Limited Exercise Capacity in Heterozygous Manganese Superoxide Dismutase Gene–Knockout Mice
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循环学杂志 2005年第3期
the Department of Physiology, New York Medical College, Valhalla, NY.
Abstract
Background— We have reported that there is a limitation of exercise capacity in mice with defects in the expression of endothelial nitric oxide (NO) synthase, which is associated with a greater increase in whole-body oxygen consumption (O2). We hypothesized that in states in which superoxide anion (O2–) is increased, especially in the mitochondria, whole-body O2 will be increased because of the inactivation of NO, and consequently, exercise capacity will be reduced.
Methods and Results— Heterozygous manganese superoxide anion dismutase (SOD2) gene–knockout mice (SOD2+/–), in which SOD2 activity is reduced by 30% to 80%, and wild-type control mice (SOD2+/+) were treadmill-tested to measure indices defining exercise capacity. Tempol was given to each mouse for 7 days by an intraperitoneal injection to scavenge O2– before a second treadmill testing. O2 and carbon dioxide production (CO2) at rest were increased in SOD2+/–. The work (vertical distance run x body weight) to exhaustion was decreased in SOD2+/–. When the maximum O2 and CO2 were corrected to per work unit, they were increased in SOD2+/–. Tempol normalized basal O2 and CO2 and improved the work to exhaustion and corrected O2 and CO2 in SOD2+/–. O2 of skeletal muscle was measured in vitro. Bradykinin-induced reduction in O2 in vitro was attenuated in SOD2+/–, and was acutely restored by Tempol. There was a decrease in SOD2 protein level and a concomitant increase in lucigenin-detectable O2– production in skeletal muscle from SOD2+/–.
Conclusions— These results suggest that exercise capacity is reduced in conditions in which superoxide anion is increased, and this is associated with a greater increase in whole-body oxygen consumption in SOD2+/– compared with SOD2+/+.
Key Words: nitric oxide ; endothelium-derived factors ; exercise ; free radicals ; metabolism
Introduction
During exercise, there is a production of nitric oxide (NO) from vascular endothelial cells in response to stimulation by blood flow/shear stress, as well as from an increased 2-adrenergic receptor activation caused by elevated sympathetic tone and plasma catecholamines.1 NO functions as an amplifier to promote vasodilation and is not directly responsible for the increase in blood flow in the coronary and skeletal muscle that occurs during exercise.2,3 NO irreversibly attenuates mitochondrial respiration by nitrosylating the iron-sulfur centers of aconitase, complexes I and II of the electron transport chain, and through a very potent reversible alteration in the activity of cytochrome c oxidase.4–6 We have shown that NO plays an important role in the modulation of oxygen consumption (O2) and oxygen extraction in hindlimb skeletal muscle at elevated metabolic states during walking or running whether or not blood flow increases.7 Given that the balance between oxygen utilization and oxygen supply is a major factor responsible for the ability to maintain long-term exercise and support maximal exercise performance,8 a loss of NO production might lead to decreased exercise capacity by increasing O2. More recently, we have shown that there is a limitation of exercise capacity in male mice with defects in the expression of endothelial NO synthase (eNOS), which is associated with a greater increase in whole-body O2 than in wild-type mice.9 Other laboratories have shown that a loss of NO production after administration of an NOS inhibitor results in an inadequate exercise-induced hyperemia and a limitation of exercise capacity.10
The limitation of exercise capacity is a major symptom in patients with heart failure (HF)11 and is independent of the degree of their cardiac dysfunction.12 Recently, increased oxidative stress has been shown to be related to the limitation of exercise capacity in patients with HF.13 Tsutsui et al14 have reported that reactive oxygen species are increased in skeletal muscle in HF after myocardial infarction and that they originate from superoxide anion (O2–) produced by mitochondrial oxidase. O2– reacts rapidly with NO, reducing NO bioactivity and producing the oxidant peroxynitrite.15 Thus, we hypothesized that in states in which O2– is increased, NO-dependent control of whole-body and skeletal-muscle O2 is decreased, and consequently exercise capacity is reduced.
To clarify the relationship between O2– and the limitation of exercise capacity, heterozygous manganese superoxide anion dismutase (SOD2) gene–knockout (SOD2+/–) and wild-type control (SOD2+/+) mice were treadmill-tested in the present study. SOD2, a family of enzymes that catalyze the dismutation of O2–, has been reported to be reduced by 30% to 80% in SOD2+/– mice, increasing O2– production in the mitochondria, associated with altered mitochondrial function and the scavenging of NO.16–20 The goals of the present study were to determine (1) whether exercise capacity is limited and whole-body O2 is altered in SOD2+/– mice; (2) whether the administration of a scavenger of O2– to SOD2+/– mice improves exercise capacity and normalizes altered whole-body O2; and (3) whether NO-dependent control of O2 in skeletal muscle tissue in vitro from SOD2+/– mice is decreased.
Methods
Animal Studies
We interbred SOD2+/– mice, which were acquired from Jackson Laboratory (Bar Harbor, Me). Mice were genotyped by polymerase chain reaction of DNA, as we described previously.17 All protocols were approved by the Institutional Animal Care and Use Committee of New York Medical College and conformed to the current National Institutes of Health and American Physiological Society guidelines for the use and care of laboratory animals.
Treadmill Testing and Indices of Exercise Capacity
Male SOD+/+ (n=5) and SOD2+/– (n=6) mice were treadmill-tested to measure indices defining exercise capacity. All mice were given 1 practice trial 3 days before the experiment to adapt to the treadmill-testing environment but otherwise were kept sedentary. At the time of treadmill testing, each mouse was placed on a treadmill at a constant 10° angle enclosed by a metabolic chamber through which air flow passes at a constant speed (Oxymax 2, Columbus Instruments). Oxygen and carbon dioxide gas fractions were monitored at both the inlet and output ports of the metabolic chamber. After a 30-minute period of acclimatization, basal measurements were obtained over a period of 5 minutes. The treadmill was then started at 4 m/min, and the speed was incrementally increased 2 m/min every 2 minutes until the mouse reached exhaustion. The treadmill protocol used in this study, which was chosen on the basis of previous data,10 was designed so that the mice would quickly attain a plateau, reaching their maximal O2 before exhaustion. Exhaustion was defined as spending time (10 seconds) on the shocker plate without attempting to reengage the treadmill.
