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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.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.

BackgroundRisk-adjusted rates of hospital readmission are a common indicator of hospital performance. There are concerns that current risk-adjustment methods do not account for the many factors outside the hospital setting that can affect readmission rates. Not accounting for these external factors could result in hospitals being unfairly penalized when they discharge patients to communities that are less able to support care transitions and disease management. While incorporating adjustments for the myriad of social and economic factors outside of the hospital setting could improve the accuracy of readmission rates as a performance measure, doing so has limited feasibility due to the number of potential variables and the paucity of data to measure them. This paper assesses a practical approach to addressing this problem: using mixed-effect regression models to estimate case-mix adjusted risk of readmission by community of patients’ residence (community risk of readmission) as a complementary performance indicator to hospital readmission rates.MethodsUsing hospital discharge data and mixed-effect regression models with a random intercept for community, we assess if case-mix adjusted community risk of readmission can be useful as a quality indicator for community-based care. Our outcome of interest was an unplanned repeat hospitalisation. Our primary exposure was community of residence.ResultsCommunity of residence is associated with case-mix adjusted risk of unplanned repeat hospitalisation. Community risk of readmission can be estimated and mapped as indicators of the ability of communities to support both care transitions and long-term disease management.ConclusionContextualising readmission rates through a community lens has the potential to help hospitals and policymakers improve discharge planning, reduce penalties to hospitals, and most importantly, provide higher quality care to the people that they serve.

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 suffering from acute critical illness, cancer, chronic inflammatory diseases, and neurological disorders.

AB 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. (C) 2016 American College of Sports Medicine

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.

As the terrestrial and marine effects of climate change continue to intensify, the value of natural habitats as a form of protection against a variety of ecological issues is becoming clear. Coastal wetlands, for example, provide protection against incoming storm surges and extreme weather, serve to improve water quality through the sequestration of various pollutants, and offer serious potential as a new source of biofuel. Unfortunately, many of the areas that have been affected by coastal habitat loss still suffer from the same problems that caused those ecosystems to change in the first place. Cities continue to release effluence into estuaries; hydrological engineering projects continue to redirect waterways that change flow and sediment patterns; and increasing populations in coastal areas all assert significant pressures on intertidal ecosystems. This ongoing changing of the landscape-and the length of time that anthropogenic factors have been influencing these habitats-has made modern-day environme...

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.

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 Nω-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.

Muscle atrophy alone is insufficient to explain the significant decline in contractile force of skeletal muscle during normal aging. One contributing factor to decreased contractile force in aging skeletal muscle could be compromised excitation-contraction (E-C) coupling, without sufficient available Ca(2+) to allow for repetitive muscle contractility, skeletal muscles naturally become weaker. Using biophysical approaches, we previously showed that store-operated Ca(2+) entry (SOCE) is compromised in aged skeletal muscle but not in young ones. While important, a missing component from previous studies is whether or not SOCE function correlates with contractile function during aging. Here we test the contribution of extracellular Ca(2+) to contractile function of skeletal muscle during aging. First, we demonstrate graded coupling between SR Ca(2+) release channel-mediated Ca(2+) release and activation of SOCE. Inhibition of SOCE produced significant reduction of contractile force in young skeletal muscle, particularly at high frequency stimulation, and such effects were completely absent in aged skeletal muscle. Our data indicate that SOCE contributes to the normal physiological contractile response of young healthy skeletal muscle and that defective extracellular Ca(2+) entry through SOCE contributes to the reduced contractile force characteristic of aged skeletal muscle.

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.

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.

Doxorubicin is a chemotherapeutic agent prescribed for a variety of tumors. While undergoing treatment, patients exhibit frequent symptoms that suggest respiratory muscle weakness. Cancer patients can receive doxorubicin chemotherapy through either intravenous (IV) or intraperitoneal (IP) injections. We hypothesized that respiratory muscle function would be depressed in a murine model of chemotherapy. We tested this hypothesis by treating C57BL/6 mice with a clinical dose of doxorubicin (20 mg/kg) via IV or IP injection. Three days later we measured contractile properties of muscle fiber bundles isolated from the diaphragm. Doxorubicin consistently depressed diaphragm force with both methods of administration (P < 0.01). Doxorubicin IP exaggerated the depression in diaphragm force and stimulated tissue inflammation and muscle fiber injury. These results suggest that clinically relevant doses of doxorubicin cause respiratory muscle weakness and that the loss of function depends, in part, on the route of administration.

Sphingomyelinase (SMase) hydrolyzes membrane sphingomyelin into ceramide, which increases oxidants in nonmuscle cells. Serum SMase activity is elevated in sepsis and heart failure, conditions where muscle oxidants are increased, maximal muscle force is diminished, and fatigue is accelerated. We tested the hypotheses that exogenous SMase and accumulation of ceramide in muscle increases oxidants in muscle cells, depresses specific force of unfatigued muscle, and accelerates the fatigue process. We also anticipated that the antioxidant N-acetylcysteine (NAC) would prevent SMase effects on muscle function. We studied the responses of C2C12 myotubes and mouse diaphragm to SMase treatment in vitro. We observed that SMase caused a 2.8-fold increase in total ceramide levels in myotubes. Exogenous ceramide and SMase elevated oxidant activity in C2C12 myotubes by 15–35% ( P < 0.05) and in diaphragm muscle fiber bundles by 58–120% ( P < 0.05). The SMase-induced increase in diaphragm oxidant activity was prevented by NAC. Exogenous ceramide depressed diaphragm force by 55% ( P < 0.05), while SMase depressed maximal force by 30% ( P < 0.05) and accelerated fatigue—effects opposed by treatment with NAC. In conclusion, our findings suggest that SMase stimulates a ceramide-oxidant signaling pathway that results in muscle weakness and fatigue.

