Journal of Applied Physiology AJP: Regulatory, Integrative and Comparative Physiology
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J Appl Physiol 92: 2429-2438, 2002. First published February 8, 2002; doi:10.1152/japplphysiol.01024.2001
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Vol. 92, Issue 6, 2429-2438, June 2002

Endurance training induces muscle-specific changes in mitochondrial function in skinned muscle fibers

Yan Burelle and Peter W. Hochachka

Departments of Zoology and Radiology, and Sports Medicine Division, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present study was conducted to investigate the potential role of changes in the apparent Km for ADP and in the functional coupling of the creatine (Cr) kinase (CK) system (CK efficiency) in explaining the tighter integration of ATP supply and demand after exercise training. Mitochondrial function was assessed in saponin-skinned fibers from the soleus and the deep red portion of the medial gastrocnemius isolated from trained (T; treadmill running, 5 days/wk, 4 wk) and control (C) female Sprague-Dawley rats. In the soleus, Vmax in the presence of 1 mM ADP was increased by 21% after training (5.9 ± 0.2 vs. 4.7 ± 0.4 nmol O2 · min-1 · mg dry wt-1, P < 0.05). This was accompanied by no change in the Km for ADP measured in the absence of Cr (146 ± 9 vs. 149 ± 13 µM in T and C, respectively) and in its presence (50 ± 4 vs. 48 ± 6 µM in T and C, respectively) and in CK efficiency [Km (+Cr)/Km (-Cr)]. In contrast, in the red gastrocnemius, training decreased, by 35%, the apparent Km for ADP in the absence (83 ± 5 vs. 129 ± 9 µM, P < 0.01) of Cr, without affecting Vmax (6.2 ± 0.4 vs. 6.7 ± 0.3 nmol O2 · min-1 · mg dry wt-1 in T and C, respectively) and CK efficiency. These results thus suggest that training induces muscle-specific adaptations of mitochondrial function and that a change in the intrinsic sensitivity of mitochondria to ADP could at least partly explain the tighter integration of ATP and demand commonly observed after training.

exercise training; oxidative phosphorylation; creatine kinase system


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ONE OF THE TYPICAL ADAPTATIONS of skeletal muscle metabolism in response to training is a tightening in the coupling between ATP supply and demand (7, 11, 18, 27). This well-described phenomenon is characterized by a lesser increase in free ADP, AMP, IMP, creatine (Cr), and Pi, by a lesser decrease in phosphocreatine (PCr), and thus by a smaller perturbation of the cytosolic phosphorylation potential in response to changes in workload. In addition, this tighter integration of ATP supply and demand is associated with less stimulation of glycolysis, resulting in a decrease in lactate production and glucose utilization, a lower cytosolic redox state, and thus an improved coupling between pyruvate oxidation and glycolytic flux (18, 28).

The mechanisms involved in this switch from a loose toward a tight metabolic control system in response to endurance training have been extensively studied over the past decades (see Ref. 18 for a review). One of the main factors thought to be responsible for this phenomenon is the improvement of muscle oxidative capacity brought about by an increase in mitochondrial volume density and in the activity of several enzymes of oxidative metabolism. Indeed, for a given workload and oxygen consumption (VO2) per unit of muscle mass, this improvement results in a lower VO2 per unit of respiratory chain. Therefore, the change in the concentration of cytosolic factors controlling mitochondrial respiration required to elicit the same VO2 at a given workload is lower in trained muscles (7, 11).

Whereas the increase in muscle oxidative capacity undoubtedly plays an important role in improving the coupling between ATP supply and demand, other mechanisms are probably involved. For example, training was shown to result in a faster increase in blood flow kinetics and oxygen delivery to the working tissue at the onset of exercise (35), presumably allowing for a quicker response of mitochondrial oxidative phosphorylation. Theories involving a faster increase in tricarboxylic acid (TCA) cycle intermediate delivery through anaplerotic pathways after training have also been advanced. However, the importance of the increase in the TCA pool size in increasing TCA flux is still undetermined, and the effect of training on the anaplerotic fluxes is unknown (16, 17).

At the mitochondrial level, an increase in the intrinsic sensitivity to cytosolic regulatory signals such as ADP and Cr could also allow for a tighter integration of ATP supply and demand. Recent studies on skinned muscle fibers support the concept that, in vivo, mitochondrial respiration may be controlled by local ADP concentration ([ADP]) in the mitochondrial intermembrane space (21, 32, 33). The [ADP] in this compartment appears to be modulated by the dynamic permeability state of the mitochondrial outer membrane (MOM) for ADP (15, 24) and also by the functional coupling of several mitochondrial kinases, among which is the mitochondrial Cr kinase (CK) (MiCK) (4, 22, 32, 33). Alteration in the permeability of the MOM to ADP and/or in the functional coupling of MiCK has been reported in a number of physiological and pathophysiological settings (3, 10, 14, 26, 39). A decrease in the apparent Km for ADP and/or an increase in the functional coupling of MiCK after training could thus partly explain the transition toward a tight metabolic control system, which is commonly observed.

