce ATP with a high capacity for complex I-linked substrates. Liver mitochondria are more connected to biosynthetic pathways, preferring fatty acids and succinate for oxidation. The physiologic diversity of mitochondria may help to understand tissue-specific disease pathologies and to develop therapies targeting mitochondrial function.

Dietary Linoleic Acid Targets Skeletal Muscle to Impact Energy Metabolism (P08-124-19)

ObjectivesInvestigate the impact of dietary linoleic acid (LA) on whole-body energy metabolism, fatty acid uptake, oxylipin metabolites, and energy production machinery in skeletal muscle. MethodsMice were fed modified AIN-93 M diets containing 6% wt of LA or palmitic acid (PA) for 5 weeks. To address energy balance, grip strength, blood glucose, and body composition were measured weekly and indirect calorimetry was performed after 4.5 weeks of diet. To address skeletal muscle architecture, cross-sectional area was quantified in quadriceps. To determine the effect of LA diet on skeletal muscle lipids, fatty acid composition was measured in quadriceps using GC. Targeted lipidomics using LC-MS/MS was utilized to measure LA-derived oxylipins in mitochondria isolated from gastrocnemius. ResultsNo difference in food intake, body weight, blood glucose, or body composition were observed between diet groups. Mice fed the LA diet exhibited higher heat production and lower respiratory exchange ratio, independent of ambulatory activity compared to mice fed the PA diet. Cross sectional area analysis in quadriceps muscle revealed that mice fed the LA diet had smaller muscle fibers compared to mice fed the PA diet. The LA diet increased LA level in quadriceps. Using hierarchal cluster analysis, we identified several unique patterns of oxylipins that impact insulin sensitivity and may be associated with increased mitochondrial capacity. Mice fed the LA diet exhibited a three-fold increase of oxylipin metabolites 9- and 13-HODE, and 12,13-diHOME. ConclusionsMice fed a diet fortified in LA have increased LA in skeletal muscle, changes to skeletal muscle architecture, favorable changes to whole body energy metabolism, and increases in LA-derived oxylipins in skeletal muscle mitochondria. Our discovery that LA generates 9-HODE and 13-HODE in muscle mitochondria suggests that downstream pathways, including PPARg transactivation, may be linked with effects on muscle energy metabolism. Funding SourcesFunding was provided by the National Institutes of Health (NIH) R21CA185140, Ohio Agriculture Research and Development Center and the Carol S. Kennedy Professorship. DBS is supported by the Center for Muscle Health and Neuromuscular Disorders Predoctoral Research Fellowship.

166-OR: AMPK-Dependent Regulation of Skeletal Muscle Mitochondrial Function by FNIP1

Skeletal muscle mitochondria are essential for maintaining metabolic homeostasis in response to a myriad of physiologic or pathophysiological stresses. The folliculin (FLCN)-interacting protein 1 (FNIP1) forms a complex with AMP-activated protein kinase (AMPK), and we previously demonstrated that FNIP1 inhibits AMPK activity in myocytes. Using gain- and loss-of-function approaches in mice, we discovered that FNIP1 is necessary, but not sufficient, to suppress AMPK-dependent mitochondrial function in skeletal muscle. Muscles in FNIP1 knockout (KO) mice show enhanced red coloration, oxidative mitochondrial activity, and type I fiber transformation. Surprisingly, no change in mitochondrial oxidative capacity and fiber type switching was observed in transgenic mice overexpressing FNIP1 in muscle (FNIP1Tg), while restoration of the expression of FNIP1 in the KO muscle (FNIP1Tg/KO) reversed the mitochondrial oxidative metabolism program. RNA-seq profiling revealed that 3328 genes showed over 1.5-fold altered in FNIP1 KO muscle, and the majority number (2856) was regulated in a FNIP1-dependent manner. Gene ontology based analysis revealed that the primary FNIP1-altered genes were mitochondrial metabolic genes. Mitochondrial respiration analysis confirmed that both pyruvate- and succinate-driven state 3 respiration rates were significantly higher in FNIP1 KO muscle, whereas restoration of the expression of FNIP1 in KO muscle reduced the mitochondrial function. Importantly, AMPK, but not mTOR, was markedly induced in FNIP1 KO muscle but reduced in FNIP1Tg/KO muscle. Muscle-specific knockout of the two AMPKα subunits (AMPKα1α2 dmKO) reduced mitochondrial function in FNIP1 KO muscle. Similar results were obtained in skeletal myocytes in culture, using AMPK loss-of-function approaches in FNIP1 KO myocyte. Thus, FNIP1 represents a previously unrecognized negative regulator of muscle mitochondrial function and AMPK is vital for FNIP1 actions in skeletal muscle. Disclosure L. Xiao: None. J. Liu: None. Z. Sun: None. Y. Yin: None. Y. Mao: None.

