Main

Respiratory muscle fatigue contributes to ventilatory pump failure in neonates(1) and adults(2). The cellular and biochemical mechanisms underlying this phenomenon, however, remain unclear. Recent studies have demonstrated that oxidative stress, an imbalance between oxidant flux and antioxidant defenses where oxidants predominate, is associated with contractile dysfunction in adult muscle(3–7). Moreover, investigators have shown that a major source of ROS in muscle is the motochondria and that oxidant flux in muscle is directly related to the rate of mitochondrial oxygen consumption(8, 9).

In previous work we have demonstrated that DIA muscle fatigue resistance is high in newborns and then declines with postnatal development at the same time that DIA oxidative capacity as indexed by the mitochondrially bound SDH activity increases(10, 11). This inverse relationship between postnatal changes in DIA fatigue resistance and oxidative capacity may seem paradoxical, but is consistent with the evidence that implicates a role for oxidative stress in the fatigue process(3–7). If skeletal muscle production of ROS is enhanced by the maturational increase in mitochondrial capacity (SDH activity) and not matched by an increase in antioxidant enzyme defense, then oxidative stress may result and contribute to an increased suceptibility to fatigue in the adult.

Little is known about the antioxidant capacity and oxidant generating potential of newborn muscle, or how these properties compare with the adult and relate to fatigue resistance. We, therefore, determined the 1) antioxidant enzyme activities (SOD, CAT, and GPX), 2) total GSH content, and 3) oxidative capacity of newborn and adult DIA. Enzymatic antioxidants provide endogenous protection against the deleterious effects of ROS generated in skeletal muscle during contractile activity. SOD scavenges the mitochondrially derived O2[horizontal line over dot] producing hydrogen peroxide(12, 13). CAT and GPX scavenge H2O2(12, 13), whereas GSH is a nonenzymatic antioxidant(14) and the substrate for GPX. Oxidative capacity was indexed by SDH activity, which defines the limits of muscle oxygen consumption(10, 11). We also determined 1) the levels of extracellular cytochrome c reduction during fatiguing activations and 2) the efficacy of exogenously administered SOD in vitro in ameliorating fatigue in both age groups to indirectly assess the role of oxidative stress in the fatigue process(6). We hypothesized that 1) the postnatal increase in DIA oxidative capacity is not matched by a postnatal increase in antioxidant enzyme activity, and 2) this imbalance between oxidative capacity and antioxidant activity in the adult is associated with an enhanced susceptibility to fatigue during repetitive activation.

METHODS

Male Sprague-Dawley rats of postnatal age d 1 and > 115 d (adult) were used in our study, and the experimental protocol was approved by the Magee-Womens Hospital Institutional Animal Care and Use Committee. Animals were anesthetized with pentobarbital sodium (50 mg/kg, intraperitoneally), and DIA muscle segments were excised for: 1) determination of SOD, CAT, and GPX activity (n = 12 at each age); 2) total glutathione content(n = 6 at each age); 3) SDH activity (n = 6 at each age); or 4) in vitro study of fatigue properties in the presence or absence of exogenous SOD (n = 6 at each age).

Determination of muscle SOD, CAT, and GPX enzyme activity. Excised DIA muscle was dissected free of fat and tendon in ice-cold saline, frozen in liquid nitrogen, and stored at -80 °C for later SOD, CAT, and GPX analysis. On the day of study, approximately 15-20 mg of each tissue were homogenized in 0.5 mL of a phosphate buffer appropriate for each enzyme assay. Homogenates were centrifuged at 20,000 × g for 10 min at 4°C, and supernatants were removed and assayed for total SOD (EC 1.15.1.1), CAT (EC 1.11.1.6), GPX (EC 1.11.1.9) activity using spectrophotometric techniques. Total SOD was determined using the procedure described by McCord and Fridovich(15) based on the cytochrome c reduction rate by xanthine oxidase. CAT activity was determined using the method of Beers and Sizer(16) based on the consumption rate of hydrogen peroxide, and GPX activity was determined using the method of Flohe and Gunzler(17) based on the NADPH oxidation rate with t-buty1 hydroperoxide substrate. SOD was fractionated into its cytosolic (Cu/Zu) and mitochondrial (Mn) forms using the diethyldithiocarbamate inhibition method(18). SOD, CAT, and GPX activities were normalized to protein content as determined by the Lowry et al.(19) method.

