INTRODUCTION

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SKELETAL MUSCLE readily adapts to alterations in contractile activity by adjusting tissue metabolic capacity to match functional demands (5, 11, 12). A primary metabolic adaptation in response to chronic increases in use or disuse is to alter skeletal muscle mitochondrial volume density and the biochemical properties of the mitochondria within the muscle (11, 12). Pioneering morphological studies by Hoppeler and colleagues (13-15) and numerous biochemical (4, 7, 17, 24-26) studies have demonstrated the presence of two distinct mitochondrial populations within both skeletal (4, 13-17, 20) and cardiac muscle (24-26). These two mitochondrial populations exhibit distinct morphological, biochemical, and functional properties and have been named with respect to their location within the myocyte. One mitochondrial population, the subsarcolemmal (SS) mitochondria, resides near the sarcolemma, whereas the other population, the intermyofibrillar (IMF) mitochondria, lies between the myofibrils.

Biochemical studies on SS and IMF mitochondrial populations have established that the maximal oxidative capacity of the SS population is markedly lower than that of the IMF population in both cardiac (24-26) and skeletal muscle (4, 17, 20, 30). However, in response to changes in functional demand, the SS population correspondingly alters oxidative capacity to a greater extent than does the IMF population (14, 15, 20). These observations were first put forth in 1973 by Hoppeler et al. (14), who demonstrated, by using electron micrographs, that the SS mitochondrial population was increased in endurance-trained subjects to a greater extent than was the IMF population with respect to untrained individuals. This response was also observed in 1980 by Krieger et al. (20), who reported a significant 33% increase in mitochondrial oxidase-specific activities in the SS population of rat hindlimb skeletal muscle in response to a chronic endurance training program. In contrast, no changes in mitochondrial oxidase-specific activities were observed in the IMF population. Moreover, with decreased muscle use elicited by immobilization (20) or microgravity (2), the SS population has been shown to significantly decrease oxidative capacity, whereas the IMF population has remained relatively unchanged. The rationale for the existence of two biochemically heterogeneous populations of mitochondria in skeletal muscle remains to be elucidated, although it has been proposed that the observed biochemical and functional differences may be because of regional differences in substrate availability or local ATP turnover rates (23).

It has been demonstrated recently that mitochondria isolated from different skeletal muscle fiber types possess different oxidase and enzymatic capacities, which are substrate dependent (16). It is therefore possible that such substrate-dependent oxidase capacities may be present in SS and IMF mitochondria. Additionally, factors such as the regional distribution of substrate, i.e., increases in intramuscular lipid stores within a muscle cell, may promote regional differences in substrate oxidase activity after an endurance training program. Thus a potential shortcoming of previous studies examining these mitochondrial populations in general and in response to endurance training has been the limited use of physiologically important oxidative substrates such as pyruvate and palmitoyl-DL-carnitine in evaluating respiratory capacity, with most papers only reporting oxidative rates with glutamate (Glu) as a substrate (4, 20). Furthermore, data on mitochondrial matrix and electron transport chain (ETC) enzyme activities are limited with respect to SS and IMF mitochondrial populations within skeletal muscle in general and how these activities may change with endurance training. Given the limited information available on the responses to endurance training in SS and IMF mitochondrial populations, the goals of the present study were 1) to examine the oxidative capacity of SS and IMF mitochondria in response to chronic endurance training by using a variety of physiologically important respiratory substrates and 2) to evaluate changes in both mitochondrial matrix and ETC enzyme activities in these two biochemically distinct mitochondrial populations.

	METHODS

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Animal care. Female Long-Evans hooded rats (n = 24) were obtained from an institutional breeding stock. Animals were maintained two per cage on a 12:12-h light-dark cycle at 25°C and had access to standard laboratory chow and water ad libitum. All procedures were in accordance with the guiding principles in the care and use of laboratory animals of Arizona State University.

