Reduction In Mitochondrial Content Research Articles

Impact of chronic muscle use and disuse on innate immune signaling in skeletal muscle

Mitochondria exhibit high levels of adaptability to various stimuli such as chronic muscle use (i.e. exercise) or disuse, such as immobilization or denervation. These changes in the mitochondrial network ultimately affect the health and functioning of skeletal muscle, which contributes to whole-body metabolism, locomotion, and postural stability. It is now acknowledged that mitochondrial dysfunction can activate inflammatory pathways through the recognition of damage-associated molecular patterns (DAMPs). The objective of this study was to examine how changes in mitochondria brought about by either chronic muscle inactivity or chronic exercise training affect innate immune system activation. We hypothesized that an inverse relationship between mitochondrial content and innate immune signaling would be evident in skeletal muscle. To investigate this, skeletal muscle from the hindlimbs of mice was collected after 7 days of sciatic muscle denervation, or after 4.5 weeks of endurance swim training involving 240 minutes of daily exercise. We analyzed the expression of the NLRP3 inflammasome, innate immune system signaling proteins, mitochondrial markers, and mitochondrial DNA (mtDNA) content using western blotting, cytochrome C oxidase (COX) activity, ELISA and qPCR. Following 7 days of denervation, there was a significant decrease in both COX-IV protein and COX activity, indicating a reduction in mitochondrial content. This was accompanied by an increase (p<0.05) in NLRP3, procaspase-1, gasdermin-D (GSDMD) and its active N-terminal fragment, GSDMD-N, as well as a notable increase in IL-1β. In addition, other possible converging pathways were also upregulated including cGAS-STING, and gasdermin-E. In comparison, following exercise training, there was a significant increase in mitochondrial content, apparent through increases in COX-IV protein, COX activity and mitochondrial RNA content. This was associated with decreases in procaspase-1 and caspase-1 protein expression alongside reduced STING activation. These findings highlight an inverse relationship between mitochondrial content and the activation of several inflammatory pathways that possibly converge upon NLRP3 inflammasome activation and pyroptotic cell death, leading to detriments in skeletal muscle health. This work aims to further the understanding of innate immune signaling pathways within muscle which can potentially highlight therapeutic targets to regulate its activation under divergent metabolic conditions. This work was supported by funds from the Natural Science and Engineering Research Council (NSERC). Priyanka Khemraj is the holder of an Ontario Graduate Scholarship. David A. Hood is the holder of a Canadian Research Chair in Cell Physiology. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Activation of the Nrf-2 pathway improves the mitochondrial phenotype in skeletal muscle cells

Aging muscle displays a reduction in mitochondrial content, along with elevated reactive oxygen species (ROS) emission and reduced levels of antioxidant enzymes. These characteristics enhance tissue susceptibility to oxidative damage, facilitating sub-optimal cellular metabolism and subsequent muscle atrophy. While these perturbations to metabolic homeostasis are a natural course of the aging process, there has been ongoing research investigating how to implement interventions to reduce this decline in muscle health. Regular exercise is one possible treatment modality, however this may be unsuitable for some populations who are unable to participate due to their health status. The objective of this study was to examine the activation of the Nrf-2 pathway using the nutraceutical Sulforaphane (SFN) and evaluate its role as a potential exercise mimetic. We hypothesized that SFN would act through similar pathways activated with chronic exercise to improve mitochondrial content, respiration, and ROS emission. To address our objective, C2C12 myotubes were treated with SFN for various timepoints basally, and in the presence of the Complex-I inhibitor rotenone to induce oxidative stress. Western blotting revealed an 8-fold increase in Nrf-2 nuclear localization following 24hrs of SFN treatment, which subsequently led to the upregulation of antioxidant enzymes NQO1, Heme Oxygenase-1, catalase, glutathione reductase and Glucose-6-Phosphate Dehydrogenase by 2-to 3-fold. These protein changes, were accompanied by dramatic reductions in both total cellular and mitochondrial ROS emission with SFN, detected with CellROX Green and MitoSOX Red respectively. Interestingly, this led to an increase in mitochondrial membrane potential, detected via MitoTracker Red (MTR), despite rotenone-induced oxidative stress. Western blotting revealed TFAM upregulation at 24hrs, suggesting an increase in mitochondrial DNA (mtDNA) replication and transcription. This was corroborated by an increase in COX subunit I protein, a mitochondrially-encoded subunit of cytochrome c oxidase, as well as MitoTracker Green and MTR staining by 48hrs. Mitochondrial respiration was evaluated using Seahorse analysis, demonstrating a marked increase in ATP production as well as basal and maximal respiration. Together these data suggest that activation of the Nrf-2 pathway via SFN may lead to adaptations similar to chronic exercise. Additionally, SFN may have applications in disorders characterized by mitochondrial loss and high ROS emission. To fully elucidate the precise mechanisms underlying this agent, SFN will be combined with chronic contractile activity to determine if these two interventions operate through independent pathways. This work was supported by funds from the Natural Science and Engineering Research Council (NSERC). Sabrina Champsi is the recipient of the NSERC Alexander Graham Bell Canada Graduate Scholarship-Master’s (CGS-M). David A. Hood is the holder of a Canadian Research Chair in Cell Physiology. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The impact of 60days of -6° head down tilt bed rest on mitochondrial content, respiration and regulators of mitochondrial dynamics.

