Free radical-mediated skeletal muscle dysfunction in inflammatory conditions.

Loss of functional capacity of skeletal muscle is a major cause of morbidity in patients with a number of acute and chronic clinical disorders, including sepsis, chronic obstructive pulmonary disease, heart failure, uremia, and cancer. Weakness in these patients can manifest as either severe limb muscle weakness (even to the point of virtual paralysis), respiratory muscle weakness requiring mechanical ventilatory support, and/or some combination of these phenomena. While factors such as nutritional deficiency and disuse may contribute to the development of muscle weakness in these conditions, systemic inflammation may be the major factor producing skeletal muscle dysfunction in these disorders. Importantly, studies conducted over the past 15 years indicate that free radical species (superoxide, hydroxyl radicals, nitric oxide, peroxynitrite, and the free radical-derived product hydrogen peroxide) play an key role in modulating inflammation and/or infection-induced alterations in skeletal muscle function. Substantial evidence exists indicating that several free radical species can directly alter contractile protein function, and evidence suggests that free radicals also have important effects on sarcoplasmic reticulum function, on mitochondrial function, and on sarcolemmal integrity. Free radicals also modulate activation of several proteolytic pathways, including proteosomally mediated protein degradation and, at least theoretically, may also influence pathways of protein synthesis. As a result, free radicals appear to play an important role in regulating a number of downstream processes that collectively act to impair muscle function and lead to reductions in muscle strength and mass in inflammatory conditions.     

Measurement of superoxide-derived free radicals in the reperfused heart. Evidence for a free radical mechanism of reperfusion injury

There has been considerable controversy regarding the role of oxygen free radicals as important mediators of cell damage in reperfused myocardium. This controversy regards whether superoxide and hydroxyl free radicals are generated on reperfusion and if these radicals actually cause impaired contractile function. In this study, EPR studies using the spin trap 5,5-dimethyl-1-pyroline-n-oxide (DMPO) demonstrate the formation of .OH and R. free radicals in the reperfused heart. EPR signals of DMPO-OH, aN = aH = 14.9 G, and DMPO-R aN = 15.8 G aH = 22.8 G are observed, with peak concentrations during the first minute of reperfusion. It is demonstrated that these radicals are derived from .O2- since reperfusion in the presence of enzymatically active recombinant human superoxide dismutase markedly reduced the formation of these signals while inactive recombinant human superoxide dismutase had no effect. On reperfusion with perfusate pretreated to remove adventitial iron, the concentration of the DMPO-OH signal was increased 2-fold and a 4-fold decrease in the DMPO-R signal was observed demonstrating that iron-mediated Fenton chemistry occurs. Hearts reperfused with recombinant human superoxide dismutase exhibited improved contractile function in parallel with the marked reduction in measured free radicals. In order to determine if the reperfusion free radical burst results in impaired contractile function, simultaneous measurements of free radical generation and contractile function were performed. A direct relationship between free radical generation and subsequent impaired contractile function was observed. These studies suggest that superoxide derived .OH and R. free radicals are generated in the reperfused heart via Fenton chemistry. These radicals appear to be key mediators of myocardial reperfusion injury.     

Use of Spin Traps in Intact Animals Undergoing Myocardial Ischemia/Reperfusion: A New Approach to Assessing the Role of Oxygen Radicals in Myocardial “Stunning”