In the present study, the saponin-skinned muscle fiber technique was thus used to investigate the effects of training on mitochondrial function in the soleus and the deep red portion of the medial gastrocnemius in rats. The first objective was to determine whether training would result in an alteration in the mitochondrial sensitivity to ADP and/or in the functional coupling of the CK system. The second objective was to address the question of whether or not training could result in muscle-specific adaptations of mitochondrial function. Different adaptations to the same training stimulus could be possible, based on the fact that, during locomotion, the recruitment pattern of the soleus and the gastrocnemius is different (19, 30) and that the regulation of mitochondrial respiration is known to be strikingly different among various muscles, depending on fiber type (20, 21, 23, 41).


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animal care and training program. The experiments reported in the present study were approved by the institutional ethics committee on the use of laboratory animals in research. The studies were conducted on female Sprague-Dawley rats (initial weight, 180 g) obtained from an institutional breeding stock. Animals were housed (4 per cage) in a room with a 12:12-h light-dark cycle at 22°C and were fed regular rodent laboratory chow with water ad libitum. Trained (T) animals were run on a motorized treadmill (22 m/min, 15% grade) 5 days/wk for a total of 4 wk (20 days of training). Training duration was progressively increased from 30 min during the first week to 90 min during the fourth week. Control (C) animals were handled daily and run on the treadmill for 10 min at low intensity (10 m/min, 0% grade).

Preparation of skinned muscle fibers. All experiments were performed 48 h after the last exercise bout. Skinned fibers were prepared according to the method of Veksler et al. (40), with slight modifications (31). Briefly, rats were anesthetized with pentobarbital sodium (5 mg/100 g body wt ip) and treated with heparin (150 IU/100 g body wt iv). The soleus, the medial gastrocnemius, and the heart were removed, placed into precooled solution A (in mM: 1.9 CaK2 EGTA, 8.1 K2EGTA, 9.5 MgCl2, 3.0 KH2PO4, 0.5 dithiothreitol, 20 imidazole, 49 methanesulfonate, 20 taurine, 2.5 ATP, and 15 PCr, pH 7.1, at 22°C), and weighed. The soleus, the deep red, and the superficial white portions of the medial gastrocnemius were quickly freeze-clamped in liquid nitrogen and stored at -80°C for subsequent enzyme analysis. Thin fiber bundles from the contralateral soleus and the red portion of the medial gastrocnemius were cut along fiber orientation (in solution A at 4°C). Muscle fibers were then separated from each other by using needles and incubated with vigorous shaking for 30 min in solution A supplemented with saponin (50 µg/ml). After this permeabilization procedure, fiber bundles were washed three times for 10 min in solution B (in mM: 1.9CaK2 EGTA, 8.1 K2EGTA, 1.4 MgCl2, 3.0 KH2PO4, 0.5 dithiothreitol, 20 imidazole, 100 methanesulfonate, 20 taurine, 5 glutamate, and 2 malate, pH 7.1, at 22°C) supplemented with BSA (2 mg/ml), to completely remove saponin and residual ADP. Fiber bundles were then kept on ice in the same solution until analysis.

Complete removal of residual ADP was evaluated in preliminary experiments (results not shown). In both muscles, low and reproducible rates of respiration were obtained. Furthermore, the basal rate of respiration (V0) could not be inhibited by carboxyatractyloside (35 µM), a potent inhibitor of the adenine nucleotide translocator (ANT). The quality of the fiber preparations, especially the intactness of the outer mitochondrial membrane, was also assessed in preliminary experiments by evaluating the effect of adding cytochrome c (0.01 mM) on the respiration rate of skinned fibers incubated in a high-KCl medium (125 mM) in the presence of 1 mM ADP (34). Addition of exogenous cytochrome c to fiber bundles isolated from the soleus and the red gastrocnemius did not increase maximal respiration rate, suggesting that the outer mitochondrial membrane was intact.