It is unclear how skeletal muscle metabolism and mitochondrial function adapt to long duration bed rest and whether changes can be prevented by nutritional intervention. The present study aimed (1) to assess the effect of prolonged bed rest on skeletal muscle mitochondrial function and dynamics and (2) to determine whether micronutrient supplementation would mitigate the adverse metabolic effect of bed rest. Participants were maintained in energy balance throughout 60days of bed rest with micronutrient supplementation (INT) (body mass index: 23.747±1.877kgm-2 ; 34.80±7.451years; n=10) or without (control) (body mass index: 24.087±2.088kgm-2 ; 33.50±8.541years; n=10). Indirect calorimetry and dual-energy x-ray absorptiometry were used for measures of energy expenditure, exercise capacity and body composition. Mitochondrial respiration was determined by high-resolution respirometry in permeabilized muscle fibre bundles from vastus lateralis biopsies. Protein and mRNA analysis further examined the metabolic changes relating to regulators of mitochondrial dynamics induced by bed rest. INT was not sufficient in preserving whole body metabolic changes conducive of a decrease in body mass, fat-free mass and exercise capacity within both groups. Mitochondrial respiration, OPA1 and Drp1 protein expression decreased with bed rest, with an increase pDrp1s616 . This reduction in mitochondrial respiration was explained through an observed decrease in mitochondrial content (mtDNA:nDNA). Changes in regulators of mitochondrial dynamics indicate an increase in mitochondrial fission driven by a decrease in inner mitochondrial membrane fusion (OPA1) and increased pDrp1s616 . KEY POINTS: Sixty days of -6° head down tilt bed rest leads to significant changes in body composition, exercise capacity and whole-body substrate metabolism. Micronutrient supplementation throughout bed rest did not preserve whole body metabolic changes. Bed rest results in a decrease in skeletal muscle mitochondrial respiratory capacity, mainly as a result of an observed decrease in mitochondrial content. Prolonged bed rest ensues changes in key regulators of mitochondrial dynamics. OPA1 and Drp1 are significantly reduced, with an increase in pDrp1s616 following bed rest indicative of an increase in mitochondrial fission. Given the reduction in mitochondrial content following 60days of bed rest, the maintenance of regulators of mitophagy in line with the increase in regulators of mitochondrial fission may act to maintain mitochondrial respiration to meet energy demands.

5-Heptadecylresorcinol Ameliorates Obesity-Associated Skeletal Muscle Mitochondrial Dysfunction through SIRT3-Mediated Mitophagy.

Skeletal muscle dysfunction caused by obesity is characterized by the decline in mitochondrial content and function. 5-Heptadecylresorcinol (AR-C17) is a specific bioactive component derived from whole wheat and rye, which has been evidenced to improve obesity-associated skeletal muscle dysregulation. However, the mechanism underlying its protective activity requires further exploration. Herein, we found that AR-C17 (5, 10, and 20 μM) intervention reversed PA-induced (0.5 mM) reduction in mitochondrial content, mitochondrial membrane potential, and mitochondrial energy metabolism in C2C12 cells. Meanwhile, AR-C17 evidently alleviated PA-mediated myotube mitochondrial dysfunction via elevating mitochondria autophagy flux and upregulating the expression level of autophagy-related protein, while this effect was abolished by an autophagy inhibitor (3-MA). Further analysis showed that SIRT3-FOXO3A-PINK-Parkin-mediated mitophagy was involved in the modulation of myocyte mitochondrial dysfunction by AR-C17. In addition, AR-C17 administration (30 and 150 mg/kg/day) significantly improved high-fat-diet-induced mitochondrial dysregulation in mice skeletal muscle tissue via SIRT3-dependent mitophagy. Our findings indicate that skeletal muscle cells are responsive to AR-C17, which improves myogenesis and mitophagy in vitro and in vivo.

Insights into the development of insulin resistance: Unraveling the interaction of physical inactivity, lipid metabolism and mitochondrial biology.