Determination of muscle GSH content. Frozen DIA were analyzed for total GSH content within 24 h of sampling using the 5,5′ -dithiobis(2-nitrobenzoic acid)-oxidized GSH reductase assay as modified by Griffith(20) and Anderson(21). Briefly, homogenized muscle (5% sulfosalicylic acid) was centrifuged at 0 °C and 200 × g for 8 min. The supernatant was centrifuged for 2 min at 12 000 × g, and the total amount of GSH was determined from a standard curve using GSH equivalents plotted against the rate of change in absorbance at 412 nm. Total muscle GSH content was normalized to protein content as determined by the Lowry method(19).

Determination of muscle SDH activity. Total muscle SDH activity was calculated on the basis of 1) the relative contribution of fiber types to total muscle CSA and 2) specific fiber type SDH measurements, the latter determined using a micro-densitometric procedure(22, 23). This quantitative SDH technique has been described in detail in previous papers(10, 11, 22–24). Briefly, muscle segments for SDH analysis were excised and quickly frozen at optimal muscle length (1.5 times excised length)(25) in isopentane cooled to its melting point by liquid nitrogen. DIA muscle samples were taken from the right mid-costal region. Serial cross-sections of muscle fibers were cut at 10-μm thickness using a cryostat (model 2800E, Reichert-Jung, Buffalo, NY) kept at -20 °C, classified as type I, IIc, IIa, IIx, and IIb based on pH lability of myofibrillar ATPase staining(26, 27), and reacted for SDH in the presence or absence (i.e., tissue blank) of enzymatic substrate (succinate). The stoichiometric reduction of nitro blue tetrazolium to its diformazan was used as an indicator of the SDH reaction. Nitro blue tetrazolium-diformazan is a colored compound (peak absorbance wavelength of 570 nm) whose concentration is directly proportional to measured light absorbance (OD). Microscopic images of the muscle fibers were digitized into an array of 1024 × 1024 picture elements (pixels) at eight-bit resolution (256 gray levels) using an image processing system calibrated for photometry (0.02-2.00 OD units) (Mega Vision 1024XM). From the digitized images, the boundaries of individual muscle fibers(100-150 per image) were outlined, and the average fiber OD was calculated. In previous studies(22, 23) the SDH reaction has been shown to be linear for at least a 9-min period, and the fiber OD at time 0 is the same as that of the tissue blank. On this basis a single end point reaction time of 6-min was used to determine the Vmax of SDH enzyme activity within individual DIA fibers, expressed as millimoles of fumarate/L of tissue/min.

The image processing system was also calibrated for morphometry using a microscope stage micrometer. Using a 20 × microscope objective, the area of each pixel was 0.08 μm2. Boundaries of individual muscle fibers were delineated, and the CSA of each fiber was calculated from the number of pixels within the outlined region. The relative contribution of each fiber type to total CSA of the muscle was calculated based on 1) the proportion of individual fiber types in a given muscle, and 2) the mean CSA of each fiber type(28).

In vitro measurement of fatigue resistance. The methods for measuring in vitro mechanical properties of the DIA muscle have been previously described(11, 29, 30). Muscle strips (≈ 2 mm wide) were cut from the midcostal region of the right hemidiaphragm with fiber attachments at the rib and central tendon left intact. The muscle strips were mounted in a vertical tissue chamber with the rib origin of the fibers anchored by a vascular clamp mounted in series to a digital micropositioner (Daedal Instruments). A small piece of aluminum foil was glued to the DIA central tendon using cyanoacrylate and then attached via fine wire to a force transducer (model 300B, Cambridge Dual Mode). The tissue chamber was constantly perfused (5 mL/min using a peristaltic pump) with mammalian Ringer's solution of the following composition (in mM): 135 Na+, 5 K+, 2 Ca2+, 1 Mg2+, 121 Cl-, 25 HCO3-, 11 glucose, 0.3 glutamic acid, 0.4 glutamate, 5 2-[bis(2-hydroxyethy1)amino]ethanesulfonic acid buffer, and 0.008%d- tubocurarine chloride(31), and aerated with 95% O2-5% CO2. The Po2, Pco2, and pH of the aerated solution were monitored: Po2, ≈60 kPa; Paco2, ≈5.3 kPa; pH ≈7.40. The temperature of the bath was maintained at 37 °C, by circulating water through the outer jacket of the tissue chamber.

