Red Gastrocnemius Research Articles

Effects of An Omega‐3 High‐Fat Diet on Skeletal Muscle Protein Degradation in Glucocorticoid‐Induced Muscle Wasting

Treatment with synthetic glucocorticoids induces muscle atrophy and are commonly used in the treatment of a variety of inflammatory and autoimmune diseases. Omega‐3 (n‐3) polyunsaturated fatty acids elicit beneficial effects in several muscle atrophy conditions. Therefore, the purpose of this study was to determine if a high‐fat diet rich in n‐3 is protective of glucocorticoid‐induced protein degradation. Male wild type C57BL/6 mice were randomized into two groups: n‐6 (45% fat 177.5 g lard) and n‐3 (45% fat 177.5 g Menhaden oil). After 4 weeks on their diets, groups were divided to receive either daily injections of 3 mg/kg/day dexamethasone (Dex) or sterile phosphate buffered saline, for 1 week while continuing diets. At sacrifice the gastrocnemius muscle was weighed and dissected into red and white portions before being snap frozen and processed for RNA extraction and protein extraction. Dex reduced gastrocnemius weight by 12% independently of diet. Protein degradation signaling in the white gastrocnemius was altered by Dex with increased atrogin‐1 expression (p=0.004) without a change in muscle RING finger 1 (MuRF‐1) expression (p=0.15). There was a large effect of Dex to decreased phosphorylation of forkhead box transcription factor O3a (FOXO3a) (Cohen’s d=1.42), increased phosphorylation of glycogen synthase kinase 3β (GSK‐3β) (Cohen’s d=1.42). However, the negative effects of dexamethasone were not attenuated by an n‐3 high‐fat diet. There was no effect of Dex or diet on phosphorylation of FOXO3a (p=0.17) or GSK3β (p=0.60) in red gastrocnemius muscle. These data support the detrimental effects of dexamethasone on muscle atrophy and report no benefit of an n‐3 high‐fat diet.Support or Funding InformationFunding provided by University of Memphis School of Health Studies

Strenuous Acute Exercise Induces Slow and Fast Twitch-Dependent NADPH Oxidase Expression in Rat Skeletal Muscle.

The enzymatic complex Nicotinamide Adenine Dinucleotide Phosphate (NADPH) oxidase (NOx) may be the principal source of reactive oxygen species (ROS). The NOX2 and NOX4 isoforms are tissue-dependent and are differentially expressed in slow-twitch fibers (type I fibers) and fast-twitch fibers (type II fibers) of skeletal muscle, making them different markers of ROS metabolism induced by physical exercise. The aim of this study was to investigate NOx signaling, as a non-adaptive and non-cumulative response, in the predominant fiber types of rat skeletal muscles 24 h after one strenuous treadmill exercise session. The levels of mRNA, reduced glycogen, thiol content, NOx, superoxide dismutase, catalase, glutathione peroxidase activity, and PPARGC1α and SLC2A4 gene expression were measured in the white gastrocnemius (WG) portion, the red gastrocnemius (RG) portion, and the soleus muscle (SOL). NOx activity showed higher values in the SOL muscle compared to the RG and WG portions. The same was true of the NOX2 and NOX4 mRNA levels, antioxidant enzymatic activities, glycogen content. Twenty-four hours after the strenuous exercise session, NOx expression increased in slow-twitch oxidative fibers. The acute strenuous exercise condition showed an attenuation of oxidative stress and an upregulation of antioxidant activity through PPARGC1α gene activity, antioxidant defense adaptations, and differential gene expression according to the predominant fiber type. The most prominent location of detoxification (indicated by NOX4 activation) in the slow-twitch oxidative SOL muscle was the mitochondria, while the fast-twitch oxidative RG portion showed a more cytosolic location. Glycolytic metabolism in the WG portion suggested possible NOX2/NOX4 non-regulation, indicating other possible ROS regulation pathways.

PGC-1α plays a pivotal role in simvastatin-induced exercise impairment in mice.

Statins decrease cardiovascular complications, but can induce myopathy. Here, we explored the implication of PGC-1α in statin-associated myotoxicity. We treated PGC-1α knockout (KO), PGC-1α overexpression (OE) and wild-type (WT) mice orally with 5mg simvastatin kg-1 day-1 for 3weeks and assessed muscle function and metabolism. In WT and KO mice, but not in OE mice, simvastatin decreased grip strength, maximal running distance and vertical power assessed by ergometry. Post-exercise plasma lactate concentrations were higher in WT and KO compared to OE mice. In glycolytic gastrocnemius, simvastatin decreased mitochondrial respiration, increased mitochondrial ROS production and free radical leak in WT and KO, but not in OE mice. Simvastatin increased mRNA expression of Sod1 and Sod2 in glycolytic and oxidative gastrocnemius of WT, but decreased it in KO mice. OE mice had a higher mitochondrial DNA content in both gastrocnemius than WT or KO mice and simvastatin exhibited a trend to decrease the citrate synthase activity in white and red gastrocnemius in all treatment groups. Simvastatin showed a trend to decrease the mitochondrial volume fraction in both muscle types of all treatment groups. Mitochondria were smaller in WT and KO compared to OE mice and simvastatin further reduced the mitochondrial size in WT and KO mice, but not in OE mice. Simvastatin impairs skeletal muscle function, muscle oxidative metabolism and mitochondrial morphology preferentially in WT and KO mice, whereas OE mice appear to be protected, suggesting a role of PGC-1α in preventing simvastatin-associated myotoxicity.