Numerous studies have indirectly, suggested that oxygen-derived free radicals play an important path-ogenetic role in the prolonged depression of contractile function observed in myocardium reperfused after reversible ischemia (myocardial “stunning”). In order to provide direct evidence for the oxy-radical hypothesis of stunning, we administered the spin trap, α-phenyl N-tert-butyl nitrone (PBN), to open-chest dogs undergoing a 15-min coronary artery occlusion followed by reperfusion. Plasma of local coronary venous blood was analyzed by electron paramagnetic resonance (EPR) spectroscopy. EPR signals characteristic of radical adducts of PBN appeared during ischemia and increased dramatically in the first few minutes after reperfusion. After this initial burst, the production of adducts abated but did not cease, persisting up to 3 h after reflow. The production of PBN adducts after reperfusion was inversely related to collateral flow during ischemia. PBN itself enhanced recovery of contractile function. indicating that the radicals trapped may play a pathogenetic role in myocardial stunning. Superoxide dismutase plus catalase attenuated PBN adduct production and, at the same time, improved recovery of contractile function. Antioxidant therapy given 1 min before reperfusion suppressed PBN adduct production and improved contractile recovery; however, the same therapy given 1 min after reperfusion did not suppress early radical production and did not attenuate contractile dysfunction. After i.v. administration, the elimination half-life of PBN was estimated to be approximately 4–5 h. The results demonstrate that 1) free radicals are produced in the stunned myocardium in intact animals; 2) inhibition of free radical production results in improved contractile recovery; and 3) the free radicals important in causing dysfunction are produced in the first few minutes of reperfusion. Taken together, these studies provide cogent evidence supporting the oxy-radical hypothesis of stunning in open-chest dogs. It is now critical to determine whether these results can be reproduced in conscious animal preparations.     

Central nervous system trauma and stroke. I. Biochemical considerations for oxygen radical formation and lipid peroxidation

The generation of oxygen radicals and the process of lipid peroxidation have become a focus of attention for investigators in the fields of central nervous system (CNS) trauma and stroke (e.g., ischemia). Considering our level of understanding of free radical and lipid peroxidation chemistry, absolute proof for their involvement in the pathophysiology of traumatic and ischemic damage to the CNS has been meager. While direct, unequivocal evidence for the participation of free radicals and lipid peroxidation as primary contributors to the death of neuronal tissue waits to be established, numerous recent studies have provided considerable support for the occurrence of free radical and lipid peroxidation reactions in the injured or ischemic CNS. In addition, the pharmacological use of antioxidants and free radical scavengers in the treatment of experimental CNS trauma and ischemia has provided convincing, although indirect evidence, for the involvement of oxygen radicals and lipid peroxidation in these conditions. The intent of this and its companion paper is to review: 1) the biochemical processes which may give rise to free radical reactions in the CNS, 2) the environment of the ischemic cell as it may affect the generation of oxygen radicals and the catalysis of lipid peroxidation reactions, 3) the evidence for the involvement of free radical mechanisms in CNS trauma and ischemia, and 4) the pathophysiological consequences of these phenomena.     

Free radicals, lipid peroxidation and muscular ischemia]

The role of oxygen free radicals in ischemia and reperfusion injury of skeletal muscle has not been well defined, partly because of the relative resistance of this tissue to normothermic ischemia. Under normal conditions small quantities of oxygen free radicals are produced but they are quenched by intracellular free radical scavenging enzymes (superoxide dismutase, catalase and glutathione peroxidase) or alpha-tocopherol. The increase in malondialdehyde suggests increased lipid peroxidation initiated by free radical reactions. Lipid peroxidation is potentially a very damaging process to the organized structure and function of membranes. The results of recent studies indicate that: a) oxygen free-radicals mediates, at least in part, the increased microvascular permeability produced by reoxygenation, b) free radical scavengers can reduce skeletal muscle necrosis occurring after prolonged ischemia. Additional evidence support the hypothesis of the interrelationship between ischemic tissue and inflammatory cells. So capillary plugging by granulocytes and oxygen free radical formation may contribute to the ischemic injury.     

Vitamin E analogue Trolox C. E.s.r. and pulse-radiolysis studies of free-radical reactions.

The reactions between Trolox C, a water-soluble vitamin E analogue, and several oxidizing free radicals including the hydroxyl radical and various peroxy radicals were examined by using the pulse-radiolysis technique. The results demonstrate that Trolox C may undergo rapid one-electron-transfer reactions as well as hydrogen-transfer processes; the resulting phenoxyl radical is shown to be relatively stable, in common with the phenoxyl radical derived from vitamin E. The reactions between the Trolox C phenoxyl radical and a variety of biologically relevant reducing compounds were examined by using both pulse radiolysis and e.s.r. The results demonstrate that the Trolox C phenoxyl radical is readily repaired by ascorbate (k = 8.3 x 10(6) dm3.mol-1.s-1) and certain thiols (k less than 10(5) dm3.mol-1.s-1) but not by urate, NADH or propyl gallate. Evidence from e.s.r. studies indicates that thiol-containing compounds may also enter into similar repair reactions with the alpha-tocopherol phenoxyl radical. Kinetic evidence is presented that suggests that Trolox C may 'repair' proteins that have been oxidized by free radicals.     

