Age-Dependent Changes in the Production of Mitochondrial Reactive Oxygen Species in Human Skeletal Muscle.

A decrease in muscle mass and its functionality (strength, endurance, and insulin sensitivity) is one of the integral signs of aging. One of the triggers of aging is an increase in the production of mitochondrial reactive oxygen species. Our study was the first to examine age-dependent changes in the production of mitochondrial reactive oxygen species related to a decrease in the proportion of mitochondria-associated hexokinase-2 in human skeletal muscle. For this purpose, a biopsy was taken from m. vastus lateralis in 10 young healthy volunteers and 70 patients (26-85 years old) with long-term primary arthrosis of the knee/hip joint. It turned out that aging (comparing different groups of patients), in contrast to inactivity/chronic inflammation (comparing young healthy people and young patients), causes a pronounced increase in peroxide production by isolated mitochondria. This correlated with the age-dependent distribution of hexokinase-2 between mitochondrial and cytosolic fractions, a decrease in the rate of coupled respiration of isolated mitochondria and respiration when stimulated with glucose (a hexokinase substrate). It is discussed that these changes may be caused by an age-dependent decrease in the content of cardiolipin, a potential regulator of the mitochondrial microcompartment containing hexokinase. The results obtained contribute to a deeper understanding of age-related pathogenetic processes in skeletal muscles and open prospects for the search for pharmacological/physiological approaches to the correction of these pathologies.

Dimorphic effect of TFE3 in determining mitochondrial and lysosomal content in muscle following denervation.

BACKGROUND: Muscle atrophy is a common consequence of the loss of innervation and is accompanied by mitochondrial dysfunction. Mitophagy is the adaptive process through which damaged mitochondria are removed via the lysosomes, which are regulated in part by the transcription factor TFE3. The role of lysosomes and TFE3 are poorly understood in muscle atrophy, and the effect of biological sex is widely underreported. METHODS: Wild-type (WT) mice, along with mice lacking TFE3 (KO), a transcriptional regulator of lysosomal and autophagy-related genes, were subjected to unilateral sciatic nerve denervation for up to 7 days, while the contralateral limb was sham-operated and served as an internal control. A subset of animals was treated with colchicine to capture mitophagy flux. RESULTS: WT females exhibited elevated oxygen consumption rates during active respiratory states compared to males, however this was blunted in the absence of TFE3. Females exhibited higher mitophagy flux rates and greater lysosomal content basally compared to males that was independent of TFE3 expression. Following denervation, female mice exhibited less muscle atrophy compared to male counterparts. Intriguingly, this sex-dependent muscle sparing was lost in the absence of TFE3. Denervation resulted in 45% and 27% losses of mitochondrial content in WT and KO males respectively, however females were completely protected against this decline. Decreases in mitochondrial function were more severe in WT females compared to males following denervation, as ROS emission was 2.4-fold higher. In response to denervation, LC3-II mitophagy flux was reduced by 44% in females, likely contributing to the maintenance of mitochondrial content and elevated ROS emission, however this response was dysregulated in the absence of TFE3. While both males and females exhibited increased lysosomal content following denervation, this response was augmented in females in a TFE3-dependent manner. CONCLUSIONS: Females have higher lysosomal content and mitophagy flux basally compared to males, likely contributing to the improved mitochondrial phenotype. Denervation-induced mitochondrial adaptations were sexually dimorphic, as females preferentially preserve content at the expense of function, while males display a tendency to maintain mitochondrial function. Our data illustrate that TFE3 is vital for the sex-dependent differences in mitochondrial function, and in determining the denervation-induced atrophy phenotype.

Reorganization of mitochondria-organelle interactions during postnatal development in skeletal muscle.