Maternal diet-induced obesity alters muscle mitochondrial function in offspring without changing insulin sensitivity.

In utero overnutrition can predispose offspring to metabolic disease. Although the mechanisms are unclear, increased oxidative stress accelerating cellular aging has been shown to play a role. Mitochondria are the main site of reactive oxygen species (ROS) production in most cell types. Levels of ROS and the risk for oxidative damage are dictated by the balance between ROS production and antioxidant defense mechanisms. Originally considered as toxic species, physiologic levels of ROS are now known to be essential cell signaling molecules. Using a model of maternal overnutrition in C57BL6N mice, we investigate the mechanisms involved in the development of insulin resistance (IR) in muscle. In red and white gastrocnemius muscles of offspring, we are the first to report characteristics of oxidative phosphorylation, H2O2 production, activity of mitoflashes, and electron transport chain supercomplex formation. Results demonstrate altered mitochondrial function with reduced response to glucose in offspring of mice fed a high-fat and high-sucrose diet, increases in mitochondrial leak respiration, and a reduction in ROS production in red gastrocnemius in response to palmitoyl carnitine. We also demonstrate differences in supercomplex formation between red and white gastrocnemius, which may be integral to fiber-type specialization. We conclude that in this model of maternal overnutrition, mitochondrial alterations occur before the development of IR.-McMurray, F., MacFarlane, M., Kim, K., Patten, D. A., Wei-LaPierre, L., Fullerton, M. D., Harper, M. E. Maternal diet-induced obesity alters muscle mitochondrial function in offspring without changing insulin sensitivity.

Mitochondrial respiration and H2O2 emission in saponin-permeabilized murine diaphragm fibers: optimization of fiber separation and comparison to limb muscle.

Diaphragm abnormalities in aging or chronic diseases include impaired mitochondrial respiration and H2O2 emission, which can be measured using saponin-permeabilized muscle fibers. Mouse diaphragm presents a challenge for isolation of fibers due to relatively high abundance of connective tissue in healthy muscle that is exacerbated in disease states. We tested a new approach to process mouse diaphragm for assessment of intact mitochondria respiration and ROS emission in saponin-permeabilized fibers. We used the red gastrocnemius (RG) as "standard" limb muscle. Markers of mitochondrial content were two- to fourfold higher in diaphragm (Dia) than in RG (P < 0.05). Maximal O2 consumption (JO2: pmol·s-1·mg-1) in Dia was higher with glutamate, malate, and succinate (Dia 399 ± 127, RG 148 ± 60; P < 0.05) and palmitoyl-CoA + carnitine (Dia 15 ± 5, RG 7 ± 1; P < 0.05) than in RG, but not different between muscles when JO2 was normalized to citrate synthase activity. Absolute JO2 for Dia was two- to fourfold higher than reported in previous studies. Mitochondrial JH2O2 was higher in Dia than in RG (P < 0.05), but lower in Dia than in RG when JH2O2 was normalized to citrate synthase activity. Our findings are consistent with an optimized diaphragm preparation for assessment of intact mitochondria in permeabilized fiber bundles. The data also suggest that higher mitochondrial content potentially makes the diaphragm more susceptible to "mitochondrial onset" myopathy. Overall, the new approach will facilitate testing and understanding of diaphragm mitochondrial function in mouse models that are used to advance biomedical research and human health.

H1/H2 Histamine Receptor Blockade Alters the Glutathione Redox Status in Skeletal Muscle Following a Bout of Prolonged Exercise