O2, carbon dioxide production (CO2), and the respiratory exchange ratio (RER) were calculated automatically every 30 seconds by the Oxymax system. O2 and CO2 were calculated by taking the difference between the input and output gas flow. RER was calculated as CO2/O2. The maximal value from each mouse was corrected by the work performed. The work is product of the vertical running distance to exhaustion and body weight.
4-Hydroxy-2,2,6,6-tetramethyl-piperidine 1-oxyl (Tempol, 1.5 mmol · kg– · d– for 7 days) was given to all mice by an intraperitoneal injection. Tempol is the stable, metal-independent, membrane-permeable SOD mimetic compound21 and has been used as a spin trap for O2–.22 The concentration of Tempol was chosen on the basis of previous data.23,24 The mice underwent a second treadmill test after 7 days. For comparison, the vehicle, saline, was given for 7 days before the first treadmill test. In preliminary studies, the same animals underwent a second run without Tempol treatment 7 days after the first run. There was no difference in indices of exercise capacity between the first and second runs in both SOD2+/+ (n=4) and SOD2+/– mice (n=4). For example, the work performed by SOD2+/+ mice was 38.4±7.0 meter kilograms (mkg) at the first and 39.9±2.4 mkg at the second runs (P=NS), and that by SOD2+/– mice was 26.5±6.4 mkg at the first and 24.9±3.7 mkg at the second run (P=NS).
Measurement of O2
Mice were anesthetized with pentobarbital sodium (50 mg/kg IP). Whole skeletal muscles were removed immediately from all 4 legs from SOD2+/+ (n=5) and SOD2+/– (n=5) mice. To exclude the effects of administration of Tempol, different mice were used for in vitro measurement of O2 and for in vivo treadmill testing. These skeletal muscles were also used for Western blot analyses and measurement of O2– production. O2 in skeletal muscle in vitro was measured as we described previously.17,25–27 The muscle tissues (about 50 mg) were incubated in Krebs solution (mol/L: 118 NaCl, 4.7 KCl, 1.5 CaCl2, 25 NaHCO3, 1.1 MgSO4, 1.2 KH2PO4, and 5.6 glucose) at 37°C for 2 hours and bubbled continuously with 20% O2–5% CO2–75% N2. At the end of the incubation period, each piece of tissue was placed in a stirred bath with 3 mL of air-saturated Krebs bicarbonate solution containing 10 mmol/L HEPES (pH 7.4). The bath was sealed by use of a Clark-type platinum oxygen electrode (Yellow Springs Instruments) that was connected to an oxygen monitor (model YSI 5331). Oxygen uptake by tissues was recorded. Tissue respiration was calculated as the rate of decrease in oxygen concentration, assuming an initial oxygen concentration of 224 nmol/mL and was expressed as nanomoles of oxygen consumed per minute per gram of tissue. The effect of all drugs on tissue oxygen uptake is expressed as a percentage of change in baseline MO2. Bradykinin (BK) stimulates kinin B2-receptors on the endothelium to stimulate NO production. S-Nitroso-N-acetylpenicillamine (SNAP) was used as an NO donor in the present study. After baselines were recorded, cumulative concentrations of BK or SNAP at 10–7 to 10–4 mol/L were added to the chambers in the presence or absence of 10–4 mol/L NG-nitro-L-arginine methyl ester (L-NAME). To assess the effects of O2–, skeletal muscle from SOD2+/– mice was preincubated with 10–3 mol/L Tempol for 30 minutes before O2 measurements in separate experiments.
Immunoblotting for eNOS, nNOS, and SOD2 Protein in Skeletal Muscle
eNOS, neuronal NOS (nNOS), and SOD2 protein in skeletal muscle from SOD2+/+ (n=4) and SOD2+/– (n=4) mice were measured by Western blot analysis with antibodies against eNOS, nNOS (Transduction Laboratories), and SOD2 (Santa Cruz Chemicals) followed by densitometry as we described previously.17,25
O2– Production
The chemiluminescence elicited by O2– in the presence of lucigenin (5 μmol/L) was measured in skeletal muscle tissues from SOD2+/+ (n=5) and SOD2+/– (n=5) mice as we described previously.28 To validate that the chemiluminescence signals we measured were derived from O2–, the experiments were performed in the presence of 10–3 mol/L Tempol.
Chemicals
All drugs were purchased from Sigma Chemical Co.
Data Analysis
All data are presented as mean±SEM. Comparisons of all exercise capacity indices and O2– production were made using 1-way ANOVA followed by Scheffe’s t test. The changes in O2 caused by BK or SNAP in vitro were analyzed using repeated-measures 2-way ANOVA followed by Scheffe’s t test. Statistical significance of differences for baseline O2 in vitro and protein level of eNOS, nNOS, and SOD2 in the skeletal muscle was determined with an unpaired t test. Significant changes were considered at a value of P<0.05.
Results
Effect of Tempol on O2, CO2, and RER at Rest in SOD2+/+ and SOD2+/– Mice
O2 and CO2 at rest were significantly increased in SOD2+/– (62.0±1.4 and 51.0±1.6 mL · min–1 · kg–1) compared with SOD+/+ (45.1±3.2 and 37.9±2.0 mL · min–1 · kg–1) mice. There was no change in RER at rest between SOD+/+ and SOD2+/– mice (Table). Intraperitoneal administration of Tempol for 7 days normalized O2 and CO2 at rest in SOD2+/– mice (Table). Tempol did not affect either index in SOD2+/+ mice (Table).
Effect of Tempol on O2, CO2, and RER at Rest in SOD2+/+ and SOD2+/– Mice
Effect of Tempol on Exercise Capacity in SOD2+/+ and SOD2+/– Mice
The running distance and work to exhaustion (Figure 2) were significantly less in SOD2+/– (592±54 m and 27.5±2.1 mkg) compared with SOD2+/+ (971±183 m and 42.5±3.4 mkg) mice. SOD2+/– mice had less exercise capacity than SOD2+/+ mice. Therefore, maximal O2, CO2, and RER were corrected by work performed for each mouse. Both maximal O2/work and CO2/work (Figure 3) were significantly increased in SOD2+/– mice. There was a trend (P=0.059) toward increase in RER/work in SOD2+/– mice compared with SOD2+/+ (0.0035±0.003 versus 0.0025±0.003/mkg).