The muscle strip was activated by electrical field stimulation using 0.5-ms duration rectangular current pulses generated by a Grass model S88 stimulator and amplified using a current amplifier (Mayo Foundation, Section of Engineering). Stimulus intensity was set at 120% of that yielding maximal twitch force (Pt) responses (≈200 mA). Muscle preload force was incrementally adjusted using the micropositioner until optimal fiber length(Lo) was achieved (i.e. length at which Pt was obtained). The Lo of the DIA was measured with a microcaliper accurate to 0.1 mm (Fisher Scientific, Pittsburgh, PA). Maximum tetanic force at Lo (Po) was determined at 75 Hz in a 1-s duration train. Previous studies in our laboratory demonstrated that this stimulation frequency is optimal for generating peak tetanic forces across the study age groups(29). Force responses were monitored on a digital oscilloscope (model 1602, Gould) and recorded. Specific force generation was defined as the force [Newtons (N)] normalized for muscle CSA, the latter of which was estimated by dividing the muscle weight by its length and specific gravity (1.056 g/cm3).

Fatigue resistance of the DIA was determined using a standard 2-min period of isometric stimulation that used activation at 40 Hz in trains of 330-ms duration repeated each second(32). Control DIA muscle strips equilibrated in mammalian Ringer's solution aerated with 95 O2-5% CO2 for 30 min before repetitive isometric activation. Exogenous SOD-treated DIA muscle strips equilibrated in mammalian Ringer's solution with SOD (S-7008, Sigma Chemical Co.) at a bath concentration of 500 U/mL aerated with 95% O2-5% CO2 for 30 min before repetitive isometric activation. This concentration of SOD in vitro has been previously demonstrated to attenuate fatigue in adult DIA(6).

Cytochrome c assay. In six separate animals from each age group we measured the reduction of cytochrome c in the muscle bathing medium(15, 33). Briefly, cytochrome c was added to the mammalian Ringer's solution to establish a concentration of 10-5 M in the bathing solution. Cytochrome c was incubated for 2 min under the following test conditions: 1) no muscle, 2) passive muscle, 3) active muscle (2-min stimulation paradigm as described above), and 4) no muscle plus electrical stimulation (2-min stimulation paradigm as described above). The test medium was then removed, and absorbance at 550 nm was read in triplicate on a spectrophotometer. The magnitude of the mean absorbance change was used to index the amount of cytochrome c reduction in the test medium. Temperature, experimental timing, number of electrical stimulations, if any, and DIA muscle strip weight were standardized across conditions and age groups. The muscle bath was foil-wrapped, and studies were performed in a darkened laboratory to minimize photoreduction of cytochrome c.

Statistical analysis. Statistical methods included the t test to compare newborn and adult antioxidant enzyme activities, GSH content, SDH activity, extracellular cytochrome c reduction, and specific force measurements. A two-way analysis of variance was used to compare temporal changes in force output during repetitive isometric activation as a function of age group and exposure to exogenous SOD in vitro (Minitab Statistical Software, PC Version Release 8.0, Minitab Inc., State College, PA)(34). Where significant interactions between grouping factors were detected, a Duncan multiple range test was used as the post hoc analysis to define differences(35). Statistical significance was established at p < 0.05. Data are reported as a mean ± SD.