Skeletal muscle cellular development requires the integrated assembly of mitochondria and other organelles adjacent to the sarcomere in support of muscle contractile performance. However, it remains unclear how interactions among organelles and with the sarcomere relates to the development of muscle cell function. Here, we combine 3D volume electron microscopy, proteomic analyses, and live cell functional imaging to investigate the postnatal reorganization of mitochondria-organelle interactions in skeletal muscle. We show that while mitochondrial networks are disorganized and loosely associated with the contractile apparatus at birth, contact sites among mitochondria, lipid droplets and the sarcoplasmic reticulum are highly abundant in neonatal muscles. The maturation process is characterized by a transition to highly organized mitochondrial networks wrapped tightly around the muscle sarcomere but also to less frequent interactions with both lipid droplets and the sarcoplasmic reticulum. Concomitantly, expression of proteins involved in mitochondria-organelle membrane contact sites decreases during postnatal development in tandem with a decrease in abundance of proteins associated with sarcomere assembly despite an overall increase in contractile protein abundance. Functionally, parallel measures of mitochondrial membrane potential, NADH redox status, and NADH flux within intact cells revealed that mitochondria in adult skeletal muscle fibres maintain a more activated electron transport chain compared with neonatal muscle mitochondria. These data demonstrate a developmental redesign reflecting a shift from muscle cell assembly and frequent inter-organelle communication toward a muscle fibre with mitochondrial structure, interactions, composition and function specialized to support contractile function. KEY POINTS: Mitochondrial network organization is remodelled during skeletal muscle postnatal development. The mitochondrial outer membrane is in frequent contact with other organelles at birth and transitions to more close associations with the contractile apparatus in mature muscles. Mitochondrial energy metabolism becomes more activated during postnatal development. Understanding the developmental redesign process within skeletal muscle cells may help pinpoint specific areas of deficit in muscles with developmental disorders.

Effects of short-term exercise and endurance training on skeletal muscle mitochondria damage induced by particular matter, atmospherically relevant artificial PM2.5.

Introduction: This study aimed to investigate the potential of short-term aerobic exercise to mitigate skeletal muscle mitochondrial damage following ambient PM2.5 exposure, and how 12 weeks of endurance training can enhance aerobic fitness to protect against such damage. Methods: Twenty-four male C57BL/6 J mice were split into sedentary (SED, n = 12) and endurance training (ETR, n = 12) groups. The ETR group underwent 12 weeks of training (10-15 m/min, 60 min/day, 4 times/week), confirmed by an Endurance Exercise Capacity (EEC) test. Post-initial training, the SED group was further divided into SSED (SED and sedentary, n = 6) and SPE (SED and PM2.5 + Exercise, n = 6). Similarly, the ETR group was divided into EEX (ETR and Exercise, n = 6) and EPE (ETR and PM2.5 + Exercise, n = 6). These groups underwent 1 week of atmospherically relevant artificial PM2.5 exposure and treadmill running (3 times/week). Following treatments, an EEC test was conducted, and mice were sacrificed for blood and skeletal muscle extraction. Blood samples were analyzed for oxidative stress indicators, while skeletal muscles were assessed for mitochondrial oxidative metabolism, antioxidant capacity, and mitochondrial damage using western blot and transmission electron microscopy (TEM). Results: After 12 weeks of endurance training, the EEC significantly increased (p < 0.000) in the ETR group compared to the SED group. Following a one-week comparison among the four groups with atmospherically relevant artificial PM2.5 exposure and exercise treatment post-endurance training, the EEX group showed improvements in EEC, oxidative metabolism, mitochondrial dynamics, and antioxidant functions. Conversely, these factors decreased in the EPE group compared to the EEX. Additionally, within the SPE group, exercise effects were evident in HK2, LDH, SOD2, and GPX4, while no impact of short-term exercise was observed in all other factors. TEM images revealed no evidence of mitochondrial damage in both the SED and EEX groups, while the majority of mitochondria were damaged in the SPE group. The EPE group also exhibited damaged mitochondria, although significantly less than the SPE group. Conclusion: Atmospherically relevant artificial PM2.5 exposure can elevate oxidative stress, potentially disrupting the benefits of short-term endurance exercise and leading to mitochondrial damage. Nonetheless, increased aerobic fitness through endurance training can mitigate PM2.5-induced mitochondrial damage.

l-norleucine on high glucose-induced insulin sensitivity and mitochondrial function in skeletal muscle cells.

l-norleucine, an isomer of leucine, stimulates the anabolic process of insulin. However, it is not known if and how it improves insulin sensitivity and insulin resistance. This experiment describes the generation of an insulin resistance model using high glucose-induced cells and the administration of 1.0 mmol/L l-norleucine for 48 h, to observe the effects on metabolism and gene expression in skeletal muscle cells. The results showed that l-norleucine significantly increased mitochondrial ATP content, decreased the amount of reactive oxygen species (ROS) and promoted the expression of mitochondrial generation-related genes TFAM, AMPK, PGC-1α in cells under high glucose treatment; at the same time, l-norleucine also increased glucose uptake, suggesting that l-norleucine increased insulin sensitivity and improved insulin resistance. This study suggesting that l-norleucine improves insulin resistance by ameliorating oxidative stress damage of mitochondria, improving mitochondrial function, and improving insulin sensitivity in skeletal muscle cell caused by high glucose, rather than by altering mitochondrial efficiency.

