Redox Balance Differentially Affects Biomechanics in Permeabilized Single Muscle Fibres-Active and Passive Force Assessments with the Myorobot.

An oxidizing redox state imposes unique effects on the contractile properties of muscle. Permeabilized fibres show reduced active force generation in the presence of H2O2. However, our knowledge about the muscle fibre's elasticity or flexibility is limited due to shortcomings in assessing the passive stress-strain properties, mostly due to technically limited experimental setups. The MyoRobot is an automated biomechatronics platform that is well-capable of not only investigating calcium responsiveness of active contraction but also features precise stretch actuation to examine the passive stress-strain behaviour. Both were carried out in a consecutive recording sequence on the same fibre for 10 single fibres in total. We denote a significantly diminished maximum calcium-saturated force for fibres exposed to ≥500 µM H2O2, with no marked alteration of the pCa50 value. In contrast to active contraction (e.g., maximum isometric force activation), passive restoration stress (force per area) significantly increases for fibres exposed to an oxidizing environment, as they showed a non-linear stress-strain relationship. Our data support the idea that a highly oxidizing environment promotes non-linear fibre stiffening and confirms that our MyoRobot platform is a suitable tool for investigating redox-related changes in muscle biomechanics.

Redox Implications of Extreme Task Performance: The Case in Driver Athletes.

Redox homeostasis and redox-mediated signaling mechanisms are fundamental elements of human biology. Physiological levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS) modulate a range of functional processes at the cellular, tissue, and systemic levels in healthy humans. Conversely, excess ROS or RNS activity can disrupt function, impairing the performance of daily activities. This article analyzes the impact of redox mechanisms on extreme task performance. Such activities (a) require complex motor skills, (b) are physically demanding, (c) are performed in an extreme environment, (d) require high-level executive function, and (e) pose an imminent risk of injury or death. The current analysis utilizes race car driving as a representative example. The physiological challenges of this extreme task include physical exertion, g loading, vibration, heat exposure, dehydration, noise, mental demands, and emotional factors. Each of these challenges stimulates ROS signaling, RNS signaling, or both, alters redox homeostasis, and exerts pro-oxidant effects at either the tissue or systemic levels. These redox mechanisms appear to promote physiological stress during race car driving and impair the performance of driver athletes.

Single muscle fibre biomechanics and biomechatronics - The challenges, the pitfalls and the future.

Interest in muscle biomechanics is growing with availabilities of patient biopsies and animal models related to muscle diseases, muscle wasting (sarcopenia, cachexia), exercise and drug effects. However, development of technologies or facilitated systems required to measure biomechanical and contractile properties of single fibres has not kept pace with this demand. Most studies use manual mechatronics systems that have not changed in decades and are confined to a few labs worldwide. Available commercial systems are expensive and limited in versatility, throughput and user-friendliness. We review major standard systems available from research labs and commercial sources, and benchmark those to our recently developed automated MyoRobot biomechatronics platform that provides versatility to cover multiple organ scales, is flexible in programming for active/passive muscle biomechanics using custom-made graphics user interfaces, employs on-the-fly data analyses and does not rely on external research microscopes. With higher throughput, this system blends Industry 4.0 automation principles into myology.

The Physiology of Auto Racing.

INTRODUCTION: Auto racing poses a unique set of physiologic challenges for athletes who compete in this sport. These challenges are not widely recognized due to the limited amount of original research in this field and the diffuse nature of this literature. The purpose of this article is to review the major physiologic challenges of auto racing and summarize what is currently known about athletes in this sport. CONCLUSIONS: The physical stressors of either driving or servicing the race car are overlaid with particular environmental challenges associated with racing (e.g., thermal, noise, carbon monoxide exposure) that increase the physiological stress on motorsport athletes. Physical stress reflects the muscular work required for car control and control of posture during high gravitational (g) loads: factors that predispose athletes to fatigue. The physiologic effects of these stressors include cardiovascular stress as reflected by prolonged elevation of heart rate, cardiac output, and oxygen consumption in both driver and pit athletes during competition. Psychological stress is evident in autonomic and endocrine responses of athletes during competition. The thermal stress of having to compete wearing multilayer fire suits and closed helmets in ambient temperatures of 50°C to 60°C results in the ubiquitous risk of dehydration. Published data show that both drivers and pit crew members are accomplished athletes with distinct challenges and abilities. There are gaps in the literature, especially in regard to female, older adult, and child participants. Additionally, minimal literature is available on appropriate training programs to offset the physiological challenges of auto racing.