170-OR: Tamoxifen Treatment Causes Insulin Resistance in Obese Mice

Breast cancer survivors treated with the selective estrogen receptor modulator tamoxifen have an elevated risk for developing type 2 diabetes (T2D) compared with cancer-free individuals, especially in the context of obesity. Half of T2D diagnoses in breast cancer survivors are attributable to the cancer therapy itself. The underlying mechanisms of tamoxifen-induced T2D are unknown. We hypothesized that the detrimental impact of tamoxifen on insulin sensitivity was mediated by changes in the liver and skeletal muscle, and was exacerbated by high fat diet (HFD). Ovariectomized female mice supplemented with estradiol were randomized to low fat diet (LFD), LFD + tamoxifen, HFD, or HFD + tamoxifen. In general, our preclinical model recapitulated the observations from epidemiological studies on breast cancer survivors. HFD + tamoxifen mice had greater body fat percentage and fasting glucose (P &amp;lt; 0.02 for the HFD-Tamoxifen interaction). The HFD + tamoxifen mice developed insulin resistance determined by HOMA-IR (P &amp;lt; 0.01) and tended towards impaired glucose tolerance measured by glucose AUC from a glucose tolerance test (P = 0.1). In the liver of HFD-fed mice, tamoxifen resulted in greater accumulation of triacylglycerol (P = 0.01) which correlated positively with HOMA-IR (R2= 0.4; P &amp;lt; 0.001). Gastrocnemius citrate synthase activity was elevated with tamoxifen treatment independent of diet, as was phosphorylation of AMP-activated kinase (P &amp;lt; 0.05). HFD + tamoxifen mice had lower content of muscle mitochondrial complex II (P = 0.05) and complex V (P = 0.1). These data suggest that the potential mechanisms underlying tamoxifen-induced T2D is ectopic hepatic fat accumulation and lowered skeletal muscle mitochondrial content. Importantly, this study reveals a difference in response to tamoxifen between lean and obese females. Future studies will explore interventions that can be combined with tamoxifen to prevent hepatosteatosis and protect skeletal muscle mitochondria with the goal of reducing T2D in breast cancer survivors. Disclosure R.L. Scalzo: None. S.E. Hull: None. J.E.B. Reusch: Board Member; Self; American Diabetes Association. Other Relationship; Self; Merck &amp; Co., Inc. P. MacLean: Advisory Panel; Self; Habit LLC. E. Wellberg: None. Funding Susan G. Komen Foundation; National Institutes of Health

Transmission electron microscopic analysis of mitochondria changes in pulmonary arterial hypertension

Mitochondria are dynamic organelles, which play crucial roles in many cellular processes. Their severe damage results in alterations of other ultrastructural components and leads to loss of cellular function. In pulmonary hypertension (PH), increasing of the pulmonary vascular resistance puts the strain on the right ventricle, leading to the demand for ATP production by mitochondria to ensure cardiomyocyte function. The failure of cardiac pump increases the peripheral resistance by inducing the neurohormonal activation and influences the skeletal muscle structure and function, leading to the muscle weakness. In a rat PH model, our previous morphometric study reported changes in the mitochondrial types. Type I–II mitochondria are defined as mitochondria with the preserved ultrastructure and Type III–IV mitochondria are defined as mitochondria with the severe cristae damage that are not able to produce the required amount of ATP for energy‐depended processes in cardiomyocytes. Fischer rats had 100% mortality because of right heart failure in PH. In their RV cardiomyocytes, the types III–IV of mitochondria were predominant, along with a drastic decrease in coefficient of energy efficiency of mitochondria (CEEM), while in LV types II–III of mitochondria were present. Whereas the SD rats that do not die of PH, mostly had mitochondria Type I–II in RV and LV, along with slight decrease in CEEM in RV. In both strains of rats with PH, the treatment with a Bcl‐2 inhibitor, Navitoclax (ABT‐263), lead to severe damage of the mitochondria structure, causing rats to die because of hearth failure. The transmission electron microscopy (TEM) analysis of mitochondria in skeletal muscle cells in PH revealed the decrease in mitochondria volume and the damage to the mitochondria structure. In skeletal muscle cells, Type III mitochondria were present in rats, which correlates with the reduction in aerobic capacities. These results suggest that the ultrastructural defects in mitochondria in both cardiac and skeletal muscle in PH impact the development of the hearth failure and death, thus PH can be described as a systemic disease.Support or Funding InformationNIHThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Uremic Toxins Decrease Skeletal Muscle Mitochondrial Energy Transfer Through Disruption of the Electron Transport System