The effects of an acute bout of prolonged exercise on muscle include increases in oxidative metabolism and altered cellular redox environment. Histamine mediates many acute exercise responses, including sustained vasodilation and associated delivery of substrates to muscle. Previously, we demonstrated that H1 and H2 histamine receptor blockade with oral antihistamines resulted in a diminished capacity for mitochondrial H2O2 emission in skeletal muscle following an acute bout of prolonged exercise. Here, we tested the hypothesis that along with diminishing mitochondrial H2O2 emission, H1 and H2 histamine receptor blockade would attenuate exercise‐induced changes in skeletal muscle glutathione redox status. Adult male Wistar rats (83 ± 5.5 days old; 440.04 ± 40.70 g) were assigned to one of four groups (n = 12/group): exercise, in the form of one hour of continuous treadmill running at 20 cm/sec (Group 1); mass‐specific oral gavage with H1 antagonist fexofenadine (7.11 mg/kg) and H2 antagonist ranitidine (3.95 mg/kg) one hour prior to the same exercise protocol (Group 2); a group receiving oral H1 and H2 antagonists, but not exercised (Group 3); and a control group, which received neither antihistamines nor exercise (Group 4). An hour post‐exercise, total, reduced (GSH) and oxidized glutathione (GSSG) were assessed in red gastrocnemius (mixed fiber types) and soleus (oxidative) skeletal muscles. Redox status was expressed as GSH/GSSG. While no changes were observed in total glutathione for any treatment in either muscle, in soleus, antihistamine treatment, but not exercise decreased GSH/GSSG (main effect; p &lt; 0.05). In red gastrocnemius, exercise, but not antihistamine treatment decreased GSH/GSSH (main effect; p &lt; 0.05). These observations trend with those of a blunted capacity for mitochondrial H2O2 emission in skeletal muscle with antihistamine treatment and suggest an altered redox response to exercise following H1/H2 histamine receptor blockade in soleus. Given the widespread and routine use of antihistamines, further research into the effects of these antagonists on redox status in muscle is warranted.Support or Funding InformationNatural Sciences and Engineering Research Council of Canada, Nova Scotia Health Research Foundation, Canada Foundation for Innovation.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mitochondrial Respiration and H2O2 Emission in Saponin‐permeabilized Murine Diaphragm Fibers: Optimization of Fiber Separation and Comparison to Limb Muscle

Chronic diseases such as cancer, COPD, and heart failure impair diaphragm metabolism and mitochondrial function. Mice are used extensively to replicate disease conditions and offer the advantage of studying genetically modified animals. Mouse diaphragm contains a high abundance of connective tissue, with relatively thin (15–25 μm diameter) and fragile fibers. In preliminary studies, we discovered that standard fiber separation approaches utilized for limb muscles are unsuitable for the diaphragm. Thus, the purpose of this study was to optimize a protocol for diaphragm (Dia) fiber bundle preparation to evaluate the mitochondrial respiration and reactive oxygen species emission. We used the red gastrocnemius (Gast) muscle as a ‘standard’ for comparison.Michaelis‐Menten kinetics of ADP‐stimulated O2 consumption with complex I substrates (JO2 in pmol/s/mg wet weight) showed 3‐fold higher Vmax in Dia than Gast (Gast 69 ± 18, Dia 211 ± 20; p &lt; 0.05, n = 9 mice), whereas Km was not significantly different. Maximal JO2 in Dia was 3‐fold higher with complex I + II substrates (Gast 148 ± 25, Dia 387 ± 45; p &lt; 0.05), and 2‐fold higher with palmitoyl‐CoA + carnitine (Gast 7 ± 0, Dia 15 ± 2, p &lt; 0.05) compared to Gast. Baseline JH2O2 (pmol/min/mg wet weight) was higher in Dia (Gast 0.8 ± 0.2, Dia 1.7 ± 0.3; p &lt; 0.05), but succinate‐induced JH2O2 was not different between muscles. Citrate synthase activity was 3‐fold higher in Dia (μmol/min/mg protein: Gast 43 ± 3, Dia 118 ± 4; p &lt; 0.05). The protein abundance of electron transport chain complexes I‐V were 2–4 fold higher in the Dia than Gast (p &lt; 0.05). When we normalized maximal JO2 by citrate synthase activity, there were no differences between Dia and Gast for complex I and complex I + II substrates. JH2O2 normalized to citrate synthase activity was higher for Gast at baseline (pmol/min/mg wet weight/U CS: Gast 2.1 ± 0.3, Dia 0.3 ± 0.1; p &lt; 0.05), but there was no difference in succinate‐induced JH2O2 normalized to citrate synthase activity.The values for maximal diaphragm mitochondria JO2 in our study are 2–3 fold higher than reported in the literature using the standard ‘limb muscle’ approach for the diaphragm fiber preparation. The similar maximal JO2 normalized to citrate synthase activity between diaphragm and red gastrocnemius muscles suggest that our new approach is valid for the assessment of intact diaphragm mitochondrial function in permeabilized fiber bundles.Support or Funding InformationFunding source: NIH 1R01HL130318‐01This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

6-Gingerol Improves Ectopic Lipid Accumulation, Mitochondrial Dysfunction, and Insulin Resistance in Skeletal Muscle of Ageing Rats: Dual Stimulation of the AMPK/PGC-1α Signaling Pathway via Plasma Adiponectin and Muscular AdipoR1.