Reactive Oxygen Species as Agents of Fatigue.

INTRODUCTION: For more than three decades, muscle biologists have been fascinated by reactive oxygen species (ROS) generated in exercising muscle and the potential role that ROS may play in fatigue. METHODS: Reports in the peer-reviewed literature were analyzed and published findings integrated to synthesize an overview of ROS as agents of fatigue. RESULTS: Muscle tissue contains multiple sources of ROS, and specific ROS molecules have been detected in muscle, including superoxide anions, hydrogen peroxide, and hydroxyl radicals. These species are present throughout the tissue, i.e., myofiber organelles and cytosol, extracellular space, and intravascular compartment, and ROS concentrations increase during strenuous contractions. Direct ROS exposure evokes many of the same changes that occur in muscle during fatigue, suggesting a possible relationship. The hypothesis that ROS play a causal role in fatigue has been tested extensively, a large body of data have been compiled, and the once-controversial verdict is now in: ROS accumulation in working muscle clearly contributes to the loss of function that occurs in fatigue. This is evident in a range of experimental settings ranging from muscle fiber bundles in vitro to neuromuscular preparations in situ, from volitional exercise of small muscle groups to whole-body exercise by elite athletes. CONCLUSION: The robust capacity of antioxidant pretreatment to delay fatigue provides compelling evidence that ROS play a causal role in this process. There are caveats to this story of course, issues related to the type of antioxidant and mode of administration. Also, the translation of this laboratory concept into clinical practice has been slow. Still, antioxidant therapy has the potential to benefit individuals who experience premature fatigue and this remains a promising area for future research.

Disease-Induced Skeletal Muscle Atrophy and Fatigue.

Numerous health problems, including acute critical illness, cancer, diseases associated with chronic inflammation, and neurological disorders, often result in skeletal muscle weakness and fatigue. Disease-related muscle atrophy and fatigue is an important clinical problem because acquired skeletal muscle weakness can increase the duration of hospitalization, result in exercise limitation, and contribute to a poor quality of life. Importantly, skeletal muscle atrophy is also associated with increased morbidity and mortality of patients. Therefore, improving our understanding of the mechanism(s) responsible for skeletal muscle weakness and fatigue in patients is a required first step to develop clinical protocols to prevent these skeletal muscle problems. This review will highlight the consequences and potential mechanisms responsible for skeletal muscle atrophy and fatigue in patients experiencing acute critical illness, cancer, chronic inflammatory diseases, and neurological disorders.

Redox interventions to increase exercise performance.

Skeletal muscle continually produces reactive oxygen species (ROS) and nitric oxide (NO) derivatives. Both oxidant cascades have complex effects on muscle contraction, metabolic function and tissue perfusion. Strenuous exercise increases oxidant production by muscle, limiting performance during endurance exercise tasks. Conversely, redox interventions that modulate ROS or NO activity have the potential to improve performance. Antioxidants have long been known to buffer ROS activity and lessen oxidative perturbations during exercise. The capacity to enhance human performance varies among antioxidant categories. Vitamins, provitamins and nutriceuticals often blunt oxidative changes at the biochemical level but do not enhance performance. In contrast, reduced thiol donors have been shown to delay fatigue or increase endurance under a variety of experimental conditions. Dietary nitrate supplementation has recently emerged as a second redox strategy for increasing endurance. Purified nitrate salts and nitrate-rich foods, notably beetroot and beetroot juice, are reported to lessen the oxygen cost of exercise, increase efficiency, and enhance performance during endurance tasks. These findings are exciting but enigmatic since nitrate per se has little bioactivity and cannot be converted to NO by mammalian cells. Overall, the available data suggest exercise endurance can be augmented by redox-active supplements, either reduced thiol donors or dietary nitrates. These findings have clear implications for athletes seeking a competitive edge. More importantly, interventions that increase endurance may benefit individuals whose physical activity is limited by illness, ageing, or frailty.

