J Appl Physiol 92: 2429–2438, 2002.
First published February 8, 2002; 10.1152/japplphysiol.01024.2001.
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
Received 11 October 2001; accepted in final form 4 February 2002
Burelle, Yan, and Peter W. Hochachka. Endurance
training induces muscle-specific changes in mitochondrial
function in skinned muscle fibers. J Appl Physiol 92:
2429–2438, 2002. First published February 8, 2002; 10.1152/
japplphysiol.01024.2001.—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 train-
ing. 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, V̇max in the presence of 1 mM ADP was
increased by 21% after training (5.9 ⫾ 0.2 vs. 4.7 ⫾ 0.4 nmol
O2 䡠 min⫺1 䡠 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 ␮M in T and C, respectively) and in its
presence (50 ⫾ 4 vs. 48 ⫾ 6 ␮M 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 ␮M, P ⬍ 0.01) of
Cr, without affecting V̇max (6.2 ⫾ 0.4 vs. 6.7 ⫾ 0.3 nmol
O2 䡠 min⫺1 䡠 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
ONE OF THE TYPICAL ADAPTATIONS of skeletal muscle me-
tabolism 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 cyto-
solic phosphorylation potential in response to changes
in workload. In addition, this tighter integration of
ATP supply and demand is associated with less stim-
ulation of glycolysis, resulting in a decrease in lactate
production and glucose utilization, a lower cytosolic
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@mrl.ubc.ca).
http://www.jap.org 8750-7587/02 $5.00 Copyright © 2002 the American Physiological Society
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 phenom-
enon is the improvement of muscle oxidative capacity
brought about by an increase in mitochondrial volume
density and in the activity of several enzymes of oxi-
dative metabolism. Indeed, for a given workload and
oxygen consumption (V̇O2) per unit of muscle mass, this
improvement results in a lower V̇O2 per unit of respi-
ratory chain. Therefore, the change in the concentra-
tion of cytosolic factors controlling mitochondrial res-
piration required to elicit the same V̇O2 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, train-
ing 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 phosphor-
ylation. Theories involving a faster increase in tricar-
boxylic 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 unde-
termined, and the effect of training on the anaplerotic
fluxes is unknown (16, 17).
At the mitochondrial level, an increase in the intrin-
sic 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, mito-
chondrial respiration may be controlled by local ADP
concentration ([ADP]) in the mitochondrial intermem-
brane space (21, 32, 33). The [ADP] in this compart-
ment appears to be modulated by the dynamic perme-
ability state of the mitochondrial outer membrane
(MOM) for ADP (15, 24) and also by the functional
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solely to indicate this fact.
2429
2430 TRAINING AND MITOCHONDRIA
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 patho-
physiological 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 mitochon-
drial sensitivity to ADP and/or in the functional cou-
pling 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 so-
leus and the gastrocnemius is different (19, 30) and
that the regulation of mitochondrial respiration is
known to be strikingly different among various mus-
cles, depending on fiber type (20, 21, 23, 41).
METHODS
Animal care and training program. The experiments re-
ported in the present study were approved by the institu-
tional 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 institu-
tional 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 imid-
azole, 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 ␮g/ml). After this permeabilization procedure, fiber bun-
dles were washed three times for 10 min in solution B (in
J Appl Physiol • VOL
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) supple-
mented 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 pre-
liminary experiments (results not shown). In both muscles,
low and reproducible rates of respiration were obtained.
Furthermore, the basal rate of respiration (V̇0) could not be
inhibited by carboxyatractyloside (35 ␮M), 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 bun-
dles 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. Addition-
ally, to avoid any experimental bias due to the possible
deterioration of the fiber bundles with time, the tests per-
formed on both muscles were randomized. The rate of V̇O2
was recorded by using a Clark electrode (Yellow Springs
Instruments, Yellow Springs, OH) connected to a data-acqui-
sition system (Datacan V, Sable Systems International, Hen-
derson, NV). Fiber bundles (1.5–2.5 mg dry wt) were incu-
bated 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 consid-
ered to be 230 nmol O2/ml. At the end of each test, fibers were
carefully removed from the oxygraphic cell, rinsed with dis-
tilled water, and evaporated to dryness (24 h, 100°C). Rates
of V̇O2 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 V̇O2 (V̇0) was plotted as a
function of [ADP]. The apparent Km for ADP and V̇max was
calculated by using the linearization method of Hanes (13).
The same kinetic experiments were performed in the pres-
ence 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 (V̇max ⫹ V̇0)/V̇0, 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.
Enzyme analysis. Frozen tissue samples were homoge-
nized 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,
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 TRAINING AND MITOCHONDRIA 2431

Fig. 1. Representative traces of oxygen consumption (V̇O2) from the soleus (A) and red gastrocnemius (B). Typical
V̇O2 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 (V̇0) was recorded, ADP was cumulatively added to the chamber to reach the final
concentration indicated on the traces (in mM). [O2], oxygen concentration.