Respirometric investigation of the dependence of oxidative phosphorylation on [ADP] and on functional coupling of MiCK. Respirometric experiments were performed within 4-6 h after fiber preparation. Preliminary experiments have shown that the respiratory parameters measured 1 and 6 h after preparation were not significantly different. Additionally, to avoid any experimental bias due to the possible deterioration of the fiber bundles with time, the tests performed on both muscles were randomized. The rate of VO2 was recorded by using a Clark electrode (Yellow Springs Instruments, Yellow Springs, OH) connected to a data-acquisition system (Datacan V, Sable Systems International, Henderson, NV). Fiber bundles (1.5-2.5 mg dry wt) were incubated at 22°C under continuous stirring in an oxygraphic chamber containing 2 ml of solution B supplemented with BSA (2 mg/ml). The solubility of oxygen at 22°C was considered to be 230 nmol O2/ml. At the end of each test, fibers were carefully removed from the oxygraphic cell, rinsed with distilled water, and evaporated to dryness (24 h, 100°C). Rates of VO2 were expressed in nanomoles of O2 per minute per milligram dry weight.

The kinetics of regulation of respiration by ADP were determined by cumulatively increasing the [ADP] in the incubation medium from 0 to 1 mM (Fig. 1). The ADP-stimulated respiration above basal VO2 (V0) was plotted as a function of [ADP]. The apparent Km for ADP and Vmax was calculated by using the linearization method of Hanes (13). The same kinetic experiments were performed in the presence of Cr (20 mM). The relative decrease in the Km values for ADP due to the presence of Cr was expressed as the ratio Km (+Cr)/Km (-Cr) (3, 10). This ratio, termed CK efficiency, was used to measure the extent of functional coupling of MiCK, located in the mitochondrial intermembrane space, with ANT and the porin channel, located in the inner and outer mitochondrial membranes, respectively. The acceptor control ratio (ACR), defined as (Vmax + V0)/V0, was used to evaluate the coupling of respiration to phosphorylation (34). In all of the experiments, the exact [ADP] in the stock solutions of ADP was determined spectrophotometrically at 259 nm.


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  		Fig. 1.   Representative traces of oxygen consumption (VO2) from the soleus (A) and red gastrocnemius (B). Typical VO2 traces were obtained during the ADP titration experiments performed on the soleus (A) and the deep red portion of the medial gastrocnemius (B) of control rats. For each muscle, kinetics performed in the presence (+Cr) and absence (-Cr) of 20 mM creatine are shown. Respiration was initiated by adding the fiber bundles (F). After the basal rate of respiration (V0) was recorded, ADP was cumulatively added to the chamber to reach the final concentration indicated on the traces (in mM). [O2], oxygen concentration.

Enzyme analysis. Frozen tissue samples were homogenized in 20 volumes of an ice-cold buffer [in mM: 5 HEPES, 1 EGTA, 5 MgCl2, 1 dithiothreitol, and Triton X-100 (0.1%), pH 8.7] by using an Ultra-turrax homogenizer and an ultrasonic cell disrupter. The homogenate was centrifuged (10 min, 14,000 g, 4°C), and the supernatant was used for enzymatic activity measurements.

All enzyme activity measurements were performed spectrophotometrically (Perkin-Elmer Lambda 2, Perkin-Elmer, Shelton, CT) at 37°C. Adenylate kinase (AK) and CK activity were assayed by using the coupled enzyme assay composed of glucose-6-phosphate dehydrogenase and hexokinase (HK) producing NADPH. NADPH production was measured at 340 nm in a buffer containing (in mM) 20 HEPES, 5 MgCl2, 0.5 dithiothreitol, 20 glucose, 1.0 ADP, 0.5 NADP, and 2 IU/ml each of HK and glucose-6-phosphate dehydrogenase, pH 7.4. AK activity was determined by measuring the rate of reaction in the absence of PCr. CK activity was measured by the difference between total activity, measured after adding PCr, and AK activity.

Citrate synthase (CS) activity was measured at 412 nm to detect the transfer of sulfhydryl groups of CoA to DTNB. The assay was performed in a buffer containing (in mM) 50 Tris · HCl, 0.1 DTNB, 0.3 acetyl-CoA, and 0.5 oxaloacetate, pH 8.0. Cytochrome-c oxidase (COX) activity was measured at 550 nm to follow the oxidation of reduced cytochrome c. The assay was performed in a phosphate buffer containing (in mM) 50 mM KH2PO4/K2HPO4 and 0.05 mM cytochrome c reduced with sodium hydrosulfite (Na2S2O4).

The activities of lactate dehydrogenase (LDH) and HK were assayed at 340 nm to follow the consumption of NADH and the production of NADPH, respectively. For LDH, the assay was performed in a buffer containing (in mM) 50 imidazole, 0.15 NADH, and 4 pyruvate, pH 7.5. For HK, the coupled enzyme assay composed of glucose-6-phosphate dehydrogenase was used to follow the production of NADPH. The assay was performed in a buffer containing (in mM) 50 HEPES, 10 MgCl2, 5 dithiothreitol, 100 KCl, 0.5 NADP, 1 ATP, 20 glucose, and 5 U/ml glucose-6-phosphate dehydrogenase. The enzyme activities were expressed in international units per milligram wet weight.