This study investigates the dual actions of 6-gingerol in stimulating both plasma adiponectin and muscular adiponectin receptor signaling in naturally ageing rats. Twenty-two-month-old male SD rats were treated with 6-gingerol (0.2 mgkg-1 , once daily) for 7 weeks. 6-Gingerol can attenuate age-associated high plasma triglyceride, glucose, and insulin concentrations under fasting conditions as well as suppress the increase in the HOMA-IR index and inhibit the decrease of muscular p-Akt/Akt protein in ageing rats, which indicates an improvement of systemic and muscular insulin sensitivity. Accompanying these changes, treatment with 6-gingerol attenuates excessive triglyceride accumulation, enhances mitochondrial function, and promotes a transition from a fast- to slow-fiber type and muscle oxidative metabolism in the red gastrocnemius of ageing rats. More importantly, 6-gingerol not only increases the plasma and adipose tissue adiponectin concentrations, but also elevates muscular AdipoR1 expression and activates downstream AMPK phosphorylation as well as upregulates PGC-1α in vivo and in vitro. 6-Gingerol may improve ectopic lipid accumulation, mitochondrial dysfunction, and insulin resistance in skeletal muscle of ageing rats. These effects rely, at least in part, on the elevated plasma adiponectin concentration and muscle AdipoR1 level to dually activate the AMPK/PGC-1α signaling pathway.

Contrasting actions of ginsenosides Rb1 and Rg1 on glucose tolerance in rats.

Ginsenoside profile of Panax ginseng is changing with season and cultivated soil. Yet, dose-response relationship of main ginsenosides on metabolic measures has not been documented in vivo. Here, we examined glucose and insulin responses after an oral glucose challenge (0.5 g/kg body weight) at various doses (0.01, 0.1, 1, and 10 mg/kg of body weight) under acute and chronic Rb1 and Rg1 supplemented conditions. The results show that Rb1 (0.01 and 0.1 mg/kg body weight) increased, whereas Rg1 (0.01 mg/kg body weight) decreased postprandial glucose levels compared with the Vehicle group (P < 0.05). This contrasting effect reduced as dose increased. Both Rb1 and Rg1 decreased the mitochondrial enzyme citrate synthase activity (P < 0.05) together with decreases in glycogen content in red gastrocnemius muscle and body temperature at low doses (P < 0.05), compared with the Vehicle group. These differences also diminished as dosage increases. For reliable ginseng research, dose standardization on Rg1 and Rb1 is essential based on their opposing action and peculiar dose-response relationship. Both major ginsenosides may influence dynamics of mitochondria turnover and alter muscle metabolism.

Effects of intermittent hyperbaric exposure on endurance and interval exercise performance in well-trained mice.

What is the central question of this study? Intermittent hyperbaric exposure (1.3 atmospheres absolute with 20.9% O2 ) enhances endurance capacity by facilitating oxidative and glycolytic capacities in skeletal muscle. It remains unclear whether this strategy enhances endurance performance in well-trained individuals. What is the main finding and its importance? Hyperbaric exposure with endurance training enhanced oxidative and glycolytic capacities and protein levels of mitochondrial transcription factorA, dynamin-related protein-1 and heat shock protein70. Hyperbaric exposure with sprint interval training increased the proportion of typeI muscle fibres and promoted capillary growth and muscle fibre hypertrophy. These results may lead to a new strategy for enhancing exercise capacity in well-trained mice. The study was designed to clarify the mechanisms by which hyperbaric exposure (1.3 atmospheres absolute with 20.9% O2 ) improves endurance and interval exercise capacities in highly trained mice. Male mice in the training group were housed in a cage with a wheel activity device for 7weeks from 5weeks old. Voluntary running markedly increased maximal endurance capacity by 6.4-fold. Trained mice were then subjected to either endurance treadmill training (20-32.5mmin-1 ) or sprint interval training (5s run-10s rest, 30-42.5mmin-1 ) with (HypET or HypSIT, respectively) and without (ET or SIT, respectively) 1h hyperbaric exposure for 4weeks. Maximal endurance capacity was significantly increased by HypET and HypSIT, and maximal interval capacity was significantly enhanced by HypSIT. Peroxisome proliferator-activated receptor gamma coactivator 1-alpha expression levels were markedly increased after HypET and HypSIT. Activity levels of 3-hydroxyacyl-CoA-dehydrogenase, citrate synthase and phosphofructokinase in the red gastrocnemius muscle were increased more by HypET than by ET. Protein levels of mitochondrial transcription factorA, dynamin-related protein-1 and heat shock protein70 were increased more by HypET than by ET. The proportion of type I fibres in the soleus muscle was remarkably increased by HypSIT. Capillary-to-fibre ratio values in the white gastrocnemius were increased more by HypSIT than by SIT. These results suggest that hyperbaric exposure has beneficial effects for endurance and interval training to improve exercise capacity in highly trained mice.

Rapid regulation of substrate use for oxidative phosphorylation during a single session of high intensity interval or aerobic exercises in different rat skeletal muscles.