Alternatives to the canonical insulin-stimulated pathway for glucose uptake are exercise- and exogenous reactive oxygen species (ROS)-stimulated glucose uptake. We proposed a model wherein mechanical loading, i.e. stretch, stimulates production of ROS to activate AMP-activated kinase (AMPK) to increase glucose uptake. Immunoblotting was used to measure protein phosphorylation; the fluorochrome probe 2′7′-dichlorofluorescin diacetate was used to measure cytosolic oxidant activity and 2-deoxy-d[1,2-3H]glucose was used to measure glucose uptake. The current studies demonstrate that stretch increases ROS, AMPKα phosphorylation and glucose transport in murine extensor digitorum longus (EDL) muscle (+121%, +164% and +184%, respectively; P 0.16). We also demonstrate that stretch-stimulated glucose uptake persists in the presence of the phosphatidylinositol 3-kinase (PI3-K) inhibitors wortmannin and LY294001 (P 0.99). These data indicate that stretch-stimulated glucose uptake in skeletal muscle is mediated by a ROS- and p38 MAPK-dependent mechanism that appears to be AMPKα2- and PI3-K-independent.

Acid sphingomyelinase plays important roles in ceramide homeostasis, which has been proposed to be linked to insulin resistance. To test this association in vivo, acid sphingomyelinase deletion (asm–/–) was transferred to mice lacking the low density lipoprotein receptor (ldlr–/–), and then offsprings were placed on control or modified (enriched in saturated fat and cholesterol) diets for 10 weeks. The modified diet caused hypercholesterolemia in all genotypes; however, in contrast to asm+/+/ldlr–/–, the acid sphingomyelinase-deficient littermates did not display hepatic triacylglyceride accumulation, although sphingomyelin and other sphingolipids were substantially elevated, and the liver was enlarged. asm–/–/ldlr–/– mice on a modified diet did not accumulate body fat and were protected against diet-induced hyperglycemia and insulin resistance. Experiments with hepatocytes revealed that acid sphingomyelinase regulates the partitioning of the major fatty acid in the modified diet, palmitate, into two competitive and inversely related pools, triacylglycerides and sphingolipids, apparently via modulation of serine palmitoyltransferase, a rate-limiting enzyme in de novo sphingolipid synthesis. These studies provide evidence that acid sphingomyelinase activity plays an essential role in the regulation of glucose metabolism by regulating the hepatic accumulation of triacylglycerides and sphingolipids during consumption of a diet rich in saturated fats.

Murine models of starvation-induced muscle atrophy demonstrate that reduced protein kinase B (AKT) function upregulates the atrophy-related gene atrogin-1/MAFbx (atrogin). The mechanism involves release of inhibition of Forkhead transcription factors, namely Foxo1 and Foxo3. Elevated atrogin mRNA also corresponds with elevated TNF in inflammatory catabolic states, including cancer and chronic heart failure. Exogenous tumor necrosis factor (TNF) increases atrogin mRNA in vivo and in vitro. We used TNF-treated C2C12 myotubes to test the hypothesis that AKT-Foxo1/3 signaling mediates TNF regulation of atrogin mRNA. Here we confirm that exposure to TNF increases atrogin mRNA (+125%). We also confirm that canonical AKT-mediated regulation of atrogin is active in C2C12 myotubes. Inhibition of phosphoinositol-3 kinase (PI3K)/AKT signaling with wortmannin reduces AKT phosphorylation (−87%) and increases atrogin mRNA (+340%). Activation with insulin-like growth factor (IGF) increases AKT phosphorylation (+126%) and reduces atrogin mRNA (−15%). Although AKT regulation is intact, our data suggest it does not mediate TNF effects on atrogin. TNF increases AKT phosphorylation (+50%) and stimulation of AKT with IGF does not prevent TNF induction of atrogin mRNA. Nor does TNF appear to signal through Foxo1/3 proteins. TNF has no effect on Foxo1/3 mRNA or Foxo1/3 nuclear localization. Instead, TNF increases nuclear Foxo4 protein (+55%). Small interfering RNA oligos targeted to two distinct regions of Foxo4 mRNA reduce the TNF-induced increase in atrogin mRNA (−34% and −32%). We conclude that TNF increases atrogin mRNA independent of AKT via Foxo4. These results suggest a mechanism by which inflammatory catabolic states may persist in the presence of adequate growth factors and nutrition.

A number of physiological changes follow prolonged skeletal muscle unloading as occurs in spaceflight, bed rest, and hindlimb suspension (HLS) and also in aging. These include muscle atrophy, fiber type switching, and loss of the ability to switch between lipid and glucose usage, or metabolic inflexibility. The signaling and genomic events that precede these physiological manifestations have not been investigated in detail, particularly in regard to loss of metabolic flexibility. Here we used gene arrays to determine the effects of 24-h HLS on metabolic remodeling in mouse muscle. Acute unloading resulted in differential expression of a number of transcripts in soleus and gastrocnemius muscle, including many involved in lipid and glucose metabolism. These include the peroxisome proliferator-activated receptors (PPARs). In contrast to Ppar-alpha and Ppar-gamma, which were downregulated by acute HLS, Ppar-delta was upregulated concomitant with increased expression of its downstream target, uncoupling protein-3 (Ucp-3). However, differential expression of Ppar-delta was both acute and transient in nature, suggesting that regulation of PPARdelta may represent an adaptive, compensatory response aimed at regulating fuel utilization and maintaining metabolic flexibility.