Tempol normalized the running distance and work (Figure 2) accompanied by a decrease in maximal O2/work and CO2/work (Figure 3). There was no effect of Tempol on exercise capacity in SOD2+/+ mice (Figure 2).
O2 in Skeletal Muscle Tissue From SOD2+/+ and SOD2+/– Mice in Response to BK and SNAP
Because whole-body O2 primarily reflects O2 in exercising muscle during maximal exercise,29 we measured O2 in skeletal muscle tissue in vitro from SOD2+/+ and SOD2+/– mice. Baseline tissue O2 was not different between SOD2+/+ and SOD2+/– (101±12 versus 100 ± 13 nmol · min–1 · g–1) mice. Cumulative doses of BK caused concentration-dependent decreases in O2 in SOD2+/+ mice. BK-induced reduction in O2 in SOD2+/+ mice was significantly attenuated by L-NAME (at 10–4 mol/L BK, –31±3% in SOD2+/+ versus –15±2% in SOD2+/+ with L-NAME, P<0.01). The extent of BK-induced reduction in O2 was significantly less in SOD2+/– than SOD2+/+ mice (Figure 4A). In contrast to SOD2+/+, BK-induced reduction in O2 in SOD2+/– mice was not affected by L-NAME (at 10–4 mol/L BK, –14±4% in SOD2+/– versus –16±3% in SOD2+/– with L-NAME, P=NS). Responses to BK in SOD2+/– mice were restored by preincubation with Tempol (Figure 4A). In contrast to BK, there were no differences in SNAP-induced reduction in O2 between SOD2+/+ and SOD2+/– mice (Figure 4B).
eNOS, nNOS, and SOD2 Protein in Skeletal Muscle From SOD2+/+ and SOD2+/– Mice
There was no difference in eNOS (Figure 5A) and nNOS (Figure 5B) protein between SOD2+/+ and SOD+/– mice (eNOS, 19.5±2.6 versus 21.4±10.7; nNOS, 94.6±2.8 versus 92.4±3.5 [x103 relative optical density, P=NS]). Figure 6A shows a representative Western blot for SOD2. There was a marked reduction in SOD2 protein in skeletal muscle from SOD2+/– compared with SOD2+/+ mice (5.8±5.8 versus 24.2±0.7 [x103 relative optical density, P<0.05]).
O2– Production in Skeletal Muscle From SOD2+/+ and SOD2+/– Mice
There was an increase in lucigenin (5x10–6 mol/L)–detectable O2– production in skeletal muscle from SOD2+/– compared with SOD2+/+ (451±45 versus 235±54 cpm/mg tissue, P<0.05) mice. Tempol decreased O2– in all groups to levels that were not different from each other (Figure 6B).
Discussion
The most important finding of the present study is that the exercise capacity is limited in SOD2+/– mice. This finding was supported by the decreases in running distance and work to exhaustion, which were associated with a greater increase in the whole-body O2 in SOD2+/– compared with SOD2+/+ mice. Furthermore, administration of Tempol to SOD2+/– mice improved exercise capacity. In in vitro experiments, NO-dependent control of O2 in skeletal muscle from SOD2+/– mice was reduced compared with SOD2+/+ mice and restored within minutes by preincubation with Tempol. There was a decrease in SOD2 protein level and a concomitant increase in lucigenin-detectable O2– production in skeletal muscle from SOD2+/– compared with SOD2+/+ mice. Therefore, O2– may have an important role in reducing exercise capacity in SOD2+/– mice, in part by attenuating NO-dependent control of O2.
Rats with vitamin E deficiency have been reported to demonstrate exacerbated muscle and liver free radical production and excessive lipid peroxidation and mitochondrial dysfunction after exhaustive exercise compared with normal rats.30 Endurance performance has also been reported to be decreased in rats fed vitamin E–deficient diets.30,31 Oxidative stress has been shown to be related to exercise intolerance in patients with HF.13 Furthermore, Tsutsui et al14 have reported that reactive oxygen species are increased in skeletal muscle in HF after myocardial infarction and that they originate from O2– produced by mitochondrial oxidase. Thus, oxygen free radicals play an important role in the limitation of exercise capacity. However, there are no available data concerning the interaction with NO, which is important for exercise performance, and oxygen free radicals during treadmill exercise.
We clearly demonstrated that SOD2+/– mice have limited exercise capacity, which was accompanied by reduced control of whole-body O2 in the present study. In SOD2+/– mice, there is a 30% to 80% reduction of SOD2 protein in liver, resulting in reduced O2– metabolism and increased interaction of O2– with NO.17 O2– reacts rapidly with NO, reducing NO bioavailability, producing the oxidant peroxynitrite.15 Recently, we have focused on the relationship between O2– and NO-dependent control of O2, which is another important function of NO. NO-dependent control of O2 in cardiac muscle from old Fisher 344 rats, a model of accelerated aging, was reduced and was associated with NAD(P)H oxidase–generated O2–.25 Angiotensin II at pathophysiological concentrations stimulates an increase in O2– through the activation of NAD(P)H oxidase and attenuates NO-dependent control of O2 in cardiac muscle from normal dogs.26 Furthermore, we reported that NO-dependent control of O2 was attenuated in cardiac muscle from SOD2+/– mice, which was reversed by the freely membrane-permeable O2– scavenger Tiron.17 Our previous in vitro studies17,25,26 have shown that O2– plays an important role in the regulation of NO-dependent control of O2. These studies support our present finding that increased O2– in skeletal muscle reduces NO bioavailability and NO-dependent control of O2 in vivo.
In the present study, intraperitoneal administration of Tempol over a period of 1 week improves exercise capacity and the control of whole-body O2 in SOD2+/– mice, whereas it has no effect in wild-type mice. Tempol is a stable, membrane-permeable, metal-independent SOD mimetic.21 Tempol does not act as a catalase mimetic or alter hydrogen peroxide concentrations32 and does not directly bind NO or produce O2–.33 The concentration of Tempol used in the present study has been shown to normalize blood pressure and decrease a marker of oxidative stress in spontaneously hypertensive rats.23,24 Those data and our data using lucigenin chemiluminescence strongly suggest that the limitation of exercise capacity in SOD2+/– mice is associated with increased O2–.