Statistical analysis. All results are expressed as means ± SE. Differences between T and C rats, as well as between muscles, were compared by means of a two-way ANOVA (SPSS Base 10.0 package, SPSS). Newman-Keuls post hoc tests were used to identify the location of significant difference when the ANOVA yielded a significant F ratio. A P value of <0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Morphometric data. The results presented in Table 1 indicate that 20 days of training had no significant morphological impact. Body and heart weight, as well as heart weight-to-body weight ratios, were similar in T and C groups, indicating an absence of training-induced myocardial hypertrophy. No signs of significant muscle hypertrophy were observed, as the weight of the soleus and the gastrocnemius, expressed in absolute term and relative to body weight, was similar in both groups.

                              
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  		Table 1.   Morphometric data of control and trained rats

Enzyme activity. Table 2 shows the activity of selected enzymes, measured in the soleus and the red and white portions of the gastrocnemius. In the soleus, 4 wk of training resulted in significant change in the activities of several enzymes. The activities of mitochondrial enzymes CS and COX were increased by 14 and 34%, respectively. The activities of glycolytic enzymes HK and LDH were also significantly higher in the T group vs. C (32 and 22%, respectively), whereas the activities of CK and AK were unchanged. In contrast to these changes in the soleus, training did not modify the activities of any of the enzymes measured in the red and white portions of the gastrocnemius (except for the AK activity in the white gastrocnemius). As expected, marked differences in the activities of most of the enzymes were observed between the red and white portions of this muscle in both groups.

                              
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  		Table 2.   Activity of selected enzymes in the soleus and gastrocnemius of control and trained rats

Mitochondrial function in skinned fibers. Figure 1 shows typical recordings of VO2 of fiber bundles obtained from the soleus and the red gastrocnemius of C rats. Similar traces were obtained in the muscles from T rats. Initial rates of VO2 in the absence of ADP were low and reproducible (compare traces -Cr and +Cr) in both the soleus and the red gastrocnemius, indicating the complete removal of residual adenylates during the preparation of the fiber bundles. In response to the cumulative addition of ADP in the respiration chamber, a stepwise increase in VO2 up to maximal rates was clearly observed, indicating that mitochondria were well coupled in both muscles.

Representative examples of ADP vs. VO2 kinetics obtained in the soleus of C and T rats are shown in Fig. 2. In every experimental condition, ADP exerted a strong stimulating effect on respiration, and the ADP-stimulated respiration above V0 was well fitted by the Michaelis-Menten equation (Fig. 2, A and C). The Hanes plot of ADP vs. ADP-to-VO2 ratio (ADP/VO2) also showed a good fit of the data to a linear equation (Fig. 2, B and D), which was used to determine the value of Vmax and apparent Km for ADP in each experiment. As expected, the addition of Cr to the incubation media led to a significant decrease in the apparent Km for ADP, due to the functional coupling of MiCK with ANT and the porin channels.


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  		Fig. 2.   Kinetics of ADP-stimulated respiration in the soleus of control and trained rats. Representative ADP concentration ([ADP]) vs. VO2 relationships obtained in single experiments performed on the soleus in -Cr (A) and +Cr (C). Linearization of the data with the Hanes method yielded the [ADP]/VO2 vs. [ADP] plots (B and D), where the slope = 1/Vmax, the y-intercept = Km/Vmax, and the x-intercept = -Km. The detailed analysis of the full set of data is summarized in Fig. 3. dw, Dry weight.

Figure 3 shows the detailed analyses of the full set of data obtained in the soleus. Maximal ADP-stimulated respiration was increased by 21-24% after training (Fig. 3A). The V0 measured in the absence of ADP was ~55% higher in the soleus of T rats. No significant differences between both groups were observed for the ACR, although slightly lower values were obtained in the T group (without Cr: 7.6 ± 0.6; with Cr: 8.4 ± 0.5) compared with the C (without Cr: 8.7 ± 0.8; with Cr: 9.2 ± 0.9). The apparent Km of oxidative phosphorylation for ADP measured in the absence of Cr was in the 100-150 µM range (Fig. 3B), with no significant difference between the C and the T group (149 ± 13 vs. 146 ± 9 µM in C and T, respectively). As expected, the addition of Cr to the incubation media caused a marked decrease in the Km for ADP in both groups. However, the efficiency of Cr in lowering the Km for ADP was unchanged after training, as indicated by comparable ratios of CK efficiency (Fig. 3C).