While impairments in peripheral tissue insulin signalling have a well-characterized role in the development of insulin resistance and type 2 diabetes (T2D), the specific mechanisms that contribute to these impairments remain debatable. Nonetheless, a prominent hypothesis implicates the presence of a high-lipid environment, resulting in both reactive lipid accumulation and increased mitochondrial reactive oxygen species (ROS) production in the induction of peripheral tissue insulin resistance. While the etiology of insulin resistance in a high lipid environment is rapid and well documented, physical inactivity promotes insulin resistance in the absence of redox stress/lipid-mediated mechanisms, suggesting alternative mechanisms-of-action. One possible mechanism is a reduction in protein synthesis and the resultant decrease in key metabolic proteins, including canonical insulin signaling and mitochondrial proteins. While reductions in mitochondrial content associated with physical inactivity are not required for the induction of insulin resistance, this could predispose individuals to the detrimental effects of a high-lipid environment. Conversely, exercise-training induced mitochondrial biogenesis has been implicated in the protective effects of exercise. Given mitochondrial biology may represent a point of convergence linking impaired insulin sensitivity in both scenarios of chronic overfeeding and physical inactivity, this review aims to describe the interaction between mitochondrial biology, physical (in)activity and lipid metabolism within the context of insulin signalling.

ANKLE1 cleaves mitochondrial DNA and contributes to cancer risk by promoting apoptosis resistance and metabolic dysregulation

Alleles within the chr19p13.1 locus are associated with increased risk of both ovarian and breast cancer and increased expression of the ANKLE1 gene. ANKLE1 is molecularly characterized as an endonuclease that efficiently cuts branched DNA and shuttles between the nucleus and cytoplasm. However, the role of ANKLE1 in mammalian development and homeostasis remains unknown. In normal development ANKLE1 expression is limited to the erythroblast lineage and we found that ANKLE1’s role is to cleave the mitochondrial genome during erythropoiesis. We show that ectopic expression of ANKLE1 in breast epithelial-derived cells leads to genome instability and mitochondrial DNA (mtDNA) cleavage. mtDNA degradation then leads to mitophagy and causes a shift from oxidative phosphorylation to glycolysis (Warburg effect). Moreover, mtDNA degradation activates STAT1 and expression of epithelial-mesenchymal transition (EMT) genes. Reduction in mitochondrial content contributes to apoptosis resistance, which may allow precancerous cells to avoid apoptotic checkpoints and proliferate. These findings provide evidence that ANKLE1 is the causal cancer susceptibility gene in the chr19p13.1 locus and describe mechanisms by which higher ANKLE1 expression promotes cancer risk.

The phosphorylation of AMPKβ1 is critical for increasing autophagy and maintaining mitochondrial homeostasis in response to fatty acids

Fatty acids are vital for the survival of eukaryotes, but when present in excess can have deleterious consequences. The AMP-activated protein kinase (AMPK) is an important regulator of multiple branches of metabolism. Studies in purified enzyme preparations and cultured cells have shown that AMPK is allosterically activated by small molecules as well as fatty acyl-CoAs through a mechanism involving Ser108 within the regulatory AMPK β1 isoform. However, the in vivo physiological significance of this residue has not been evaluated. In the current study, we generated mice with a targeted germline knock-in (KI) mutation of AMPKβ1 Ser108 to Ala (S108A-KI), which renders the site phospho-deficient. S108A-KI mice had reduced AMPK activity (50 to 75%) in the liver but not in the skeletal muscle. On a chow diet, S108A-KI mice had impairments in exogenous lipid-induced fatty acid oxidation. Studies in mice fed a high-fat diet found that S108A-KI mice had a tendency for greater glucose intolerance and elevated liver triglycerides. Consistent with increased liver triglycerides, livers of S108A-KI mice had reductions in mitochondrial content and respiration that were accompanied by enlarged mitochondria, suggestive of impairments in mitophagy. Subsequent studies in primary hepatocytes found that S108A-KI mice had reductions in palmitate- stimulated Cpt1a and Ppargc1a mRNA, ULK1 phosphorylation and autophagic/mitophagic flux. These data demonstrate an important physiological role of AMPKβ1 Ser108 phosphorylation in promoting fatty acid oxidation, mitochondrial biogenesis and autophagy under conditions of high lipid availability. As both ketogenic diets and intermittent fasting increase circulating free fatty acid levels, AMPK activity, mitochondrial biogenesis, and mitophagy, these data suggest a potential unifying mechanism which may be important in mediating these effects.

The Impact of Training on the Loss of Cardiorespiratory Fitness in Aging Masters Endurance Athletes.