Exercise training. Animals were randomly assigned to either the exercise training group (n = 12) or a sedentary control group (n = 12) at the beginning of the study. The exercise training group was subjected to running on a motor-driven treadmill by using a progressive exercise training regimen. The duration and intensity of exercise were gradually increased until exercise duration was 60 min and intensity was 30 m/min at a 15% grade by the end of the second week. This final intensity and duration were maintained for the final 4 wk of the 6-wk training period. Animals were not run for 24 h before being euthanized.

Preparation of mitochondrial populations. Animals were euthanized by CO₂ inhalation. The gastrocnemius, soleus, and quadriceps muscles were quickly excised and placed into ice-cold solution 1 that contained (in mM) 100 KCl, 40 Tris · HCl, 10 Tris base, 5 MgCl₂, 1 EDTA, and 1 ATP, pH 7.5. All subsequent steps were performed at 0-4°C. The excised muscles were trimmed to remove excess fat and connective tissue, finely minced, suspended 10-fold (wt/vol) in solution 1, and homogenized in an Ultra-Turrax (Cincinnati, OH) tissue homogenizer at 40% power for 10 s. Preparation of mitochondrial populations was then carried out by a modification of the procedures of Palmer et al. (24-26), as described by Krieger et al. (20). The homogenate was centrifuged at 800 g for 10 min in a refrigerated centrifuge (model J2-21 M/E, Beckman) to pellet the myofibrils, which were subsequently used for the preparation of the IMF mitochondria. The 800-g supernatant was filtered through a double layer of cheese cloth and centrifuged at 9,000 g for 10 min to pellet the SS mitochondrial population. The SS mitochondrial pellet was then resuspended, washed in solution 2 [in mM; 100 KCl, 10 Tris · HCl, 10 Tris, 1 MgSO₄, 0.1 EDTA, 0.02 ATP, and 1.5% BSA (no. A-7030, Sigma Chemical, St. Louis, MO; fatty acid content <0.01%), pH 7.4], and centrifuged at 8,000 g for 10 min. The supernatant was discarded, and the pellet was resuspended, washed in 10 ml of solution 3 (same as solution 2 but without the BSA), and centrifuged at 6,000 g for 10 min. The final mitochondrial pellet was then suspended in a small volume (0.5-1 ml) of a suspension buffer containing (in mM) 220 sucrose, 70 mannitol, 10 Tris · HCl, and 1 EDTA, pH 7.4.

Mitochondrial respiration. Mitochondrial respiration measurements were conducted in a temperature-controlled respiration chamber (Rank Brothers, Cambridge, UK) at 37°C with constant stirring. The respiration medium contained (in mM) 10 KH₂PO₄, 15 KCl, 25 Tris, 45 sucrose, 12 mannitol, 1 EDTA, 0.2% BSA, 20 glucose, and 5 MgCl₂, pH 7.4 (6), in a final volume of 2 ml. The oxidative substrates used were 1 mM pyruvate + 1 mM malate (P+M), 10 mM 2-oxoglutarate (2-OG), 45 µM palmitoyl-DL-carnitine + 1 mM malate (PC+M), and 10 mM Glu.

Enzyme activities. All spectrophotometric assays were carried out at 25°C. The maximal activities of two ETC enzyme pathways, succinate- and glycerol-3-phosphate (G3P)-cytochrome-c reductase, were assayed spectrophotometrically by using 10 mM succinate and 10 mM G3P as substrates, respectively, according to the methods of Gohil et al. (9) with the use of a Hewlett-Packard 8452A diode-array spectrophotometer. Mitochondria were diluted in a 10 mM KH₂PO₄ buffer, pH 7.5, and subjected to five freeze-thaw cycles to fully expose the dehydrogenase enzymes. After the addition of either succinate or G3P to the assay medium, the linear reduction of oxidized cytochrome c (type VI; Sigma Chemical) was followed at 550 nm. Maximal activity of the mitochondrial matrix enzyme isocitrate dehydrogenase (IDH) was assayed according to the methods of Chen and Plaut (3). Undiluted mitochondrial suspension from freshly isolated mitochondria was added to the assay medium with 0.5% Triton X-100 and incubated in the presence of 0.5 mM ADP for 2 min before the addition of isocitrate. The linear increase in absorbance was recorded after the addition of the isocitrate. The presence of nonmitochondrial ATPase activity in both the IMF and SS populations was measured as an evaluation of contamination of the samples by other ATPase-containing structures. Contamination of the preparation by nonmitochondrial ATPase was quantified as the amount of ATPase activity observed in the presence of 1 µl of oligomycin by using a modification of the procedure described by Pullman et al. (27) measuring the release of P_i in the presence of an ATP-regenerating system.