Cachexia is common in chronic inflammatory diseases and is attributed, in part, to an elevation of circulating proinflammatory cytokines. TNF-α is the prototype in this category. IFN-γ is also thought to play a role, but the evidence supporting this model is primarily indirect. To determine the direct effects of IFN-γ stimulation on muscle cells, we selected key components of the procatabolic signaling pathways by which TNF-α stimulates protein loss. We tested two hypotheses: 1) IFN-γ mimics TNF-α signaling by increasing intracellular oxidant activity and activating MAPKs and NF-κB and 2) IFN-γ increases the expression of the ubiquitin ligases atrogin1/MAFbx and muscle-specific ring finger protein 1 (MuRF1). Results showed that treatment with IFN-γ at 60 ng/ml increased Stat1 phosphorylation after 15 min, indicating receptor activation. IFN-γ had no effect on cytosolic oxidant activity, as measured by 2′,7′-dichlorofluorescein oxidation. Nor did IFN-γ activate JNK, ERK1/2, or p38 MAPK, as assessed by Western blot. Treatment for up to 60 min did not decrease IκB-α protein levels, as measured by Western blot analysis, or the DNA binding activity of NF-κB, as measured by EMSA. After 6 h, IFN-γ decreased Akt phosphorylation and increased atrogin1/MAFbx and MuRF1 mRNA. Daily treatment for up to 72 h did not alter adult fast-type myosin heavy chain content or the total protein-to-DNA ratio. These data show that responses of myotubes to IFN-γ and TNF-α differ markedly and provide little evidence for a direct catabolic effect of IFN-γ on muscle.

Antigravity muscles atrophy and weaken during prolonged mechanical unloading caused by bed rest or spaceflight. Unloading also induces oxidative stress in muscle, a putative cause of weakness. We tested the hypothesis that dietary supplementation with Bowman-Birk inhibitor concentrate (BBIC), a soy protein extract, would oppose these changes. Adult mice were fed a diet supplemented with 1% BBIC during hindlimb unloading for up to 12 days. Soleus muscles of mice fed the BBIC-supplemented diet weighed less, developed less force per cross-sectional area, and developed less total force after unloading than controls. BBIC supplementation was protective, blunting decrements in soleus muscle weight and force. Cytosolic oxidant activity was assessed using 2′,7′-dichlorofluorescin diacetate. Oxidant activity increased in unloaded muscle, peaking at 3 days and remaining elevated through 12 days of unloading. Increases in oxidant activity correlated directly with loss of muscle mass and were abolished by BBIC supplementation. In vitro assays established that BBIC directly buffers reactive oxygen species and also inhibits serine protease activity. We conclude that dietary supplementation with BBIC protects skeletal muscle during prolonged unloading, promoting redox homeostasis in muscle fibers and blunting atrophy-induced weakness.

Underlying the pathogenesis of chronic disease is the state of oxidative stress. Oxidative stress is an imbalance in oxidant and antioxidant levels. If an overproduction of oxidants overwhelms the antioxidant defenses, oxidative damage of cells, tissues, and organs ensues. In some cases, oxidative stress is assigned a causal role in disease pathogenesis, whereas in others the link is less certain. Along with underlying oxidative stress, chronic disease is often accompanied by muscle wasting. It has been hypothesized that catabolic programs leading to muscle wasting are mediated by oxidative stress. In cases where disease is localized to the muscle, this concept is easy to appreciate. Transmission of oxidative stress from diseased remote organs to skeletal muscle is thought to be mediated by humoral factors such as inflammatory cytokines. This review examines the relationship between oxidative stress, chronic disease, and muscle wasting, and the mechanisms by which oxidative stress acts as a catabolic signal.

Critical illness polyneuropathies (CIP) and myopathies (CIM) are common complications of critical illness. Several weakness syndromes are summarized under the term intensive care unit-acquired weakness (ICUAW). We propose a classification of different ICUAW forms (CIM, CIP, sepsis-induced, steroid-denervation myopathy) and pathophysiological mechanisms from clinical and animal model data. Triggers include sepsis, mechanical ventilation, muscle unloading, steroid treatment, or denervation. Some ICUAW forms require stringent diagnostic features; CIM is marked by membrane hypoexcitability, severe atrophy, preferential myosin loss, ultrastructural alterations, and inadequate autophagy activation while myopathies in pure sepsis do not reproduce marked myosin loss. Reduced membrane excitability results from depolarization and ion channel dysfunction. Mitochondrial dysfunction contributes to energy-dependent processes. Ubiquitin proteasome and calpain activation trigger muscle proteolysis and atrophy while protein synthesis is impaired. Myosin loss is more pronounced than actin loss in CIM. Protein quality control is altered by inadequate autophagy. Ca(2+) dysregulation is present through altered Ca(2+) homeostasis. We highlight clinical hallmarks, trigger factors, and potential mechanisms from human studies and animal models that allow separation of risk factors that may trigger distinct mechanisms contributing to weakness. During critical illness, altered inflammatory (cytokines) and metabolic pathways deteriorate muscle function. ICUAW prevention/treatment is limited, e.g., tight glycemic control, delaying nutrition, and early mobilization. Future challenges include identification of primary/secondary events during the time course of critical illness, the interplay between membrane excitability, bioenergetic failure and differential proteolysis, and finding new therapeutic targets by help of tailored animal models. ispartof: Physiological Reviews vol:95 issue:3 pages:1025-1109 ispartof: location:United States status: published

© 2015 Elsevier Inc. 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.