Elite masters endurance athletes are considered models of optimal healthy aging due to the maintenance of high cardiorespiratory fitness (CRF) until old age. Whereas a drop in VO2max in masters athletes has been broadly investigated, the modifying impact of training still remains a matter of debate. Longitudinal observations in masters endurance athletes demonstrated VO2max declines between −5% and −46% per decade that were closely related to changes in training volume. Here, using regression analyses, we show that 54% and 39% of the variance in observed VO2max decline in male and female athletes, respectively is explained by changes in training volume. An almost linear VO2max decrease was observed in studies on young and older athletes, as well as non-athletes, starting a few days after training cessation, with a decline of as much as −20% after 12 weeks. Besides a decline in stroke volume and cardiac output, training cessation was accompanied by considerable reductions in citrate synthase and succinate dehydrogenase activity (reduction in mitochondrial content and oxidative capacity). This reduction could largely be rescued within similar time periods of training (re)uptake. It is evident that training reduction or cessation leads to a considerably accelerated VO2max drop, as compared to the gradual aging-related VO2max decline, which can rapidly nullify many of the benefits of preceding long-term training efforts.

Effects of total knee arthroplasty on skeletal muscle structure and function at the cellular, organellar, and molecular levels.

Total knee arthroplasty (TKA) is an important treatment option for knee osteoarthritis (OA) that improves self-reported pain and physical function, but objectively measured physical function typically remains reduced for years after surgery due, in part, to precipitous reductions in lower extremity neuromuscular function early after surgery. The present study examined intrinsic skeletal muscle adaptations during the first 5 weeks post-TKA to identify skeletal muscle attributes that may contribute to functional disability. Patients with advanced stage knee OA were evaluated prior to TKA and 5 weeks after surgery. Biopsies of the vastus lateralis were performed to assess muscle fiber size, contractility, and mitochondrial content, along with assessments of whole muscle size and function. TKA was accompanied by marked reductions in whole muscle size and strength. At the fiber (i.e., cellular) level, TKA caused profound muscle atrophy that was approximately twofold higher than that observed at the whole muscle level. TKA markedly reduced muscle fiber force production, contractile velocity, and power production, with force deficits persisting in myosin heavy chain (MHC) II fibers after expression relative to fiber size. Molecular level assessments suggest reduced strongly bound myosin-actin cross bridges and myofilament lattice stiffness as a mechanism underlying reduced force per unit fiber size. Finally, marked reductions in mitochondrial content were apparent and more prominent in the subsarcolemmal compartment. Our study represents the most comprehensive evaluation of skeletal muscle cellular adaptations to TKA and uncovers novel effects of TKA on muscle fiber size and intrinsic contractility early after surgery that may contribute to functional disability.NEW & NOTEWORTHY We report the first evaluation of the effects of total knee arthroplasty (TKA) on skeletal muscle at the cellular and subcellular levels. We found marked effects of TKA to cause skeletal muscle fiber atrophy and contractile dysfunction in older adults, as well as molecular mechanisms underlying impaired contractility. Our results reveal profound effects of TKA on muscle fiber size and intrinsic contractility early after surgery that may contribute to functional disability.

Role of TFE3 in Mitochondrial Adaptations to Skeletal Muscle Disuse

Prolonged muscle disuse is accompanied by a phenotypic shift characterized by declines in mitochondrial content and function within skeletal muscle. This loss in mitochondrial content can be partially attributed to elevations in various catabolic processes that occur with atrophy. One of these is termed mitophagy, a selective form of cellular recycling (autophagy) whereby dysfunctional mitochondria are degraded via lysosomes. TFEB and TFE3 are key transcription factors that regulate lysosomes by activating the expression of various lysosomal genes. Loss of TFE3 has been associated with depressed levels of autophagy and with mitochondrial dysfunction. It is speculated that these functional impairments are due to diminished clearance via the lysosomes, however to date this has not been examined. We hypothesized that the loss of TFE3 would amplify the mitochondrial dysfunction associated with muscle disuse, while paradoxically preserving muscle mass and mitochondrial content. Using a severe model of muscle disuse, sciatic denervation, we observed a 10% loss in hindlimb muscle mass within 7 days of denervation. In the absence of TFE3, a trend for muscle preservation was observed, as these animals lost 20% less muscle mass than WT counterparts. Reduced rates of respiration supported by complex I and II were observed irrespective of genotype, concurrent with elevated ROS emissions, suggesting an impaired oxidative capacity following 7 days of denervation. Surprisingly, TFE3 KO animals did exhibit lower levels of oxidative stress basally, but this too increased following 7 days of denervation, and taken relative to baseline the fold induction of ROS emission was 3 times greater than WT animals. Higher levels of mitophagy flux were observed in the absence of TFE3 basally, which supports the observed 35% decline in mitochondrial content as measured by COX activity, as well as the lower levels of ROS emissions. Following only 1 day of denervation, increases in mitophagy flux were observed in WT animals, while the response in KO animals was clearly attenuated. In the WT animals, denervation led to a 30% reduction in mitochondrial content, however no change was observed in the absence of TFE3. Finally, increases in a number of autophagy‐related markers such Beclin‐1 and ATG7 were observed following denervation irrespective of genotype. However, the mature form of Cathepsin B, a hydrolytic enzyme, was markedly reduced by 55% in the absence of TFE3 and did not increase to the same extent as WT following 7 days of denervation suggesting an impairment in lysosomal function. Together, our results suggest that TFE3 exerts multiple roles in skeletal muscle plasticity, as a partial mediator of muscle mass, and in the control of lysosomal function and mitophagy in response to the acute stress of muscle disuse.