Statistical analysis. Data were analyzed by using a one-way analysis of variance. If the overall F value was significant, comparisons between mean values were made by using a Student-Newman-Keuls test. Significance was set at P < 0.05 for all comparisons.

	RESULTS

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Mitochondrial isolation. By using previously validated isolation techniques (4, 20, 24-26), contamination of the SS and IMF mitochondria by other ATPase-containing membranes has proven to be minimal. The presence of contamination was assessed by measuring nonmitochondrial ATPase activity in the presence of oligomycin. There was no difference in oligomycin-sensitive ATPase activity among any of the groups, with total contamination in all groups being <9% (Table 1).

Oxidative capacities of SS and IMF mitochondria. Maximal (state III) rates of oxygen consumption (O₂) were greater in IMF vs. SS mitochondria (P < 0.05) with any given substrate or substrate combination, regardless of training status (cf. Figs. 1 and 2). In SS mitochondria, endurance training elicited significant (P < 0.05) 26 and 36% increases in state III respiratory rates by using Glu and P+M as oxidative substrates, respectively (Fig. 1). There were no significant differences in the oxidative capacity induced by training in SS mitochondria by using either PC+M or 2-OG as oxidative substrates. Contrary to the results found in SS mitochondria, in IMF mitochondria, endurance training increased (P < 0.05) maximal oxidative capacity with PC+M and 2-OG by 27 and 33%, respectively, over control IMF mitochondria from sedentary rats (Fig. 2). As has been previously demonstrated when Glu (Fig. 2) is used as the oxidative substrate (18), no significant difference in oxidative capacity resulted in the IMF population with chronic endurance training. However, with P+M, endurance training resulted in a significant (P < 0.05) 20% increase in maximal O₂ vs. the control group (Fig. 2).

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Maximal (state III) respiratory rates for isolated subsarcolemmal (SS) mitochondria from control (open bars) and endurance-trained animals (solid bars). Values are means ± SE for 10 rats. Substrates used were pyruvate + malate (P+M), palmitoly-DL-carnitine + malate (PC+M), 2-oxoglutarate (2-OG), and glutamate (Glu). *P < 0.05 between control and endurance trained rats.

View larger version (26K): [in this window] [in a new window]   Fig. 2.   Maximal (state III) respiratory rates for isolated intermyofibrillar (IMF) mitochondria from control (open bars) and endurance-trained animals (solid bars). Values are means ± SE for 10 rats. *P < 0.05 between control and endurance trained rats.

Enzyme activities. As was the case for oxidase activities, the maximal activities of all enzymes measured were higher in the IMF mitochondria regardless of training status (Fig. 3). In SS mitochondria, endurance training resulted in a significant 30% increase in the activity of succinate-cytochrome c reductase, whereas no significant differences were detected in either G3P-cytochrome c reductase or IDH activities. Similarly, in IMF mitochondria, training increased succinate-cytochrome c reductase by 28% for the trained and control groups. Similar to SS mitochondria, G3P-linked cytochrome c reductase activity was not increased with endurance training. The maximal activity of IDH, unlike that of the SS mitochondria, was increased by 32% as a result of endurance training in the IMF mitochondria. In all cases, both ETC and matrix enzyme maximal activity were higher in IMF vs. SS mitochondria.