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  		Fig. 3.   Respiratory parameters of the soleus of control (open bars) and trained (solid bars) rats. A: V0 and maximal ADP-stimulated respiration above basal value (Vmax) measured in -Cr and +Cr. B: apparent Km for ADP measured in -Cr and +Cr. C: relative decrease in the Km values for ADP due to the presence of Cr [creatine kinase (CK) efficiency], expressed as the ratio Km (+Cr)/Km (-Cr). Values are means ± SE for n = 6-8 rats. Measurements were carried in 2 separate fiber bundles from the same muscle in each experiments. Significantly different from control: * P <=  0.05, ** P <=  0.01. dagger  Significantly different from -Cr within the same experimental group: P <=  0.01.

Figure 4 shows the ADP vs. VO2 relationships obtained in the kinetics experiments performed on the red gastrocnemius. As it was observed in the soleus, ADP exerted a strong stimulating effect on respiration, and the ADP-stimulated respiration above V0 was reasonably well fitted by the Michaelis-Menten equation (Fig. 4, A and D). However, in contrast to what was observed in the soleus, the Hanes plot revealed a clear and systematic deviation from linearity in the experiments performed without Cr (Fig. 4, B and C). Indeed, in both the T and the C groups, two distinct linear segments could be identified, with a break point appearing at ~100 µM ADP. Such a phenomenon has previously been attributed to the presence of two functionally different populations of mitochondria within the fiber bundles (21, 34).


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  		Fig. 4.   Kinetics of ADP-stimulated respiration in the red gastrocnemius of control () and trained () rats. Representative [ADP] vs. VO2 relationships obtained in single experiments performed on the deep red portion of the medial gastrocnemius in -Cr (A) and +Cr (D). The corresponding Hanes plots (see Fig. 2 legend for details) for the experiments performed in -Cr and +Cr are shown in panels B-C and E, respectively.

Regression analysis of the two linear segments observed in the kinetics without Cr indicated that, in both experimental groups, one population of mitochondria was characterized by low values of Vmax (3.4 ± 0.2 and 3.0 ± 0.16 nmol O2 · min-1 · mg dry wt-1 in C and T, respectively) and Km for ADP (38 ± 5 and 13 ± 2 µM in C and T, respectively), whereas the other population displayed a comparatively much higher Vmax (7.1 ± 0.4 and 6.6 ± 0.4 nmol O2 · min-1 · mg dry wt-1 in C and T, respectively) and apparent Km for ADP (182 ± 19 and 140 ± 12 µM in C and T, respectively). However, when Cr was present in the incubation medium, the evidence for two mitochondrial populations could no longer be observed (Fig. 4E). This was likely due to the stimulating effect of Cr, which selectively lowered the Km for ADP of the high-Km-high-Vmax population. Indeed, in the presence of Cr, the apparent Km for ADP, computed with the whole set of data points (25-35 µM), was close to that observed in the low-Km-low-Vmax population.

Although the presence of functionally different mitochondrial populations within fiber bundles of the red gastrocnemius was detected, the skinned fiber technique only allowed a rough estimation of the respiratory parameters of each subpopulation of mitochondria, as these were determined from experiments on the mixed mitochondrial population. For this reason, the effect of training was primarily evaluated by using the "average" respiratory parameters for the mixed mitochondrial population, which was computed by using the whole set of data point (Fig. 5).


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  		Fig. 5.   Respiratory parameters of the red gastrocnemius of control (open bars) and trained (solid bars) rats. A: V0 and maximal ADP-stimulated respiration above basal value (Vmax) measured in -Cr and +Cr. B: apparent Km for ADP measured in -Cr and +Cr. C: relative decrease in the Km values for ADP due to the presence of Cr (CK efficiency), expressed as the ratio Km (+Cr)/Km (-Cr). Values are means ± SE for n = 6-8 rats. Measurements were carried in 2 separate fiber bundles from the same muscle in each experiments. ** Significantly different from control, P <=  0.01. dagger  Significantly different from -Cr within the same experimental group, P <=  0.01.

Consistent with the fact that, in rats, the oxidative capacity of fast-twitch red fibers is higher than that of slow-twitch red fibers (1, 18), the values obtained for the average Vmax and V0 were significantly higher in the red gastrocnemius than in the soleus in C rats (Fig. 5A). This difference was less apparent in the T group. Indeed, in contrast to the increase in oxidative capacity observed in the soleus, neither Vmax nor V0 was modified by training in the red gastrocnemius (Fig. 5A), thus reducing the difference in oxidative capacity between both muscles. As for the ACRs, the values obtained in the red gastrocnemius were lower than those observed in the soleus, with no significant difference between the T group (without Cr: 4.7 ± 0.2; with Cr: 4.7 ± 0.3) and the C group (without Cr: 5.8 ± 0.4; with Cr: 5.1 ± 0.2).

