Markers Of Mitochondrial Content Research Articles

Time-dependent changes in autophagy, mitophagy and lysosomes in skeletal muscle during denervation-induced disuse.

Deficits in skeletal muscle mitochondrial content and quality are observed following denervation-atrophy. This is due to alterations in the biogenesis of new mitochondria as well as their degradation via mitophagy. The regulation of autophagy and mitophagy over the course of denervation (Den) remains unknown. Further, the time-dependent changes in lysosome content, the end-stage organelle for mitophagy, remain unexplored. Here, we studied autophagic as well as mitophagic pre-lysosomal flux in subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria from rat muscle subjected to Den for 1, 3 or 7days. We also assessed flux at 1day post-denervation in transgenic mt-keima mice. Markers of mitochondrial content were reduced at 7days following Den, and Den further resulted in rapid decrements in mitochondrial respiration, along with increased ROS emission. Pre-lysosomal autophagy flux was upregulated at 1 and 3days post-Den but was reduced compared to time-matched sham-operated controls at 7days post-Den. Similarly, pre-lysosomal mitophagy flux was enhanced in SS mitochondria as early as 1 and 3days of Den but decreased in both SS and IMF subfractions following 7days of Den. Lysosome protein content and transcriptional regulators TFEB and TFE3 were progressively enhanced with Den, an adaptation designed to enhance autophagic capacity. However, evidence for lysosome dysfunction was apparent by 7days, which may limit degradation capacity. This may contribute to an inability to clear dysfunctional mitochondria and increased ROS signalling, thereby accelerating muscle atrophy. Thus, therapeutic targeting of lysosome function may help to maintain autophagy and muscle health during conditions of muscle disuse or denervation. KEY POINTS: Denervation is an experimental model of peripheral neuropathies as well as muscle disuse, and it helps us understand some aspects of the sarcopenia of ageing. Muscle disuse is associated with reduced mitochondrial content and function, leading to metabolic impairments within the tissue. Although the processes that regulate mitochondrial biogenesis are understood, those that govern mitochondrial breakdown (i.e. mitophagy) are not well characterized in this context. Autophagy and mitophagy flux, measured up to the point of the lysosome (pre-lysosomal flux rates), were increased in the early stages of denervation, along with mitochondrial dysfunction, but were reduced at later time points when the degree of muscle atrophy was highest. Denervation led to progressive increases in lysosomal proteins to accommodate mitophagy flux, yet evidence for lysosomal impairment at later stages may limit the removal of dysfunctional mitochondria, stimulate reactive oxygen species signalling, and reduce muscle health as denervation time progresses.

Measurement of Protein Import Capacity of Skeletal Muscle Mitochondria.

Mitochondria are key metabolic and regulatory organelles that determine the energy supply as well as the overall health of the cell. In skeletal muscle, mitochondria exist in a series of complex morphologies, ranging from small oval organelles to a broad, reticulum-like network. Understanding how the mitochondrial reticulum expands and develops in response to diverse stimuli such as alterations in energy demand has long been a topic of research. A key aspect of this growth, or biogenesis, is the import of precursor proteins, originally encoded by the nuclear genome, synthesized in the cytosol, and translocated into various mitochondrial sub-compartments. Mitochondria have developed a sophisticated mechanism for this import process, involving many selective inner and outer membrane channels, known as the protein import machinery (PIM). Import into the mitochondrion is dependent on viable membrane potential and the availability of organelle-derived ATP through oxidative phosphorylation. Therefore its measurement can serve as a measure of organelle health. The PIM also exhibits a high level of adaptive plasticity in skeletal muscle that is tightly coupled to the energy status of the cell. For example, exercise training has been shown to increase import capacity, while muscle disuse reduces it, coincident with changes in markers of mitochondrial content. Although protein import is a critical step in the biogenesis and expansion of mitochondria, the process is not widely studied in skeletal muscle. Thus, this paper outlines how to use isolated and fully functional mitochondria from skeletal muscle to measure protein import capacity in order to promote a greater understanding of the methods involved and an appreciation of the importance of the pathway for organelle turnover in exercise, health, and disease.

Influence of Immune Cell Subtypes on Mitochondrial Measurements in Peripheral Blood Mononuclear Cells From Children with Sepsis.