View larger version (32K): [in this window] [in a new window]   Fig. 3.   Maximal enzyme activities for isolated SS and IMF mitochondria from control (open bars) and endurance-trained animals (solid bars). Values are means ± SE for 10 rats. S-CR, succinate-cytochrome c reductase; IDH, isocitrate dehydrogenase; G3P-CR, glycerol-3-phosphate-cytochrome c reductase. * P < 0.05 between control and endurance trained rats.

	DISCUSSION

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Biochemically distinct populations of mitochondria have previously been isolated in cardiac (24-26) and skeletal muscle (4, 17, 20, 30) and have been named according to their intracellular location. By using isolation techniques described in previous reports (20, 25), we have isolated two biochemically distinct populations of mitochondria from skeletal muscle of endurance-trained and untrained animals that exhibit both high respiratory control ratio values and respiration rates. Concerns have been raised (22) that cytochrome c is preferentially lost from the SS population during isolation. We addressed this in preliminary studies when 4 µmol of cytochrome c were added to the respiration medium. This manipulation increased state III rates by <10% in both mitochondrial populations (data not shown). Similar results were obtained by Cogswell et al. (4) and by Palmer et al. (26). Although it cannot be ruled out that differences in isolation procedures may contribute to the observed differences in functionality of these mitochondrial populations, the majority of evidence suggests that biochemical and functional differences in SS and IMF mitochondria are in fact physiologically relevant differences, not artifacts of the isolation procedure. Contamination of the isolated mitochondrial fractions by nonmitochondrial ATPase-containing membranes as assessed by oligomycin-insensitive ATPase activity was <9%. This value is similar to those reported by both Krieger et al. (20) and Cogswell et al. (4), indicating that the isolation procedure is in fact delivering a relatively pure mitochondrial fraction. Six weeks of treadmill running increased the mitochondrial yields and the maximal activity of cytochrome-c reductase in both the SS and IMF populations and thus indicates the effectiveness of the endurance training program. The results presented in the present study are in agreement with previous studies that have evaluated the oxidative capacities of SS and IMF mitochondria where, in all conditions tested, IMF mitochondria exhibited significantly higher state II O₂ rates compared with SS mitochondria, regardless of training status (4, 17, 20, 30).

Only one previous study has attempted to examine the effects of endurance training on the adaptability of SS and IMF mitochondria. Krieger et al. (20) concluded that SS mitochondria showed significant increases in Glu oxidative capacity after an endurance training program, whereas IMF mitochondria did not. However, the use of only Glu in that study led us to further characterize the oxidative capacities of these mitochondrial populations using a variety of substrates that are more similar to those encountered by the mitochondria during respiration in vivo. When Glu was used as the respiratory substrate, our results were in agreement to those reported by Krieger et al. (20); i.e., IMF mitochondria exhibited nonsignificant numerical increases (16%) in state III rates after training. In contrast, the present study demonstrates that state III respiration was significantly increased by training in IMF mitochondria when P+M (20%), PC+M (27%), and 2-OG (33%) were used as respiratory substrates. Once more in agreement with Krieger et al. (20), we report a more marked increase in Glu oxidase in SS mitochondria compared with IMF mitochondria. However, when respiration was evaluated with other substrates in the SS population, endurance training increased only P+M oxidase. Thus there appears to be a differential response in the adaptation of oxidative capacity in these two mitochondrial populations that is substrate specific. This finding differs from previous reports (20) that have concluded IMF have a limited adaptability in response to endurance training. This results from evaluating respiration using a wide variety of substrates.