Oxygen-derived free radicals and 'stunned myocardium'

A brief, transient period of coronary artery occlusion (less than 20 minutes in duration) followed by reperfusion does not result in irreversible myocyte injury or death, yet the regional contractile function and high energy phosphate content of the previously ischemic tissue remains depressed or 'stunned' for hours to days following reperfusion. It has been suggested that this prolonged postischemic dysfunction of viable, previously ischemic myocardium may be a consequence of oxygen-derived free radicals generated during occlusion or at the time of reperfusion. Recent evidence demonstrates that free radical scavenging agents such as superoxide dismutase (SOD) + catalase, N-2-mercaptopropionylglycine, and allopurinol, administered prior to coronary artery occlusion, significantly enhance recovery of regional contractile function of the stunned, previously ischemic tissue. This improved contractile function was not, however, accompanied by improvements in high energy phosphate metabolism: infusion of SOD + catalase did not preserve ATP stores in the previously ischemic tissue. These data support the hypothesis that oxygen-derived free radicals contribute, at least in part, to the phenomenon of the stunned myocardium. The source or mechanisms of free radical production in the setting of brief, transient ischemia, however, remains to be elucidated.     

Free radicals and anticancer drug resistance: Oxygen free radicals in the mechanisms of drug cytotoxicity and resistance by certain tumors

Certain anticancer agents form free radical intermediates during enzymatic activation. Recent studies have indicated that free radicals generated from adriamycin and mitomycin C may play a critical role in their toxicity to human tumor cells. Furthermore, it is becoming increasingly apparent that reduced drug activation and or enhanced detoxification of reactive oxygen species may be related to the resistance to these anticancer agents by certain tumor cell lines. The purposes of this review are to summarize the evidence pointing toward the significance of free radicals formation in drug toxicity and to evaluate the role of decreased free radical formation and enhanced free radical scavenging and detoxification in the development of anticancer drug resistance by a spectrum of tumor cell types. Studies failing to support the participation of oxyradicals in the cytotoxicity and resistance of adriamycin are also discussed.     

Production, detection, and adaptive responses to free radicals in exercise

Free radicals (particularly oxygen- and nitrogen-centered radicals), and related reactive oxygen and nitrogen species, are generated in cells and tissues during exercise. Mitochondria (actually, 'leakage' of electrons from ubisemiquinone and other electron transport chain components), xanthine oxidase, and phagocytes such as neutrophils may all contribute to free radical production. In this article we review mechanisms of free radical production during exercise and methods for detecting free radicals and related reactive species, during, or immediately following exercise. The evidence presented strongly suggests that free radicals generated during mild to moderate endurance-type exercise actually form part of the mechanism of exercise adaptation that includes extensive biogenesis of muscle mitochondria, increased muscle blood supply, and altered fuel consumption patterns. We suggest, as originally proposed [1], that (at moderately increased levels) free radicals actually act as intracellular signaling molecules to initiate exercise adaptation. In contrast, endurance exercise of extreme duration and extreme intensity appears to generate much higher levels of free radicals that overwhelm cellular antioxidant defenses, and cause tissue damage. Such free radical damage requires effective protein, lipid, and DNA repair systems, and sufficient recuperation, before exercise adaptation can recommence.     

Extracellular calcium modulates generation of reactive oxygen species by the contracting diaphragm.