High-dose atorvastatin therapy progressively decreases skeletal muscle mitochondrial respiratory capacity in humans.

BACKGROUNDWhile the benefits of statin therapy on atherosclerotic cardiovascular disease are clear, patients often experience mild to moderate skeletal myopathic symptoms, the mechanism for which is unknown. This study investigated the potential effect of high-dose atorvastatin therapy on skeletal muscle mitochondrial function and whole-body aerobic capacity in humans.METHODSEight overweight (BMI, 31.9 ± 2.0) but otherwise healthy sedentary adults (4 females, 4 males) were studied before (day 0) and 14, 28, and 56 days after initiating atorvastatin (80 mg/d) therapy.RESULTSMaximal ADP-stimulated respiration, measured in permeabilized fiber bundles from muscle biopsies taken at each time point, declined gradually over the course of atorvastatin treatment, resulting in > 30% loss of skeletal muscle mitochondrial oxidative phosphorylation capacity by day 56. Indices of in vivo muscle oxidative capacity (via near-infrared spectroscopy) decreased by 23% to 45%. In whole muscle homogenates from day 0 biopsies, atorvastatin inhibited complex III activity at midmicromolar concentrations, whereas complex IV activity was inhibited at low nanomolar concentrations.CONCLUSIONThese findings demonstrate that high-dose atorvastatin treatment elicits a striking progressive decline in skeletal muscle mitochondrial respiratory capacity, highlighting the need for longer-term dose-response studies in different patient populations to thoroughly define the effect of statin therapy on skeletal muscle health.FUNDINGNIH R01 AR071263.

Functional and Morphological Differences of Muscle Mitochondria in Chronic Fatigue Syndrome and Post-COVID Syndrome.

Patients suffering from chronic fatigue syndrome (CFS) or post-COVID syndrome (PCS) exhibit a reduced physiological performance capability. Impaired mitochondrial function and morphology may play a pivotal role. Thus, we aimed to measure the muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity and assess mitochondrial morphology in CFS and PCS patients in comparison to healthy controls (HCs). Mitochondrial OXPHOS capacity was measured in permeabilized muscle fibers using high-resolution respirometry. Mitochondrial morphology (subsarcolemmal/intermyofibrillar mitochondrial form/cristae/diameter/circumference/area) and content (number and proportion/cell) were assessed via electron microscopy. Analyses included differences in OXPHOS between HC, CFS, and PCS, whereas comparisons in morphology/content were made for CFS vs. PCS. OXPHOS capacity of complex I, which was reduced in PCS compared to HC. While the subsarcolemmal area, volume/cell, diameter, and perimeter were higher in PCS vs. CFS, no difference was observed for these variables in intermyofibrillar mitochondria. Both the intermyofibrillar and subsarcolemmal cristae integrity was higher in PCS compared to CFS. Both CFS and PCS exhibit increased fatigue and impaired mitochondrial function, but the progressed pathological morphological changes in CFS suggest structural changes due to prolonged inactivity or unknown molecular causes. Instead, the significantly lower complex I activity in PCS suggests probably direct virus-induced alterations.

Effect of skeletal muscle mitochondrial phenotype on H2O2 emission.