Exercise increases glucose transport into skeletal muscle via a pathway that is poorly understood. We investigated the role of endogenously produced reactive oxygen species (ROS) in contraction-mediated glucose transport. Repeated contractions increased 2-deoxyglucose (2-DG) uptake roughly threefold in isolated, mouse extensor digitorum longus (fast-twitch) muscle. N-Acetylcysteine (NAC), a non-specific antioxidant, inhibited contraction-mediated 2-DG uptake by ∼50% (P < 0.05 versus control values), but did not significantly affect basal 2-DG uptake or the uptake induced by insulin, hypoxia or 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR, which mimics AMP-mediated activation of AMP-activated protein kinase, AMPK). Ebselen, a glutathione peroxidase mimetic, also inhibited contraction-mediated 2-DG uptake (by almost 60%, P < 0.001 versus control values). Muscles from mice overexpressing Mn2+-dependent superoxide dismutase, which catalyses H2O2 production from superoxide anions, exhibited a ∼25% higher rate of contraction-mediated 2-DG uptake versus muscles from wild-type control mice (P < 0.05). Exogenous H2O2 induced oxidative stress, as judged by an increase in the [GSSG]/[GSH + GSSG] (reduced glutathione + oxidized glutathione) ratio to 2.5 times control values, and this increase was substantially blocked by NAC. Similarly, NAC significantly attenuated contraction-mediated oxidative stress as judged by measurements of glutathione status and the intracellular ROS level with the fluorescent indicator 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein (P < 0.05). Finally, contraction increased AMPK activity and phosphorylation ∼10-fold, and NAC blocked ∼50% of these changes. These data indicate that endogenously produced ROS, possibly H2O2 or its derivatives, play an important role in contraction-mediated activation of glucose transport in fast-twitch muscle.

Phospholipase A2(PLA2) activity supports production of reactive oxygen species (ROS) by mammalian cells. In skeletal muscle, endogenous ROS modulate the force of muscle contraction. We tested the hypothesis that skeletal muscle cells constitutively express the calcium-independent PLA2(iPLA2) isoform and that iPLA2modulates both cytosolic oxidant activity and contractile function. Experiments utilized differentiated C2C12myotubes and a panel of striated muscles isolated from adult mice. Muscle preparations were processed for measurement of mRNA by real-time PCR, protein by immunoblot, cytosolic oxidant activity by the dichlorofluorescein oxidation assay, and contractile function by in vitro testing. We found that iPLA2was constitutively expressed by all muscles tested (myotubes, diaphragm, soleus, extensor digitorum longus, gastrocnemius, heart) and that mRNA and protein levels were generally similar among muscles. Selective iPLA2blockade by use of bromoenol lactone (10 μM) decreased cytosolic oxidant activity in myotubes and intact soleus muscle fibers. iPLA2blockade also inhibited contractile function of unfatigued soleus muscles, shifting the force-frequency relationship rightward and depressing force production during acute fatigue. Each of these changes could be reproduced by selective depletion of superoxide anions using superoxide dismutase (1 kU/ml). These findings suggest that constitutively expressed iPLA2modulates oxidant activity in skeletal muscle fibers by supporting ROS production, thereby influencing contractile properties and fatigue characteristics.

Free radicals first were detected in skeletal muscle by Commoner et al [1] in 1952 using the emerging technology of electron paramagnetic resonance spectroscopy. The pathophysiologic importance of this finding was largely unappreciated until the mid-1980s, when scattered reports began to link muscle disease with oxidative stress [2–6]. In the ensuing 2 decades, an increasing number of publications has made it clear that oxidative stress is a fundamental part of inflammatory disease and is linked inextricably to muscle dysfunction under pathologic conditions. Oxidative stress has been linked to loss of muscle performance in widely diverse pathologies that include heart failure [7,8], cancer [9,10], muscular dystrophy [11–13], chronic obstructive pulmonary disease [14], rheumatoid arthritis [15], obstructive sleep apnea [16–18], rhabdomyolysis [19], renal insufficiency [20], and AIDS [21]. The prevalence of this association and the disparate nature of the underlying diseases raise obvious questions about the factors that induce oxidative stress in muscle and the mechanisms whereby oxidative stress compromise muscle function.

The ubiquitin-proteasome pathway plays a critical role in the adaptation of skeletal muscle to persistent decreases or increases in muscle activity. This article outlines the basics of pathway function and reviews what we know about pathway responses to altered muscle use. The ubiquitin-proteasome pathway regulates proteolysis in mammalian cells by attaching ubiquitin polymers to damaged proteins; this targets the protein for degradation via the 26S proteasome. The pathway is constitutively active in muscle and continually regulates protein turnover. Conditions of decreased muscle use, e.g., unloading, denervation, or immobilization, stimulate general pathway activity. This activity increase is caused by upregulation of regulatory components in the pathway and leads to accelerated proteolysis, resulting in net loss of muscle protein. Pathway activity is also increased in response to exercise, a two-phase response. An immediate increase in selective ubiquitin conjugation by constitutive pathway components contributes to exercise-stimulated signal transduction. Over hours-to-days, exercise also stimulates a delayed increase in general ubiquitin conjugating activity by inducing expression of key components in the pathway. This increase mediates a late-phase rise in protein degradation that is required for muscle adaptation to exercise. Thus the ubiquitin-proteasome pathway functions as an essential mediator of muscle remodeling, both in atrophic states and exercise training.

Atrogin1/MAFbx is an ubiquitin ligase that mediates muscle atrophy in a variety of catabolic states. We recently found that H2O2 stimulates atrogin1/MAFbx gene expression. Since the cytokine tumor necrosis factor-alpha (TNF-alpha) stimulates both reactive oxygen production and general activity of the ubiquitin conjugating pathway, we hypothesized that TNF-alpha would also increase atrogin1/MAFbx gene expression. As with H2O2, we found that TNF-alpha exposure up-regulates atrogin1/MAFbx mRNA within 2 h in C2C12 myotubes. Intraperitoneal injection of TNF-alpha increased atrogin1/MAFbx mRNA in skeletal muscle of adult mice within 4 h. Exposing myotubes to either TNF-alpha or H2O2 also produced general activation of the mitogen-activated protein kinases (MAPKs): p38, ERK1/2, and JNK. The increase in atrogin1/MAFbx gene expression induced by TNF-alpha was not altered significantly by ERK inhibitor PD98059 or the JNK inhibitor SP600125. In contrast, atrogin1/MAFbx up-regulation and the associated increase in ubiquitin conjugating activity were both blunted by p38 inhibitors, either SB203580 or curcumin. These data suggest that TNF-alpha acts via p38 to increase atrogin1/MAFbx gene expression in skeletal muscle.