Different exercise protocols lead to long-term adaptations that are related to increased mitochondrial content through the activation of mitochondrial biogenesis. However, immediate mitochondrial response to exercise and energetic substrate utilization is still unknown. We evaluate the mitochondrial physiology of two types rat skeletal muscle fibres immediately after a single session of high intensity interval exercise (HIIE) or aerobic exercise (AER). We found AER was able to reduce the ATP synthesis dependent oxygen flux in the tibialis (TA) when stimulated by complex I and II substrates. On the other hand, there was an increase of the maximum velocity (Vmax) for glycerol-phosphate oxidation and Vmax and affinity (KM) of palmitoyl-carnitine oxidation (PC). The exercise did not affect oxygen flux coupled to ATP synthesis in red gastrocnemius (RG) but, surprisingly, reduced its affinity for PC, decreasing the apparent catalytic efficiency (Vmax/KM) of oxidation for PC. Neither exercise protocol was able to change the electron transfer system capacity of the mitochondria or markers of mitochondrial content. The AER group had increased H2O2 production compared to the SED and HIIE groups, with the mechanism being predominantly the escape of electrons through reverse flux in complex I and other sites in TA, and only through other sites in RG. There were no changes in the activities of antioxidant enzymes. Our results show that mitochondria from different muscles submitted to distinct exercise protocols show alterations in the specific fluxes of substrate utilization and oxygen metabolism, indicating that the dynamics of mitochondria are linked to the metabolic flexibility.

49 - MnSod deficiency causes significant mitochondrial abnormalities and contractile dysfunction in skeletal muscle, but does not decrease muscle mass

Loss of innervation and consequent disruption of the neuromuscular junction (NMJ) in skeletal muscle results in mitochondrial dysfunction and increased ROS that are associated with muscle atrophy and weakness. To directly test the role of mitochondrial dysfunction and ROS in this series of events independent of loss of innervation, we generated a muscle specific knockout mice in which MnSOD, the primary superoxide anion scavenger in the mitochondrial matrix, was deleted using a Cre-Lox approach with Cre driven by a constitutively active human skeletal actin promoter (HSA-Cre). We measured mitochondrial ROS production and respiration using permeabilized fibers from red gastrocnemius muscle in wild type and MnSod deficient (mSod2 KO) mice at 6-10 months of age. In mSod2 KO mice, ROS generation was elevated by ~5 fold, and significant defects in mitochondrial respiration were observed-most pronounced in complex II-mediated respiration. Mitochondrial calcium retention capacity was also decreased by 70% in mSod2 KO mice. To determine the effect of reduced MnSOD and increased superoxide on muscle function, we measured in situ contractile function in gastrocnemius muscle. Maximum isometric specific force was decreased by ~30% in mutant mice with direct stimulation on muscle, but the reduction in force was greater (~50%) after stimulation of the sciatic nerve. Surprisingly, gastrocnemius mass was increased in mSod2 KO mice potentially due to compensatory mechanism by fiber branching. Our findings suggest that increased mitochondrial ROS generation and subsequent oxidative stress play an important role in regulating contractile function of skeletal muscle but did not cause muscle atrophy. This result supports the concept that mitochondrial ROS and dysfunction are not critical mediators of denervation-induced atrophy.

Endurance performance is enhanced by intermittent hyperbaric exposure via up-regulation of proteins involved in mitochondrial biogenesis in mice.

This study was designed to (1) investigate the effects of acute hyperbaric exposure on muscle mRNA expression levels, and (2) clarify the mechanisms by which intermittent hyperbaric exposure improves endurance capacity. Experiment 1: Male mice were subjected to acute 1‐h hyperbaric exposure (1.3 atmospheres absolute with 20.9% O2). The expression of peroxisome proliferator‐activated receptor gamma coactivator 1‐alpha (PGC‐1α) mRNA significantly increased in the soleus (7.2‐fold) and red gastrocnemius muscles (Gr, 5.1‐fold) 3 h after hyperbaric exposure. Peroxisome proliferator‐activated receptor alpha (PPAR α) mRNA levels significantly increased in the plantaris (PL, 2.9‐fold) and Gr (2.3‐fold) 3 h after hyperbaric exposure. Experiment 2: Mice were subjected to exercise training with (HypTr) and without (Tr) 1‐h hyperbaric exposure for 4 weeks. Increases in maximal exercise capacity were significantly greater in HypTr than in Tr. In PL, activity levels of 3‐hydroxyacyl‐CoA‐dehydrogenase and citrate synthase (CS) were significantly greater in HypTr than in Tr. CS and phosphofructokinase activities both markedly increased in the white gastrocnemius muscle (Gw) in HypTr only. PGC‐1α expression in the nucleus was significantly greater in HypTr than in Tr in PL (4.8‐fold), Gr (3.2‐fold), and Gw (15‐fold). Protein levels of mitochondrial transcription factor A and heat shock protein 70 significantly increased after training with hyperbaric exposure. These results suggest that exercise training with intermittent hyperbaric exposure represents a beneficial strategy for increasing endurance performance by facilitating oxidative and glycolytic capacities and the expression of proteins involved in mitochondrial biogenesis in the hindlimb muscles.