14,000 g, 4°C), and the supernatant was used for enzymatic
activity measurements.
All enzyme activity measurements were performed spec-
trophotometrically (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 reac-
tion 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 䡠 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 de-
hydrogenase 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 dehydroge-
nase. 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 differ-
ence when the ANOVA yielded a significant F ratio. A P
value of ⬍0.05 was considered significant.
RESULTS
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 signifi-
cant muscle hypertrophy were observed, as the weight

Table 1. Morphometric data of control and trained rats

Soleus, Gastrocnemius, Soleus/BW, Gastrocnemius/BW, Heart/BW,
n BW, g mg mg Heart, g mg/g mg/g mg/g

Control 6 257 ⫾ 5 147 ⫾ 8 751 ⫾ 35 1.03 ⫾ 0.04 0.57 ⫾ 0.03 2.91 ⫾ 0.13 4.00 ⫾ 0.12
Trained 8 253 ⫾ 6 158 ⫾ 6 767 ⫾ 34 1.02 ⫾ 0.03 0.62 ⫾ 0.02 3.03 ⫾ 0.11 4.05 ⫾ 0.11
Values are means ⫾ SE; n, no. of rats. BW, body weight; soleus/BW, Gastrocnemius/BW, Heart/BW: ratio of soleus, gastrocnemius, and
heart, respectively, to BW.

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2432 TRAINING AND MITOCHONDRIA

of the soleus and the gastrocnemius, expressed in ab-
solute term and relative to body weight, was similar in
both groups.
Enzyme activity. Table 2 shows the activity of se-
lected 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 mito-
chondrial enzymes CS and COX were increased by 14
and 34%, respectively. The activities of glycolytic en-
zymes 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 con-
trast 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 (ex-
cept 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.
Mitochondrial function in skinned fibers. Figure 1
shows typical recordings of V̇O2 of fiber bundles ob-
tained from the soleus and the red gastrocnemius of C
rats. Similar traces were obtained in the muscles from
T rats. Initial rates of V̇O2 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 cham-
ber, a stepwise increase in V̇O2 up to maximal rates
was clearly observed, indicating that mitochondria
were well coupled in both muscles.
Representative examples of ADP vs. V̇O2 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 V̇0 was well fitted by the
Michaelis-Menten equation (Fig. 2, A and C). The
Hanes plot of ADP vs. ADP-to-V̇O2 ratio (ADP/V̇O2) 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
V̇max 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.
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 V̇0 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 phosphoryla-
tion for ADP measured in the absence of Cr was in the
100–150 ␮M range (Fig. 3B), with no significant dif-
ference between the C and the T group (149 ⫾ 13 vs.
146 ⫾ 9 ␮M 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).
Figure 4 shows the ADP vs. V̇O2 relationships ob-
tained 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 V̇0 was rea-
sonably 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 experi-
ments 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 ap-
pearing at ⬃100 ␮M ADP. Such a phenomenon has
previously been attributed to the presence of two func-
tionally different populations of mitochondria within
the fiber bundles (21, 34).
Regression analysis of the two linear segments ob-
served in the kinetics without Cr indicated that, in
both experimental groups, one population of mitochon-
dria was characterized by low values of V̇max (3.4 ⫾ 0.2
and 3.0 ⫾ 0.16 nmol O2 䡠 min⫺1 䡠 mg dry wt⫺1 in C and T,
respectively) and Km for ADP (38 ⫾ 5 and 13 ⫾ 2 ␮M in
C and T, respectively), whereas the other population
displayed a comparatively much higher V̇max (7.1 ⫾ 0.4

Table 2. Activity of selected enzymes in the soleus and gastrocnemius of control and trained rats
Soleus Red Gastrocnemius White Gastrocnemius