Peripheral blood mononuclear cells (PBMCs) are commonly used to compare mitochondrial function in patients with versus without sepsis, but how these measurements in this mixed cell population vary by composition of immune cell subtypes is not known, especially in children. We determined the effect of changing immune cell composition on PBMC mitochondrial respiration and content in children with and without sepsis. PBMC mitochondrial respiration and citrate synthase (CS) activity, a marker of mitochondrial content, were measured in 167 children with sepsis at three timepoints (day 1-2, 3-5, and 8-14) and once in 19 nonseptic controls. The proportion of lymphocytes and monocytes and T, B, and NK cells was measured using flow cytometry. More specific CD4+ and CD8+ T cell subsets were measured from 13 sepsis patients and 6 controls. Spearman's correlation and simple and mixed effects linear regression were used to determine the association of PBMC mitochondrial measures with proportion of immune cell subtypes. PBMC mitochondrial respiration and CS activity were correlated with proportion of monocytes, lymphocytes, T B, and NK cells in controls, but not in sepsis patients. PBMC mitochondrial respiration was correlated with CD4+ and CD8+ T cell subsets in both groups. After controlling for differences in immune cell composition between groups using linear regression models, PBMC respiration and CS activity remained lower in sepsis patients than controls. Mitochondrial measurements from PBMCs varied with changes in immune cell composition in children with and without sepsis. However, differences in PBMC mitochondrial measurements between sepsis patients and controls were at least partially attributable to the effects of sepsis rather than solely an epiphenomena of variable immune cell composition.

Blood-based mitochondrial respiratory chain function in major depression

Mitochondrial dysfunction has been implicated in major depressive disorder (MDD). A measure of mitochondrial respiratory chain (RC) enzymatic activity—the Mitochondrial Health Index (MHI)—has previously been found to correlate with stress and emotional states in caregivers. We here report mitochondrial RC activities, mitochondrial DNA copy number (mtDNAcn), and the composite MHI in unmedicated and somatically healthy subjects with MDD (n = 47) and healthy controls (HC) (n = 11). We also explore, in a subset of the MDD sample (n = 33), whether these markers are associated with response to 8 weeks of SSRI treatment. Mitochondrial RC complexes I, II, IV, citrate synthase (CS), mtDNAcn, and the MHI were assayed in peripheral blood mononuclear cells. Treatment response was defined as >50% decrease on the 25-item Hamilton Depression Rating Scale (HRDS-25). There were no significant differences in MHI or any of the mitochondrial markers between MDD subjects and HCs. Compared to SSRI nonresponders, SSRI responders had significantly higher baseline mitochondrial content markers CS (p = 0.02) and mtDNAcn (p = 0.02), and higher complex I activity (p = 0.01). Complex II activity increased significantly over treatment, irrespective of clinical response (p = 0.03). Complex I activity decreased in responders (n = 9), but increased in nonresponders (n = 18) (group x time interaction, p = 0.02). Absolute treatment-associated change in HDRS-25 scores correlated significantly with change in complex I activity between baseline and week 8 (r = 0.47, p = 0.01). Although mitochondrial markers did not distinguish MDD from controls, they did distinguish SSRI responders from nonresponders. If larger studies validate these mitochondrial differences, they may become useful biomarkers and identify new drug targets.

Glucose 6-P dehydrogenase delays the onset of frailty by protecting against muscle damage.

BackgroundFrailty is a major age‐associated syndrome leading to disability. Oxidative damage plays a significant role in the promotion of frailty. The cellular antioxidant system relies on reduced nicotinamide adenine dinucleotide phosphate (NADPH) that is highly dependent on glucose 6‐P dehydrogenase (G6PD). The G6PD‐overexpressing mouse (G6PD‐Tg) is protected against metabolic stresses. Our aim was to examine whether this protection delays frailty.MethodsOld wild‐type (WT) and G6PD‐Tg mice were evaluated longitudinally in terms of frailty. Indirect calorimetry, transcriptomic profile, and different skeletal muscle quality markers and muscle regenerative capacity were also investigated.ResultsThe percentage of frail mice was significantly lower in the G6PD‐Tg than in the WT genotype, especially in 26‐month‐old mice where 50% of the WT were frail vs. only 13% of the Tg ones (P < 0.001). Skeletal muscle transcriptomic analysis showed an up‐regulation of respiratory chain and oxidative phosphorylation (P = 0.009) as well as glutathione metabolism (P = 0.035) pathways in the G6PD‐Tg mice. Accordingly, the Tg animals exhibited an increase in reduced glutathione (34.5%, P < 0.01) and a decrease on its oxidized form (−69%, P < 0.05) and in lipid peroxidation (4‐HNE: −20.5%, P < 0.05). The G6PD‐Tg mice also showed reduced apoptosis (BAX/Bcl2: −25.5%, P < 0.05; and Bcl‐xL: −20.5%, P < 0.05), lower levels of the intramuscular adipocyte marker FABP4 (−54.7%, P < 0.05), and increased markers of mitochondrial content (COX IV: 89.7%, P < 0.05; Grp75: 37.8%, P < 0.05) and mitochondrial OXPHOS complexes (CII: 81.25%, P < 0.01; CIII: 52.5%, P < 0.01; and CV: 37.2%, P < 0.05). Energy expenditure (−4.29%, P < 0.001) and the respiratory exchange ratio were lower (−13.4%, P < 0.0001) while the locomotor activity was higher (43.4%, P < 0.0001) in the 20‐month‐old Tg, indicating a major energetic advantage in these mice. Short‐term exercise training in young C57BL76J mice induced a robust activation of G6PD in skeletal muscle (203.4%, P < 0.05), similar to that achieved in the G6PD‐Tg mice (142.3%, P < 0.01).ConclusionsGlucose 6‐P dehydrogenase deficiency can be an underestimated risk factor for several human pathologies and even frailty. By overexpressing G6PD, we provide the first molecular model of robustness. Because G6PD is regulated by pharmacological and physiological interventions like exercise, our results provide molecular bases for interventions that by increasing G6PD will delay the onset of frailty.