Reactive oxygen species (ROS) are a key output of the skeletal muscle mitochondrial information processing system both at rest and during exercise. In skeletal muscle, mitochondrial ROS release depends on multiple factors; however, fiber-type specific differences remain ambiguous in part owing to the use of mitochondria from mammalian muscle that consist of mixed fibers. To elucidate fiber-type specific differences, we used mitochondria isolated from rainbow trout (Oncorhynchus mykiss) red and white skeletal muscles that consist of spatially distinct essentially pure red and white fibers. We first characterized the assay conditions for measuring ROS production (as H2O2) in isolated fish red and white skeletal muscle mitochondria (RMM and WMM) and thereafter compared the rates of emission during oxidation of different substrates and the responses to mitochondrial electron transport system (ETS) pharmacological modulators. Our results showed that H2O2 emission rates by RMM and WMM can be quantified using the same protein concentration and composition of the Amplex UltraRed-horseradish peroxidase (AUR-HRP) detection system. For both RMM and WMM, protein normalized H2O2 emission rates were highest at the lowest protein concentration tested and decreased exponentially thereafter. However, the absolute values of H2O2 emission rates depended on the calibration curves used to convert fluorescent signals to H2O2 while the trends depended on the normalization strategy. We found substantial qualitative and quantitative differences between RMM and WMM in the H2O2 emission rates depending on the substrates being oxidized and their concentrations. Similarly, pharmacological modulators of the ETS altered the magnitudes and trends of the H2O2 emission differently in RMM and WMM. While comparable concentrations of substrates elicited maximal albeit quantitively different emission rates in RMM and WMM, different concentrations of pharmacological ETS modulators may be required for maximal H2O2 emission rates depending on muscle fiber-type. Taken together, our study suggests that biochemical differences exist in RMM compared with WMM that alter substrate oxidation and responses to ETS modulators resulting in fiber-type specific mitochondrial H2O2 emission rates.

Actovegin improves skeletal muscle mitochondrial respiration and functional aerobic capacity in a type 1 diabetic male murine model.

Insulin deficiency in type 1 diabetes (T1D) leads to an impairment of glucose metabolism and mitochondrial function. Actovegin is a hemodialysate of calf blood, which has been shown to enhance glucose uptake and cell metabolism in healthy human skeletal muscle. The objectives of this study were to determine the effects of Actovegin on skeletal muscle mitochondrial respiration and functional aerobic capacity in a T1D mouse model. Effects on the expression of mitochondrial proteins, body mass, and food and water consumption were also investigated. Streptozotocin-induced T1D male C57B1/6 mice (aged 3-4 months) were randomized to an Actovegin group and a control group. Every third day, the Actovegin and control groups were injected intraperitoneally with (0.1 mL) Actovegin and (0.1 mL) physiological salt solution, respectively. Oxidative phosphorylation (OXPHOS) capacity of the vastus lateralis muscle was measured by high resolution respirometry in addition to the expression levels of the mitochondrial complexes as well as voltage-dependent anion channel. Functional aerobic capacity was measured using a rodent treadmill protocol. Body mass and food and water consumption were also measured. After 13 days, in comparison to the control group, the Actovegin group demonstrated a significantly higher skeletal muscle mitochondrial respiratory capacity in an ADP-restricted and ADP-stimulated environment. The Actovegin group displayed a significantly lesser decline in functional aerobic capacity and baseline body mass after 13 days. There were no significant differences in food or water consumption between groups. Actovegin could act as an effective agent for facilitating glucose metabolism and improving OXPHOS capacity and functional aerobic capacity in T1D. Further investigation is warranted to establish Actovegin's potential as an alternative therapeutic drug for T1D.

The contribution of mitochondria to age-related skeletal muscle wasting: A sex-specific perspective.

As people age, their skeletal muscle (SkM) experiences a decline in mitochondrial functionality and density, which leads to decreased energy production and increased generation of reactive oxygen species. This cascade of events, in turn, might determine the loss of SkM mass, strength and quality. Even though the mitochondrial processes dysregulated by aging, such as oxidative phosphorylation, mitophagy, antioxidant defenses and mtDNA transcription, are the same in both sexes, mitochondria age differently in the SkM of men and women. Indeed, the onset and magnitude of the impairment of these processes seem to be influenced by sex-specific factors. Sexual hormones play a pivotal role in the regulation of SkM mass through both genomic and non-genomic mechanisms. However, the precise mechanisms by which these hormones regulate mitochondrial plasticity in SkM are not fully understood. Although the presence of estrogen receptors in mitochondria is recognized, it remains unclear whether androgen receptors affect mitochondrial function. This comprehensive review critically dissects the current knowledge on the interplay of sex in the aging of SkM, focusing on the role of sex hormones and the corresponding signaling pathways in shaping mitochondrial plasticity. Improved knowledge on the sex dimorphism of mitochondrial aging may lead to sex-tailored interventions that target mitochondrial health, which could be effective in slowing or preventing age-related muscle loss.