Control Trained Control Trained Control Trained

CS 30.3 ⫾ 1.1 34.4 ⫾ 1.4* 38.7 ⫾ 3.1 39.1 ⫾ 2.4 17.0 ⫾ 1.9‡ 16.5 ⫾ 0.9‡
COX 91.4 ⫾ 5.9 122.4 ⫾ 8.6† 116.2 ⫾ 7.9 123.5 ⫾ 8.3 41.0 ⫾ 6.3‡ 39.1 ⫾ 4.6‡
HK 6.16 ⫾ 0.51 8.15 ⫾ 0.40† 6.17 ⫾ 0.76 6.42 ⫾ 0.62 2.49 ⫾ 0.22‡ 2.59 ⫾ 0.13‡
LDH 315 ⫾ 21 385 ⫾ 17* 820 ⫾ 44 826 ⫾ 45 1,234 ⫾ 36‡ 1,230 ⫾ 42‡
CK 1,786 ⫾ 153 1,829 ⫾ 187 3,811 ⫾ 365 3,848 ⫾ 364 7,046 ⫾ 337‡ 6,670 ⫾ 482‡
AK 281 ⫾ 6.8 278 ⫾ 45 682 ⫾ 66 720 ⫾ 64 704 ⫾ 25 996 ⫾ 92
Values are means ⫾ SE in IU/g wet wt; n ⫽ 6–8 rats in soleus and red gastrocnemius groups and n ⫽ 4–5 rats in white gastrocnemius
group. CS, citrate synthase; COX, cytochrome-c oxidase; HK, hexokinase; LDH, lactate dehydrogenase; CK, creatine kinase; AK, adenylate
kinase. All measurements were carried out in duplicate in each muscle homogenate. Significantly different from control: * P ⬍ 0.05; † P ⬍
0.01. Significantly different from red gastrocnemius within the same experimental group: ‡ P ⬍ 0.01.

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 TRAINING AND MITOCHONDRIA 2433

Fig. 2. Kinetics of ADP-stimulated respi-

ration in the soleus of control and trained
rats. Representative ADP concentration
([ADP]) vs. V̇O2 relationships obtained in
single experiments performed on the so-
leus in ⫺Cr (A) and ⫹Cr (C). Linearization
of the data with the Hanes method yielded
the [ADP]/V̇O2 vs. [ADP] plots (B and D),
where the slope ⫽ 1/V̇max, the y-inter-
cept ⫽ Km/V̇max, and the x-intercept ⫽
⫺Km. The detailed analysis of the full set
of data is summarized in Fig. 3. dw, Dry
weight.

and 6.6 ⫾ 0.4 nmol O2 䡠 min⫺1 䡠 mg dry wt⫺1 in C and T,
respectively) and apparent Km for ADP (182 ⫾ 19 and
140 ⫾ 12 ␮M 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 stim-
ulating effect of Cr, which selectively lowered the Km
for ADP of the high-Km-high-V̇max population. In-
deed, in the presence of Cr, the apparent Km for ADP,
computed with the whole set of data points (25–35

Fig. 3. Respiratory parameters of the soleus of control (open bars) and trained (solid bars) rats. A: V̇0 and maximal
ADP-stimulated respiration above basal value (V̇max) 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 ⱕ 0.05, **P ⱕ 0.01. †Significantly different from ⫺Cr within the same experimental group: P ⱕ 0.01.

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2434 TRAINING AND MITOCHONDRIA

Fig. 4. Kinetics of ADP-stimulated respiration in the red gastrocnemius of control (䊐) and trained (F) rats.
Representative [ADP] vs. V̇O2 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.

␮M), was close to that observed in the low-Km-low-
V̇max population.
Although the presence of functionally different mito-
chondrial populations within fiber bundles of the red
gastrocnemius was detected, the skinned fiber tech-
nique only allowed a rough estimation of the respira-
tory parameters of each subpopulation of mitochon-
dria, 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 us-
ing the whole set of data point (Fig. 5).
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 V̇max and V̇0 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 V̇max nor V̇0 was modi-
fied by training in the red gastrocnemius (Fig. 5A),
thus reducing the difference in oxidative capacity be-
tween both muscles. As for the ACRs, the values ob-
tained 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:
J Appl Physiol • VOL
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 ␮M range in the red
gastrocnemius (Fig. 5B). However, in contrast to what
was observed in the soleus, the Km for ADP was signif-
icantly 35% lower in the T compared with the C group
(83 ⫾ 5 vs. 129 ⫾ 9 ␮M). 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 ␮M). As a conse-
quence, the CK efficiency was not significantly affected
by training (Fig. 5C).
DISCUSSION
Muscle-specific change in oxidative capacity in re-
sponse 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 V̇0 per unit
of muscle mass was increased by training in the ab-
sence of significant change in the ACR, suggesting that
92 • JUNE 2002 • www.jap.org
 TRAINING AND MITOCHONDRIA 2435

Fig. 5. Respiratory parameters of the red gastrocnemius of control (open bars) and trained (solid bars) rats. A: V̇0
and maximal ADP-stimulated respiration above basal value (V̇max) 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 ⱕ 0.01. † Significantly different from ⫺Cr within the same experimental group, P ⱕ 0.01.