Fatigue of hand and forearm muscle groups can limit task performance by astronauts wearing space suits. Countermeasures to delay fatigue would therefore be useful to the space program. N-acetylcysteine (NAC) has been shown to inhibit fatigue during other tasks so we tested its effects during handgrip exercise. Volunteers practiced isometric handgrip maneuvers until performance was reproducible over three successive sessions (baseline). Performance then was retested after ingesting NAC (150 mg.kg(-1)) or saline. Drug administration increased NAC and cysteine blood levels (P < 0.001). Performance of sustained maximal efforts was unaffected. During repetitive submaximal efforts, NAC delayed fatigue (130% baseline) and inhibited glutathione oxidation. Saline did not alter glutathione status or performance of sustained maneuvers; repetitive task performance was increased by 15% (P < 0.05), a placebo effect. These data indicate that NAC supports glutathione homeostasis in exercising humans and may delay muscle fatigue during repetitive handgrip exercise. Our findings support oxidative stress as a causal factor in human muscle fatigue and argue for larger translational studies to define NAC effects on human performance.

Background— Mechanical unloading of the heart results in atrophic remodeling. In skeletal muscle, atrophy is associated with inactivation of the mammalian target of rapamycin (mTOR) pathway and upregulation of critical components of the ubiquitin proteosome proteolytic (UPP) pathway. The hypothesis is that mechanical unloading of the mammalian heart has differential effects on pathways of protein synthesis and degradation. Methods and Results— In a model of atrophic remodeling induced by heterotopic transplantation of the rat heart, we measured gene transcription, protein expression, polyubiquitin content, and regulators of the mTOR pathway at 2, 4, 7, and 28 days. In atrophic hearts, there was an increase in polyubiquitin content that peaked at 7 days and decreased by 28 days. Furthermore, gene and protein expression of UbcH2, a ubiquitin conjugating enzyme, was also increased early in the course of unloading. Transcript levels of TNF-α, a known regulator of UbcH2-dependent ubiquitin conjugating activity, were upregulated early and transiently in the atrophying rat heart. Unexpectedly, p70S6K and 4EBP1, downstream components of mTOR, were activated in atrophic rat heart. This activation was independent of Akt, a known upstream regulator of mTOR. Rapamycin treatment of the unloaded rat hearts inhibited the activation of p70S6K and 4EBP1 and subsequently augmented atrophy in these hearts compared with vehicle-treated, unloaded hearts. Conclusions— Atrophy of the heart, secondary to mechanical unloading, is associated with early activation of the UPP. The simultaneous activation of the mTOR pathway suggests active remodeling, involving both protein synthesis and degradation.

The classical definition of “heart failure” (HF) holds that “heart failure occurs when an abnormality of cardiac function causes the heart to fail to pump blood at a rate required by the metabolizing tissues or when the heart can do so only with an elevated pressure” [(1)][1]. However, the

Since the early 1980s biologists have recognized that skeletal muscle generates free radicals. Of particular interest are two closely related redox cascades--reactive oxygen species (ROS) and nitric oxide (NO) derivatives. The ROS cascade is initiated by superoxide anion radicals derived from the mitochondrial electron transport chain, the membrane-associated NAD(P)H oxidase complex, or other sources. NO is produced by two NO synthase isoforms constitutively expressed by muscle fibers. ROS and NO derivatives are produced continually and are detectable in both the cytosolic and extracellular compartments. Production increases during strenuous exercise. Both ROS and NO modulate contractile function. Under basal conditions, low levels of ROS enhance force production. Excessive ROS accumulation inhibits force, for example, during fatiguing exercise. NO inhibits skeletal muscle contraction, an effect that is partially mediated by cyclic GMP as a second messenger. With aging, redox modulation of muscle contraction may be altered by changes in the rates of ROS and NO production, the levels of endogenous antioxidants that buffer ROS and NO, and the sensitivities of regulatory proteins to ROS and NO action. The impact of aging on contractile regulation depends on the relative magnitude of these changes and their net effects on ROS and NO activities at the cellular level.

Skeletal muscle adapts to a prolonged reduction in physical activity by decreasing muscle fibre size and protein content. This physiological response is commonly termed disuse atrophy. It is most clearly stimulated by conditions that reduce mechanical loading of postural or respiratory muscles in the absence of disease and neural deficits. Examples of such conditions include bed rest, joint immobilization, limb suspension, microgravity and mechanical ventilation. In muscle disuse, as in all atrophic processes, the rate of proteolysis exceeds the rate of protein synthesis causing net loss of protein. A dispassionate review of the literature shows that both aspects of muscle protein homeostasis are disrupted by prolonged disuse. Proteolysis increases and synthesis declines. These changes have additive effects, contributing jointly to protein loss and muscle atrophy. The relative magnitudes of these contributions have not been measured directly and are not known. Therefore, like most scientists, we are resolutely ecumenical on the issue of proteolysis vs. synthesis. In contrast, a few individuals fervently argue that disuse atrophy is caused by decreased protein synthesis and that proteolysis is unimportant. This viewpoint has created unnecessary controversy in the literature and heated discussion at scientific conferences. This controversy is the focus of our CrossTalk debate. To be clear, the ‘dominant mechanism’ of disuse atrophy is not known. Rates of proteolysis vs. protein synthesis cannot be quantified using existing methods nor can differences in these rates be measured directly. Attempts to estimate these values must be based on first principles, must involve untested assumptions, and cannot be validated experimentally. This makes it impossible to assign ‘dominance’ with scientific rigor. Instead of speculating on this issue, we will present evidence for the biological importance of proteolysis in disuse atrophy. The extent of peer-reviewed research, the diversity of experimental approaches, and the level of mechanistic detail make a compelling case for our position that proteolysis is essential for disuse atrophy.