Chronic kidney disease (CKD) impacts more than 25 million Americans and is associated with higher risk of all‐cause and cardiovascular mortality. Impaired kidney function leads to retention of metabolic waste products, termed uremic toxins, that negatively impact skeletal muscle resulting in increased fatigue, weakness, and muscle atrophy. Previous evidence has implicated mitochondria within the skeletal muscle as a primary mediator of muscle dysfunction in CKD, yet the underlying mechanisms are unknown. Therefore, the purpose of this study was to define the impact of uremic toxins on mitochondrial energetics. Skeletal muscle mitochondria were isolated from healthy C57BL/6N mice and exposed to vehicle (DMSO) or varying doses of the following uremic toxins: indoxyl sulfate, indole‐3‐acetic‐acid, L‐kynurenine, kynurenic acid, and methylguanidine. We employed a comprehensive mitochondrial phenotyping platform that included assessments of mitochondrial OXPHOS conductance across several levels of energy demand, hydrogen peroxide production (JH2O2), and dehydrogenase flux (using NADH autofluorescence). Exposure to uremic toxins resulted in a dose‐dependent decrease in OXPHOS conductance for all toxins, with 100nM exposure resulting in an average decrease of ~22% supported by pyruvate/malate (all P&lt;0.05, n= 5–6/group). Uremic toxins did not decrease pyruvate dehydrogenase activity even at millimolar concentrations (all P&gt;0.64), suggesting the decreased OXPHOS conductance occurs downstream of matrix dehydrogenases. Consistent with decreased OXPHOS conductance, uremic toxins dose‐dependently increased JH2O2 by 2–5‐fold (all P&lt;0.01, n=4/group). These findings provide direct evidence that uremic toxins negatively impact skeletal muscle mitochondrial energetics, resulting in decreased energy transfer. Impaired mitochondrial energetics appears to be mediated downstream of matrix dehydrogenases, through either direct interaction within the electron transport system or ATP synthase.Support or Funding InformationPartially supported by AHA Grant 18CDA34110044 to TERThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Molecular Toxicological Mechanisms of Synthetic Cathinones on C2C12 Myoblasts.

Synthetic cathinones are popular psychoactive substances that may cause skeletal muscle damage. In addition to indirect sympathomimetic myotoxicity, these substances could be directly myotoxic. Since studies in myocytes are currently lacking, the aim of the present study was to investigate potential toxicological effects by synthetic cathinones on C2C12 myoblasts (mouse skeletal muscle cell line). We exposed C2C12 myoblasts to 3-methylmethcathinone, 4-methylmethcathinone (mephedrone), 3,4-methylenedioxymethcathinone (methylone), 3,4-methylenedioxypyrovalerone (MDPV), alpha-pyrrolidinovalerophenone (α-PVP), and naphthylpyrovalerone (naphyrone) for 1 or 24 h before cell membrane integrity, ATP content, mitochondrial oxygen consumption, and mitochondrial superoxide production was measured. 3,4-Methylenedioxymethamphetamine (MDMA) was included as a reference compound. All investigated synthetic cathinones, as well as MDMA, impaired cell membrane integrity, depleted ATP levels, and increased mitochondrial superoxide concentrations in a concentration-dependent manner in the range of 50–2000 μM. The two pyrovalerone derivatives α-PVP and naphyrone, and MDMA, additionally impaired basal and maximal cellular respiration, suggesting mitochondrial dysfunction. Alpha-PVP inhibited complex I, naphyrone complex II, and MDMA complex I and III, whereas complex IV was not affected. We conclude that, in addition to sympathetic nervous system effects and strenuous muscle exercise, direct effects of some cathinones on skeletal muscle mitochondria may contribute to myotoxicity in susceptible synthetic cathinone drugs users.