Fission accomplished: Uncovering the role of Drp1 in regulating mitochondrial dysfunction and age-related muscle atrophy.

Fission accomplished: Uncovering the role of Drp1 in regulating mitochondrial dysfunction and age-related muscle atrophy.

Voluntary exercise prevents abnormal muscle mitochondrial morphology in cancer cachexia mice.

This study aimed to examine the effects of voluntary wheel running on cancer cachexia‐induced mitochondrial alterations in mouse skeletal muscle. Mice bearing colon 26 adenocarcinoma (C26) were used as a model of cancer cachexia. C26 mice showed a lower gastrocnemius and plantaris muscle weight, but 4 weeks of voluntary exercise rescued these changes. Further, voluntary exercise attenuated observed declines in the levels of oxidative phosphorylation proteins and activities of citrate synthase and cytochrome c oxidase in the skeletal muscle of C26 mice. Among mitochondrial morphology regulatory proteins, mitofusin 2 (Mfn2) and dynamin‐related protein 1 (Drp1) were decreased in the skeletal muscle of C26 mice, but exercise resulted in similar improvements as seen in markers of mitochondrial content. In isolated mitochondria, 4‐hydroxynonenal and protein carbonyls were elevated in C26 mice, but exercise blunted the increases in these markers of oxidative stress. In addition, electron microscopy revealed that exercise alleviated the observed increase in the percentage of damaged mitochondria in C26 mice. These results suggest that voluntary exercise effectively counteracts mitochondrial dysfunction to mitigate muscle loss in cachexia.

AUF1 gene transfer increases exercise performance and improves skeletal muscle deficit in adult mice

Muscle function and mass begin declining in adults long before evidence of sarcopenia and include reduced mitochondrial function, although much remains to be characterized. We found that mRNA decay factor AU-rich mRNA binding factor 1 (AUF1), which stimulates myogenesis, is strongly reduced in skeletal muscle of adult and older mice in the absence of evidence of sarcopenia. Muscle-specific adeno-associated virus (AAV)8-AUF1 gene therapy increased expression of AUF1, muscle function, and mass. AAV8 AUF1 muscle gene transfer in 12-month-old mice increased the levels of activated muscle stem (satellite) cells, increased muscle mass, reduced markers of muscle atrophy, increased markers of mitochondrial content and muscle fiber oxidative capacity, and enhanced exercise performance to levels of 3-month-old mice. With wild-type and AUF1 knockout mice and cultured myoblasts, AUF1 supplementation of muscle fibers was found to increase expression of Peroxisome Proliferator-activated Receptor Gamma Co-activator 1-alpha (PGC1α), a major effector of skeletal muscle mitochondrial oxidative metabolism. AUF1 stabilized and increased translation of the pgc1α mRNA, which is strongly reduced in adult muscle in the absence of AUF1 supplementation. Skeletal muscle-specific gene transfer of AUF1 therefore restores muscle mass, increases exercise endurance, and may provide a therapeutic strategy for age-related muscle loss.

A 1-Month Ketogenic Diet Increased Mitochondrial Mass in Red Gastrocnemius Muscle, but Not in the Brain or Liver of Middle-Aged Mice.