Skeletal muscle mitochondria demonstrate similar respiration per cristae surface area independent of training status and sex in healthy humans.

The impact of training status and sex on intrinsic skeletal muscle mitochondrial respiratory capacity remains unclear. We examined this by analysing human skeletal muscle mitochondrial respiration relative to mitochondrial volume and cristae density across training statuses and sexes. Mitochondrial cristae density was estimated in skeletal muscle biopsies originating from previous independent studies. Participants included females (n = 12) and males (n = 41) across training statuses ranging from untrained (UT, n = 8), recreationally active (RA, n = 9), active-to-elite runners (RUN, n = 27) and cross-country skiers (XC, n = 9). The XC and RUN groups demonstrated higher mitochondrial volume density than the RA and UT groups while all active groups (RA, RUN and XC) displayed higher mass-specific capacity of oxidative phosphorylation (OXPHOS) and mitochondrial cristae density than UT. Differences in OXPHOS diminished between active groups and UT when normalising to mitochondrial volume density and were lost when normalising to muscle cristae surface area density. Moreover, active females (n = 6-9) and males (n = 15-18) did not differ in mitochondrial volume and cristae density, OXPHOS, or when normalising OXPHOS to mitochondrial volume density and muscle cristae surface area density. These findings demonstrate: (1) differences in OXPHOS between active and untrained individuals may be explained by both higher mitochondrial volume and cristae density in active individuals, with no difference in intrinsic mitochondrial respiratory capacity (OXPHOS per muscle cristae surface area density); and (2) no sex differences in mitochondrial volume and cristae density or mass-specific and normalised OXPHOS. This highlights the importance of normalising OXPHOS to muscle cristae surface area density when studying skeletal muscle mitochondrial biology. KEY POINTS: Oxidative phosphorylation is the mitochondrial process by which ATP is produced, governed by the electrochemical gradient across the inner mitochondrial membrane with infoldings named cristae. In human skeletal muscle, the mass-specific capacity of oxidative phosphorylation (OXPHOS) can change independently of shifts in mitochondrial volume density, which may be attributed to variations in cristae density. We demonstrate that differences in skeletal muscle OXPHOS between healthy females and males, ranging from untrained to elite endurance athletes, are matched by differences in cristae density. This suggests that higher OXPHOS in skeletal muscles of active individuals is attributable to an increase in the density of cristae. These findings broaden our understanding of the variability in human skeletal muscle OXPHOS and highlight the significance of cristae, specific to mitochondrial respiration.

Combined changes in temperature and pH mimicking exercise result in decreased efficiency in muscle mitochondria.

It is well known that exercise efficiency declines at intensities above the lactate threshold, yet the underlying mechanisms are poorly understood. Some have suggested it is due to a decline in mitochondrial efficiency, but this is difficult to examine in vivo. Therefore, the aim of the current study was to examine how changes in temperature and pH, mimicking those that occur during exercise, affect mitochondrial efficiency in skeletal muscle mitochondria. This study was performed on quadriceps muscle of 20 wild-type mice. Muscle tissue was dissected and either permeabilized (n = 10) or homogenized for isolation of mitochondria (n = 10), and oxidative phosphorylation capacity and P/O ratio were assessed using high-resolution respirometry. Samples from each muscle were analyzed in both normal physiological conditions (37°C, pH 7.4), decreased pH (6.8), increased temperature (40°C), and a combination of both. The combination of increased temperature and decreased pH resulted in a significantly lower P/O ratio, mirrored by an increase in leak respiration and a decrease in respiratory control ratio (RCR), in isolated mitochondria. In permeabilized fibers, RCR and leak were relatively unaffected, though a main effect of temperature was observed. Oxidative phosphorylation capacity was unaffected by changes in pH and temperature in both isolated mitochondria and permeabilized fibers. These results indicate that exercise-like changes in temperature and pH lead to impaired mitochondrial efficiency. These findings offer some degree of support to the concept of decreased mitochondrial efficiency during exercise, and may have implications for the assessment of mitochondrial function related to exercise.NEW & NOTEWORTHY To the best of our knowledge, this is the first study to examine the effects of combined changes in temperature and pH, mimicking intramuscular alterations during exercise. Our findings suggest that mitochondrial efficiency is impaired during exercise of moderate to high intensity, which could be a possible mechanism contributing to the decline in exercise efficiency at intensities above the lactate threshold.