The average apparent Km for ADP measured in the absence of Cr was also in the 100 µM range in the red gastrocnemius (Fig. 5B). However, in contrast to what was observed in the soleus, the Km for ADP was significantly 35% lower in the T compared with the C group (83 ± 5 vs. 129 ± 9 µM). This 30-35% difference was also apparent when the Km for ADP was measured in the presence of Cr, although statistical significance was not reached (25 ± 3 vs. 35 ± 5 µM). As a consequence, the CK efficiency was not significantly affected by training (Fig. 5C).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Muscle-specific change in oxidative capacity in response to training. In the present study, the effect of 20 days of treadmill running on the oxidative capacity was found to be different in the soleus and the red portion of the gastrocnemius. In the soleus, training clearly increased the oxidative capacity, as indicated by an increase in the maximal ADP-stimulated respiration rate measured in skinned fibers and by the increase in the activity of CS and COX. In addition, the V0 per unit of muscle mass was increased by training in the absence of significant change in the ACR, suggesting that the mitochondrial fraction was increased. These results are thus in line with previous reports showing that an increase in the oxidative capacity of skeletal muscles can be observed after only 5-14 days of training in both humans (5, 36, 37) and rats (2, 29, 38) and that the half-life of several mitochondrial proteins is in the order of 5-10 days (2, 12).

In contrast to what was observed in the soleus, the training protocol used in the present experiment did not result in an increase in oxidative capacity in the red gastrocnemius, as indicated by the absence of change in the maximal rate of ADP-stimulated respiration and in the activities of the mitochondrial marker enzymes CS and COX. The activities of these enzymes were also unaltered by training in the white gastrocnemius, suggesting that the oxidative capacity of this portion was not increased either.

One likely explanation for this differential effect of training on oxidative capacity is that the exercise intensity selected for the training regimen led to a proportionally lower level of fiber recruitment in the gastrocnemius compared with the soleus. As a consequence, the total amount of contractile activity performed over the 20 days of training, which is a major determinant of mitochondrial biogenesis (12, 18), may have been sufficient to increase oxidative capacity in the soleus but not in the gastrocnemius. Studies on the electromyogram patterns of these ankle extensors during treadmill locomotion support this hypothesis (30). Indeed, at an exercise intensity comparable to what was used in the present study (26.8 m/min, 0% vs. 22 m/min, 15% in the present study), Roy et al. (30) have shown that the activation level of the soleus was near maximal, whereas that of the medial gastrocnemius was proportionately much lower. Furthermore, these authors have shown that, even at twice the slope used in the present study (26.8 m/min and 30%), the integrated electromyogram and integrated electromyogram per minute of the gastrocnemius were still 4.5- to 5-fold lower than those of the soleus (30). This can be expected on the basis that both muscles have markedly different types of motor units, with the former being mostly composed of low-threshold motoneurons with fibers expressing mainly type I myosin heavy chains (MHCs), whereas, in the latter, higher threshold motoneurons and type IIa and IIx MHCs are predominant (8, 9, 30). Therefore, the use of higher intensity exercise might have been necessary to observe an increase in oxidative potential in the gastrocnemius, given the duration of the training program used in the present study.

Apparent affinity of oxidative phosphorylation for ADP and functional coupling of MiCK. Several studies have shown that, in slow oxidative muscles such as the heart and the soleus, the apparent Km for ADP is in the 200-500 µM range, a value that is typically ~10-fold higher than that observed in isolated mitochondria (21, 23, 32, 33). This low sensitivity for ADP observed in skinned fibers is thought to be due to the low permeability of the mitochondrial outer membrane to ADP (21, 23, 32, 33). Indeed, it has been shown that, in skinned fibers submitted to a well-controlled hypoosmotic shock, which results in the disruption of the MOM, the Km for ADP can be decreased to values similar to those observed in isolated mitochondria (23). The low permeability of the MOM for ADP is considered to be due to the maintenance of the porin channel (voltage-dependent anion channel) in its low-conductance state. The conductance of this channel appears to be modulated by the cytosolic oncotic pressure (15, 24), the membrane potential (6), and by a yet unidentified cytoskeletal protein (21, 23, 32, 33).

Another feature of mitochondria in slow-twitch oxidative muscles in vivo is the capacity of Cr to decrease the Km for ADP to 30-100 µM, which is closer to the physiological [ADP] prevailing in the muscle (21, 23, 32, 33). This amplifying effect of Cr on ADP-stimulated respiration is the result of the functional coupling of MiCK, located in the mitochondrial intermembrane space, with ANT and porin, located in the inner and outer membranes, respectively. When Cr is present, this functional coupling allows for an increase in local ADP production at the vicinity of ANT at the expense of mitochondrial ATP, thus stimulating oxidative phosphorylation.