The arguments put forward by Phillips and McGlory do not take into consideration advances in the field over the past 5 years. These new discoveries have provided critical information on disuse atrophy in both human and rodent muscle. They also call into question key elements of our opponents’ reasoning. The assertion that disuse atrophy largely reflects a decline in protein synthesis is based solely on data from human vastus lateralis muscle. Recent studies demonstrate the limitations of this approach. For example, during cast immobilization, disuse atrophy occurs in a variety of leg muscles at least 2 weeks before significant atrophy is detectable in vastus lateralis (Psatha et al. 2012), suggesting the response of vastus lateralis is delayed relative to other muscles. New data also show that atrophy of vastus lateralis is non-uniform, affecting some regions more than others (Miokovic et al. 2012). Such confounding variables preclude the use of localized measurements in vastus lateralis, indeed any single muscle, as the sole source of information on disuse atrophy. Our opponents argue for limiting research to human vastus lateralis because ‘inherent species-specific differences’ exist between rodent and human muscles. Again, a plethora of recent research has debunked this misconception. As in rodent limb muscle, new data show that human type I fibres undergo greater atrophy than human type II fibres in soleus, gastrocnemius and vastus lateralis muscles after prolonged unloading (Fitts et al. 2010; Brocca et al. 2012). As in rodents, disuse atrophy of human muscle can be detected in as few as 4–5 days (Suetta et al. 2012; Wall et al. 2014), causes losses of up to 2.5–6% per day (Suetta et al. 2012; Grosu et al. 2012; Wall et al. 2014), and occurs at rates that differ widely among human muscles (Belavý et al. 2009; Miokovic et al. 2012; Psatha et al. 2012). Recent data also confirm that human muscle mimics rodent muscle by upregulating proteolytic gene products (Suetta et al. 2012). This consistency among species is fundamental, broad based, and compelling. It provides a solid scientific foundation for translational research in the field. Preclinical studies in rodents have been instrumental in defining the mechanisms of disuse atrophy, identifying atrophy-related pathways, and discovering genes that regulate the process. Rodent research has also demonstrated that proteolytic blockade can inhibit disuse atrophy (Jamart et al. 2011; Talbert et al. 2013), establishing a causal role for proteolysis. Modern evidence leads to the inexorable conclusion that proteolysis is not simply important for disuse atrophy. It is essential.

Nitric oxide (NO) derivatives and reactive oxygen species (ROS) modulate contractile function of respiratory and limb skeletal muscle. The intracellular processes regulated by NO and ROS remain enigmatic, however. Studies of reduced preparations have identified a number of regulatory proteins that exhibit altered function when exposed to exogenous NO or ROS donors ex vivo. The relative importance of these targets in the intact cell is not known and conflicting theories abound regarding the mechanism(s) whereby NO and ROS regulate contraction. This review article provides a personal perspective on the processes regulated by NO and ROS by addressing three major topics: 1) the regulatory mechanisms by which endogenous NO depresses force production, 2) the processes whereby endogenous ROS modulate contraction of unfatigued muscle, and 3) the site(s) of action and reversibility of ROS effects in muscle fatigue.

Over the past decade, reactive oxygen species (ROS) and nitric oxide (NO) derivatives have been established as physiological modulators of skeletal muscle function. This mini-review addresses the roles of these molecules as endogenous regulators of muscle contraction. The article is organized in two parts. First, established concepts are briefly outlined. This section provides an overview of ROS production by muscle, antioxidant buffers that oppose ROS effects, enzymatic synthesis of NO in muscle, the effects of endogenous ROS on contractile function, and NO as a contractile modulator. Second, a selected group of unresolved topics are highlighted. These more controversial issues include putative source(s) of regulatory ROS, the relative importance of the two NO synthase isoforms constitutively coexpressed by muscle fibers, molecular mechanisms of ROS and NO action, and the physiological relevance of redox regulation. By discussing current questions, as well as the established paradigm, this article is intended to further debate and stimulate research in this area.

Background —We have developed a transgenic mouse with cardiac-restricted overexpression of tumor necrosis factor-α (TNF-α). These mice develop a heart failure phenotype characterized by left ventricular dysfunction and remodeling, pulmonary edema, and elevated levels of TNF-α in the peripheral circulation from cardiac spillover. Given that TNF-α causes atrophy and loss of function in respiratory muscle, we asked whether transgenic mice developed diaphragm dysfunction and whether contractile losses were caused by oxidative stress or tissue remodeling. Methods and Results —Muscles excised from transgenic mice and littermate controls were studied in vitro with direct electrical stimulation. Cytosolic oxidant levels were measured with 2′,7′-dichlorofluorescin diacetate; emissions of the oxidized product were detected by fluorescence microscopy. Force generation by the diaphragm of transgenic animals was 47% less than control (13.2±0.8 [±SEM] versus 25.1±0.6 N/cm 2 ; P P N -acetylcysteine 10 mmol/L ( P N -acetylcysteine, suggesting that changes observed in the diaphragm of transgenic animals were mediated by TNF-α. There were no differences in body or diaphragm weights between transgenic and control animals, nor was there evidence of muscle injury or apoptosis. Conclusions —Elevated circulating levels of TNF-α provoke contractile dysfunction in the diaphragm through an endocrine mechanism thought to be mediated by oxidative stress.