Changes of whole-body O2 during exercise closely reflect those occurring within the exercising muscle.29 Therefore, we investigated NO-dependent control of O2, the expression of eNOS, nNOS, and SOD2 protein, and lucigenin-detectable O2– production in limb skeletal muscle from SOD2+/+ and SOD2+/– mice. There was no difference in eNOS or nNOS protein levels. The expression of SOD2 protein was decreased, O2– production is increased, and BK-induced reduction in O2 is attenuated in skeletal muscle in vitro from SOD2+/– compared with SOD2+/+ mice. L-NAME, a nonselective inhibitor of NOS, attenuated BK-induced reduction in MO2 in SOD2+/+ mice, whereas it did not affect that in SOD2+/– mice. These results suggest that NO-dependent control of MO2 is attenuated in SOD2+/– mice. We have previously shown that BK-induced reduction in tissue O2 in eNOS–/– mice is almost completely abolished.27 L-NAME did not completely abolish BK-induced reduction in O2 in skeletal muscle in the present study. These results suggest that NOS, including eNOS, was not completely inhibited by L-NAME. This effect of L-NAME depends on incubation time or concentration used. Nevertheless, our conclusions are based on the amount of inhibition of NO-dependent responses. Furthermore, acute preincubation of tissue ex vivo with Tempol enhanced the NO-dependent control of O2 in response to BK in SOD2+/– mice. These results are consistent with our previous data from hearts in SOD2+/– mice17 and support our present in vivo data. We also investigated the role of exogenous NO. However, there was no difference in NO-dependent control of O2 in response to increasing concentrations of SNAP in skeletal muscle from SOD2+/+ and SOD2+/– mice. Although mitochondrial function of heart and liver has been reported to be altered in SOD2+/– mice,19,20 our data with SNAP suggest that the sensitivity of cytochrome c oxidase for NO is unchanged.
CO2 also was increased in SOD2+/– mice under resting conditions and during maximal exercise. Our previous studies showed that NO can regulate substrate utilization. Blockade of NOS resulted in reductions in myocardial free fatty acid consumption for comparable levels of cardiac work.34 The acute inhibition of NOS by nitro-L-arginine causes a switch from fatty acids to lactate and glucose utilization in the heart, which can be reversed by an NO donor.35 Glucose uptake was increased in the mice with defects in the expression of eNOS and mice with L-NAME in a Langendorff heart preparation.36 The ATP yield from glucose oxidation is greater for a given rate of O2 because of the higher ATP/O2 ratio compared with that from fatty acid oxidation.37 Therefore, a switch of substrate utilization occurring in the absence of NO is thought to be a compensatory mechanism during an imbalance of energy demand and supply. We demonstrated that basal CO2 and maximal CO2/work were increased and that there was a trend (P<0.059) toward an increase in maximal RER/work in SOD2+/– mice compared with SOD2+/+ mice. These findings suggest that a switch of substrate utilization from free fatty acid to glucose may occur in SOD2+/– mice.
In many animal studies, maximal exercise capacity or distance to exhaustion is used as a surrogate or index of oxygen consumption. In human exercise testing, individuals with greater O2max are typically able to achieve more exercise/work. This seems to be inconsistent with our results. Shen et al38 have shown that inhibition of endogenous NO production increased O2 in heart without a change in the ATP synthesis rate. They investigated whether endogenous NO modulates myocardial O2, ATP synthesis, and metabolic efficiency using isolated isovolumic guinea pig hearts perfused at a constant flow. N-nitro-L-arginine (L-NNA, an inhibitor of NOS) increased O2 without an increase in cardiac work. When the relationship between contractile performance and O2 was measured at different levels of cardiac work, it showed an upward shift during treatment with L-NNA. Conversely, L-NNA did not alter ATP contents and ATP synthesis rates. Shen et al concluded that the heart wasted oxygen on ATP production in the absence of NO. We have previously shown that body temperature was increased after inhibition of NOS in conscious dogs.39 These data may suggest that the increased O2 in the absence of NO is associated with tissue heat production. Therefore, more oxygen is consumed/split during ATP production in SOD2+/– compared with SOD2+/+ mice by a decrease in NO bioavailability.
There are other possible mechanisms for the limitation of exercise capacity to be addressed. First, impaired oxidative energy production in mitochondria in SOD2+/– mice can limit exercise. The respiratory control ratio and state 3 respiration for substrates metabolized by complex I are decreased in mitochondria of heart isolated from SOD2+/– mice.19 Second, muscle atrophy through the induction of apoptosis can cause a limitation of exercise capacity. The induction of apoptosis because of alterations in the mitochondrial permeability transition has been reported to be increased in heart from SOD2+/– mice.19 Because there were no differences in body weight and limb muscle weight between SOD2+/+ and SOD2+/– mice (data not shown), the contribution of muscle atrophy seems to be small. Third, impaired myoplasmic calcium homeostasis by reactive oxygen species may contribute to muscle contractile dysfunction.40 It should be remembered that all the effects on exercise capacity were reversed by Tempol, indicating that structural changes are most likely not responsible. Further studies that focus on the mechanisms for the limitation of exercise capacity in SOD2+/– mice may be necessary. Nevertheless, we have clearly shown the importance of both O2– and NO in the present study.
We demonstrated that exercise capacity is limited in SOD2+/– mice and that the limitation is associated with a greater increase in whole-body O2. Furthermore, Tempol improved exercise capacity and O2 to normal levels. In in vitro experiments, NO-dependent control of O2 in skeletal muscle from SOD2+/– mice was reduced compared with wild-type mice and was restored within minutes by preincubation with Tempol. There was a decrease in the expression of SOD2 protein and a concomitant increase in lucigenin-detectable O2– production in skeletal muscle from SOD2+/– compared with SOD2+/+ mice. Therefore, O2– plays an important role in the limitation of exercise capacity, perhaps in part by reducing NO availability. The present results suggest that scavenging O2– may improve exercise capacity in conditions in which O2– is increased, such as HF.
Acknowledgments
This study was supported by grants PO-1-HL-43023, HL-50412, HL-61290 (Dr Hintze), HL-31069, and HL-66331 (Dr Wolin) from the National Heart, Lung, and Blood Institute. We thank K. Rafalski for genotyping the SOD mice.
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Shen W, Tian R, Saupe KW, Spindler M, Ingwall JS. Endogenous nitric oxide enhances coupling between O2 consumption and ATP synthesis in guinea pig hearts. Am J Physiol. 2001; 281: H838–H846.