Pyruvate Dehydrogenase Activity in Red and Superficial White Gastrocnemius Muscles of the Rat during Contraction

The increase in glucose oxidation during exercise is dependent upon the mitochondrial enzyme pyruvate dehydrogenase (PDH) which is allosterically regulated by reversible phosphorylation through a family of PDH kinases and phosphatases. However, the relationship between PDH activation and muscle fiber type during exercise has not been well described because fiber specific muscles have not been reported, and, because voluntary exercise involves motor unit recruitment which further complicates interpretation. To determine whether PDH activation differs in red versus superficial white muscle in vivo, gastrocnemius muscle contractile activity was controlled by electrical pacing via surgically implanted electrodes on the sciatic nerve of anesthetized male Wistar rats. Muscle force production of the triceps surae muscle group was recorded from the Achilles tendon and the knee pinned in place with a tungsten rod through the head of the femur. Gastrocnemius muscle samples were collected from the superficial white (predominantly Type IIb fibers) and deep red (predominantly Type IIb fibers) portions and freeze clamped at rest and during 5 min of twitch stimulation at 0.5Hz and 1.0Hz intensities. PDH activity was assayed in vitro using a radioisotopic tracer assay with [2‐14C]‐pyruvate as the substrate. Total PDH activity in the deep red gastrocnemius was almost two‐fold greater than that of the superficial white (1.9±0.1 vs 1.1±0.1 U/gWW, n=15). Fractional PDH activity (% total activity) was significantly increased in muscle samples collected from superficial white relative to deep red gastrocnemius at rest (15.0±2.0 vs 9.5±0.7%), and during 0.5Hz (60.9±8.1 vs 37.4±3.9%), and 1.0Hz stimulation frequencies (68.3±2.8 vs 40.5±2.8%) (n=4–5 for all groups). However, the active fraction of PDH activity was not different when comparing superficial white to deep red gastrocnemius at rest (0.12±0.02 vs 0.15±0.02 U/gWW) or during stimulation at 0.5Hz (0.67±0.08 vs 0.71±0.04 U/gWW), or 1.0Hz intensities (3±0.08 vs 0.82±0.02 U/gWW). These data suggest that the active fraction of PDH is dependent on the contraction intensity and therefore muscle fiber type differences do not appear to play a role in the number of PDH active sites during contractile activity. Therefore future designs of therapeutic exercise to optimize glucose disposal should be chosen based on the maximal intensity that can be maintained without fatigue to maximize the working muscle mass and PDH activity.Support or Funding InformationSupported by NIH DK095210

Vascular cell transcriptomic changes to exercise training differ directionally along and between skeletal muscle arteriolar trees.

EXT-induced arteriolar adaptations in skeletal muscle are heterogeneous because of spatial variations in muscle fiber type composition and fiber recruitment patterns during exercise. The purpose of this report is to summarize a series of experiments conducted to test the hypothesis that changes in vascular gene expression are signaled by alterations in shear stress resulting from increases in blood flow, muscle fiber type composition, and fiber recruitment patterns. We also report results from a follow-up study of Ankrd23, one gene whose expression was changed by EXT. We expected to see differences in magnitude of changes in gene expression along arteriolar trees and between/among arteriolar trees but similar directional changes. However, transcriptional profiles of arterioles/arteries from OLETF rats exposed to END or SIT reveal that EXT does not lead to similar directional changes in the transcriptome among arteriolar trees of different skeletal muscles or along arteriolar trees within a particular muscle. END caused the most changes in gene expression in 2A arterioles of soleus and white gastrocnemius with little to no changes in the FAs. Ingenuity Pathway Analysis across vessels revealed significant changes in gene expression in 18 pathways. EXT increased expression of some genes (Shc1, desert hedgehog protein (Dhh), adenylate cyclase 4 (Adcy4), G protein-binding protein, alpha (Gnat1), and Bcl2l1) in all arterioles examined, but decreased expression of ubiquitin D (Ubd) and cAMP response element modulator (Crem). Many contractile and/or structural protein genes were increased by SIT in the gastrocnemius FA, but the same genes exhibited decreased expression in red gastrocnemius arterioles. Ankrd23 mRNA levels increased with increasing branch order in the gastrocnemius arteriolar tree and were increased 19-fold in gastrocnemius muscle FA by SIT. Follow-up experiments indicate that Ankrd23 mRNA level was increased 14-fold in cannulated gastrocnemius FA when intraluminal pressure was increased from 90 and 180cm H2O for 4hours. Also, Ankrd23-/- mice exhibit limited ability to form collateral arteries following femoral artery occlusion compared to WT mice (angioscore WT=0.18±0.03; Ankrd23-/- =0.04±0.01). Further research will be required to determine whether Ankrd23 plays an important role in mechanically induced vascular remodeling of the arterial tree in skeletal muscle.

