INTRODUCTION

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ONE CONSEQUENCE OF EMPHYSEMA or chronic obstructive pulmonary disease (COPD) is a diminished exercise capacity. Historically, this limitation has been attributed primarily to decrements in lung function and blood-gas perturbations. However, several lines of evidence suggest that peripheral (nonventilatory) muscles are also affected. Specifically, human studies demonstrate muscle weakness (6, 28), reduced oxidative enzyme activities (10, 16), and elevated exercising muscle P_i-to-phophocreatine (PCr) ratios (P_i/PCr) evaluated by ³¹P-NMR (26, 27). Unfortunately, in humans it has not been possible to determine whether these alterations result from reduced physical activity or some other aspect of the disease condition per se.

At the same moderate submaximal work rate, the energy requirement and oxygen uptake (O₂) are the same in trained and untrained states (9). However, the degree of metabolic perturbation required to elicit that O₂ is reduced after training, consequent to a greater mitochondrial volume and oxidative enzyme activity (20). Accordingly, if mitochondrial volumes and oxidative enzyme activities are reduced in the skeletal muscles of emphysema or COPD patients, the P_i/PCr (and also the ADP-to-ATP ratio) required to drive a given O₂ must be higher. Because the activity of the primary rate-limiting enzyme of glycolysis (phosphofructokinase) is sensitive to intracellular concentrations of ADP, PCr, and P_i, glycolytic flux during exercise will be increased and intracellular pH and glycogen levels will fall to a greater extent. This scenario is expected to enhance muscle fatigability and impair exercise tolerance (9).

What is uncertain is whether skeletal muscle oxidative enzyme activity decrements in emphysema result from the disease condition itself or are secondary to the reduced physical activity (i.e., detraining) of these patients. There is certainly evidence that chronic heart failure per se rather than reduced activity levels is responsible for decreased muscle oxidative enzymes in rats after coronary artery ligation (24). Therefore, the purpose of this investigation was to determine the effect of emphysema on hindlimb skeletal muscle oxidative enzyme activity in an accepted animal model in which activity levels between control and emphysema groups can be equated better than is possible with human studies. Moreover, with this model it is feasible to analyze the entire muscle of interest.

	METHODS

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The protocols used in this investigation were approved by the Kansas State University Institutional Animal Care and Use Committee. In all respects, the protocols conform with the Guide for the Care and Use of Laboratory Animals [DHEW Publication No. (NIH) 86-23, Revised 1985, Office of Science and Health Reports, DRR/NIH, Bethesda, MD 20892].

Emphysema model. Adolescent male Syrian Golden hamsters (9 wk old, 80-120 g body wt) were housed individually in 7.5 × 8.5-in. cages, maintained on a 12:12-h light-dark cycle, and supplied with rodent chow and water ad libitum. After a 1-wk habituation period, the animals were divided into control (Con; 111 ± 3 g body wt) and emphysema (Emp; 99 ± 3 g body wt) groups at random. There was no significant difference between groups with respect to body weight (P = 0.36). Under deep ketamine/xylazine anesthesia (150/7.5 mg/kg im), either saline (0.3 ml/100 g body wt) or porcine elastase [25 IU/100 g body wt (Sigma Chemical, St. Louis, MO) in 0.3 ml of normal saline] was instilled intratracheally by using a 27-gauge hypodermic needle (12, 14). To ensure a more uniform elastase distribution throughout the lungs, each hamster was supported in a vertical head-up position and rotated gently from side to side during injection. This procedure has previously been proven to be effective in producing panacinar emphysema with increased lung compliance, elevated lung volumes, and reduced pulmonary internal surface area (25). After the surgery, the animals were returned to their cages and monitored (appearance and body weight) daily for the first 2 wk and weekly thereafter.

Tissue harvesting. Five to six months after elastase injection, the hamsters were euthanized and the lungs, gastrocnemius, vastus lateralis, plantaris, and diaphragm muscles were excised. The gastrocnemius was not subdivided into white, red, and mixed portions because, unlike in rats, there is no gross difference among muscle regions. Rather, the muscle color appears uniform throughout the entire thickness. The diaphragm was further dissected into costal and crural portions. Each muscle was frozen in liquid N₂ and stored separately at 70°C for subsequent determination of citrate synthase (CS) activity. In addition, a saline-displacement technique was used to measure excised lung volume at 0 cmH₂O airway pressure (22). In emphysematous hamsters, this technique provides a measurement of lung volume that is proportional to and highly correlated with (n = 32, r = 0.724, P < 0.001) the augmented passive vital capacity (23).