Recent studies have indicated that free radicals may play an important role in the development of muscle dysfunction in many pathophysiological conditions. Because the degree of muscle dysfunction observed in some of these conditions appears to be both free radical dependent and modulated by extracellular calcium concentrations, we thought that there may be a link between these two phenomena; i.e., the propensity of a muscle to generate free radicals may be dependent on extracellular calcium concentrations. For this reason, we compared formation of reactive oxygen species (ROS; i.e., free radicals) by electrically stimulated rat diaphragms (trains of 20-Hz stimuli for 10 min, train rate 0.25 trains/s) incubated in organ baths filled with physiological solutions containing low (1 mM), normal (2.5 mM), or high (5 mM) calcium levels. Generation of ROS was assessed by measuring the conversion of hydroethidine to ethidium. We found ROS generation with contraction varied with the extracellular calcium level, with low ROS production (3.18 +/- 0.40 ng ethidium/mg tissue) for low-calcium studies and with much higher ROS generation for normal-calcium (18. 90 +/- 2.70 ng/mg) or high-calcium (19.30 +/- 4.50 ng/mg) studies (P < 0.001). Control, noncontracting diaphragms (in 2.5 mM calcium) had little ROS production (3.40 +/- 0.80 ng/mg; P < 0.001). To further investigate this issue, we added nimodipine (20 microM), an L-type calcium channel blocker, to contracting diaphragms (2.5 mM calcium bath) and found that nimodipine also suppressed ROS formation (2.56 +/- 0.85 ng ethidium/mg tissue). These data indicate that ROS generation by the contracting diaphragm is strongly influenced by extracellular calcium concentrations and may be dependent on calcium transport through L-type calcium channels.     

Comparison of protein expression in human deltoideus and vastus lateralis muscles using two-dimensional gel electrophoresis

We have used two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) to study the expression of contractile and regulatory proteins in human vastus lateralis and deltoideus muscles, in order to understand protein turnover and isoform switching in muscles with the same fiber-type composition but different functional properties. We demonstrate a two- to six-fold overexpression of enzymes associated with glycolysis, the tricarboxylic acid cycle, oxidative phosphorylation, and substrate transport in vastus lateralis compared to deltoideus. Expression levels of contractile protein isoforms correlated to the proportion of slow-twitch fibers in deltoideus compared to vastus lateralis are consistent with the different contractile properties of the two muscles. Two proteins involved in free radical homeostasis were differentially expressed, suggesting a direct relationship between radical scavenging and the muscle function. The application of 2-DE and MS to studies of muscle physiology thus offers a more comprehensive assessment of the molecular determinants of muscle function than traditional approaches.     

Measurement of free radical production by in vivo microdialysis during ischemia/reperfusion injury to skeletal muscle

Microdialysis techniques have been used to detect hydroxyl radical and superoxide release into the interstitial space of anaesthetized rat anterior tibialis muscles during a period of prolonged (4 h) limb ischemia and subsequent reperfusion. Data indicate that reperfusion of the ischemic skeletal muscle was associated with a large increase in hydroxyl radical activity in the interstitial space, which may contribute to the significant oxidation of muscle glutathione, protein thiols, and lipids also seen in this model. No evidence for release of superoxide into the interstitial space was found during reperfusion, although this was observed during electrically stimulated contractile activity of the rat limb muscle. These data imply that therapeutic approaches aimed at reduction of hydroxyl radical generation in the interstitial fluid are more likely to be beneficial in reduction of skeletal muscle reperfusion injury than approaches designed to scavenge superoxide radicals.     

Endogenous antioxidant changes in the myocardium in response to acute and chronic stress conditions

Oxygen is a diradical and because of its unique electronic configuration, it has the potential to form strong oxidants (e.g. superoxide radical, hydrogen peroxide and hydroxyl radical) called oxygen free radicals or partially reduced forms of oxygen (PRFO). These highly reactive oxygen species can cause cellular injury by oxidizing lipids and proteins as well as by causing strand breaks in nucleic acids. PRFO are produced in the cell during normal redox reactions including respiration and there are various antioxidants in the cell which scavenge these radicals. Thus in order to maintain a normal cell structure and function, a proper balance between free radical production and antioxidant levels is absolutely essential. Production of PRFO in the myocardium is increased during variousin vivo as well asin vitro pathological conditions and these toxic radicals are responsible for causing functional, biochemical and ultrastructural changes in cardiac myocytes. Indirect evidence of free radical involvement in myocardial injury is provided by studies in which protection against these alterations is seen in the presence of exogenous administration of antioxidants. Endogenous myocardial antioxidants have also been reported to change under various physiological as well as pathophysiological conditions. It appears that endogenous antioxidants respond and adjust to different stress conditions and failure of these compensatory changes may also contribute in cardiac dysfunction. Thus endogenous and/or exogenous increase in antioxidants might have a therapeutic potential in various pathological conditions which result from increased free radical production.     