Skeletal muscle abnormalities in heart failure with preserved ejection fraction.

Almost half of all heart failure (HF) disease burden is due to HF with preserved ejection fraction (HFpEF). The primary symptom in patients with HFpEF, even when well compensated, is severe exercise intolerance and is associated with their reduced quality of life. Recently, studies showed that HFpEF patients have multiple skeletal muscle (SM) abnormalities, and these are associated with decreased exercise intolerance. The SM abnormalities are likely intrinsic to the HFpEF syndrome, not a secondary consequence of an epiphenomenon. These abnormalities are decreased muscle mass, reduced type I (oxidative) muscle fibers, and reduced type I-to-type II fiber ratio as well as a reduced capillary-to-fiber ratio, abnormal fat infiltration into the thigh SM, increased levels of atrophy genes and proteins, reduction in mitochondrial content, and rapid depletion of high-energy phosphate during exercise with markedly delayed repletion of high-energy phosphate during recovery in mitochondria. In addition, patients with HFpEF have impaired nitric oxide bioavailability, particularly in the microvasculature. These SM abnormalities may be responsible for impaired diffusive oxygen transport and/or impaired SM oxygen extraction. To date, exercise training (ET) and caloric restriction are some of the interventions shown to improve outcomes in HFpEF patients. Improvements in exercise tolerance following aerobic ET are largely mediated through peripheral SM adaptations with minimal change in central hemodynamics and highlight the importance of targeting SM to improve exercise intolerance in HFpEF. Focusing on the abnormalities mentioned above may improve the clinical condition of patients with HFpEF.

Revisiting the contribution of mitochondrial biology to the pathophysiology of skeletal muscle insulin resistance.

While the etiology of type 2 diabetes is multifaceted, the induction of insulin resistance in skeletal muscle is a key phenomenon, and impairments in insulin signaling in this tissue directly contribute to hyperglycemia. Despite the lack of clarity regarding the specific mechanisms whereby insulin signaling is impaired, the key role of a high lipid environment within skeletal muscle has been recognized for decades. Many of the proposed mechanisms leading to the attenuation of insulin signaling - namely the accumulation of reactive lipids and the pathological production of reactive oxygen species (ROS), appear to rely on this high lipid environment. Mitochondrial biology is a central component to these processes, as these organelles are almost exclusively responsible for the oxidation and metabolism of lipids within skeletal muscle and are a primary source of ROS production. Classic studies have suggested that reductions in skeletal muscle mitochondrial content and/or function contribute to lipid-induced insulin resistance; however, in recent years the role of mitochondria in the pathophysiology of insulin resistance has been gradually re-evaluated to consider the biological effects of alterations in mitochondrial content. In this respect, while reductions in mitochondrial content are not required for the induction of insulin resistance, mechanisms that increase mitochondrial content are thought to enhance mitochondrial substrate sensitivity and submaximal adenosine diphosphate (ADP) kinetics. Thus, this review will describe the central role of a high lipid environment in the pathophysiology of insulin resistance, and present both classic and contemporary views of how mitochondrial biology contributes to insulin resistance in skeletal muscle.

Effect of inflammation-mediated endothelial metabolic shift on endothelial barrier function