Enzyme assay. CS activity was determined in frozen muscle samples according to the methods described by Reichmann et al. (19). Briefly, the frozen whole muscle was pulverized under liquid N₂, and total cellular enzymes were extracted by homogenizing the muscle powder in a cold extraction buffer [(in mM) 174 KCl, 104 glutathione, and 2 EDTA]. Enzyme activities, expressed as micromoles per minute per gram wet weight, were measured spectophotometrically in 1 ml of assay mixture consisting of 0.6 ml 100 mM Tris buffer, 0.1 ml 3.0 mM acetyl-CoA, 0.1 ml 1.0 mM DTNB, 0.1 ml diluted homogenate, and 0.1 ml 5 mM oxaloacetate at room temperature (i.e., 25°C).

Activity monitoring. Hamster ambulatory and total activity were determined 6-18 wk after induction of the emphysematous condition in separate groups of Con (n = 8) and Emp (n = 13) hamsters. By using the Columbus Instruments Opto-varimex Minor activity monitor (24), measurements were made for four consecutive 12-h periods, corresponding to two light and dark cycles. The monitoring cage was the same size as the regular housing (7.5 × 8.5 in.), and photocells were placed at ~1-in. intervals ~1.5 in. above the floor along the length of the cage. Interruption of a single photocell beam was recorded as total activity. When two beams were interrupted sequentially, ambulatory activity was recorded. In pilot experiments, there was no detectable difference between the first and second light or first and second dark cycles. Thus each animal was placed in the monitor at 6 AM, and recordings of ambulatory and total activity were made every 12 h for the next two light and dark cycles.

Statistical analysis. For each hindlimb muscle, differences between Emp and Con animals were compared by using an unpaired t-test. A significance level of P < 0.05 was accepted. A one-tailed test was used for diaphragm enzyme comparisons because an increased activity was expected on the basis of previous reports (3, 14). All values are presented as means ± SE.

	RESULTS

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Emphysema condition. The final body weight of Emp (111 ± 5 g; n = 8) vs. Con (116 ± 7 g; n = 7) hamsters was not different. The presence of lung pathology and air trapping was supported by the large increase (102%; P < 0.002) in saline-displacement lung volume weight of Emp (3.0 ± 0.3 g) vs. Con (1.5 ± 0.3 g) groups.

Activity level. Of the two activity measurements made, i.e., ambulatory and total, that of ambulatory activity, which required the sequential interruption of two photocell beams per one count, best describes whole body movement. Table 1 demonstrates that there were no differences between Con and Emp groups with respect to either ambulatory or total activity. As expected in nocturnal animals, nighttime (dark cycle) activity increased two- to fourfold over daytime values (light cycle).

Oxidative enzymes. The activity of CS was 13% (P < 0.05) lower in the gastrocnemius (Emp, 39.2 ± 0.8; Con, 45.1 ± 2.0 µmol · min¹ · g wet wt¹) and 7% (P < 0.05) lower in the vastus lateralis (Emp, 44.9 ± 0.8; Con, 48.5 ± 1.5 µmol · min¹ · g wet wt¹) muscle of Emp compared with Con hamsters (Fig. 1). There was no significant correlation between lung volume and CS activity in any muscle at the P < 0.05 level. However, with respect to lung volume vs. CS activity in the gastrocnemius muscle, the correlation coefficient of r = 0.69 achieved a P value of 0.086. In contrast, the CS activity of plantaris muscle was similar between groups studied (Fig. 1). In keeping with the findings of others (3, 14), the oxidative enzyme activity was increased in Emp diaphragms. Specifically, there was a 12% (P < 0.05) increase in the CS activity in the crural region (Emp, 65.7 ± 2.2; Con, 58.5 ± 2.0 µmol · min¹ · g wet wt¹) and a 7% (P < 0.05) increase in the costal region (Emp, 65.1 ± 1.5; Con, 61.1 ± 1.8 µmol · min¹ · g wet wt¹) of diaphragms from Emp hamsters (Fig. 2).

View larger version (33K): [in this window] [in a new window]   Fig. 1.   Citrate synthase (CS) activity in limb (i.e., nonventilatory) muscle of control (Con) and emphysematous (Emp) hamsters. Gastroc, gastrocnemius muscle. * P < 0.05 compared with Con.

View larger version (37K): [in this window] [in a new window]   Fig. 2.   CS activity in crural and costal regions of diaphragms from Con and Emp hamsters. * P < 0.05 compared with Con.

	DISCUSSION

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The present study demonstrates that emphysema promotes a modest reduction in the oxidative capacity of nonventilatory muscles. Conversely, as demonstrated previously (3, 14), emphysema induces an increased oxidative enzyme capacity in the diaphragm. To our knowledge, this is the first study to demonstrate reductions in skeletal muscle oxidative enzyme capacity in an animal model of emphysema in which it was demonstrated that there were no differences in physical activity levels between Emp and Con groups.