RESULTS

Antioxidant enzyme activities and GSH content. Total SOD, CAT, and GPX activities of newborn and adult DIA are shown in Figure 1. Newborn and adult DIA total SOD activities were not significantly different, and their Cu/Zn-Mn subfraction phenotypes were identical (Cu/Zn SOD, newborn, 70 ± 5% total activity; adult, 69± 4% total activity; Mn SOD, newborn, 30 ± 5% total activity; adult, 31 ± 4% total SOD activity). In contrast, newborn CAT activity was greater than the adult (p < 0.05) than adult DIA (Fig. 1). Total GSH content, like GPX, was lower in newborn (2272 ± 352 nmol/mg protein) compared with adult (3779 ± 325 nmol/mg protein) DIA (p < 0.05).

Figure 1
figure 1

SOD, CAT, and GPX (10-2) antioxidant enzyme activities in expressed in units/mg of protein in newborn (solid bars) and adult (open bars) DIA. *p < 0.05 newborn vs adult.

Full size image

Muscle SDH activity. Table 1 demonstrates the fiber type proportions, fiber CSA, and fiber-specific SDH activities of newborn and adult rat DIA muscle. The majority of fibers in the newborn DIA were classified as type IIc, whereas the adult DIA was comprised of a mixture of type I, IIa, IIx, and IIb fibers. Moreover, the SDH activity of adult type I and IIa fibers was significantly greater than that of newborn type I and IIc fibers (p < 0.05) (Table 1). The SDH activity of adult type IIx and IIb fibers was comparable to newborns fiber types (Table 1). As a result of these developmental differences in fibers SDH activities, total muscle SDH activity was significantly higher in adult DIA (5.5 ± 0.4 mmol of fumarate/L of tissue/min) as compared with the newborn (3.5 ± 0.2 mmol of fumarate/L of tissue/min) (p < 0.05).

Table 1 Fiber type proportions, CSA, and SDH activity of newborn and adult rat diaphragm muscle
Full size table

Diaphragm fatigue resistance. Maximum specific force of the adult DIA was significantly greater than that of the newborn DIA (22.8± 3.0 versus 6.6 ± 1.4 N/cm2, p < 0.05). Similarly, specific force at 40-Hz stimulation used during the repetitive isometric fatigue protocol was significantly greater in the adult(16.5 ± 3.5 N/cm2) as compared with the newborn (5.1 ± 1.8 N/cm2). DIA force declined during repetitive isometric activation in both age groups; the relative magnitude of this decline after 2 min of stimulation was greater in the adult (78 ± 4%) compared with the neonate (28 ± 8%) (Fig. 2). Exogenous SOD had no effect on the decline in force in newborn DIA during repetitive activation (Fig. 2) but significantly attenuated fatigue in the adult DIA (Fig. 2).

Figure 2
figure 2

Force production expressed as a percent of baseline of newborn and adult DIA during repetitive isometric activation (40-Hz, 330-ms stimulus repeated each second for 2 min). Control DIA, solid line; SOD treated DIA, dashed line. *p < 0.05, control vs SOD.

Full size image

Extracellular cytochrome c reduction. Figure 3 demonstrates the cytochrome c reduction in the bathing medium of passive and active DIA from newborn and adult animals. Passive muscle increased absorbance in both age groups, and this increase was significantly greater in adult compared with newborn DIA (p < 0.01). Repetitive DIA activation was also associated with an increase in cytochrome c reduction in both age groups that was significantly greater than that observed in passive DIA (p < 0.05), indicating that repetitive contractions augmented extracellular cytochrome c reduction. Moreover, the increase in cytochrome c reduction during fatiguing activations was significantly greater in the adult compared with the newborn (Fig. 3) (p < 0.05). Absorbance measures in musclefree cytochrome c solutions were unaffected by electrical field stimulation.

Figure 3
figure 3

Cytochrome c reduction in newborn (solid bars) and adult (open bars) DIA under passive conditions and during repetitive isometric activation (40-Hz, 330-ms stimulus repeated each second for 2 min).*p < 0.05 adult vs newborn; #p < 0.05 active muscle vs passive muscle for each respective age group.