The role of TFE3 in mediating skeletal muscle mitochondrial adaptations to exercise training.

Transcription factor E3 (TFE3) is a transcription factor that activates the expression of lysosomal genes involved in the clearance of dysfunctional mitochondria, termed mitophagy. With exercise, TFE3 is presumed to optimize the mitochondrial pool through the removal of organelles via lysosomes. However, the molecular mechanisms of the involved pathways remain unknown. Wild-type (WT) and TFE3 knockout (KO) mice were subjected to 6 wk of voluntary wheel running as an endurance training regimen. This was followed by a 45-min bout of in situ stimulation of the sciatic nerve innervating hindlimb muscles to evaluate muscle fatigue and contractile properties. A subset of animals was treated with colchicine to measure autophagy and mitophagy flux. Fatigability during stimulation was reduced with training in WT animals, as seen by a 13% increase in the percentage of maximum force at 5 min of stimulation, and a 30% increase at 30 minutes. Permeabilized fiber oxygen consumption was also improved with training. Concurrent with improved muscle and mitochondrial function, cytochrome c oxidase (COX) activity and COX I protein expression were increased in trained WT animals compared to untrained animals, signifying an increase in mitochondrial content. These training adaptations were abolished with the loss of TFE3. Surprisingly, the absence of TFE3 did not affect lysosomal content nor did it blunt the induction of mitophagy flux with contractile activity compared to WT mice. Our results suggest that the loss of TFE3 compromises beneficial training adaptations that lead to improved muscle endurance and mitochondrial function.NEW & NOTEWORTHY Our understanding of the role of transcription factor E3 (TFE3) in skeletal muscle is very limited. This research shows that TFE3 plays a direct role in skeletal muscle mitochondrial enhancement with exercise training, thereby introducing a paradigm shift in our perception of the function of TFE3 in mitochondrial maintenance, beyond mitophagy. This research serves to introduce TFE3 as a protein that holds promise as a future therapeutic target for metabolic diseases and skeletal muscle dysfunction.

Mitochondrial density in skeletal and cardiac muscle.

Kubat et al. provide a review on the role Mitochondrial density in skeletal and cardiac muscle of mitochondrial dysfunction in muscle atrophy. They stress mitochondria's pivotal function, citing a 52 % density in skeletal muscle. However, the reference to Park et al.'s work misinterprets their findings. Park et al. report citrate synthase (CS) activity, indicating mitochondrial density as 222 ± 13 µmol.min-1.mg-1 for cardiac muscle and 115 ± 2 µmol.min-1.mg-1 for skeletal muscle. Thus, the authors should clarify that skeletal muscle density is approximately 52 % of cardiac muscle, not an absolute 52 %. Mitochondrial volume density assessment, predominantly through TEM, establishes cardiomyocytes at 25-30 % and untrained skeletal muscle at 2-6 %, increasing to 11 % in trained athletes. However, this remains modest compared to myofibrils' 75 %-85 % of muscle fiber volume. Although the utility of CS activity is evident, TEM and other novel approaches such as three-dimensional focused ion beam scanning electron microscopy are likely superior for assessing mitochondrial volume density and morphology.

Translating In Vitro Models of Exercise in Human Muscle Cells: A Mitocentric View.

Human skeletal muscle cell (HSkMC) models provide the opportunity to examine in vivo training-induced muscle-specific mitochondrial adaptations, additionally allowing for deeper interrogation into the effect of in vitro exercise models on myocellular mitochondrial quality and quantity. As such, this review will compare and contrast the effects of in vivo and in vitro models of exercise on mitochondrial adaptations in HSkMCs.

GADD45A is a mediator of mitochondrial loss, atrophy, and weakness in skeletal muscle.