Calcium signaling and oxidant stress in the vasculature

Recent evidence suggests that oxidant stress plays a major role in several aspects of vascular biology. Oxygen free radicals are implicated as important factors in signaling mechanisms leading to vascular pathologies such as postischemic reperfusion injury and atherosclerosis. The role of intracellular Ca(2+) in these signaling events is an emerging area of vascular research that is providing insights into the mechanisms mediating these complex physiological processes. This review explores sources of free radicals in the vasculature, as well as effects of free radicals on Ca(2+) signaling in vascular endothelial and smooth muscle cells. In the endothelium, superoxides enhance and peroxides attenuate agonist-stimulated Ca(2+) responses, suggesting differential signaling mechanisms depending on radical species. In smooth muscle cells, both superoxides and peroxides disrupt the sarcoplasmic reticulum Ca(2+)-ATPase, leading to both short- and long-term effects on smooth muscle Ca(2+) handling. Because vascular Ca(2+) signaling is altered by oxidant stress in ischemia-related disease states, understanding these pathways may lead to new strategies for preventing or treating arterial disease.     

The role of free radicals in the pathophysiology of muscular dystrophy.

Null mutation of any one of several members of the dystrophin protein complex can cause progressive, and possibly fatal, muscle wasting. Although these muscular dystrophies arise from mutation of a single gene that is expressed primarily in muscle, the resulting pathology is complex and multisystemic, which shows a broader disruption of homeostasis than would be predicted by deletion of a single-gene product. Before the identification of the deficient proteins that underlie muscular dystrophies, such as Duchenne muscular dystrophy (DMD), oxidative stress was proposed as a major cause of the disease. Now, current knowledge supports the likelihood that interactions between the primary genetic defect and disruptions in the normal production of free radicals contribute to the pathophysiology of muscular dystrophies. In this review, we focus on the pathophysiology that results from dystrophin deficiency in humans with DMD and the mdx mouse model of DMD. Current evidence indicates three general routes through which free radical production can be disrupted in dystrophin deficiency to contribute to the ensuing pathology. First, constitutive differences in free radical production can disrupt signaling processes in muscle and other tissues and thereby exacerbate pathology. Second, tissue responses to the presence of pathology can cause a shift in free radical production that can promote cellular injury and dysfunction. Finally, behavioral differences in the affected individual can cause further changes in the production and stoichiometry of free radicals and thereby contribute to disease. Unfortunately, the complexity of the free radical-mediated processes that are perturbed in complex pathologies such as DMD will make it difficult to develop therapeutic approaches founded on systemic administration of antioxidants. More mechanistic knowledge of the specific disruptions of free radicals that underlie major features of muscular dystrophy is needed to develop more targeted and successful therapeutic approaches.     

Free radicals and neuronal cell death

Production of oxygen free radicals is a natural consequence of aerobic metabolism and they are constantly generated in vivo by chemical reactions and metabolic processes. Antioxidant defence systems scavenge and minimise the formation of oxygen-radical-derived biochemical products, however, these defences are not completely effective even under normal physiological conditions. In pathologic situations, oxygen free radicals can be generated in excess of a cell's antioxidant capacity resulting in severe damage to cellular constituents including proteins, DNA and lipids. The inherent biochemical and physiological charateristics of the brain, including high lipid concentrations and energy requirements, make it particularly susceptible to free radical mediated insult. Increasing evidence indicates that many neurological disorders may have components of free radical and oxidative stress induce injury.     