Abstract Background The integrity of the endothelial cell barrier of the microvasculature is compromised by inflammation. The increased vascular permeability leads to tissue injury and organ dysfunction. In recent years, considerable advances have been made in the understanding of signalling mechanisms regulating the endothelial barrier integrity. The role of endothelial metabolism as a modulator of endothelial barrier integrity is not yet well-studied. The aim of the present study was to investigate the effect of inflammation on endothelial metabolism and its role in the maintenance of endothelial barrier integrity. Methods The study was carried out on cultured human umbilical vein endothelial cells and rat coronary microvascular endothelial cells. Inflammatory condition was simulated by treating cells with low concentrations (1 ng/mL) of TNFα for 24h. Endothelial barrier function was analysed by measuring the flux of albumen through endothelial monolayers cultured on filter membranes. Gene expression was analysed by qPCR-based assays. The capacity of endothelial cells for maximal ATP synthesis rate was investigated by the real-time live-cell imaging using FRET-based ATP-biosensor (live cell FRET). Total cellular ATP concentration was measured using luminescence-based commercial kit (ATPLite, PerkinElmer). Mitochondrial mass was analysed by the ratio of mitochondrial DNA (mtDNA) to nuclear DNA (nDNA). The cellular glucose uptake was measured by fluorescent microscopy using a fluorescent analogue of glucose (2-NBDG). Results Treatment of human endothelial cells with TNFα resulted in significant suppression of mitochondrial and upregulation of glycolytic ATP synthesis rate, suggesting a metabolic switch. This was accompanied by a reduction in mitochondrial content (mtDNA/nDNA), reduction in total cellular ATP levels, an enhanced expression of glycolytic enzymes 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) and phosphofructokinase 1 (PFK1), and enhanced glucose uptake by endothelial cells (n=5; p<0.05 for all parameters tested). Moreover, TNFα caused a 3-fold increase in endothelial permeability. Pharmacological inhibition of glycolysis either by partial replacement of glucose with 2-deoxy glucose (2DG) or an inhibition of PFKFB3 resulted in further worsening (a 5-fold increase in permeability) of TNFα-induced endothelial barrier failure. On the other hand pharmacological activation of AMPK, a potent inducer of mitochondrial biogenesis, could attenuate TNFα-induced but not 2DG-induced endothelial hyperpermeability. Conclusion The study demonstrates that TNFα induces metabolic switch towards glycolysis in endothelial cells. Moreover, the data suggest that upregulation of glycolysis may serve as an endogenous metabolic adaptation to the TNFα-induced suppression of mitochondrial ATP synthesis, which protects endothelial barrier integrity. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Justus-Liebig University GiessenDZHK (German Centre for Cardiovascular Research), partner site Rhein-Main, Bad Nauheim, Germany

The erythropoietin receptor expressed in skeletal muscle is essential for mitochondrial biogenesis and physiological exercise

Erythropoietin (EPO) is a haematopoietic hormone that regulates erythropoiesis, but the EPO-receptor (EpoR) is also expressed in non-haematopoietic tissues. Stimulation of the EpoR in cardiac and skeletal muscle provides protection from various forms of pathological stress, but its relevance for normal muscle physiology remains unclear. We aimed to determine the contribution of the tissue-specific EpoR to exercise-induced remodelling of cardiac and skeletal muscle. Baseline phenotyping was performed on left ventricle and m. gastrocnemius of mice that only express the EpoR in haematopoietic tissues (EpoR-tKO). Subsequently, mice were caged in the presence or absence of a running wheel for 4 weeks and exercise performance, cardiac function and histological and molecular markers for physiological adaptation were assessed. While gross morphology of both muscles was normal in EpoR-tKO mice, mitochondrial content in skeletal muscle was decreased by 50%, associated with similar reductions in mitochondrial biogenesis, while mitophagy was unaltered. When subjected to exercise, EpoR-tKO mice ran slower and covered less distance than wild-type (WT) mice (5.5 ± 0.6 vs. 8.0 ± 0.4 km/day, p < 0.01). The impaired exercise performance was paralleled by reductions in myocyte growth and angiogenesis in both muscle types. Our findings indicate that the endogenous EPO-EpoR system controls mitochondrial biogenesis in skeletal muscle. The reductions in mitochondrial content were associated with reduced exercise capacity in response to voluntary exercise, supporting a critical role for the extra-haematopoietic EpoR in exercise performance.

Investigation of Skeletal Muscle Mitochondrial Function Following an Ultramarathon: A Case Study in Monozygotic Twins