Understanding the Cellular and Molecular Mechanisms of Physical Activity-Induced Health Benefits.

The beneficial effects of physical activity (PA) are well documented, yet the mechanisms by which PA prevents disease and improves health outcomes are poorly understood. To identify major gaps in knowledge and potential strategies for catalyzing progress in the field, the NIH convened a workshop in late October 2014 entitled "Understanding the Cellular and Molecular Mechanisms of Physical Activity-Induced Health Benefits." Presentations and discussions emphasized the challenges imposed by the integrative and intermittent nature of PA, the tremendous discovery potential of applying "-omics" technologies to understand interorgan crosstalk and biological networking systems during PA, and the need to establish an infrastructure of clinical trial sites with sufficient expertise to incorporate mechanistic outcome measures into adequately sized human PA trials. Identification of the mechanisms that underlie the link between PA and improved health holds extraordinary promise for discovery of novel therapeutic targets and development of personalized exercise medicine.

Neutral sphingomyelinase-3 mediates TNF-stimulated oxidant activity in skeletal muscle.

AIMS: Sphingolipid and oxidant signaling affect glucose uptake, atrophy, and force production of skeletal muscle similarly and both are stimulated by tumor necrosis factor (TNF), suggesting a connection between systems. Sphingolipid signaling is initiated by neutral sphingomyelinase (nSMase), a family of agonist-activated effector enzymes. Northern blot analyses suggest that nSMase3 may be a striated muscle-specific nSMase. The present study tested the hypothesis that nSMase3 protein is expressed in skeletal muscle and functions to regulate TNF-stimulated oxidant production. RESULTS: We demonstrate constitutive nSMase activity in skeletal muscles of healthy mice and humans and in differentiated C2C12 myotubes. nSMase3 (Smpd4 gene) mRNA is highly expressed in muscle. An nSMase3 protein doublet (88 and 85 kD) is derived from alternative mRNA splicing of exon 11. The proteins partition differently. The full-length 88 kD isoform (nSMase3a) fractionates with membrane proteins that are resistant to detergent extraction; the 85 kD isoform lacking exon 11 (nSMase3b) is more readily extracted and fractionates with detergent soluble membrane proteins; neither variant is detected in the cytosol. By immunofluorescence microscopy, nSMase3 resides in both internal and sarcolemmal membranes. Finally, myotube nSMase activity and cytosolic oxidant activity are stimulated by TNF. Both if these responses are inhibited by nSMase3 knockdown. INNOVATION: These findings identify nSMase3 as an intermediate that links TNF receptor activation, sphingolipid signaling, and skeletal muscle oxidant production. CONCLUSION: Our data show that nSMase3 acts as a signaling nSMase in skeletal muscle that is essential for TNF-stimulated oxidant activity.

Mechanical properties of respiratory muscles.

Striated respiratory muscles are necessary for lung ventilation and to maintain the patency of the upper airway. The basic structural and functional properties of respiratory muscles are similar to those of other striated muscles (both skeletal and cardiac). The sarcomere is the fundamental organizational unit of striated muscles and sarcomeric proteins underlie the passive and active mechanical properties of muscle fibers. In this respect, the functional categorization of different fiber types provides a conceptual framework to understand the physiological properties of respiratory muscles. Within the sarcomere, the interaction between the thick and thin filaments at the level of cross-bridges provides the elementary unit of force generation and contraction. Key to an understanding of the unique functional differences across muscle fiber types are differences in cross-bridge recruitment and cycling that relate to the expression of different myosin heavy chain isoforms in the thick filament. The active mechanical properties of muscle fibers are characterized by the relationship between myoplasmic Ca2+ and cross-bridge recruitment, force generation and sarcomere length (also cross-bridge recruitment), external load and shortening velocity (cross-bridge cycling rate), and cross-bridge cycling rate and ATP consumption. Passive mechanical properties are also important reflecting viscoelastic elements within sarcomeres as well as the extracellular matrix. Conditions that affect respiratory muscle performance may have a range of underlying pathophysiological causes, but their manifestations will depend on their impact on these basic elemental structures.