3540 Effects of Local Interleukin-6 on Mitochondrial Physiology in Skeletal Muscle

OBJECTIVES/SPECIFIC AIMS: In the context of skeletal muscle, IL-6 plays a major role in muscle quality. The goal of this project was to study the influence of systemic IL-6 on skeletal muscle mitochondrial physiology, most notably mitochondrial function (respiration and ROS production) and mitochondrial content. METHODS/STUDY POPULATION: To determine the influence of interleukin-6 (IL-6) on skeletal muscle mitochondria, high-resolution respirometry was performed to simultaneously measure oxygen consumption (JO2) and ROS production in differentiated myotubes incubated with increasing IL-6 (0, 10, 50, 100 ng/mL) for 18 hours in serum free conditions. To evaluate the impact of IL-6 on mitochondrial content we performed western blots on cell lysates from treated cells, measuring proteins of the mitochondrial electron transport chain (ETC) using a cocktail antibody and PGC-1α/PGC-1ß for mitochondrial biogenesis. To determine the role of mitochondrial ROS production on JO2 and mitochondrial content, we co-treated differentiated myotubes for 18 hours with 50 and 100ng/mL IL-6 and the mitochondrial specific antioxidant, MitoQ and performed respirometry for mitochondrial functional measurements and western blots for mitochondrial content.Statistical significance was evaluated by using a 2-tailed Student’s t-test and two-way ANOVA. Post hoc all-group analyses were conducted to determine which groups were different when the model was significant. RESULTS/ANTICIPATED RESULTS: Mitochondrial functional measurements show increased JO2 and increased ROS production in an IL-6 dose-dependent manner. Targeting mitochondrial ROS production with 0.5µm MitoQ attenuated IL-6 induced increases in JO2 and ROS production. Complexes I and II (CI, CII) of the ETC increased significantly in an IL-6 dose-wise fashion, and co-treatment with MitoQ normalized increases at 100ng/mL Il-6. 100ng/mL IL-6 significantly increased protein expression of PGC-1α and PGC-1ß. Co-treatment with MitoQ normalized IL-6 induced increase in PGC-1α. DISCUSSION/SIGNIFICANCE OF IMPACT: Our data suggest that when treated chronically at a high dose, IL-6 increases mitochondrial respiration, ROS production, and content. Targeting mitochondrial ROS production normalizes these mitochondrial adaptations. The present study provides new insights into mitochondrial physiology in the context of inflammation. Therapeutically targeting mitochondrial ROS production may impact skeletal muscle quality in certain populations.

TIGAR regulates mitochondrial functions through SIRT1-PGC1α pathway and translocation of TIGAR into mitochondria in skeletal muscle.

TP53-induced glycolysis and apoptosis regulator (TIGAR), a glycolytic inhibitor, plays vital roles in regulating cellular metabolism and oxidative stress. However, the role of highly expressed TIGAR in skeletal muscle remains unexplored. In the present study, TIGAR levels varied in different skeletal muscles and fibers. An exhaustive swimming test with a load corresponding to 5% of body weight was utilized in mice to assess the effects of TIGAR on exercise-induced fatigue and muscle damage. The running time and metabolic indicators were significantly greater in wild-type (WT) mice compared with TIGAR knockout (KO) mice. Poor exercise capacity was accompanied by decreased type IIA fibers in TIGAR KO mice. Decreased mitochondrial number and mitochondrial oxidative phosphorylation were observed more in TIGAR KO mice than in WT mice, which were involved in sirtuin 1 (SIRT1)-mediated deacetylation of peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α), and resveratrol treatment in TIGAR KO mice can increase mitochondrial content and exercise time. Much more TIGAR was also detected in mitochondria during exhaustive exercise. In addition, TIGAR, rather than mitochondria-targeted TIGAR achieved by in vitro plasmid transfection, promoted SIRT1-PGC1α pathway. Glutathione S-transferase-TIGAR pull-down assay followed by liquid chromatography mass spectrometry found that TIGAR interacted with ATP synthase F1 subunit α (ATP5A1), and its binding to ATP5A1 increased during exhaustive exercise. Overexpression of mitochondrial-TIGAR enhanced ATP generation, maintained mitochondrial membrane potential and reduced mitochondrial oxidative stress under hypoxia condition. Taken together, our results uncovered a novel role for TIGAR in mitochondrial regulation in fast-twitch oxidative skeletal muscle through SIRT1-PGC1α and translocation into mitochondria, which contribute to the increase in exercise endurance of mice.-Geng, J., Wei, M., Yuan, X., Liu, Z., Wang, X., Zhang, D., Luo, L., Wu, J., Guo, W., Qin, Z.-H. TIGAR regulates mitochondrial functions through SIRT1-PGC1α pathway and translocation of TIGAR into mitochondria in skeletal muscle.