Alterations in markers of mitochondrial content with ketogenic diets (KD) have been reported in tissues of rodents, but morphological quantification of mitochondrial mass using transmission electron microscopy (TEM), the gold standard for mitochondrial quantification, is needed to further validate these findings and look at specific regions of interest within a tissue. In this study, red gastrocnemius muscle, the prefrontal cortex, the hippocampus, and the liver left lobe were used to investigate the impact of a 1-month KD on mitochondrial content in healthy middle-aged mice. The results showed that in red gastrocnemius muscle, the fractional area of both subsarcolemmal (SSM) and intermyofibrillar (IMM) mitochondria was increased, and this was driven by an increase in the number of mitochondria. Mitochondrial fractional area or number was not altered in the liver, prefrontal cortex, or hippocampus following 1 month of a KD. These results demonstrate tissue-specific changes in mitochondrial mass with a short-term KD and highlight the need to study different muscle groups or tissue regions with TEM to thoroughly determine the effects of a KD on mitochondrial mass.

Mitochondria in epithelial ovarian carcinoma exhibit abnormal phenotypes and blunted associations with biobehavioral factors

Malignant tumor cells exhibit mitochondrial alterations and are also influenced by biobehavioral processes, but the intersection of biobehavioral factors and mitochondria in malignant tumors remains unexplored. Here we examined multiple biochemical and molecular markers of mitochondrial content and function in benign tissue and in high-grade epithelial ovarian carcinoma (EOC) in parallel with exploratory analyses of biobehavioral factors. First, analysis of a publicly-available database (n = 1435) showed that gene expression of specific mitochondrial proteins in EOC is associated with survival. Quantifying multiple biochemical and molecular markers of mitochondrial content and function in tissue from 51 patients with benign ovarian masses and 128 patients with high-grade EOC revealed that compared to benign tissue, EOCs exhibit 3.3–8.4-fold higher mitochondrial content and respiratory chain enzymatic activities (P < 0.001) but similar mitochondrial DNA (mtDNA) levels (− 3.1%), documenting abnormal mitochondrial phenotypes in EOC. Mitochondrial respiratory chain activity was also associated with interleukin-6 (IL-6) levels in ascites. In benign tissue, negative biobehavioral factors were inversely correlated with mitochondrial content and respiratory chain activities, whereas positive biobehavioral factors tended to be positively correlated with mitochondrial measures, although effect sizes were small to medium (r = − 0.43 to 0.47). In contrast, serous EOCs showed less pronounced biobehavioral-mitochondrial correlations. These results document abnormal mitochondrial functional phenotypes in EOC and warrant further research on the link between biobehavioral factors and mitochondria in cancer.

A single session of high‐intensity interval exercise in hypoxia did not change mitochondrial respiration or content in human skeletal muscle biopsies

Both high-intensity interval exercise (HIIE) and hypoxia have been suggested to affect mitochondria. However, it is not yet fully elucidated how a single session of HIIE in hypoxia affects mitochondrial respiration and content in human skeletal muscle. In this current study, we investigated the effects of a single session of HIIE combined with simulated hypoxia (3200 m, oxygen fraction of 14%) on mitochondrial respiration and content. We hypothesized that HIIE in hypoxia would induce substantial changes in mitochondrial respiration, but not in mitochondrial content. Ten healthy male participants (age, 28 ± 5 years; BMI, 26.0 ± 3.4) were recruited and completed three HIIE sessions (six 4-minute exercise bouts, separated by 2 minutes of resting) in a random order, separated by at least one week. Exercise sessions include an HIIE in hypoxia (88.4% of peak oxygen uptake (VO2peak) determined in hypoxia), an HIIE in normoxia matched for relative intensity (88.7% of VO2peak determined in normoxia), and an HIIE in normoxia matched for absolute intensity (74.1% of VO2peak determined in normoxia). Skeletal muscle samples were collected prior to, 3 hours, and 24 hours post-exercise to determine mitochondrial respiration and content. Hypoxia decreased VO2peak (by 20.2 ± 9.1%, p < 0.01), peak power output (PPO, by 9.4 ± 2.1%, p<0.01), and lactate threshold (LT, by 13.1 ± 3.0%, p<0.01). In the skeletal muscle biopsies, mitochondrial respiration, measured by Oroboros O2k with high-resolution respirometry, was similar among the three conditions prior to the HIIE interventions (p > 0.05) and remained unchanged both 3 and 24 hours after HIIE interventions performed in different conditions (p > 0.05). Citrate synthase activity, as a marker of mitochondrial content, did not differ prior to, or 3 and 24 hours after HIIE interventions among the three conditions (p > 0.05) . In conclusion, a single session of HIIE in hypoxia did not alter mitochondrial respiration and content. Gene expression and protein content of mitochondrial biogenesis markers are currently being analyzed.