TNF signals via neuronal-type nitric oxide synthase and reactive oxygen species to depress specific force of skeletal muscle.

TNF promotes skeletal muscle weakness, in part, by depressing specific force of muscle fibers. This is a rapid, receptor-mediated response, in which TNF stimulates cellular oxidant production, causing myofilament dysfunction. The oxidants appear to include nitric oxide (NO); otherwise, the redox mechanisms that underlie this response remain undefined. The current study tested the hypotheses that 1) TNF signals via neuronal-type NO synthase (nNOS) to depress specific force, and 2) muscle-derived reactive oxygen species (ROS) are essential co-mediators of this response. Mouse diaphragm fiber bundles were studied using live cell assays. TNF exposure increased general oxidant activity (P < 0.05; 2',7'-dichlorodihydrofluorescein diacetate assay) and NO activity (P < 0.05; 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate assay) and depressed specific force across the full range of stimulus frequencies (1-300 Hz; P < 0.05). These responses were abolished by pretreatment with N(ω)-nitro-L-arginine methyl ester (L-NAME; a nonspecific inhibitor of NOS activity), confirming NO involvement. Genetic nNOS deficiency replicated L-NAME effects on TNF-treated muscle, diminishing NO activity (-80%; P < 0.05) and preventing the decrement in specific force (P < 0.05). Comparable protection was achieved by selective depletion of muscle-derived ROS. Pretreatment with either SOD (degrades superoxide anion) or catalase (degrades hydrogen peroxide) depressed oxidant activity in TNF-treated muscle and abolished the decrement in specific force. These findings indicate that TNF signals via nNOS to depress contractile function, a response that requires ROS and NO as obligate co-mediators.

Sphingomyelinase depresses force and calcium sensitivity of the contractile apparatus in mouse diaphragm muscle fibers.

Diseases that result in muscle weakness, e.g., heart failure, are characterized by elevated sphingomyelinase (SMase) activity. In intact muscle, SMase increases oxidants that contribute to diminished muscle force. However, the source of oxidants, specific processes of muscle contraction that are dysfunctional, and biochemical changes underlying the weakness elicited by SMase remain unknown. We tested three hypotheses: 1) SMase-induced depression of muscle force is mediated by mitochondrial reactive oxygen species (ROS), 2) SMase depresses force and calcium sensitivity of the contractile apparatus, and 3) SMase promotes oxidation and phosphorylation of myofibrillar proteins. Our experiments included intact muscle bundles, permeabilized single fibers, and isolated myofibrillar proteins. The mitochondrial-targeted antioxidant d-Arg-2',6'-dimethyl-Tyr-Lys-Phe-NH(2), decreased cytosolic oxidants and protected intact muscle bundles from weakness stimulated by SMase. SMase depressed maximal calcium-activated force by 20% in permeabilized single fibers (in kN/m(2): control 117 ± 6; SMase 93 ± 8; P < 0.05). Calcium sensitivity of permeabilized single fibers decreased from 5.98 ± 0.03 (control) to 5.91 ± 0.02 (SMase; P < 0.05). Myofibrillar protein nitrotyrosines, carbonyls, and phosphorylation were unaltered by SMase. Our study shows that the fall in specific force of intact muscle elicited by SMase is mediated by mitochondrial ROS and can be attributed largely to dysfunction of the contractile apparatus.

Doxorubicin acts via mitochondrial ROS to stimulate catabolism in C2C12 myotubes.