The results of this study build on previous findings that COPD reduces the oxidative capacity of hindlimb musculature (11, 16). Both of the aforementioned previous investigations found decreases in CS and 3-hydroxyacyl CoA dehydrogenase activity in vastus lateralis muscle biopsies of COPD patients. Neither the present investigation nor that of Farkas and Roussos (3) found any alteration of CS activity in the plantaris muscle of emphysematous hamsters. In addition, oxidative enzyme activity was not altered in the latissimus dorsi of COPD patients (21). In the present investigation, it is possible that the disparate response of CS activity among hindlimb muscles is a function of their fiber type composition and/or recruitment profiles. To our knowledge, of the hindlimb muscles sampled in this investigation, only the fiber type of the hamster plantaris is known (3). Interestingly, Farkas and Roussos (3) have determined that the percentage of fast glycolytic (IIb) fibers (47%) within the plantaris is exactly the same for hamsters as for rats (1). If this similarity in fiber type between these species holds true for the gastrocnemius and vastus lateralis muscles, an interesting relationship emerges. Specifically, the change in CS activity in emphysema across these three muscles would be significantly correlated with %type I + IIa + IId/x fiber type composition (r = 0.999, P < 0.05). This is a provocative observation, and one that provides a compelling rationale for fiber typing all the principal hindlimb muscles in Con and Emp hamsters. It is noteworthy that, in rats, CS activity among muscles is most highly correlated with %type I + IIa + IId/x fiber type composition than with any other fiber combination (1).

With respect to the physiological importance of the hamster plantaris muscle to locomotor activity, it is an extremely small muscle. Therefore, it is expected to contribute quantitatively less work or power to foot and hindlimb extension during running, particularly compared with the gastrocnemius (>10 times the mass of the plantaris).

The mean final body weights of the animals in this investigation (i.e., Con: 116 ± 7 g; Emp: 111 ± 5 g) inclined toward the lower end of the spectrum for published values in adult Syrian Golden hamsters, i.e., from 105 (25) to 191 g (14). In a review of data from two previous investigations (17, 23), in which the reported mean values were ~150 g, it was apparent that the presence of two or three exceptionally large (>200 g) individuals substantially elevated the mean body weight. In the present investigation, there were no such animals. As in previous studies, all animals were fed and watered ad libitum. There was a modest although significant weight gain during the course of the study, and neither initial nor final body weights were significantly different between groups.

As presented in the introduction to this study, decreases in muscle oxidative capacity are expected to result in a greater stimulation of glycolysis at a given moderate submaximal workload or O₂. Our results are consistent with the observation that COPD patients have a higher P_i/PCr at submaximal work rates (5, 10, 13, 27), which indicates a greater stimulation of glycolysis. Enhanced glycolytic flux, due to reduced oxidative enzyme activity combined with arterial hypoxemia, is expected to exacerbate the exercise-induced intracellular acidosis. Indeed, this is what Fiaccadori et al. (5) reported in the quadriceps femoris muscle of COPD patients. Such an effect likely accounts for at least a portion of the decreased fatigue resistance (28) and isometric strength (6) observed in COPD patients. It would also be of interest to know whether skeletal muscle glycolytic enzyme activity is increased in the emphysematous hamster hindlimb, which could further exacerbate these effects.

The present investigation negates the possibility that physical activity differences between Con and Emp hamsters represent a potential cause for reduced enzyme activity. Other mediators that can, in certain circumstances, reduce oxidative enzyme activity include respiratory acidosis (7) and muscle wasting. However, in our experience and that of others, there is no difference in either arterial PCO₂ (15, 23) or body weight (2, 3, 14, 15, 17, 23, 25) between Con and Emp hamsters. In contrast, arterial hypoxemia is substantially present in emphysematous animals (15, 23) and humans (4, 5). Chronic hypoxemia in healthy human climbers has been associated with reduced skeletal muscle oxidative enzyme capacity (8) and therefore represents one putative mechanism for the reduced CS activity observed herein. It would also be of interest to determine in future studies whether elastase-induced emphysema affects thyroid hormone levels, which are known to be important for achieving and sustaining mitochondrial enzyme function.

The increase in CS activity found in the Emp diaphragm reported herein is in agreement with previous findings of increased diaphragm oxidative enzyme capacity (3, 14) and will augment diaphragm fatigue resistance (2, 5) as found in emphysematous hamsters. One consequence of emphysema is that the diaphragm becomes mechanically disadvantaged and is thought to perform more work in the emphysematous condition. Combined with the increased diaphragm capillarity (14, 17), these findings suggest that the diaphragm in emphysematous hamsters undergoes a training response. However, in certain instances, a preferential atrophy of fast oxidative glycolytic fibers (IIa) in the costal diaphragm has been found (3). This response is not consistent with a pure training response within the diaphragm.

In summary, emphysema induces reductions in CS activity in vastus lateralis and gastrocnemius muscle, but not in plantaris muscle. This response in the vastus lateralis and gastrocnemius is diametrically opposed to that found in the diaphragm, where chronic ventilatory overload may increase costal and crural oxidative capacity similar to that observed after endurance training in limb skeletal muscles.