Human skeletal muscle fiber type-specific responses to sprint interval and moderate-intensity continuous exercise: acute and training-induced changes

There are limited and equivocal data regarding potential fiber type-specific differences in the human skeletal muscle response to sprint interval training (SIT), including how this compares to moderate-intensity continuous training (MICT). We examined mixed muscle and fiber type-specific responses to a single session (study 1) and 12 wk (study 2) of MICT and SIT using Western blotting. MICT consisted of 45 min of cycling at ~70% of maximal heart rate and SIT involved 3 x 20-sec 'all-out' sprints interspersed with 2 min of recovery. Changes in signaling proteins involved in mitochondrial biogenesis in mixed muscle and pooled fiber samples were similar after acute MICT and SIT. This included increases in the ratios of phosphorylated to total acetyl CoA carboxylase and p38 mitogen activated protein kinase protein content (main effects, p<0.05). Following training, mitochondrial content markers including the protein content of cytochrome c oxidase subunit IV and NADH:ubiquinone oxidoreductase subunit A9 were increased similarly in mixed muscle and type IIa fibers (main effects, p<0.05). In contrast, only MICT increased these markers of mitochondrial content in type I fibers (interactions, p<0.05). MICT and SIT also similarly increased the content of mitochondrial fusion proteins optic atrophy 1 (OPA1) and mitofusin 2 in mixed muscle, and OPA1 in pooled fibre samples (main effects, p<0.05). In summary, acute MICT and SIT elicited similar fiber type-specific responses of signaling proteins involved in mitochondrial biogenesis, whereas 12 wk of training revealed differential responses of mitochondrial content markers in type I but not type IIa fibers.

Branched-chain amino acid metabolism, insulin sensitivity and liver fat response to exercise training in sedentary dysglycaemic and normoglycaemic men

Aims/hypothesisObesity and insulin resistance may be associated with elevated plasma concentration of branched-chain amino acids (BCAAs) and impaired BCAA metabolism. However, it is unknown whether the insulin-sensitising effect of long-term exercise can be explained by concomitant change in BCAAs and their metabolism.MethodsWe included 26 sedentary overweight and normal-weight middle-aged men from the MyoGlu clinical trial, with or without dysglycaemia, for 12 weeks of supervised intensive exercise intervention, including two endurance and two resistance sessions weekly. Insulin sensitivity was measured as the glucose infusion rate (GIR) from a hyperinsulinaemic−euglycaemic clamp. In addition, maximum oxygen uptake, upper and lower body strength and adipose tissue depots (using MRI and spectroscopy) were measured, and subcutaneous white adipose tissue (ScWAT) and skeletal muscle (SkM) biopsies were harvested both before and after the 12 week intervention. In the present study we have measured plasma BCAAs and related metabolites using CG-MS/MS and HPLC-MS/MS, and performed global mRNA-sequencing pathway analysis on ScWAT and SkM.ResultsIn MyoGlu, men with dysglycaemia displayed lower GIR, more fat mass and higher liver fat content than normoglycaemic men at baseline, and 12 weeks of exercise increased GIR, improved body composition and reduced liver fat content similarly for both groups. In our current study we observed higher plasma concentrations of BCAAs (14.4%, p = 0.01) and related metabolites, such as 3-hydroxyisobutyrate (19.4%, p = 0.034) in dysglycaemic vs normoglycaemic men at baseline. Baseline plasma BCAA levels correlated negatively to the change in GIR (ρ = −0.41, p = 0.037) and dot{V}{mathrm{O}}_{2max } (ρ = −0.47, p = 0.015) after 12 weeks of exercise and positively to amounts of intraperitoneal fat (ρ = 0.40, p = 0.044) and liver fat (ρ = 0.58, p = 0.01). However, circulating BCAAs and related metabolites did not respond to 12 weeks of exercise, with the exception of isoleucine, which increased in normoglycaemic men (10 μmol/l, p = 0.01). Pathway analyses of mRNA-sequencing data implied reduced BCAA catabolism in both SkM and ScWAT in men with dysglycaemia compared with men with normoglycaemia at baseline. Gene expression levels related to BCAA metabolism correlated positively with GIR and markers of mitochondrial content in both SkM and ScWAT, and negatively with fat mass generally, and particularly with intraperitoneal fat mass. mRNA-sequencing pathway analysis also implied increased BCAA metabolism after 12 weeks of exercise in both groups and in both tissues, including enhanced expression of the gene encoding branched-chain α-ketoacid dehydrogenase (BCKDH) and reduced expression of the BCKDH phosphatase in both groups and tissues. Gene expression of SLC25A44, which encodes a mitochondrial BCAA transporter, was increased in SkM in both groups, and gene expression of BCKDK, which encodes BCKDH kinase, was reduced in ScWAT in dysglycaemic men. Mediation analyses indicated a pronounced effect of enhanced SkM (~53%, p = 0.022), and a moderate effect of enhanced ScWAT (~18%, p = 0.018) BCAA metabolism on improved insulin sensitivity after 12 weeks of exercise, based on mRNA sequencing. In comparison, plasma concentration of BCAAs did not mediate any effect in this regard.Conclusion/interpretationPlasma BCAA concentration was largely unresponsive to long-term exercise and unrelated to exercise-induced insulin sensitivity. On the other hand, the insulin-sensitising effect of long-term exercise in men may be explained by enhanced SkM and, to a lesser degree, also by enhanced ScWAT BCAA catabolism.Graphical abstract