Doxorubicin, a commonly prescribed chemotherapeutic agent, causes skeletal muscle wasting in cancer patients undergoing treatment and increases mitochondrial reactive oxygen species (ROS) production. ROS stimulate protein degradation in muscle by activating proteolytic systems that include caspase-3 and the ubiquitin-proteasome pathway. We hypothesized that doxorubicin causes skeletal muscle catabolism through ROS, causing upregulation of E3 ubiquitin ligases and caspase-3. We tested this hypothesis by exposing differentiated C2C12 myotubes to doxorubicin (0.2 µM). Doxorubicin decreased myotube width 48 h following exposure, along with a 40-50% reduction in myosin and sarcomeric actin. Cytosolic oxidant activity was elevated in myotubes 2 h following doxorubicin exposure. This increase in oxidants was followed by an increase in the E3 ubiquitin ligase atrogin-1/muscle atrophy F-box (MAFbx) and caspase-3. Treating myotubes with SS31 (opposes mitochondrial ROS) inhibited expression of ROS-sensitive atrogin-1/MAFbx and protected against doxorubicin-stimulated catabolism. These findings suggest doxorubicin acts via mitochondrial ROS to stimulate myotube atrophy.

N-acetylcysteine in handgrip exercise: plasma thiols and adverse reactions.

UNLABELLED: N-acetylcysteine (NAC) is a thiol donor with antioxidant properties that has potential use as an ergogenic aid. However, NAC is associated with adverse reactions that limit its use in humans. PURPOSE: The authors evaluated NAC efficacy as a thiol donor before handgrip exercise, measuring changes in serum cysteine and glutathione status and recording adverse reactions in adult subjects across a range of doses. METHODS: Healthy individuals ingested NAC capsules (9 ± 2 or 18 ± 4 mg/kg) or solution (0, 35, 70, or 140 mg/kg). Venous blood samples were collected and subjects answered a questionnaire about adverse reactions. RESULTS: Low doses of NAC (capsules) did not affect plasma cysteine or glutathione or cause adverse reactions. Adverse reactions to NAC solution were predominantly mild and gastrointestinal (GI). Intensity of GI reactions to 140 mg/kg NAC was significantly higher than placebo (in a.u., 0.67 ± 0.16 vs. 0.07 ± 0.04; p < .05). Plasma cysteine concentration increased with NAC dose from 9.3 ± 0.7 µM (placebo) to 65.3 ± 6.7 µM (140 mg/kg); however, there was no difference (p > .05) in plasma cysteine for 70 mg/kg vs. 140 mg/kg. Similar increases were observed for the ratio of cysteine to total cysteine, which was directly related to handgrip exercise performance. Plasma glutathione was elevated and oxidized glutathione diminished (p < .05) with NAC 140 mg/kg vs. placebo. CONCLUSION: NAC effects on plasma thiols are maximized by oral administration of 70 mg/kg, a dose that does not cause significant adverse reactions.

Sphingolipid metabolism, oxidant signaling, and contractile function of skeletal muscle.

SIGNIFICANCE: Sphingolipids are a class of bioactive lipids that regulate diverse cell functions. Ceramide, sphingosine, and sphingosine-1-phosphate accumulate in tissues such as liver, brain, and lung under conditions of cellular stress, including oxidative stress. The activity of some sphingolipid metabolizing enzymes, chiefly the sphingomyelinases, is stimulated during inflammation and in response to oxidative stress. Ceramide, the sphingomyelinase product, as well as the ceramide metabolite, sphingosine-1-phosphate, can induce the generation of more reactive oxygen species, propagating further inflammation. RECENT ADVANCES: This review article summarizes information on sphingolipid biochemistry and signaling pertinent to skeletal muscle and describes the potential influence of sphingolipids on contractile function. CRITICAL ISSUES: It encompasses topics related to (1) the pathways for complex sphingolipid biosynthesis and degradation, emphasizing sphingolipid regulation in various muscle fiber types and subcellular compartments; (2) the emerging evidence that implicates ceramide, sphingosine, and sphingosine-1-phosphate as regulators of muscle oxidant activity, and (3) sphingolipid effects on contractile function and fatigue. FUTURE DIRECTIONS: We propose that prolonged inflammatory conditions alter ceramide, sphingosine, and sphingosine-1-phosphate levels in skeletal muscle and that these changes promote the weakness, premature fatigue, and cachexia that plague individuals with heart failure, cancer, diabetes, and other chronic inflammatory diseases.

Prion protein expression and functional importance in skeletal muscle.