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De Sousa E, Veksler V, Bigard X, Mateo P, Ventura-Clapier R. Heart failure affects mitochondrial but not myofibrillar intrinsic properties of skeletal muscle. Circulation. 2000; 102: 1847–1853.(Shintaro Kinugawa, MD, Ph)
Abstract
Background— We have reported that there is a limitation of exercise capacity in mice with defects in the expression of endothelial nitric oxide (NO) synthase, which is associated with a greater increase in whole-body oxygen consumption (O2). We hypothesized that in states in which superoxide anion (O2–) is increased, especially in the mitochondria, whole-body O2 will be increased because of the inactivation of NO, and consequently, exercise capacity will be reduced.
Methods and Results— Heterozygous manganese superoxide anion dismutase (SOD2) gene–knockout mice (SOD2+/–), in which SOD2 activity is reduced by 30% to 80%, and wild-type control mice (SOD2+/+) were treadmill-tested to measure indices defining exercise capacity. Tempol was given to each mouse for 7 days by an intraperitoneal injection to scavenge O2– before a second treadmill testing. O2 and carbon dioxide production (CO2) at rest were increased in SOD2+/–. The work (vertical distance run x body weight) to exhaustion was decreased in SOD2+/–. When the maximum O2 and CO2 were corrected to per work unit, they were increased in SOD2+/–. Tempol normalized basal O2 and CO2 and improved the work to exhaustion and corrected O2 and CO2 in SOD2+/–. O2 of skeletal muscle was measured in vitro. Bradykinin-induced reduction in O2 in vitro was attenuated in SOD2+/–, and was acutely restored by Tempol. There was a decrease in SOD2 protein level and a concomitant increase in lucigenin-detectable O2– production in skeletal muscle from SOD2+/–.
Conclusions— These results suggest that exercise capacity is reduced in conditions in which superoxide anion is increased, and this is associated with a greater increase in whole-body oxygen consumption in SOD2+/– compared with SOD2+/+.
Key Words: nitric oxide ; endothelium-derived factors ; exercise ; free radicals ; metabolism
Introduction
During exercise, there is a production of nitric oxide (NO) from vascular endothelial cells in response to stimulation by blood flow/shear stress, as well as from an increased 2-adrenergic receptor activation caused by elevated sympathetic tone and plasma catecholamines.1 NO functions as an amplifier to promote vasodilation and is not directly responsible for the increase in blood flow in the coronary and skeletal muscle that occurs during exercise.2,3 NO irreversibly attenuates mitochondrial respiration by nitrosylating the iron-sulfur centers of aconitase, complexes I and II of the electron transport chain, and through a very potent reversible alteration in the activity of cytochrome c oxidase.4–6 We have shown that NO plays an important role in the modulation of oxygen consumption (O2) and oxygen extraction in hindlimb skeletal muscle at elevated metabolic states during walking or running whether or not blood flow increases.7 Given that the balance between oxygen utilization and oxygen supply is a major factor responsible for the ability to maintain long-term exercise and support maximal exercise performance,8 a loss of NO production might lead to decreased exercise capacity by increasing O2. More recently, we have shown that there is a limitation of exercise capacity in male mice with defects in the expression of endothelial NO synthase (eNOS), which is associated with a greater increase in whole-body O2 than in wild-type mice.9 Other laboratories have shown that a loss of NO production after administration of an NOS inhibitor results in an inadequate exercise-induced hyperemia and a limitation of exercise capacity.10
The limitation of exercise capacity is a major symptom in patients with heart failure (HF)11 and is independent of the degree of their cardiac dysfunction.12 Recently, increased oxidative stress has been shown to be related to the limitation of exercise capacity in patients with HF.13 Tsutsui et al14 have reported that reactive oxygen species are increased in skeletal muscle in HF after myocardial infarction and that they originate from superoxide anion (O2–) produced by mitochondrial oxidase. O2– reacts rapidly with NO, reducing NO bioactivity and producing the oxidant peroxynitrite.15 Thus, we hypothesized that in states in which O2– is increased, NO-dependent control of whole-body and skeletal-muscle O2 is decreased, and consequently exercise capacity is reduced.
To clarify the relationship between O2– and the limitation of exercise capacity, heterozygous manganese superoxide anion dismutase (SOD2) gene–knockout (SOD2+/–) and wild-type control (SOD2+/+) mice were treadmill-tested in the present study. SOD2, a family of enzymes that catalyze the dismutation of O2–, has been reported to be reduced by 30% to 80% in SOD2+/– mice, increasing O2– production in the mitochondria, associated with altered mitochondrial function and the scavenging of NO.16–20 The goals of the present study were to determine (1) whether exercise capacity is limited and whole-body O2 is altered in SOD2+/– mice; (2) whether the administration of a scavenger of O2– to SOD2+/– mice improves exercise capacity and normalizes altered whole-body O2; and (3) whether NO-dependent control of O2 in skeletal muscle tissue in vitro from SOD2+/– mice is decreased.
Methods
Animal Studies
We interbred SOD2+/– mice, which were acquired from Jackson Laboratory (Bar Harbor, Me). Mice were genotyped by polymerase chain reaction of DNA, as we described previously.17 All protocols were approved by the Institutional Animal Care and Use Committee of New York Medical College and conformed to the current National Institutes of Health and American Physiological Society guidelines for the use and care of laboratory animals.
Treadmill Testing and Indices of Exercise Capacity
Male SOD+/+ (n=5) and SOD2+/– (n=6) mice were treadmill-tested to measure indices defining exercise capacity. All mice were given 1 practice trial 3 days before the experiment to adapt to the treadmill-testing environment but otherwise were kept sedentary. At the time of treadmill testing, each mouse was placed on a treadmill at a constant 10° angle enclosed by a metabolic chamber through which air flow passes at a constant speed (Oxymax 2, Columbus Instruments). Oxygen and carbon dioxide gas fractions were monitored at both the inlet and output ports of the metabolic chamber. After a 30-minute period of acclimatization, basal measurements were obtained over a period of 5 minutes. The treadmill was then started at 4 m/min, and the speed was incrementally increased 2 m/min every 2 minutes until the mouse reached exhaustion. The treadmill protocol used in this study, which was chosen on the basis of previous data,10 was designed so that the mice would quickly attain a plateau, reaching their maximal O2 before exhaustion. Exhaustion was defined as spending time (10 seconds) on the shocker plate without attempting to reengage the treadmill.