Mitochondrial Function and Protein Turnover in the Diaphragm are Altered in LLC Tumor Model of Cancer Cachexia.

It is established that cancer cachexia causes limb muscle atrophy and is strongly associated with morbidity and mortality; less is known about how the development of cachexia impacts the diaphragm. The purpose of this study was to investigate cellular signaling mechanisms related to mitochondrial function, reactive oxygen species (ROS) production, and protein synthesis during the development of cancer cachexia. C57BL/J6 mice developed Lewis Lung Carcinoma for either 0 weeks (Control), 1 week, 2 weeks, 3 weeks, or 4 weeks. At designated time points, diaphragms were harvested and analyzed. Mitochondrial respiratory control ratio was ~50% lower in experimental groups, which was significant by 2 weeks of cancer development, with no difference in mitochondrial content markers COXIV or VDAC. Compared to the controls, ROS was 4-fold elevated in 2-week animals but then was not different at later time points. Only one antioxidant protein, GPX3, was altered by cancer development (~70% lower in experimental groups). Protein synthesis, measured by a fractional synthesis rate, appeared to become progressively lower with the cancer duration, but the mean difference was not significant. The development and progression of cancer cachexia induces marked alterations to mitochondrial function and ROS production in the diaphragm and may contribute to increased cachexia-associated morbidity and mortality.

Effects of long-term intradialytic oral nutrition and exercise on muscle protein homeostasis and markers of mitochondrial content in patients on hemodialysis.

Patients with end-stage kidney disease on maintenance hemodialysis commonly develop protein-energy wasting, a syndrome characterized by nutritional and metabolic abnormalities. Nutritional supplementation and exercise are recommended to prevent protein-energy wasting. In a 6-mo prospective randomized, open-label, clinical trial, we reported that the combination of resistance exercise and nutritional supplementation does not have an additive effect on lean body mass measured by dual-energy X-ray absorptiometry. To provide more mechanistic data, we performed a secondary analysis where we hypothesized that the combination of nutritional supplementation and resistance exercise would have additive effects on muscle protein accretion by stable isotope protein kinetic experiments, muscle mass by MRI, and mitochondrial content markers in muscle. We found that 6 mo of nutritional supplementation during hemodialysis increased muscle protein net balance [baseline: 2.5 (-17.8, 13.0) µg·100 mL-1·min-1 vs. 6 mo: 43.7 (13.0, 98.5) µg·100 mL-1·min-1, median (interquartile range), P = 0.04] and mid-thigh fat area [baseline: 162.3 (104.7, 226.6) cm2 vs. 6 mo: 181.9 (126.3, 279.2) cm2, median (interquartile range), P = 0.04]. Three months of nutritional supplementation also increased markers of mitochondrial content in muscle. Although the study is underpowered to detected differences, the combination of nutritional supplementation and exercise failed to show further benefit in protein accretion or muscle cross-sectional area. We conclude that long-term nutritional supplementation increases the skeletal muscle anabolic effect, the fat cross-sectional area of the thigh, and markers of mitochondrial content in skeletal muscle.