The primary objective of this case study was to investigate the changes in skeletal muscle mitochondria that occur in response to a 100-km ultramarathon in monozygotic twins. Secondarily, we sought to determine if periodically consuming a low-carbohydrate, high-fat diet (LCHFD) during training would affect these changes. To accomplish these objectives, one pair of male monozygotic twins ran 100 km side-by-side on treadmills. During 8 weeks of training leading up to this event, twin 1 (T1) and twin 2 (T2) consumed a calorie and macronutrient-matched, high-carbohydrate diet, with the exception of 2 consecutive days per week for the final 4 weeks where T2 consumed a calorie and protein-matched LCHFD. Training load was matched between the subjects, most of which they completed together. The subjects were extremely well-matched in VO2 max (61.2 and 60.3 ml/kg/min), BMI (20.4 and 20.6 kg/m2), and body mass (62 and 61.9 kg). During the 100-km run, food and fluid intake were matched, as well as the pace of the run. The run took 16 hours and 3 minutes to complete. Blood draws and muscle biopsies from the vastus lateralis were collected 4 weeks prior to the run, as well as 4- and 52-hours post-run. During the last 4 weeks of training, T2 lost an average of ~1 kg of body weight (BW) over the two days of LCHFD each week (comparing BW of 4 consecutive Mondays to Wednesdays, p=0.02), which was regained two days after reintroduction of carbohydrate, consistent with fluctuating glycogen levels. Though T2 trained with periodically low glycogen levels, respiratory-exchange ratio was no different from T1 throughout the 100-km run. Four hours post-run, state 3, uncoupled, and fatty acid-supported mitochondrial respiration were reduced in T1 by 22.3%, 34.1% and 25.7%, respectively, and by 28.4%, 49.4% and 39.9% in T2, which was associated with a decrease of 15-20% in citrate synthase (CS) activity. Both twins exhibited decreases in mitochondrial complex proteins, though this was more marked in T2. Blood CPK, CRP, and AST levels were also elevated post-run. Possibly the most novel finding from this study was that 52 hours post-run, respiratory capacity, mitochondrial proteins, CS activity and blood markers showed signs of rapid recovery. It is currently unclear by what mechanism mitochondrial respiration and content was reduced 4 hours post-run, or how it recovered in 2 days. Interestingly, maximal mitochondrial ROS production (glutamate, malate and succinate-supported) was decreased post-run at both timepoints but was increased compared to baseline with the addition of ADP (12.5 μM – 100 μM). This suggests that the mitochondria were less sensitive to ADP-induced reductions in ROS. Neither diet resulted in better preservation of mitochondrial function 4-hours post-run, though T2 showed some signs of supercompensation 52 hours-post run. This study is valuable because it provides evidence of an acute reduction in mitochondrial content and function following ultra-endurance activity, followed by rapid recovery. This study was approved by the Brigham Young University Institutional Review Board and conformed to the principles of human experimentation set by the Declaration of Helsinki.

The lifecycle of skeletal muscle mitochondria in obesity.

Skeletal muscle possesses dramatic metabolic plasticity that allows for the rapid adaptation in cellular energy transduction to meet the demands of the organism. Obesity elicits changes in skeletal muscle structure and function, resulting in the accumulation of intramuscular lipids. The accumulation of intramuscular lipids in obesity is associated with impaired skeletal muscle mitochondrial content and function. Mitochondria exist as a dynamic network that is regulated by the processes of biogenesis, fusion, fission, and mitophagy. In this review, we outline adaptations in molecular pathways that regulate mitochondrial structure and function in obesity. We highlight the emerging role of dysregulated skeletal muscle macroautophagy and mitochondrial turnover in obesity. Future research should further elucidate the role of mitophagy in observed reductions in mitochondrial content and function during obesity.

Hypoxia-induced mitochondrial abnormalities in cells of the placenta.

Impaired utero-placental perfusion is a well-known feature of early preeclampsia and is associated with placental hypoxia and oxidative stress. Although aberrations at the level of the mitochondrion have been implicated in PE pathophysiology, whether or not hypoxia-induced mitochondrial abnormalities contribute to placental oxidative stress is unknown. We explored whether abnormalities in mitochondrial metabolism contribute to hypoxia-induced placental oxidative stress by using both healthy term placentae as well as a trophoblast cell line (BeWo cells) exposed to hypoxia. Furthermore, we explored the therapeutic potential of the antioxidants MitoQ and quercetin in preventing hypoxia-induced placental oxidative stress. Both in placental explants as well as BeWo cells, hypoxia resulted in reductions in mitochondrial content, decreased abundance of key molecules involved in the electron transport chain and increased expression and activity of glycolytic enzymes. Furthermore, expression levels of key regulators of mitochondrial biogenesis were decreased while the abundance of constituents of the mitophagy, autophagy and mitochondrial fission machinery was increased in response to hypoxia. In addition, placental hypoxia was associated with increased oxidative stress, inflammation, and apoptosis. Moreover, experiments with MitoQ revealed that hypoxia-induced reactive oxygen species originated from the mitochondria in the trophoblasts. This study is the first to demonstrate that placental hypoxia is associated with mitochondrial-generated reactive oxygen species and significant alterations in the molecular pathways controlling mitochondrial content and function. Furthermore, our data indicate that targeting mitochondrial oxidative stress may have therapeutic benefit in the management of pathologies related to placental hypoxia.

Rosiglitazone Protects Endothelial Cells From Irradiation-Induced Mitochondrial Dysfunction.