Transcriptomic effects of metformin in skeletal muscle arteries of obese insulin-resistant rats.

We examined the effects of metformin, a commonly used antidiabetic drug, on gene expression in multiple arteries. Specifically, transcriptional profiles of feed arteries and second branch order arterioles in the soleus, gastrocnemius, and diaphragm muscles as well as aortic endothelial scrapes were examined from obese insulin-resistant Otsuka Long-Evans Tokushima Fatty rats treated with ( n = 9) or without ( n = 10) metformin from 20 to 32 weeks of age. Metformin-treated rats exhibited a reduction in body weight, adiposity, and HbA1c ( P < 0.05). The greatest number of differentially expressed genes (FDR < 15%) between those treated with and without metformin was found in the red gastrocnemius 2a arterioles (93 genes), followed by the diaphragm 2a arterioles (62 genes), and soleus 2a arterioles (15 genes). We also found that two genes were differentially expressed in aortic endothelial cells (LETMD1 and HMGCS2, both downregulated), one gene in the gastrocnemius feed artery (BLNK, downregulated), and no genes in the soleus and diaphragm feed arteries and white gastrocnemius 2a arterioles. No single gene was altered by metformin across all vessels examined. This study provides evidence that metformin treatment produces distinct gene expression effects throughout the arterial tree in a rat model of obesity and insulin resistance. Genes whose expression was modulated with metformin do not appear to have a clear connection with its known mechanisms of action. These findings support the notion that vascular gene regulation in response to oral pharmacological therapy, such as metformin, is vessel specific. Impact statement This study provides evidence that metformin treatment produces artery-specific gene expression effects. The genes whose expression was modulated with metformin do not appear to have a clear connection with its known mechanisms of action.

Attempting to Compensate for Reduced Neuronal Nitric Oxide Synthase Protein with Nitrate Supplementation Cannot Overcome Metabolic Dysfunction but Rather Has Detrimental Effects in Dystrophin-Deficient mdx Muscle

Duchenne muscular dystrophy arises from the loss of dystrophin and is characterized by calcium dysregulation, muscular atrophy, and metabolic dysfunction. The secondary reduction of neuronal nitric oxide synthase (nNOS) from the sarcolemma reduces NO production and bioavailability. As NO modulates glucose uptake, metabolism, and mitochondrial bioenergetics, we investigated whether an 8-week nitrate supplementation regimen could overcome metabolic dysfunction in the mdx mouse. Dystrophin-positive control (C57BL/10) and dystrophin-deficient mdx mice were supplemented with sodium nitrate (85 mg/l) in drinking water. Following the supplementation period, extensor digitorum longus and soleus were excised and radioactive glucose uptake was measured at rest (basal) and during contraction. Gastrocnemius was excised and mitochondrial respiration was measured using the Oroboros Oxygraph. Tibialis anterior was analyzed immunohistochemically for the presence of dystrophin, nNOS, nitrotyrosine, IgG and CD45+ cells, and histologically to assess areas of damage and regeneration. Glucose uptake in the basal and contracting states was normal in unsupplemented mdx muscles but was reduced following nitrate supplementation in mdx muscles only. The mitochondrial utilization of substrates was also impaired in mdx gastrocnemius during phosphorylating and maximal uncoupled respiration, and nitrate could not improve respiration in mdx muscle. Although nitrate supplementation reduced mitochondrial hydrogen peroxide emission, it induced mitochondrial uncoupling in red gastrocnemius, increased muscle fiber peroxynitrite (nitrotyrosine), and promoted skeletal muscle damage. Our novel data suggest that despite lower nNOS protein expression and likely lower NO production in mdx muscle, enhancing NO production with nitrate supplementation in these mice has detrimental effects on skeletal muscle. This may have important relevance for those with DMD.

Pseudouridine synthase 1 deficient mice, a model for Mitochondrial Myopathy with Sideroblastic Anemia, exhibit muscle morphology and physiology alterations.

Mitochondrial myopathy with lactic acidosis and sideroblastic anemia (MLASA) is an oxidative phosphorylation disorder, with primary clinical manifestations of myopathic exercise intolerance and a macrocytic sideroblastic anemia. One cause of MLASA is recessive mutations in PUS1, which encodes pseudouridine (Ψ) synthase 1 (Pus1p). Here we describe a mouse model of MLASA due to mutations in PUS1. As expected, certain Ψ modifications were missing in cytoplasmic and mitochondrial tRNAs from Pus1−/− animals. Pus1−/− mice were born at the expected Mendelian frequency and were non-dysmorphic. At 14 weeks the mutants displayed reduced exercise capacity. Examination of tibialis anterior (TA) muscle morphology and histochemistry demonstrated an increase in the cross sectional area and proportion of myosin heavy chain (MHC) IIB and low succinate dehydrogenase (SDH) expressing myofibers, without a change in the size of MHC IIA positive or high SDH myofibers. Cytochrome c oxidase activity was significantly reduced in extracts from red gastrocnemius muscle from Pus1−/− mice. Transmission electron microscopy on red gastrocnemius muscle demonstrated that Pus1−/− mice also had lower intermyofibrillar mitochondrial density and smaller mitochondria. Collectively, these results suggest that alterations in muscle metabolism related to mitochondrial content and oxidative capacity may account for the reduced exercise capacity in Pus1−/− mice.