O2, carbon dioxide production (CO2), and the respiratory exchange ratio (RER) were calculated automatically every 30 seconds by the Oxymax system. O2 and CO2 were calculated by taking the difference between the input and output gas flow. RER was calculated as CO2/O2. The maximal value from each mouse was corrected by the work performed. The work is product of the vertical running distance to exhaustion and body weight.
4-Hydroxy-2,2,6,6-tetramethyl-piperidine 1-oxyl (Tempol, 1.5 mmol · kg– · d– for 7 days) was given to all mice by an intraperitoneal injection. Tempol is the stable, metal-independent, membrane-permeable SOD mimetic compound21 and has been used as a spin trap for O2–.22 The concentration of Tempol was chosen on the basis of previous data.23,24 The mice underwent a second treadmill test after 7 days. For comparison, the vehicle, saline, was given for 7 days before the first treadmill test. In preliminary studies, the same animals underwent a second run without Tempol treatment 7 days after the first run. There was no difference in indices of exercise capacity between the first and second runs in both SOD2+/+ (n=4) and SOD2+/– mice (n=4). For example, the work performed by SOD2+/+ mice was 38.4±7.0 meter kilograms (mkg) at the first and 39.9±2.4 mkg at the second runs (P=NS), and that by SOD2+/– mice was 26.5±6.4 mkg at the first and 24.9±3.7 mkg at the second run (P=NS).
Measurement of O2
Mice were anesthetized with pentobarbital sodium (50 mg/kg IP). Whole skeletal muscles were removed immediately from all 4 legs from SOD2+/+ (n=5) and SOD2+/– (n=5) mice. To exclude the effects of administration of Tempol, different mice were used for in vitro measurement of O2 and for in vivo treadmill testing. These skeletal muscles were also used for Western blot analyses and measurement of O2– production. O2 in skeletal muscle in vitro was measured as we described previously.17,25–27 The muscle tissues (about 50 mg) were incubated in Krebs solution (mol/L: 118 NaCl, 4.7 KCl, 1.5 CaCl2, 25 NaHCO3, 1.1 MgSO4, 1.2 KH2PO4, and 5.6 glucose) at 37°C for 2 hours and bubbled continuously with 20% O2–5% CO2–75% N2. At the end of the incubation period, each piece of tissue was placed in a stirred bath with 3 mL of air-saturated Krebs bicarbonate solution containing 10 mmol/L HEPES (pH 7.4). The bath was sealed by use of a Clark-type platinum oxygen electrode (Yellow Springs Instruments) that was connected to an oxygen monitor (model YSI 5331). Oxygen uptake by tissues was recorded. Tissue respiration was calculated as the rate of decrease in oxygen concentration, assuming an initial oxygen concentration of 224 nmol/mL and was expressed as nanomoles of oxygen consumed per minute per gram of tissue. The effect of all drugs on tissue oxygen uptake is expressed as a percentage of change in baseline MO2. Bradykinin (BK) stimulates kinin B2-receptors on the endothelium to stimulate NO production. S-Nitroso-N-acetylpenicillamine (SNAP) was used as an NO donor in the present study. After baselines were recorded, cumulative concentrations of BK or SNAP at 10–7 to 10–4 mol/L were added to the chambers in the presence or absence of 10–4 mol/L NG-nitro-L-arginine methyl ester (L-NAME). To assess the effects of O2–, skeletal muscle from SOD2+/– mice was preincubated with 10–3 mol/L Tempol for 30 minutes before O2 measurements in separate experiments.
Immunoblotting for eNOS, nNOS, and SOD2 Protein in Skeletal Muscle
eNOS, neuronal NOS (nNOS), and SOD2 protein in skeletal muscle from SOD2+/+ (n=4) and SOD2+/– (n=4) mice were measured by Western blot analysis with antibodies against eNOS, nNOS (Transduction Laboratories), and SOD2 (Santa Cruz Chemicals) followed by densitometry as we described previously.17,25
O2– Production
The chemiluminescence elicited by O2– in the presence of lucigenin (5 μmol/L) was measured in skeletal muscle tissues from SOD2+/+ (n=5) and SOD2+/– (n=5) mice as we described previously.28 To validate that the chemiluminescence signals we measured were derived from O2–, the experiments were performed in the presence of 10–3 mol/L Tempol.
Chemicals
All drugs were purchased from Sigma Chemical Co.
Data Analysis
All data are presented as mean±SEM. Comparisons of all exercise capacity indices and O2– production were made using 1-way ANOVA followed by Scheffe’s t test. The changes in O2 caused by BK or SNAP in vitro were analyzed using repeated-measures 2-way ANOVA followed by Scheffe’s t test. Statistical significance of differences for baseline O2 in vitro and protein level of eNOS, nNOS, and SOD2 in the skeletal muscle was determined with an unpaired t test. Significant changes were considered at a value of P<0.05.
Results
Effect of Tempol on O2, CO2, and RER at Rest in SOD2+/+ and SOD2+/– Mice
O2 and CO2 at rest were significantly increased in SOD2+/– (62.0±1.4 and 51.0±1.6 mL · min–1 · kg–1) compared with SOD+/+ (45.1±3.2 and 37.9±2.0 mL · min–1 · kg–1) mice. There was no change in RER at rest between SOD+/+ and SOD2+/– mice (Table). Intraperitoneal administration of Tempol for 7 days normalized O2 and CO2 at rest in SOD2+/– mice (Table). Tempol did not affect either index in SOD2+/+ mice (Table).
Effect of Tempol on O2, CO2, and RER at Rest in SOD2+/+ and SOD2+/– Mice
Effect of Tempol on Exercise Capacity in SOD2+/+ and SOD2+/– Mice
The running distance and work to exhaustion (Figure 2) were significantly less in SOD2+/– (592±54 m and 27.5±2.1 mkg) compared with SOD2+/+ (971±183 m and 42.5±3.4 mkg) mice. SOD2+/– mice had less exercise capacity than SOD2+/+ mice. Therefore, maximal O2, CO2, and RER were corrected by work performed for each mouse. Both maximal O2/work and CO2/work (Figure 3) were significantly increased in SOD2+/– mice. There was a trend (P=0.059) toward increase in RER/work in SOD2+/– mice compared with SOD2+/+ (0.0035±0.003 versus 0.0025±0.003/mkg).