Bufei Jianpi Granules Reduce Quadriceps Muscular Cell Apoptosis by Improving Mitochondrial Function in Rats with Chronic Obstructive Pulmonary Disease.

Background Cell apoptosis is an important mechanism underlying skeletal muscle dysfunction in chronic obstructive pulmonary disease (COPD) patients, and mitochondrial dysfunction is recognized as a central aspect contributing to skeletal muscle deterioration. Bufei Jianpi granules have been confirmed effective for improving motor function in COPD patients, but the specific mechanism for this improved function remains unknown. This study explored the mechanisms by which Bufei Jianpi granules improve cell apoptosis and mitochondrial dysfunction in COPD. Methods Sprague-Dawley rats were randomized into control, model, Bufei Jianpi, and aminophylline groups. A stable COPD rat model was induced with respective repeated cigarette smoke inhalation and intragastric bacterial infection, and rats were sacrificed after 20 weeks; the quadriceps muscle was harvested from each rat. Skeletal muscle mitochondria were extracted for measurements of mitochondrial membrane potential (MMP) and mitochondrial permeability transition pore openings (mPTPs). ATP levels were determined with a firefly luciferase-based ATP assay kit. The rates of cell apoptosis were determined by the transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) method. Cyto C and caspase-3 mRNA and protein levels were measured by qPCR and western blotting. Results ATP, MMP, and mPTPs were markedly decreased in COPD rats, while cell apoptosis, caspase-3, and Cyto C were increased (P<0.01). All aforementioned parameters were improved in treatment groups (P<0.05). ATP, MMP, and mPTPs were significantly higher in the Bufei Jianpi group than in the aminophylline group, while cell apoptosis, caspase-3, and Cyto C were lower (P<0.05). Conclusions Bufei Jianpi granules can inhibit mitochondrial dysfunction and cell apoptosis in peripheral muscles, which might be the mechanism involved in improving skeletal muscle function in COPD patients.

Mitochondrial health and muscle plasticity after spinal cord injury.

Mitochondria are responsible for aerobic respiration and large-scale ATP production in almost all cells of the body. Their function is decreased in many neurodegenerative and cardiovascular disease states, in metabolic disorders such as type II diabetes and obesity, and as a normal component of aging. Disuse of skeletal muscle from immobilization or unloading triggers alterations of mitochondrial density and activity. Resultant mitochondrial dysfunction after paralysis, which precedes muscle atrophy, may augment subsequent release of reactive oxygen species leading to protein ubiquitination and degradation. Spinal cord injury is a unique form of disuse atrophy as there is a complete or partial disruption in tonic communication between the central nervous system (CNS) and skeletal muscle. Paralysis, unloading and disruption of CNS communication result in a rapid decline in skeletal muscle function and metabolic status with disruption in activity of peroxisome-proliferator-activated receptor-gamma co-activator 1 alpha and calcineurin, key regulators of mitochondrial health and function. External interventions, both acute and chronical with training using body-weight-assisted treadmill training or electrical stimulation have consistently demonstrated adaptations in skeletal muscle mitochondria, and expression of the genes and proteins required for mitochondrial oxidation of fats and carbohydrates to ATP, water, and carbon dioxide. The purpose of this mini-review is to highlight our current understanding as to how paralysis mechanistically triggers downstream regulation in mitochondrial density and activity and to discuss how mitochondrial dysfunction may contribute to skeletal muscle atrophy.

Acute High-Intensity Exercise Impairs Skeletal Muscle Respiratory Capacity.