Alteration of skeletal muscle function by reactive oxygen species and nitric oxide (NO) may involve regulation of the activity of the skeletal muscle Ca2+ release channel (also known as RyR1). We have shown that oxidants can activate RyR1 and produce inter-subunit disulfide bonds. Both effects are prevented by pretreatment with either NO donors or N-ethylmaleimide under conditions that modify less than 5% of the total sulfhydryls on RyR1. Oxidation-induced intersubunit crosslinking can also be prevented by the binding of either Ca2+ calmodulin or apocalmodulin to RyR1. Also, both Ca2+ calmodulin and apocalmodulin binding are blocked by oxidation of RyR1. In contrast, alkylation with N-ethylmaleimide or reaction with NO donors preferentially blocks apocalmodulin binding to RyR1, suggesting the existence of a regulatory cysteine within the apocalmodulin binding site. We have demonstrated that Ca2+ calmodulin and apocalmodulin bind to overlapping, but nonidentical, sites on RyR1 and that cysteine 3635 is close to or within the apocalmodulin-binding site on RyR1. This cysteine is also one of the cysteines that form the intersubunit disulfide bonds, suggesting that calmodulin binds at an intersubunit contact site. Our findings are consistent with a model in which oxidants regulate the activity of RyR1 directly by altering subunit-subunit interactions and indirectly by preventing the binding of either Ca2+-bound calmodulin or apocalmodulin. NO also has both a direct and an indirect effect: it blocks the ability of oxidants to generate intersubunit disulfide bonds and prevents apocalmodulin binding.

Skeletal muscle atrophy and weakness are thought to be stimulated by tumor necrosis factor alpha (TNF-alpha) in a variety of chronic diseases. However, little is known about the direct effects of TNF-alpha on differentiated skeletal muscle cells or the signaling mechanisms involved. We have tested the effects of TNF-alpha on the mouse-derived C2C12 muscle cell line and on primary cultures from rat skeletal muscle. TNF-alpha treatment of differentiated myotubes stimulated time- and concentration-dependent reductions in total protein content and loss of adult myosin heavy chain (MHCf) content; these changes were evident at low TNF-alpha concentrations (1-3 ng/ml) that did not alter muscle DNA content and were not associated with a decrease in MHCf synthesis. TNF-alpha activated binding of nuclear factor kappaB (NF-kappaB) to its targeted DNA sequence and stimulated degradation of I-kappaBalpha, an NF-kappaB inhibitory protein. TNF-alpha stimulated total ubiquitin conjugation whereas a 26S proteasome inhibitor (MG132 10-40 microM) blocked TNF-alpha activation of NF-kappaB. Catalase 1 kU/ml inhibited NF-kappaB activation by TNF-alpha; exogenous hydrogen peroxide 200 microM activated NF-kappaB and stimulated I-kappaBalpha degradation. These data demonstrate that TNF-alpha directly induces skeletal muscle protein loss, that NF-kappaB is rapidly activated by TNF-alpha in differentiated skeletal muscle cells, and that TNF-alpha/NF-kappaB signaling in skeletal muscle is regulated by endogenous reactive oxygen species.

1. We used intact single fibres from a mouse foot muscle to study the role of oxidation-reduction in the modulation of contractile function. 2. The oxidant hydrogen peroxide (H2O2, 100-300 microM) for brief periods did not change myoplasmic Ca2+ concentrations ([Ca2+]i) during submaximal tetani. However, force increased by 27 % during the same contractions. 3. The effects of H2O2 were time dependent. Prolonged exposures resulted in increased resting and tetanic [Ca2+]i, while force was significantly diminished. The force decline was mainly due to reduced myofibrillar Ca2+ sensitivity. There was also evidence of altered sarcoplasmic reticulum (SR) function: passive Ca2+ leak was increased and Ca2+ uptake was decreased. 4. The reductant dithiothreitol (DTT, 0.5-1 mM) did not change tetanic [Ca2+]i, but decreased force by over 40 %. This was completely reversed by subsequent incubations with H2O2. The force decline induced by prolonged exposure to H2O2 was reversed by subsequent exposure to DTT. 5. These results show that the elements of the contractile machinery are differentially responsive to changes in the oxidation-reduction balance of the muscle fibres. Myofibrillar Ca2+ sensitivity appears to be especially susceptible, while the SR functions (Ca2+ leak and uptake) are less so.

Respiratory failure is a common and often lethal complication of severe peritonitis. Because this inflammatory process develops in the abdomen, adjacent to the diaphragm, we hypothesized that peritonitis might directly compromise diaphragm function. We tested this hypothesis using male Sprague-Dawley rats. We injected oyster glycogen into the rats' peritoneum, and 16 h later the peritoneum was lavaged for leukocyte analysis and muscle samples were excised. Contractile properties of diaphragm fiber bundles were measured in vitro. We found that neutrophils and macrophages were concentrated in peritoneal lavage fluid of experimental animals (p0.01) and were adherent to the abdominal surface of the diaphragm. Immunohistochemistry showed increases in inducible nitric oxide synthase in microvessels of the diaphragm and limb skeletal muscles but not in heart or spleen. Peritonitis decreased maximal force production by the diaphragm (23.6+/-0.6 versus 21.2+/-0.6 N/cm2; p0.05) and decreased the absolute forces developed at physiologic stimulus frequencies (30 Hz; p0.01), depressing the overall force-frequency relationship (p0.001). Peritonitis had little effect on acute muscular fatigue. These data demonstrate that peritonitis weakens the diaphragm in rats and suggest that humans with peritonitis may be predisposed to respiratory muscle dysfunction.