Presently, the literature is equivocal regarding the catalytic potentials of substrate dehydrogenase enzymes in these mitochondrial populations. Krieger et al. (20) reported 65% greater succinate dehydrogenase (SDH) activity in the IMF population compared with the SS population in untrained skeletal muscle. These results are in conflict with those of Cogswell et al. (4), who reported SDH activity was 40% greater in SS compared with IMF mitochondria. In a recent study, malate dehydrogenase was measured to be 2.2-fold higher in the IMF population (30). In the present study, the matrix enzyme IDH was measured. When values from the untrained control group are used, IMF mitochondria demonstrated 70% greater IDH activity than did the SS fraction. Thus, when the present results are compared with others reporting matrix enzyme activities, it becomes apparent that IMF mitochondria have greater substrate dehydrogenase enzyme activity. Little data are available regarding substrate dehydrogenase enzyme adaptations to endurance training in the SS and IMF mitochondrial populations. In the present study, training increased IMF IDH activity by 32%, whereas the SS population did not show a significant increase. This increase is similar to the 31% training-induced increase in SDH activity in IMF mitochondria shown by Krieger et al. (20). It is puzzling that training did not increase IDH in the SS population. In the only previous study to measure the activity of a substrate dehydrogenase enzyme after a training program, Krieger et al. (20) reported a 58% increase in SDH activity in the SS population. Given the results of recent studies by Takahashi and Hood (30) quantifying protein import into these two mitochondrial populations, the results of the present study may be explained by greater rates of specific protein import into IMF mitochondria. Conversely, other factors, such as population-specific rates of protein degradation or targeting of specific proteins to individual populations, could potentially explain the observed regional differences in enzyme adaptive responses to endurance training.

To the best of our knowledge, only one previous study (4) specifically measured ETC enzymes in SS and IMF mitochondria. Cytochrome oxidase activity was reported to be 17% higher in IMF than in SS mitochondria. In the present study, the activity of cytochrome c reductase was measured by using both succinate and G3P as substrates. Similar to the results of Cogswell et al. (4), we found greater activities of an ETC enzyme: cytochrome-c reductase was ~40% in the IMF population with the use of either succinate or G3P as substrate. Succinate-cytochrome c reductase demonstrated similar 30% increases with training in both SS and IMF populations, and, similar to results presented by Davies (5) when G3P was used as substrate, there was no increase or even a slight decrease in cytochrome-c reductase activity after training.

It has been demonstrated by Kirkwood et al. (18, 19) and Skulachev (29) that rat skeletal muscle mitochondria most likely exist not as spatially distinct organelles but as a reticular structure. The presently available data on SS and IMF mitochondria do not discount a mitochondrial reticulum in vivo but only provides evidence of mitochondrial heterogeneity with skeletal muscle or within a reticulum. Because similar mitochondrial heterogeneity has been demonstrated in both cardiac (24-26) and skeletal muscle (4, 17, 20, 30) concurrently with mitochondrial reticula in these tissues, it is interesting to speculate as to the role two biochemically and functionally distinct populations of mitochondria might have in this reticular structure. One proposed hypothesis (2, 18) suggests that less oxidative mitochondria lie at the SS region of the myocyte because they function to provide energy for the less energetically demanding plasma membrane-bound ATPases and ATP for protein synthesis. An alternative view of the functional significance of a mitochondrial reticulum would be that a continuum of oxidative capacities exists with the less oxidative SS mitochondria at the exterior of a cell to establish a gradient for oxygen and substrate transport into the IMF region of a muscle cell (1, 29). Both of these views are consistent with the current biochemical and functional data on IMF and SS mitochondria, and experiments designed to specifically test these hypotheses will be required to fully elucidate the role of mitochondrial heterogeneity in skeletal muscle.

In conclusion, the findings of this study demonstrate that endurance training increases the oxidative capacity and specific activities of both ETC and matrix enzymes in both SS and IMF mitochondria. These increases are substrate dependent and differ in magnitude between mitochondrial populations. Whether regional compositional differences in skeletal muscle mitochondria serve a purely functional purpose or are a result of mitochondrial biogenesis is an interesting question and will require further research.