In contrast to what is typically observed in slow-twitch oxidative muscles, very low Km for ADP (12-15 µM) and a negligible effect of Cr have been reported in fast-twitch white muscles such as the extensor digitorum longus and the white gastrocnemius (21, 23, 41). Kuznetsov et al. (23) also reported that this phenomenon was similar in the fast-twitch red and white portions of the gastrocnemius, despite a markedly different fiber-type composition, and oxidative capacities (8, 9).

Consistent with these findings, the Km for ADP reported in the present study was comparatively high in the slow-twitch soleus in the absence of Cr and was decreased by approximately threefold in its presence (Fig. 3). In recent experiments (unpublished observations) performed on the white portion of the medial gastrocnemius, we also observed low-Km values for ADP (<25 µM) and a negligible effect of Cr, as previously reported in fast-twitch glycolytic muscles (21, 23, 41). However, results from the present experiment obtained in the red portion of the gastrocnemius are in sharp contrast with those reported by Kuznetsov et al. (23). Indeed, the average Km for ADP found in this muscle was in the 100-150 µM range, and Cr decreased the Km for ADP by approximately threefold (Fig. 5).

The reasons for the discrepancy between the results of Kuznetsov et al. (23) and the present data remain obscure. However, we believe that it may largely be due to the different range of [ADP] used in the kinetic experiments. In the study by Kuznetsov et al., the maximal [ADP] used in the kinetic experiments was 0.1 mM for both the red and white gastrocnemius (vs. 1 mM in the present study). Whereas in the white gastrocnemius this low range of [ADP] is clearly appropriate to achieve maximal respiratory rate and measure the apparent Km for ADP, results from the present experiment show that this is not the case for the red gastrocnemius. Indeed, in fiber bundles from this muscle, two functionally distinct populations of mitochondria were detected (Fig. 4). One population was characterized by low values of Vmax (3.0-3.4 nmol · min-1 · mg dry wt-1) and Km (13-38 µM) and was apparently not sensitive to Cr. In contrast, the other population displayed high values of Vmax (6.6-7.1 nmol · min-1 · mg dry wt-1) and Km (140-182 µM) and was sensitive to the action of Cr.

Although these values are only estimates of the true kinetic parameters of each population of mitochondria (as they were determined from experiments on the mixed mitochondrial population), they clearly suggest that, in the study by Kuznetsov et al. (23), the use of a low range of [ADP] allowed detection of the low-Km-low-Vmax population but probably failed to reveal the existence of the high-Km-high-Vmax population, which is sensitive to the action of Cr. This would also explain why, in this study, the maximal oxidative capacity of the red gastrocnemius (2.7 ± 0.2 nmol · min-1 · mg dry wt-1) was found to be slightly lower than that of the white gastrocnemius (3.5 ± 0.3 nmol · min-1 · mg dry wt-1) and much lower than that of the soleus (6.1 ± 0.3 nmol · min-1 · mg dry wt-1). This observation is in contradiction to the well-established fact that, in rats, the oxidative capacity of fast-twitch red fibers is up to twofold greater than that of slow-twitch fibers, and four- to eightfold greater than that of fast-twitch white fibers (1, 18). In the present study, these fundamental differences in oxidative capacity were observed. Indeed, the Vmax of the red gastrocnemius was ~40% higher than that of the soleus in the C group (6.69 ± 0.32 vs. 4.71 ± 0.41 nmol · min-1 · mg dry wt-1, P < 0.05) and 140-160% higher than that of the white gastrocnemius (2.8 ± 0.3 nmol · min-1 · mg dry wt-1, P < 0.01; unpublished observations).

Taken together, results from the present experiment thus indicate the presence of two functionally different populations of mitochondria in the red gastrocnemius. In addition, they suggest that, in one of the two populations, diffusion of ADP in the mitochondrial intermembrane space is restricted and that the coupled CK system is effective in stimulating oxidative phosphorylation in those mitochondria. However, the present study does not allow the clear identification of the origin of this mitochondrial heterogeneity. One hypothesis could be that it is the consequence of the heterogeneous fiber-type composition of the red portion of the gastrocnemius [MHC distribution: 30% type I, 20% type IIa, 4% type I-IIa, 40% type IIx, 5% type IIb (8)]. This would be consistent with the fact that dramatic differences in oxidative capacity, sensitivity to ADP, and functional coupling of the CK system are observed between muscles displaying opposite but homogenous fiber-type distributions such as the soleus [85% type I, 15% type IIa (8)] and the white gastrocnemius [95% type IIb, 5% type IIx (8)]. Alternately, it cannot be ruled out that the two functionally different populations represent the subsarcolemmal and intermyofibrillar mitochondria. However, this appears less likely because two mitochondrial populations would also have been observed in the soleus.