Impact of aerobic exercise type on blood flow, muscle energy metabolism, and mitochondrial biogenesis in experimental lower extremity artery disease

Exercise training (ET) is recommended for lower extremity artery disease (LEAD) management. However, there is still little information on the hemodynamic and metabolic adaptations by skeletal muscle with ET. We examined whether hindlimb perfusion/vascularization and muscle energy metabolism are altered differently by three types of aerobic ET. ApoE−/− mice with LEAD were assigned to one of four groups for 4 weeks: sedentary (SED), forced treadmill running (FTR), voluntary wheel running (VWR), or forced swimming (FS). Voluntary exercise capacity was improved and equally as efficient with FTR and VWR, but remained unchanged with FS. Neither ischemic hindlimb perfusion and oxygenation, nor arteriolar density and mRNA expression of arteriogenic-related genes differed between groups. 18FDG PET imaging revealed no difference in the steady-state levels of phosphorylated 18FDG in ischemic and non-ischemic hindlimb muscle between groups, nor was glycogen content or mRNA and protein expression of glucose metabolism-related genes in ischemic muscle modified. mRNA (but not protein) expression of lipid metabolism-related genes was upregulated across all exercise groups, particularly by non-ischemic muscle. Markers of mitochondrial content (mitochondrial DNA content and citrate synthase activity) as well as mRNA expression of mitochondrial biogenesis-related genes in muscle were not increased with ET. Contrary to FTR and VWR, swimming was ineffective in improving voluntary exercise capacity. The underlying hindlimb hemodynamics or muscle energy metabolism are unable to explain the benefits of running exercise.

Cancer-induced Cardiac Atrophy Adversely Affects Myocardial Redox State and Mitochondrial Oxidative Characteristics.

Determine how cancer-induced cardiac atrophy alters mitochondrial turnover, mitochondrial mRNA translation machinery and in-vitro oxidative characteristics. Lewis lung carcinoma (LLC) tumors were implanted in C57BL6/J mice and grown for 28days to induce cardiac atrophy. Endogenous metabolic species, and markers of mitochondrial function were assessed. H9c2 cardiomyocytes were cultured in LLC-conditioned media with(out) the antioxidant MitoTempo. Cells were analyzed for ROS, oxidative capacity, and hypoxic resistance. LLC heart weights were ~10% lower than controls. LLC hearts demonstrated ~15% lower optical redox ratio (FAD/FAD+NADH) compared to PBS controls. When compared to PBS, LLC hearts showed ~50% greater COX-IV and VDAC, attributed to ~50% lower mitophagy markers. mt-mRNA translation machinery was elevated similarly to markers of mitochondrial content. mitochondrial DNA-encoded Cytb was ~30% lower in LLC hearts. ROS scavengers GPx-3 and GPx-7 were ~50% lower in LLC hearts. Treatment of cardiomyocytes with LLC-conditioned media resulted in higher ROS (25%), lower oxygen consumption rates (10% at basal, 75% at maximal), and greater susceptibility to hypoxia (~25%) -- which was reversed by MitoTempo. These results substantiate metabolic cardiotoxic effects attributable to tumor-associated factors and provide insight into interactions between mitochondrial mRNA translation, ROS mitigation, oxidative capacity and hypoxia resistance.

Effects of electrical stimulation-induced resistance exercise training on white and brown adipose tissues and plasma meteorin-like concentration in rats.

Chronic endurance exercise training induces morphological and metabolic alterations including mitochondrial biogenesis in white adipose tissue (WAT) and brown adipose tissue (BAT) in rodents. A myokine called meteorin‐like (Metrnl) is associated with morphological and metabolic adaptation and increased in blood after acute resistance exercise. However, the effects of chronic resistance exercise training (RT), which aims to increase muscle mass and strength, on WAT and BAT are unclear. Therefore, we aimed to clarify the effects of RT on morphological and metabolic parameters in WAT and BAT and on plasma Metrnl concentrations. We applied electrical stimulation to both legs of rats as RT three times a week for 4 weeks. RT reduced adipocyte size in subcutaneous WAT but induced no changes in mitochondrial and thermogenesis proteins. In BAT, peroxisome proliferator‐activated receptor gamma coactivator‐1 alpha (PGC‐1α) protein levels and mitochondrial content markers were significantly higher in the RT group compared with the control group. A significant positive correlation was found between the expression of PGC‐1α in BAT and plasma Metrnl concentrations. These results suggest that plasma Metrnl is associated with PGC‐1α and mitochondrial biogenesis in BAT. This study describes a potential role of RT in preventing metabolic diseases via altering WAT and BAT and increasing plasma Mertnl concentrations.