The effect of an acute bout of exercise, especially high-intensity exercise, on the function of mitochondrial respiratory complexes is not well understood, with potential implications for both the healthy population and patients undergoing exercise-based rehabilitation. Therefore, this study sought to comprehensively examine respiratory flux through the different complexes of the electron transport chain in skeletal muscle mitochondria before and immediately after high-intensity aerobic exercise. Muscle biopsies of the vastus lateralis were obtained at baseline and immediately after a 5-km time trial performed on a cycle ergometer. Mitochondrial respiratory flux through the complexes of the electron transport chain was measured in permeabilized skeletal muscle fibers by high-resolution respirometry. Complex I + II state 3 (state 3CI + CII) respiration, a measure of oxidative phosphorylation capacity, was diminished immediately after the exercise (pre, 27 ± 3 ρm·mg·s; post, 17 ± 2 ρm·mg·s; P < 0.05). This decreased oxidative phosphorylation capacity was predominantly the consequence of attenuated complex II-driven state 3 (state 3CII) respiration (pre, 17 ± 1 ρm·mg·s; post, 9 ± 2 ρm·mg·s; P < 0.05). Although complex I-driven state 3 (3CI) respiration was also lower (pre, 20 ± 2 ρm·mg·s; post, 14 ± 4 ρm·mg·s), this did not reach statistical significance (P = 0.27). In contrast, citrate synthase activity, proton leak (state 2 respiration), and complex IV capacity were not significantly altered immediately after the exercise. These findings reveal that acute high-intensity aerobic exercise significantly inhibits skeletal muscle state 3CII and oxidative phosphorylation capacity. This, likely transient, mitochondrial defect might amplify the exercise-induced development of fatigue and play an important role in initiating exercise-induced mitochondrial adaptations.

Loss of the Mia40a oxidoreductase leads to hepato-pancreatic insufficiency in zebrafish.

Development and function of tissues and organs are powered by the activity of mitochondria. In humans, inherited genetic mutations that lead to progressive mitochondrial pathology often manifest during infancy and can lead to death, reflecting the indispensable nature of mitochondrial biogenesis and function. Here, we describe a zebrafish mutant for the gene mia40a (chchd4a), the life-essential homologue of the evolutionarily conserved Mia40 oxidoreductase which drives the biogenesis of cysteine-rich mitochondrial proteins. We report that mia40a mutant animals undergo progressive cellular respiration defects and develop enlarged mitochondria in skeletal muscles before their ultimate death at the larval stage. We generated a deep transcriptomic and proteomic resource that allowed us to identify abnormalities in the development and physiology of endodermal organs, in particular the liver and pancreas. We identify the acinar cells of the exocrine pancreas to be severely affected by mutations in the MIA pathway. Our data contribute to a better understanding of the molecular, cellular and organismal effects of mitochondrial deficiency, important for the accurate diagnosis and future treatment strategies of mitochondrial diseases.

Mitochondrial dysfunction and inhibition of myoblast differentiation in mice with high-fat-diet-induced pre-diabetes.

Pre-diabetes is characterized by impaired glucose tolerance (IGT) and/or impaired fasting glucose. Impairment of skeletal muscle function is closely associated with the progression of diabetes. However, the entire pathological characteristics and mechanisms of pre-diabetes in skeletal muscle remain fully unknown. Here, we established a mouse model of pre-diabetes, in which 6-week-old male C57BL6/J mice were fed either normal diet or high-fat diet (HFD) for 8 or 16 weeks. Both non-fasting and fasting glucose levels and the results of glucose and insulin tolerance tests showed that mice fed an 8-week HFD developed pre-diabetes with IGT; whereas mice fed a 16-week HFD presented with impaired fasting glucose and impaired glucose tolerance (IFG-IGT). Mice at both stages of pre-diabetes displayed decreased numbers of mitochondria in skeletal muscle. Moreover, IFG-IGT mice exhibited decreased mitochondrial membrane potential and ATP production in skeletal muscle and muscle degeneration characterized by a shift in muscle fibers from predominantly oxidative type I to glycolytic type II. Western blotting and histological analysis confirmed that myoblast differentiation was only inhibited in IFG-IGT mice. For primary skeletal muscle satellite cells, inhibition of differentiation was observed in palmitic acid-induced insulin resistance model. Moreover, enhanced myoblast differentiation increased glucose uptake and insulin sensitivity. These findings indicate that pre-diabetes result in mitochondrial dysfunction and inhibition of myoblast differentiation in skeletal muscle. Therefore, interventions that enhance myoblast differentiation may improve insulin resistance of diabetes at the earlier stage.