Ten Days of Free Wheel Running Alters Cortical Glutamate‐Linked Mitochondrial Respiration in Rat Brain

The mechanisms through which exercise benefits brain function are not completely understood. Appreciation for the role of lactate as a major neuroenergetic fuel, particularly during exercise, has progressed in recent decades; however, the mitochondrial involvement in brain lactate metabolism is not clear. To explore the mechanisms involved, we examined mitochondrial lactate oxidation in brain and skeletal muscle from exercised rats. Respiratory oxygen flux (JO2) was measured in saponin‐permeabilized prefrontal cortex (PFC) and hippocampus (dentate gyrus; DG) samples, as well as red and white gastrocnemius muscle samples from five male Wistar rats (age: approximately 104 days) given access to running wheels. Over ten days of free wheel running, rats attained a maximal daily running distance averaging 2.1 ± 0.5 km. The wheel‐running rats remained relatively weight stable, losing 1.4 ± 3.1 g body mass on average, whereas the five paired control rats gained an average of 39.4 ± 2.8 g over the ten days. On day eleven, high resolution respirometry was performed on exercise and control samples ex vivo. The addition of lactate failed to affect mitochondrial JO2 (ADP + malate) from either brain or muscle in either group. However, JO2 rose significantly following the addition of exogenous NAD+ in each tissue (P &lt; 0.01), implying the lactate dehydrogenase reaction operates outside of the mitochondrial matrix. This interpretation was further supported by inhibition of JO2 following addition of an inhibitor of pyruvate transport, UK‐5099 (P &lt; 0.01). Interestingly, the subsequent addition of glutamate resulted in a disparate increase in mitochondrial JO2 among tissues and treatment. Compared to controls, glutamate‐supported mitochondrial JO2 in wheel runners was significantly greater in PFC (P &lt; 0.05), but not DG. This rise in JO2 likely reflects activation of the malate‐aspartate shuttle through elevated mitochondrial glutamate transport. Paradoxically, wheel runners exhibited lower glutamate‐supported JO2 in red gastrocnemius (P &lt; 0.05). This may be due to the influence of regular exercise on the putative lactate‐malate‐aspartate shuttle. The lactate‐malate‐aspartate shuttle is the interaction between the lactate and malate‐aspartate shuttles to translocate reducing power to the mitochondria. The decline in lactate dehydrogenase activity in skeletal muscle that accompanies aerobic exercise training is expected to negatively influence lactate‐malate‐aspartate shuttling, and may explain our observations in muscle. Collectively, the results of this study suggest that electron shuttling in rat brain following ten days of voluntary exercise is upregulated in PFC. These results also provide further evidence for an indirect mechanism of mitochondrial lactate oxidation in both brain and skeletal muscle.Support or Funding InformationThe Natural Sciences and Engineering Research Council of Canada; The Nova Scotia Health Research Foundation

Differential Expression of NADPH Oxidases Depends on Skeletal Muscle Fiber Type in Rats.

NADPH oxidases (NOX) are important sources of reactive oxygen species (ROS) in skeletal muscle, being involved in excitation-contraction coupling. Thus, we aimed to investigate if NOX activity and expression in skeletal muscle are fiber type specific and the possible contribution of this difference to cellular oxidative stress. Oxygen consumption rate, NOX activity and mRNA levels, and the activity of catalase (CAT), glutathione peroxidase (GPX), and superoxide dismutase (SOD), as well as the reactive protein thiol levels, were measured in the soleus (SOL), red gastrocnemius (RG), and white gastrocnemius (WG) muscles of rats. RG showed higher oxygen consumption flow than SOL and WG, while SOL had higher oxygen consumption than WG. SOL showed higher NOX activity, as well as NOX2 and NOX4 mRNA levels, antioxidant enzymatic activities, and reactive protein thiol contents when compared to WG and RG. NOX activity and NOX4 mRNA levels as well as antioxidant enzymatic activities were higher in RG than in WG. Physical exercise increased NOX activity in SOL and RG, specifically NOX2 mRNA levels in RG and NOX4 mRNA levels in SOL. In conclusion, we demonstrated that NOX activity and expression differ according to the skeletal muscle fiber type, as well as antioxidant defense.