Contractile activity-induced oxidative stress: cellular origin and adaptive responses

Previous studies have reported that oxidizing free radical species are generated during exercise, and there has been considerable interest in the potential effects of these on exercising tissues. We hypothesized that contracting skeletal muscle was a major source of oxidizing free radical species and that untrained skeletal muscle would adapt to the oxidative stress of a single short period of contractile activity by upregulation of the activity of cytoprotective proteins in the absence of overt cellular damage. Fifteen minutes of aerobic contractile activity was found to induce a rapid release of superoxide anions from mouse skeletal muscle in vivo, and studies with contracting cultured skeletal muscle myotubes confirmed that this was due to release from myocytes rather than other cell types present within muscle tissue in vivo. This increased oxidant production caused a rapid, transient reduction in muscle protein thiol content, followed by increases in the activities of superoxide dismutase and catalase and in content of heat shock proteins. These changes occurred in the absence of overt damage to the muscle cells.     

Oxidative stress leads to cholesterol accumulation in vascular smooth muscle cells.

The transformation of macrophages and smooth muscle cells into foam cells by modified low-density lipoproteins (LDL) is one of the key events of atherogenesis. Effects of free radicals have mainly been studied in LDL, and other than toxicity, data dealing with direct action of free radicals on cells are scarce. This study focused on the direct effects of free radicals on cholesterol metabolism of smooth muscle cells. A free radical generator, azobis-amidinopropane dihydrochloride, was used, and conditions for a standardized oxidative stress were set up in vascular smooth muscle cells. After free radical action, the cells presented an accumulation of cholesterol that appeared to be the result of: (i) an increase in cholesterol biosynthesis and esterification; (ii) a decrease in cell cholesteryl ester hydrolysis; and (iii) a reduced cholesterol efflux. All these parameters were opposed by antioxidants. In addition, oxidant stress induced an increased degradation of acetyl-LDL, whereas no change was noted for native LDL. From this data, it was concluded that cholesterol metabolism of vascular smooth muscle cells was markedly altered by in vitro treatment with free radicals, although cell viability was unaffected. The resulting disturbance in cholesterol metabolism favors accumulation of cholesterol and cholesteryl esters in vascular cells, and thus may contribute to the formation of smooth muscle foam cells.     

Free radicals alter maximal diaphragmatic mitochondrial oxygen consumption in endotoxin-induced sepsis

Recent studies indicate that sepsis is associated with enhanced generation of several free radical species (nitric oxide, superoxide, hydrogen peroxide) in skeletal muscle. While studies suggest that free radical generation causes uncoupling of oxidative phosphorylation in sepsis, no previous report has examined the role of free radicals in modulating skeletal muscle oxygen consumption during State 3 respiration or inhibiting the electron transport chain in sepsis. The purpose of the present study was to examine the effects of endotoxin-induced sepsis on State 3 diaphragm mitochondrial oxygen utilization and to determine if inhibitors/scavengers of various free radical species would protect against these effects. We also examined mitochondrial protein electrophoretic patterns to determine if observed endotoxin-related physiological derangements were accompanied by overt alterations in protein composition. Studies were performed on: (a) control animals, (b) endotoxin-treated animals, (c) animals given endotoxin plus PEG-SOD, a superoxide scavenger, (d) animals given endotoxin plus L-NAME, a nitric oxide synthase inhibitor, (e) animals given only PEG-SOD or L-NAME, (f) animals given endotoxin plus D-NAME, and (g) animals given endotoxin plus denatured PEG-SOD. We found: (a) no alteration in maximal State 3 mitochondrial oxygen consumption rate at 24 h after endotoxin administration, but (b) a significant reduction in oxygen consumption rate at 48 h after endotoxin, (c) no effect of endotoxin to induce uncoupling of oxidative phosphorylation, (d) either PEG-SOD or L-NAME (but neither denatured PEG-SOD nor D-NAME) prevented endotoxin-mediated reductions in State 3 respiration rates, (e) some mitochondrial proteins underwent tyrosine nitrosylation at 24 h after endotoxin administration, and (f) SDS-page electrophoresis of mitochondria from endotoxin-treated animals revealed a selective depletion of several proteins at 48 h after endotoxin administration (but not at 24 h); (g) administration of L-NAME or PEG-SOD prevented this protein depletion. These data provide the first evidence that endotoxin-induced reductions in State 3 mitochondrial oxygen consumption are free radical-mediated.