Full size image

DISCUSSION

The principal findings of the current study are that: 1) the oxidative capacity (SDH activity) of the adult DIA is greater than that of the newborn, whereas DIA total SOD activity and its mitochondrial subfraction are comparable in these age groups; 2) extracellular cytochrome c reduction is greater in adult DIA compared with the newborn under passive and active conditions, and 3) exogenous SOD attenuates DIA fatigue in the adult but not the newborn. These data suggest that increased oxidative capacity relative to SOD activity in adult DIA may lead to oxidative stress and an enhanced susceptibility to fatigue during repetitive activation. We also observed a reciprocal pattern of change in DIA H2O2 scavenging enzyme activities (decreased CAT and increased GPX-GSH) with maturation.

Oxidative stress, defined as an imbalance between oxidant flux and antioxidant defense, where oxidants predominate, can lead to contractile dysfunction in skeletal muscle. The possibility that oxidant stress is produced in respiratory muscle during loaded conditions (both in vitro) and contributes to fatigue has been strongly suggested by a number of studies on adult DIA. These findings include evidence of glutathione oxidation(36) and lipid peroxidation(37), as well as increased production of ROS(5, 6) in the fatiguing adult DIA. Other investigations have demonstrated an attenuation of DIA fatigue and an inhibition of the aforementioned biochemical markers of oxidant stress, by the use of exogenous antioxidants(4, 6, 37). Thus, ROS are generated during respiratory muscle contractile activity and may, at times, overwhelm antioxidant defenses, resulting in oxidant stress that contributes to the genesis of fatigue in the adult.

Sources of ROS in skeletal muscle include mitochondrial electron transport(9), oxidases in the sarcoplasmic reticulum(38) and sarcolemma(39), and cytosolic xanthine oxidase(40). The mitochondria constitute the greatest source of ROS as the electron transport system consumes ≈85-90% of the oxygen used in the cell, and up to 5% of electron flux down the electron transport chain is lost to molecular O2 producing O2[horizontal line over dot](9). Strong evidence that mitochondrially derived ROS are important in the genesis of oxidative stress in skeletal muscle now exists(6, 8, 9). Specifically, data from Reid et al.(6, 33) suggest that the mitochondria are the likely source of O2[horizontal line over dot] in adult DIA and demonstrate that the magnitude of contractile dysfunction observed during repetitive activations is positively correlated with extracellular O2[horizontal line over dot] release. Moreover, Boveris and Chance(8) and Chance et al.(9) have previously documented that the rate of production of ROS, or oxidant flux, in muscle is directly related to the rate of mitochondrial oxygen consumption. These data, when taken together, strongly suggest that oxidative stress in muscle and its sequelae are related to oxidative capacity.

Our data demonstrate that the oxidative capacity of the adult DIA, as indexed by SDH activity, is significantly greater than that of the newborn. This finding is consistent with prior observations(10, 11) and defines and enhanced potential for muscle oxygen consumption and oxidant flux (O2[horizontal line over dot] anion generation) in the adult DIA during repetitive muscle activation. It is important to note that the quantitative histochemical method used in this study is not substrate-limited and is a measure of the Vmax of the SDH-catalyzed reaction(22, 23). As such, fiber SDH activity measured using this method is a valid reflection of the maximum oxidative capacity of muscle fibers.

The enhanced oxidative capacity of the adult DIA, however, was not matched by an increase in total SOD or its mitochondrial subfraction. SOD dismutates the O2[horizontal line over dot] anion to H2O2, an important first step in the defense against mitochondrially derived oxidant flux. Thus, an apparent imbalance exists between oxidative capacity and SOD activity in adult DIA and suggests that there may be an increased risk for O2[horizontal line over dot] output in this age group.