the mitochondrial fraction was increased. These re-
sults 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 train-
ing 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 respi-
ration 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 gastroc-
nemius, 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 con-
sequence, 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 electromyo-
gram and integrated electromyogram per minute of
J Appl Physiol • VOL
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 predom-
inant (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 ␮M 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 perme-
ability 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 hypoos-
motic 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 consid-
ered to be due to the maintenance of the porin channel
(voltage-dependent anion channel) in its low-conduc-
tance 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 oxi-
dative muscles in vivo is the capacity of Cr to decrease
the Km for ADP to 30–100 ␮M, which is closer to the
physiological [ADP] prevailing in the muscle (21, 23,
32, 33). This amplifying effect of Cr on ADP-stimulated
92 • JUNE 2002 • www.jap.org
2436 TRAINING AND MITOCHONDRIA
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 phos-
phorylation.
In contrast to what is typically observed in slow-
twitch oxidative muscles, very low Km for ADP (12–
15 ␮M) and a negligible effect of Cr have been
reported in fast-twitch white muscles such as the
extensor digitorum longus and the white gastrocne-
mius (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 ox-
idative capacities (8, 9).
Consistent with these findings, the Km for ADP re-
ported 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 observa-
tions) performed on the white portion of the medial
gastrocnemius, we also observed low-Km values for
ADP (⬍25 ␮M) and a negligible effect of Cr, as previ-
ously 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 ␮M range, and Cr de-
creased 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 appro-
priate to achieve maximal respiratory rate and mea-
sure 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 mus-
cle, two functionally distinct populations of mitochon-
dria were detected (Fig. 4). One population was char-
acterized by low values of V̇max (3.0–3.4 nmol 䡠
min⫺1 䡠 mg dry wt⫺1) and Km (13–38 ␮M) and was
apparently not sensitive to Cr. In contrast, the other
population displayed high values of V̇max (6.6–7.1
nmol 䡠 min⫺1 䡠 mg dry wt⫺1) and Km (140–182 ␮M) 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-
J Appl Physiol • VOL
low-V̇max population but probably failed to reveal the
existence of the high-Km-high-V̇max 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 䡠 min⫺1 䡠 mg dry
wt⫺1) was found to be slightly lower than that of the
white gastrocnemius (3.5 ⫾ 0.3 nmol 䡠 min⫺1 䡠 mg dry
wt⫺1) and much lower than that of the soleus (6.1 ⫾ 0.3
nmol 䡠 min⫺1 䡠 mg dry wt⫺1). This observation is in con-
tradiction 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. In-
deed, the V̇max 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 䡠 min⫺1 䡠 mg dry wt⫺1, P ⬍
0.05) and 140–160% higher than that of the white
gastrocnemius (2.8 ⫾ 0.3 nmol 䡠 min⫺1 䡠 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 popu-
lations, diffusion of ADP in the mitochondrial inter-
membrane space is restricted and that the coupled CK
system is effective in stimulating oxidative phosphor-
ylation in those mitochondria. However, the present
study does not allow the clear identification of the
origin of this mitochondrial heterogeneity. One hypoth-
esis could be that it is the consequence of the hetero-
geneous 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 popula-
tions represent the subsarcolemmal and intermyofi-
brillar 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 cou-
pling of the CK system (25, 42). In humans, an increase
in the Km for ADP (estimated from the ratio of respi-
ration at 0.1 and 1 mM ADP), accompanied by a ten-
dency 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 sug-
gested a shift toward a more oxidative type of control of
respiration, such as that found in the heart.
92 • JUNE 2002 • www.jap.org
 TRAINING AND MITOCHONDRIA
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 train-
ing. 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 asso-
ciated with a 35% decrease in the Km for ADP without
noticeable changes in CK efficiency, suggesting that
the diffusional restrictions on ADP in the mitochon-
drial 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 respira-
tion differ considerably among species and among var-
ious tissues within the same species (23, 41). There-
fore, it is possible that training could induce different
types of mitochondrial adaptations, depending on both
the species and the type of muscle investigated. Mus-
cle-specific adaptations could also at least partly ex-
plain 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 re-
mains 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-
V̇max mitochondria. However, we believe that this is
unlikely because this would also have resulted in a
decrease in V̇max.
Because there is no condition in which PCr and Cr
are absent intramuscularly in vivo, the functional im-
plication of a training-induced decrease in the appar-
ent 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 de-
J Appl Physiol • VOL
2437
crease in the PCr-to-Cr ratio observed during transi-
tion from rest to exercise effectively decreases the Km
for ADP by up to threefold (from ⬃300 to 100 ␮M). In
such a system, training can thus increase the intrinsic
mitochondrial sensitivity to cytosolic regulatory sig-
nals 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 re-
ported in the studies by Walsh et al. (42) and Nemi-
rovskaia 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 ade-
nylates 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).
The authors thank Laurence Kay for expert advice concerning the
saponin-skinned fiber technique.
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).
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