Aging and many illnesses and injuries impair skeletal muscle mass and function, but the molecular mechanisms are not well understood. To better understand the mechanisms, we generated and studied transgenic mice with skeletal muscle-specific expression of growth arrest and DNA damage inducible α (GADD45A), a signaling protein whose expression in skeletal muscle rises during aging and a wide range of illnesses and injuries. We found that GADD45A induced several cellular changes that are characteristic of skeletal muscle atrophy, including a reduction in skeletal muscle mitochondria and oxidative capacity, selective atrophy of glycolytic muscle fibers, and paradoxical expression of oxidative myosin heavy chains despite mitochondrial loss. These cellular changes were at least partly mediated by MAP kinase kinase kinase 4, a protein kinase that is directly activated by GADD45A. By inducing these changes, GADD45A decreased the mass of muscles that are enriched in glycolytic fibers, and it impaired strength, specific force, and endurance exercise capacity. Furthermore, as predicted by data from mouse models, we found that GADD45A expression in skeletal muscle was associated with muscle weakness in humans. Collectively, these findings identify GADD45A as a mediator of mitochondrial loss, atrophy, and weakness in mouse skeletal muscle and a potential target for muscle weakness in humans.

Effects of hyperthermia and acidosis on mitochondrial production of reactive oxygen species.

Exercise is associated with the development of oxidative stress, but the specific source and mechanism of production of pro-oxidant chemicals during exercise has not been confirmed. We used equine skeletal muscle mitochondria to test the hypothesis that hyperthermia and acidosis affect mitochondrial oxygen consumption and production of reactive oxygen species (ROS). Skeletal muscle biopsies were obtained at rest, after an acute episode of fatiguing exercise, and after a 9-wk conditioning program to increase aerobic fitness. Mitochondrial oxygen consumption and ROS production were measured simultaneously using high-resolution respirometry. Both hyperthermia and acidosis increased nonphosphorylating (LEAK) respiration (5.8× and 3.0×, respectively, P < 0.001) and decreased efficiency of oxidative phosphorylation. The combined effects of hyperthermia and acidosis resulted in large decreases in phosphorylating respiration, further decreasing oxidative phosphorylation efficiency from 97% to 86% (P < 0.01). Increased aerobic fitness reduced the effects of acidosis on LEAK respiration. Hyperthermia increased and acidosis decreased ROS production (2× and 0.23×, respectively, P < 0.001). There was no effect of acute exercise, but an aerobic conditioning program was associated with increased ROS production during both nonphosphorylating and phosphorylating respiration. Hyperthermia increased the ratio of ROS production to O2 consumption during phosphorylating respiration, suggesting that high-temperature impaired transfer of energy through the electron transfer system despite relatively low mitochondrial membrane potential. These data support the role of skeletal muscle mitochondria in the development of exercise-induced oxidative stress, particularly during forms of exercise that result in prolonged hyperthermia without acidosis.NEW & NOTEWORTHY The results of this study provide evidence for the role of mitochondria-derived ROS in the development of systemic oxidative stress during exercise as well as skeletal muscle diseases such as exertional rhabdomyolysis.

Purification of functional mouse skeletal muscle mitochondria using percoll density gradient centrifugation.

OBJECTIVE: Our goal was to isolate purified mitochondria from mouse skeletal muscle using a Percoll density gradient and to assess bioenergetic function and purity via Seahorse Extracellular Flux (XF) Analyses and mass spectrometry. RESULTS: Mitochondria isolated from murine quadriceps femoris skeletal muscle using a Percoll density gradient method allowed for minimally contaminated preparations with time from tissue harvest to mitochondrial isolation and quantification in about 3-4 h. Percoll purification from 100 to 200 mg fresh tissue yielded ~ 200-400 ug protein. Mitochondrial bioenergetics evaluated using the Seahorse XFe96 analyzer, a high-throughput respirometry platform, showed optimum mitochondrial input at 500 ng with respiratory control ratio ranging from 3.9 to 7.1 using various substrates demonstrating a high degree of functionality. Furthermore, proteomic analysis of Percoll-enriched mitochondria isolated from skeletal muscle using this method showed significant enrichment of mitochondrial proteins indicating high sample purity. This study established a methodology that ensures sufficient high quality mitochondria for downstream analyses such as mitochondrial bioenergetics and proteomics.