Effect of training on mitochondrial sensitivity to ADP and functional coupling of MiCK. Two studies have previously looked at the effect of endurance training on mitochondrial sensitivity to ADP and functional coupling of the CK system (25, 42). In humans, an increase in the Km for ADP (estimated from the ratio of respiration at 0.1 and 1 mM ADP), accompanied by a tendency toward a greater CK efficiency, was recently reported after 6 wk of training in the vastus lateralis (42). In the same muscle, but after a longer training period (4 mo), Nemirovskaia et al. (25) reported a significant increase in the CK efficiency, which suggested a shift toward a more oxidative type of control of respiration, such as that found in the heart.

In the present study, results obtained in the soleus showed no modifications in the Km for ADP and in the functional coupling of MiCK (CK efficiency) after training. These results thus indicate that there was no changes in the intrinsic sensitivity of the mitochondria to these cytosolic regulatory signals in this muscle. In contrast, in the red gastrocnemius, training was associated with a 35% decrease in the Km for ADP without noticeable changes in CK efficiency, suggesting that the diffusional restrictions on ADP in the mitochondrial intermembrane space were decreased by training.

At least two reasons could account for the differences between the present results and those reported by Walsh et al. (42) and Nemirovskaia et al. (25). The first reason is that the training program used in the present study was substantially shorter and of lower intensity. However, no data are presently available on the effect of training intensity and duration on mitochondrial sensitivity to ADP and CK efficiency to ascertain this hypothesis. The second reason is that the present study was performed on rats and on different muscles. It is indeed known that the sensitivity to ADP and the importance of coupled CK in the regulation of respiration differ considerably among species and among various tissues within the same species (23, 41). Therefore, it is possible that training could induce different types of mitochondrial adaptations, depending on both the species and the type of muscle investigated. Muscle-specific adaptations could also at least partly explain why, in contrast to the red gastrocnemius, no change in Km for ADP occurred in the soleus, but yet oxidative capacity was increased. However, this remains speculative.

The mechanisms by which training decreased the apparent Km for ADP in the red gastrocnemius are presently unknown. Studies on skinned muscle fibers (21, 23, 32, 33) indicate that the main factor explaining the high apparent Km for ADP in such preparations is the low permeability of the MOM to ADP, due to the low conductance of the porin channels. One hypothesis could thus be that training increased the conductance state of the porin channels for ADP, thus reducing the diffusion barrier to the intermembrane space. Because, in the red gastrocnemius, two functionally distinct mitochondrial populations were observed, it cannot be excluded that the decrease in the Km for ADP was also due to an increase in the proportion of low-Km-low-Vmax mitochondria. However, we believe that this is unlikely because this would also have resulted in a decrease in Vmax.

Because there is no condition in which PCr and Cr are absent intramuscularly in vivo, the functional implication of a training-induced decrease in the apparent Km for ADP measured in the absence of these guanidino compounds has to be considered. Walsh et al. (43) have recently shown that Cr and PCr were reciprocally modulating the mitochondrial sensitivity to ADP, Cr acting as an amplifier by decreasing the Km for ADP, and PCr acting as a repressor by increasing the Km for ADP. These authors have convincingly showed that, through the coupled CK system, the decrease in the PCr-to-Cr ratio observed during transition from rest to exercise effectively decreases the Km for ADP by up to threefold (from ~300 to 100 µM). In such a system, training can thus increase the intrinsic mitochondrial sensitivity to cytosolic regulatory signals in two ways. The first one is by making the system more sensitive to changes in PCr-to-Cr ratio through an improvement of the functional coupling of the CK system. Evidence for this mechanism has been reported in the studies by Walsh et al. (42) and Nemirovskaia et al. (25). The second one is by lowering the Km for ADP, as reported in the present study. Although the change in the concentration of intramuscular adenylates during transition from rest to exercise was not measured in the present and previous studies (25, 42), these mechanisms could at least partly explain the lesser perturbation of the adenylates and thus the tighter metabolic control system observed in the trained state (7, 11, 18, 27).


    ACKNOWLEDGEMENTS

The authors thank Laurence Kay for expert advice concerning the saponin-skinned fiber technique.


    FOOTNOTES

This work was supported by a Research Grant from Natural Sciences and Engineering Research Council of Canada (NSERC) (to P. W. Hochachka) and an NSERC Post-Doctoral Fellowship (to Y. Burelle).

Address for reprint requests and other correspondence: Y. Burelle, iCAPTURE, MacDonald Research Laboratories, St. Paul's Hospital, Univ. of British Columbia, Rm. 292, 1081 Burrard St., Vancouver, BC, Canada V6Z 1Y6 (E-mail: yburelle{at}mrl.ubc.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published February 8, 2002;10.1152/japplphysiol.01024.2001

Received 11 October 2001; accepted in final form 4 February 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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