Skeletal Muscle Mitochondrial Capacity Is Similar In Ambulatory And Non-ambulatory Children With Cerebral Palsy

PURPOSE: Cerebral palsy (CP) is the most common childhood movement dysfunction secondary to a brain injury around birth. These children can be classified as ambulatory or non-ambulatory based on their functional abilities. Importantly, they expend significantly increased energy expenditure during movement. Muscle mitochondria, specifically the electron transport system are responsible for oxidative capacity, energy production and are associated with functional capacity. Unfortunately, mitochondrial oxidative capacity in children with CP and its association with different functional levels is not known. We measured maximum mitochondrial respiration rates directly from biopsies in children with CP, compared across ambulatory levels. METHODS: Twenty children (6-16 years, 10 M/6F, Ambulatory-12), undergoing surgery participated in this study. Twenty-nine biopsies were obtained from adductors, vastus lateralis, gastrocnemius. Carbohydrate and fatty acid respirometry substrate-uncoupler-inhibitor titration (SUIT) protocols were performed on permeabilized muscle fiber bundles. In addition, muscle homogenate was used to measure citrate synthase activity as a marker of mitochondrial content. Ambulatory capacity was measured using 6-minute walk tests (n=7), muscle strength, and gait velocity (n=15) during routine therapy visits. RESULTS: Surprisingly, maximal mitochondrial phosphorylation capacity was similar across between functionally ambulatory and non-ambulatory children (77.1± 23.9 vs. 84.9± 24.0 pmol O2/s/mg). This was uncorrelated with mitochondrial content (p>0.1), as measured by citrate synthase. Mitochondria in children with CP still preferentially used carbohydrates over fatty acids based on state-3 respiration. Functionally ambulatory children showed positive associations between mitochondrial function and ambulatory capacity measures (r2 values for gait velocity=0.50, 6-meter walk test=0.33, p< 0.05). CONCLUSIONS: Mitochondrial function are typically associated with activity level. Surprisingly, functionally ambulatory children with CP did not have greater mitochondrial function compared to non-ambulatory children and might even be lower. Importantly, within ambulatory children walking capacity was related to maximal mitochondrial function.

Mitochondrial Utilization of Competing Fuels Is Altered in Insulin Resistant Skeletal Muscle of Non-obese Rats (Goto-Kakizaki).

AimInsulin-resistant skeletal muscle is characterized by metabolic inflexibility with associated alterations in substrate selection, mediated by peroxisome-proliferator activated receptor δ (PPARδ). Although it is established that PPARδ contributes to the alteration of energy metabolism, it is not clear whether it plays a role in mitochondrial fuel competition. While nutrient overload may impair metabolic flexibility by fuel congestion within mitochondria, in absence of obesity defects at a mitochondrial level have not yet been excluded. We sought to determine whether reduced PPARδ content in insulin-resistant rat skeletal muscle of a non-obese rat model of T2DM (Goto-Kakizaki, GK) ameliorate the inhibitory effect of fatty acid (i.e., palmitoylcarnitine) on mitochondrial carbohydrate oxidization (i.e., pyruvate) in muscle fibers.MethodsBioenergetic function was characterized in oxidative soleus (S) and glycolytic white gastrocnemius (WG) muscles with measurement of respiration rates in permeabilized fibers in the presence of complex I, II, IV, and fatty acid substrates. Mitochondrial content was measured by citrate synthase (CS) and succinate dehydrogenase activity (SDH). Western blot was used to determine protein expression of PPARδ, PDK isoform 2 and 4.ResultsCS and SDH activity, key markers of mitochondrial content, were reduced by ∼10–30% in diabetic vs. control, and the effect was evident in both oxidative and glycolytic muscles. PPARδ (p < 0.01), PDK2 (p < 0.01), and PDK4 (p = 0.06) protein content was reduced in GK animals compared to Wistar rats (N = 6 per group). Ex vivo respiration rates in permeabilized muscle fibers determined in the presence of complex I, II, IV, and fatty acid substrates, suggested unaltered mitochondrial bioenergetic function in T2DM muscle. Respiration in the presence of pyruvate was higher compared to palmitoylcarnitine in both animal groups and fiber types. Moreover, respiration rates in the presence of both palmitoylcarnitine and pyruvate were reduced by 25 ± 6% (S), 37 ± 6% (WG) and 63 ± 6% (S), 57 ± 8% (WG) compared to pyruvate for both controls and GK, respectively. The inhibitory effect of palmitoylcarnitine on respiration was significantly greater in GK than controls (p < 10–3).ConclusionWith competing fuels, the presence of fatty acids diminishes mitochondria ability to utilize carbohydrate derived substrates in insulin-resistant muscle despite reduced PPARδ content.