Tempol normalized the running distance and work (Figure 2) accompanied by a decrease in maximal O2/work and CO2/work (Figure 3). There was no effect of Tempol on exercise capacity in SOD2+/+ mice (Figure 2).
O2 in Skeletal Muscle Tissue From SOD2+/+ and SOD2+/– Mice in Response to BK and SNAP
Because whole-body O2 primarily reflects O2 in exercising muscle during maximal exercise,29 we measured O2 in skeletal muscle tissue in vitro from SOD2+/+ and SOD2+/– mice. Baseline tissue O2 was not different between SOD2+/+ and SOD2+/– (101±12 versus 100 ± 13 nmol · min–1 · g–1) mice. Cumulative doses of BK caused concentration-dependent decreases in O2 in SOD2+/+ mice. BK-induced reduction in O2 in SOD2+/+ mice was significantly attenuated by L-NAME (at 10–4 mol/L BK, –31±3% in SOD2+/+ versus –15±2% in SOD2+/+ with L-NAME, P<0.01). The extent of BK-induced reduction in O2 was significantly less in SOD2+/– than SOD2+/+ mice (Figure 4A). In contrast to SOD2+/+, BK-induced reduction in O2 in SOD2+/– mice was not affected by L-NAME (at 10–4 mol/L BK, –14±4% in SOD2+/– versus –16±3% in SOD2+/– with L-NAME, P=NS). Responses to BK in SOD2+/– mice were restored by preincubation with Tempol (Figure 4A). In contrast to BK, there were no differences in SNAP-induced reduction in O2 between SOD2+/+ and SOD2+/– mice (Figure 4B).
eNOS, nNOS, and SOD2 Protein in Skeletal Muscle From SOD2+/+ and SOD2+/– Mice
There was no difference in eNOS (Figure 5A) and nNOS (Figure 5B) protein between SOD2+/+ and SOD+/– mice (eNOS, 19.5±2.6 versus 21.4±10.7; nNOS, 94.6±2.8 versus 92.4±3.5 [x103 relative optical density, P=NS]). Figure 6A shows a representative Western blot for SOD2. There was a marked reduction in SOD2 protein in skeletal muscle from SOD2+/– compared with SOD2+/+ mice (5.8±5.8 versus 24.2±0.7 [x103 relative optical density, P<0.05]).
O2– Production in Skeletal Muscle From SOD2+/+ and SOD2+/– Mice
There was an increase in lucigenin (5x10–6 mol/L)–detectable O2– production in skeletal muscle from SOD2+/– compared with SOD2+/+ (451±45 versus 235±54 cpm/mg tissue, P<0.05) mice. Tempol decreased O2– in all groups to levels that were not different from each other (Figure 6B).
Discussion
The most important finding of the present study is that the exercise capacity is limited in SOD2+/– mice. This finding was supported by the decreases in running distance and work to exhaustion, which were associated with a greater increase in the whole-body O2 in SOD2+/– compared with SOD2+/+ mice. Furthermore, administration of Tempol to SOD2+/– mice improved exercise capacity. In in vitro experiments, NO-dependent control of O2 in skeletal muscle from SOD2+/– mice was reduced compared with SOD2+/+ mice and restored within minutes by preincubation with Tempol. There was a decrease in SOD2 protein level and a concomitant increase in lucigenin-detectable O2– production in skeletal muscle from SOD2+/– compared with SOD2+/+ mice. Therefore, O2– may have an important role in reducing exercise capacity in SOD2+/– mice, in part by attenuating NO-dependent control of O2.
Rats with vitamin E deficiency have been reported to demonstrate exacerbated muscle and liver free radical production and excessive lipid peroxidation and mitochondrial dysfunction after exhaustive exercise compared with normal rats.30 Endurance performance has also been reported to be decreased in rats fed vitamin E–deficient diets.30,31 Oxidative stress has been shown to be related to exercise intolerance in patients with HF.13 Furthermore, Tsutsui et al14 have reported that reactive oxygen species are increased in skeletal muscle in HF after myocardial infarction and that they originate from O2– produced by mitochondrial oxidase. Thus, oxygen free radicals play an important role in the limitation of exercise capacity. However, there are no available data concerning the interaction with NO, which is important for exercise performance, and oxygen free radicals during treadmill exercise.
We clearly demonstrated that SOD2+/– mice have limited exercise capacity, which was accompanied by reduced control of whole-body O2 in the present study. In SOD2+/– mice, there is a 30% to 80% reduction of SOD2 protein in liver, resulting in reduced O2– metabolism and increased interaction of O2– with NO.17 O2– reacts rapidly with NO, reducing NO bioavailability, producing the oxidant peroxynitrite.15 Recently, we have focused on the relationship between O2– and NO-dependent control of O2, which is another important function of NO. NO-dependent control of O2 in cardiac muscle from old Fisher 344 rats, a model of accelerated aging, was reduced and was associated with NAD(P)H oxidase–generated O2–.25 Angiotensin II at pathophysiological concentrations stimulates an increase in O2– through the activation of NAD(P)H oxidase and attenuates NO-dependent control of O2 in cardiac muscle from normal dogs.26 Furthermore, we reported that NO-dependent control of O2 was attenuated in cardiac muscle from SOD2+/– mice, which was reversed by the freely membrane-permeable O2– scavenger Tiron.17 Our previous in vitro studies17,25,26 have shown that O2– plays an important role in the regulation of NO-dependent control of O2. These studies support our present finding that increased O2– in skeletal muscle reduces NO bioavailability and NO-dependent control of O2 in vivo.
In the present study, intraperitoneal administration of Tempol over a period of 1 week improves exercise capacity and the control of whole-body O2 in SOD2+/– mice, whereas it has no effect in wild-type mice. Tempol is a stable, membrane-permeable, metal-independent SOD mimetic.21 Tempol does not act as a catalase mimetic or alter hydrogen peroxide concentrations32 and does not directly bind NO or produce O2–.33 The concentration of Tempol used in the present study has been shown to normalize blood pressure and decrease a marker of oxidative stress in spontaneously hypertensive rats.23,24 Those data and our data using lucigenin chemiluminescence strongly suggest that the limitation of exercise capacity in SOD2+/– mice is associated with increased O2–.