We observed that cytochrome c reduction in the muscle bathing medium was significantly greater in the adult DIA as compared with the newborn under both both passive and active conditions. This process was significantly augmented by repetitive fatiguing contractions. The extracellular cytochrome c reduction noted in the current study may be due to diphragm O2[horizontal line over dot] release, although we cannot rule out nonspecific reactions of cytochrome c reduction or oxidation. Cytochrome c reduction or oxidation. Cytochrome c can be reduced by nitric oxide, GSH, and ascorbate among other agents(41). Reid et al.(6, 33) have reported extracellular O2[horizontal line over dot] release in adult diaphragm during fatiguing contractions and suggest that such release is mitochondrial in origin given that 1) mitochondria are immediately subsarcolemmal in location (compatible with both an intracellular and extracellular distribution of O2[horizontal line over dot]), and 2) mitochondrial electron transport is linked to muscle activity(8, 9). The current data are consistent with the observations of Reid and co-workers and the notion that the extracellular O2[horizontal line over dot] release in adult DIA is related to a relative imbalance between oxidative capacity and SOD activity in this age group.

The in vitro contractile conditions used in the current study do not strictly mimic the normal physiologic situation. Nevertheless, they do permit 1) a measurement of force during repetitive direct muscle activation and 2) controlled exposure to exogenous antioxidants. We observed that when challenged in vitro the adult DIA is more susceptible to fatigue during repetitive isometric activation than the newborn DIA, a finding consistent with prior investgations(10, 11, 30, 42). Moreover, exogenous SOD added to the mammalian Ringer's solution bathing the DIA muscle attenuated the development of fatigue in the adult, but had no effect on newborn DIA. The current data further suggest there may be two phases to the pattern of DIA fatigue, an early phase independent of ROS and a later phase mediated by ROS. The differences between adult and newborn DIA in their resistance to fatigue and extracellular cytochrome c reduction, the response to exogenous SOD in the more severely fatigued group, and the partial protection of SOD would all tend to support this hypothesis.

SOD is a large protein, and when administered exogenously, it is unlikely to enter the myocyte. Instead, exogenous SOD may serve to create an extrecellular sink for O2[horizontal line over dot](6). Because the newborn DIA is significantly thinner than the adult muscle, it is unlikely that differences in O2[horizontal line over dot] diffusion could have accounted for the lack of an SOD effect on fatigue in the newborn. Instead, these results suggest that in the adult DIA repetitive activation may lead to oxidative stress and contribute to fatigue. Newborn DIA, on the other hand, may have sufficient intrinsic antioxidant scavenging capacity relative to oxidant-generating potential to adequately buffer the increased oxidant flux associated with repetitive activation.

The product of SOD is H2O2, which is also toxic and must be rapidly scavenged. In muscle, H2O2 is detoxified by both CAT and GPX by reducing it to H2O and O2. GPX uses the reducing power of GSH to detoxify H2O2. We observed that, although newborn and adult DIA SOD activities were not different, adult CAT activity was less, and adult GPX activity and GSH content greater than newborn DIA. This reciprocal pattern of change in CAT and GPX expression with postnatal maturation raises questions regarding any developmental differences in overall capacity to scavenge H2O2 between the newborn and adult DIA. Mitochondria are the most important source of H2O2 in muscle(9). Some investigators have suggested that CAT has a limited role in scavenging mitochondrially derived H2O2 because in most mammalian cell types CAT is localized to peroxisomes and is not expressed in the mitochondria or cytosol(43). CAT expression in muscle is an exception in this regard being found in the cytosol as well as microperoxisomes(44). Limited, if any, CAT expression is found in skeletal muscle mitochondria(44). In contrast, GPX is found in both the cytosol and mitochondrial matrix(9). Nevertheless, at this time it remains unclear 1) whether the adult DIA is at any disadvantage compared with the newborn DIA in its capacity to scavenge H2O2, or 2) how any developmental difference in H2O2 detoxification would affect the overall DIA antioxidant capacity between age groups. A determination of the overall endogenous antioxidant defense of DIA muscle across age groups awaits further investigation.

In summary, we conclude that the oxidative capacity of the adult DIA is greater than the newborn and not matched by a concomitant postnatal increase in SOD activity. Our data further suggest that the increased oxidative capacity relative to SOD activity in adult DIA may lead to oxidative stress and an enhanced susceptibility to fatigue during repetitive activation.