Background and PurposeUp to 50–60% of all cancer patients receive radiotherapy as part of their treatment strategy. However, the mechanisms accounting for increased vascular risks after irradiation are not completely understood. Mitochondrial dysfunction has been identified as a potential cause of radiation-induced atherosclerosis.Materials and MethodsAssays for apoptosis, cellular metabolism, mitochondrial DNA content, functionality and morphology were used to compare the response of endothelial cells to a single 2 Gy dose of X-rays under basal conditions or after pharmacological treatments that either reduced (EtBr) or increased (rosiglitazone) mitochondrial content.ResultsExposure to ionizing radiation caused a persistent reduction in mitochondrial content of endothelial cells. Pharmacological reduction of mitochondrial DNA content rendered endothelial cells more vulnerable to radiation-induced apoptosis, whereas rosiglitazone treatment increased oxidative metabolism and redox state and decreased the levels of apoptosis after irradiation.ConclusionPre-existing mitochondrial damage sensitizes endothelial cells to ionizing radiation-induced mitochondrial dysfunction. Rosiglitazone protects endothelial cells from the detrimental effects of radiation exposure on mitochondrial metabolism and oxidative stress. Thus, our findings indicate that rosiglitazone may have potential value as prophylactic for radiation-induced atherosclerosis.

Pulmonary inflammation-induced alterations in key regulators of mitophagy and mitochondrial biogenesis in murine skeletal muscle

BackgroundBoth mitophagy, a selective mechanism for clearance of mitochondria, and mitochondrial biogenesis are key processes determining mitochondrial content and oxidative capacity of the musculature. Abnormalities in these processes could therefore contribute to deterioration of peripheral muscle oxidative capacity as observed in e.g. chronic obstructive pulmonary disease. Although it has been suggested that inflammatory mediators can modulate both mitophagy and mitochondrial biogenesis, it is unknown whether acute pulmonary inflammation affects these processes in oxidative and glycolytic skeletal muscle in vivo. Therefore, we hypothesised that molecular signalling patterns of mitochondrial breakdown and biogenesis temporally shift towards increased breakdown and decreased biogenesis in the skeletal muscle of mice exposed to one single bolus of IT-LPS, as a model for acute lung injury and pulmonary inflammation.MethodsWe investigated multiple important constituents and molecular regulators of mitochondrial breakdown, biogenesis, dynamics, and mitochondrial content in skeletal muscle over time in a murine (FVB/N background) model of acute pulmonary- and systemic inflammation induced by a single bolus of intra-tracheally (IT)-instilled lipopolysaccharide (LPS). Moreover, we compared the expression of these constituents between gastrocnemius and soleus muscle.ResultsBoth in soleus and gastrocnemius muscle, IT-LPS instillation resulted in molecular patterns indicative of activation of mitophagy. This coincided with modulation of mRNA transcript abundance of genes involved in mitochondrial fusion and fission as well as an initial decrease and subsequent recovery of transcript levels of key proteins involved in the molecular regulation of mitochondrial biogenesis. Moreover, no solid differences in markers for mitochondrial content were found.ConclusionsThese data suggest that one bolus of IT-LPS results in a temporal modulation of mitochondrial clearance and biogenesis in both oxidative and glycolytic skeletal muscle, which is insufficient to result in a reduction of mitochondrial content.

Supplementation with dietary ω-3 mitigates immobilization-induced reductions in skeletal muscle mitochondrial respiration in young women.

Omega-3 (ω-3) supplementation attenuates immobilization-induced atrophy; however, the underlying mechanisms remain unclear. Since mitochondrial dysfunction and oxidative stress have been implicated in muscle atrophy, we examined whether ω-3 supplementation could mitigate disuse-mediated mitochondrial dysfunction. Healthy young women (age = 22 ± 3 yr) randomly received control (n = 9) or ω-3 supplementation (n = 11; 3 g eicosapentaenoic acid, 2 g docosahexaenoic acid) for 4 wk prior to and throughout 2 wk of single-limb immobilization. Biopsies were performed before and after 3 and 14 d of immobilization for the assessment of mitochondrial respiration, H2O2 emission, and markers of ADP transport/lipid metabolism. In controls, immobilization rapidly (3 d) reduced (∼20%) ADP-stimulated mitochondrial respiration without altering ADP sensitivity or the abundance of mitochondrial proteins. Extending immobilization to 14 d did not further reduce mitochondrial coupled respiration; however, unlike following 3 d, mitochondrial proteins were reduced ∼20%. In contrast, ω-3 supplementation prevented immobilization-induced reductions in mitochondrial content and respiration throughout the immobilization period. Regardless of dietary supplement, immobilization did not alter mitochondrial H2O2 emission in the presence or absence of ADP, markers of cellular redox state, mitochondrial lipid-supported respiration, or lipid-related metabolic proteins. These data highlight the rapidity of mitochondrial adaptations in response to muscle disuse, challenge the necessity for increased oxidative stress during inactivity, and establish that ω-3 supplementation preserves oxidative phosphorylation function and content during immobilization.-Miotto, P. M., McGlory, C., Bahniwal, R., Kamal, M., Phillips, S. M., Holloway, G. P. Supplementation with dietary ω-3 mitigates immobilization-induced reductions in skeletal muscle mitochondrial respiration in young women.