Changes of whole-body O2 during exercise closely reflect those occurring within the exercising muscle.29 Therefore, we investigated NO-dependent control of O2, the expression of eNOS, nNOS, and SOD2 protein, and lucigenin-detectable O2– production in limb skeletal muscle from SOD2+/+ and SOD2+/– mice. There was no difference in eNOS or nNOS protein levels. The expression of SOD2 protein was decreased, O2– production is increased, and BK-induced reduction in O2 is attenuated in skeletal muscle in vitro from SOD2+/– compared with SOD2+/+ mice. L-NAME, a nonselective inhibitor of NOS, attenuated BK-induced reduction in MO2 in SOD2+/+ mice, whereas it did not affect that in SOD2+/– mice. These results suggest that NO-dependent control of MO2 is attenuated in SOD2+/– mice. We have previously shown that BK-induced reduction in tissue O2 in eNOS–/– mice is almost completely abolished.27 L-NAME did not completely abolish BK-induced reduction in O2 in skeletal muscle in the present study. These results suggest that NOS, including eNOS, was not completely inhibited by L-NAME. This effect of L-NAME depends on incubation time or concentration used. Nevertheless, our conclusions are based on the amount of inhibition of NO-dependent responses. Furthermore, acute preincubation of tissue ex vivo with Tempol enhanced the NO-dependent control of O2 in response to BK in SOD2+/– mice. These results are consistent with our previous data from hearts in SOD2+/– mice17 and support our present in vivo data. We also investigated the role of exogenous NO. However, there was no difference in NO-dependent control of O2 in response to increasing concentrations of SNAP in skeletal muscle from SOD2+/+ and SOD2+/– mice. Although mitochondrial function of heart and liver has been reported to be altered in SOD2+/– mice,19,20 our data with SNAP suggest that the sensitivity of cytochrome c oxidase for NO is unchanged.
CO2 also was increased in SOD2+/– mice under resting conditions and during maximal exercise. Our previous studies showed that NO can regulate substrate utilization. Blockade of NOS resulted in reductions in myocardial free fatty acid consumption for comparable levels of cardiac work.34 The acute inhibition of NOS by nitro-L-arginine causes a switch from fatty acids to lactate and glucose utilization in the heart, which can be reversed by an NO donor.35 Glucose uptake was increased in the mice with defects in the expression of eNOS and mice with L-NAME in a Langendorff heart preparation.36 The ATP yield from glucose oxidation is greater for a given rate of O2 because of the higher ATP/O2 ratio compared with that from fatty acid oxidation.37 Therefore, a switch of substrate utilization occurring in the absence of NO is thought to be a compensatory mechanism during an imbalance of energy demand and supply. We demonstrated that basal CO2 and maximal CO2/work were increased and that there was a trend (P<0.059) toward an increase in maximal RER/work in SOD2+/– mice compared with SOD2+/+ mice. These findings suggest that a switch of substrate utilization from free fatty acid to glucose may occur in SOD2+/– mice.
In many animal studies, maximal exercise capacity or distance to exhaustion is used as a surrogate or index of oxygen consumption. In human exercise testing, individuals with greater O2max are typically able to achieve more exercise/work. This seems to be inconsistent with our results. Shen et al38 have shown that inhibition of endogenous NO production increased O2 in heart without a change in the ATP synthesis rate. They investigated whether endogenous NO modulates myocardial O2, ATP synthesis, and metabolic efficiency using isolated isovolumic guinea pig hearts perfused at a constant flow. N-nitro-L-arginine (L-NNA, an inhibitor of NOS) increased O2 without an increase in cardiac work. When the relationship between contractile performance and O2 was measured at different levels of cardiac work, it showed an upward shift during treatment with L-NNA. Conversely, L-NNA did not alter ATP contents and ATP synthesis rates. Shen et al concluded that the heart wasted oxygen on ATP production in the absence of NO. We have previously shown that body temperature was increased after inhibition of NOS in conscious dogs.39 These data may suggest that the increased O2 in the absence of NO is associated with tissue heat production. Therefore, more oxygen is consumed/split during ATP production in SOD2+/– compared with SOD2+/+ mice by a decrease in NO bioavailability.
There are other possible mechanisms for the limitation of exercise capacity to be addressed. First, impaired oxidative energy production in mitochondria in SOD2+/– mice can limit exercise. The respiratory control ratio and state 3 respiration for substrates metabolized by complex I are decreased in mitochondria of heart isolated from SOD2+/– mice.19 Second, muscle atrophy through the induction of apoptosis can cause a limitation of exercise capacity. The induction of apoptosis because of alterations in the mitochondrial permeability transition has been reported to be increased in heart from SOD2+/– mice.19 Because there were no differences in body weight and limb muscle weight between SOD2+/+ and SOD2+/– mice (data not shown), the contribution of muscle atrophy seems to be small. Third, impaired myoplasmic calcium homeostasis by reactive oxygen species may contribute to muscle contractile dysfunction.40 It should be remembered that all the effects on exercise capacity were reversed by Tempol, indicating that structural changes are most likely not responsible. Further studies that focus on the mechanisms for the limitation of exercise capacity in SOD2+/– mice may be necessary. Nevertheless, we have clearly shown the importance of both O2– and NO in the present study.
We demonstrated that exercise capacity is limited in SOD2+/– mice and that the limitation is associated with a greater increase in whole-body O2. Furthermore, Tempol improved exercise capacity and O2 to normal levels. In in vitro experiments, NO-dependent control of O2 in skeletal muscle from SOD2+/– mice was reduced compared with wild-type mice and was restored within minutes by preincubation with Tempol. There was a decrease in the expression of SOD2 protein and a concomitant increase in lucigenin-detectable O2– production in skeletal muscle from SOD2+/– compared with SOD2+/+ mice. Therefore, O2– plays an important role in the limitation of exercise capacity, perhaps in part by reducing NO availability. The present results suggest that scavenging O2– may improve exercise capacity in conditions in which O2– is increased, such as HF.
Acknowledgments
This study was supported by grants PO-1-HL-43023, HL-50412, HL-61290 (Dr Hintze), HL-31069, and HL-66331 (Dr Wolin) from the National Heart, Lung, and Blood Institute. We thank K. Rafalski for genotyping the SOD mice.
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