Over the last two decades, nitric oxide (NO) has been established as a novel mediator of biological processes, ranging from vascular control to long-term memory, from tissue inflammation to penile erection. This paper reviews recent research which shows that NO and its derivatives also are synthesized within skeletal muscle and that NO derivatives influence various aspects of muscle function. Individual muscle fibres express one or both of the constitutive NO synthase (NOS) isoforms. Type I (neuronal) NOS is localized to the sarcolemma of fast fibres; type III (endothelial) NOS is associated with mitochondria. Isolated skeletal muscle produces NO at low rates under resting conditions and at higher rates during repetitive contraction. NO appears to mediate cell-cell interactions in muscle, including vasodilation and inhibition of leucocyte adhesion. NO also acts directly on muscle fibres to alter cell function. Muscle metabolism appears to be NO-sensitive at several sites, including glucose uptake, glycolysis, mitochondrial oxygen consumption and creatine kinase activity. NO also modulates muscle contraction, inhibiting force output by altering excitation-contraction coupling. The mechanisms of NO action are likely to include direct effects on redox-sensitive regulatory proteins, interaction with endogenous reactive oxygen species, and activation of second messengers such as cyclic guanosine monophosphate (cGMP). In conclusion, research published over the past few years makes it clear that skeletal muscle produces NO and that endogenous NO modulates muscle function. Much remains to be learned, however, about the physiological importance of NO actions and about their underlying mechanisms.

The present experiments tested nitric oxide (NO) effects on shortening velocity and power production in maximally activated rat diaphragm at 37 degrees C. Diaphragm fiber bundles (n = 10/group) were incubated at 37 degrees C in Krebs-Ringer solution containing no added drug (control), the NO synthase inhibitor Nomega-nitro-L-arginine (L-NNA; 10 mM), the NO donor sodium nitroprusside (SNP; 1 mM), or a combination (L-NNA + SNP). Loaded shortening velocity was measured via the load-clamp technique over a range of afterloads. Unloaded shortening velocity (Vo) was measured in control and L-NNA-treated bundles (n = 12/group) by using the slack test. Force-velocity data fitted to the Hill equation determined a Vmax of 13.7+/-0.4 lengths/s, contradicting the notion that rat diaphragm Vmax declines at temperatures35 degrees C. In contrast, L-NNA decreased Vmax (P0.05), loaded shortening velocity (P0.001), and power production (P0.001), but did not change Vo or maximal isometric force. All L-NNA effects were prevented by coincubating fiber bundles with L-NNA + SNP. SNP alone had no effect on any variable. These data indicate that endogenous NO is essential for optimal myofilament function during active shortening.

We investigated the enzymatic source, cellular production, and functional importance of nitric oxide (NO) in rat diaphragm. Neuronal and endothelial isoforms of constituitive nitric oxide synthase (nc-NOS, ec-NOS) were identified by immunostaining. NOS activity measured in diaphragm homogenates averaged 5.1 pmol/min/mg. Passive diaphragm fiber bundles produced NO derivatives (NOx) at the rate of 0.9 pmol/min/mg as measured by the cytochrome c reduction assay; NO production was confirmed by photolysis/ chemiluminescence measurements. Endogenous NO depressed diaphragm contractile function. The force of submaximal contraction was increased by NOS inhibitors, an effect that was stable for up to 60 min and was reversed by NO donors. We conclude that diaphragm muscle fibers express nc-NOS, ec-NOS, or both; passive myocytes produce NOx; and NO or NO-derivatives inhibit force production by modulating excitation-contraction coupling.

Ventilation during exercise is near-normal in double-lung transplant recipients despite lung denervation. We tested the hypothesis that denervation effects might be unmasked during exercise by exposing these patients to an expiratory load. Eight double-lung recipients and nine intact control subjects were exercised to exhaustion. Ergometer work increased 20 Watt every 2 min; expiratory threshold loading (4 cm H2O) was imposed for five to six breaths at each exercise level; ventilation and O2 consumption were measured. Transplant recipients and control subjects increased ventilation similarly for comparable fractions of maximal work. At maximal exercise, transplant recipients achieved lower work (62 versus 155 W; p0.001) and O2 consumption (0.88 versus 2.26 L/min; p0.001) than control subjects, with proportional reductions in tidal volume (1.6 versus 2.6 L; p0.05) and ventilation (38 versus 79 L/min; p0.01). Threshold loading decreased expiratory flow, breathing frequency, and minute ventilation in both groups (p0.05). Unlike control subjects, transplant recipients also slowed inspiratory flow (p0.05) and prolonged inspiration (p0.01), exaggerating the fall in breathing frequency and ventilation (p0.01). We conclude that afferent information from pulmonary receptors modulates inspiration during expiratory loading; bilateral denervation disrupts these pathways, causing double-lung recipients to inspire more slowly.

We have shown that endogenous nitrogen oxides (NOx) modulate excitation-contraction coupling in diaphragm. Because cyclic GMP (cGMP) is a second messenger for nitric oxide (NO) inhibition of smooth muscle contraction, we rested the hypothesis that NO acts via cGMP in diaphragm. Fiber bundles from rat diaphragm were studied in vitro. Immunohistochemical analysis using a cGMP-specific monoclonal antibody confirmed the presence of cGMP in the subsarcolemmal region, near nitric oxide synthase (NOS). cGMP measured by ELISA in control muscle (0.27 pmol/mg +/- 0.01 SE) was significantly increased by the NO donor S-nitroso-N-acetylcysteine 1 mM (0.55+/-0.05; N = 6; P < 0.001). Contractile studies showed that the nitric oxide synthase inhibitor N-nitro-L-arginine (L-NNA) 10 mM increased submaximal (40 Hz) tetanic force (P < 0.0001). L-NNA effects were exaggerated by the guanylate cyclase inhibitor LY83583 5-10 microM; force at 40 Hz was increased (P < 0.001). L-NNA effects were partially reversed by 8-bromo-cGMP 1 mM (8-Br-GMP; a cell-permeable cGMP analogue; P < 0.0001) or dipyridamole 10 microM (DPM; a phosphodiesterase inhibitor; P < 0.0001). 8-Br-GMP and DPM produced more-complete L-NNA reversal in combination (P < 0.0001). We conclude that cGMP functions as a second messenger by which NO inhibits diaphragm contraction.