UNLABELLED: Skeletal muscle expresses prion protein (PrP) that buffers oxidant activity in neurons. AIMS: We hypothesize that PrP deficiency would increase oxidant activity in skeletal muscle and alter redox-sensitive functions, including contraction and glucose uptake. We used real-time polymerase chain reaction and Western blot analysis to measure PrP mRNA and protein in human diaphragm, five murine muscles, and muscle-derived C2C12 cells. Effects of PrP deficiency were tested by comparing PrP-deficient mice versus wild-type mice and morpholino-knockdown versus vehicle-treated myotubes. Oxidant activity (dichlorofluorescin oxidation) and specific force were measured in murine diaphragm fiber bundles. RESULTS: PrP content differs among mouse muscles (gastrocnemius>extensor digitorum longus, EDL>tibialis anterior, TA; soleus>diaphragm) as does glycosylation (di-, mono-, nonglycosylated; gastrocnemius, EDL, TA=60%, 30%, 10%; soleus, 30%, 40%, 30%; diaphragm, 30%, 30%, 40%). PrP is predominantly di-glycosylated in human diaphragm. PrP deficiency decreases body weight (15%) and EDL mass (9%); increases cytosolic oxidant activity (fiber bundles, 36%; C2C12 myotubes, 7%); and depresses specific force (12%) in adult (8-12 mos) but not adolescent (2 mos) mice. INNOVATION: This study is the first to directly assess a role of prion protein in skeletal muscle function. CONCLUSIONS: PrP content varies among murine skeletal muscles and is essential for maintaining normal redox homeostasis, muscle size, and contractile function in adult animals.

Beyond atrophy: redox mechanisms of muscle dysfunction in chronic inflammatory disease.

Chronic inflammatory diseases such as heart failure, cancer and arthritis have secondary effects on skeletal muscle that cause weakness and exercise intolerance. These symptoms exacerbate illness and make death more likely. Weakness is not simply a matter of muscle atrophy. Functional studies show that contractile dysfunction, i.e. a reduction in specific force, makes an equally important contribution to overall weakness. The most clearly defined mediator of contractile dysfunction is tumour necrosis factor (TNF). TNF serum levels are elevated in chronic disease, correlate with muscle weakness, and are a predictor of morbidity and mortality. Research is beginning to unravel the mechanism by which TNF depresses specific force. TNF acts via the TNFR1 receptor subtype to depress force by increasing cytosolic oxidant activity. Oxidants depress myofibrillar function, decreasing specific force without altering calcium regulation or other aspects of myofibrillar mechanics. Beyond these concepts, the intracellular mechanisms that depress specific force remain undefined. We do not know the pathway by which receptor-ligand interaction stimulates oxidant production. Nor do we know the type(s) of oxidants stimulated by TNF, their intracellular source(s), or their molecular targets. Investigators in the field are pursuing these issues with the long-term goal of preserving muscle function in individuals afflicted by chronic disease.

TNF/TNFR1 signaling mediates doxorubicin-induced diaphragm weakness.

Doxorubicin, a common chemotherapeutic agent, causes respiratory muscle weakness in both patients and rodents. Tumor necrosis factor-α (TNF), a proinflammatory cytokine that depresses diaphragm force, is elevated following doxorubicin chemotherapy. TNF-induced diaphragm weakness is mediated through TNF type 1 receptor (TNFR1). These findings lead us to hypothesize that TNF/TNFR1 signaling mediates doxorubicin-induced diaphragm muscle weakness. We tested this hypothesis by treating C57BL/6 mice with a clinical dose of doxorubicin (20 mg/kg) via intravenous injection. Three days later, we measured contractile properties of muscle fiber bundles isolated from the diaphragm. We tested the involvement of TNF/TNFR1 signaling using pharmaceutical and genetic interventions. Etanercept, a soluble TNF receptor, and TNFR1 deficiency protected against the depression in diaphragm-specific force caused by doxorubicin. Doxorubicin stimulated an increase in TNFR1 mRNA and protein (P < 0.05) in the diaphragm, along with colocalization of TNFR1 to the plasma membrane. These results suggest that doxorubicin increases diaphragm sensitivity to TNF by upregulating TNFR1, thereby causing respiratory muscle weakness.