(Received for publication, May 9, 1995)
From the
Functional properties of in situ mitochondria and of
mitochondrial creatine kinase were studied in saponin-skinned fibers
taken from normal and M-creatine kinase-deficient mice. In control
animals, apparent K values of
mitochondrial respiration for ADP in cardiac (ventricular) and
slow-twitch (soleus) muscles (137 ± 16 µM and 209
± 10 µM, respectively) were manyfold higher than
that in fast-twitch (gastrocnemius) muscle (7.5 ± 0.5
µM). Creatine substantially decreased the K
values only in cardiac and slow-twitch
muscles (73 ± 11 µM and 131 ± 21
µM, respectively). As compared to control, in situ mitochondria in transgenic ventricular and slow-twitch muscles
showed two times lower K
values for ADP,
and the presence of creatine only slightly decreased the K
values. In mutant fast-twitch muscle, a
decrease rather than increase in mitochondrial sensitivity to ADP
occurred, but creatine still had no effect. Furthermore, in these
muscles, relatively low oxidative capacity was considerably elevated.
It is suggested that in the mutant mice, impairment of energy transport
function in ventricular and slow-twitch muscles is compensated by a
facilitation of adenine nucleotide transportation between mitochondria
and cellular ATPases; in fast-twitch muscle, mainly energy buffering
function is depressed, and that is overcome by an increase in
energy-producing potential.
Intracellular integration of muscle contractile activity and
energy metabolism is one of the most intriguing problems in
bioenergetics. Under physiological conditions, functional requirements
of muscle and cellular energy supply are well coordinated, but
mechanisms for maintaining this balance are still ill-defined. A number
of works indicated a very important role of creatine kinase (CK) ()in cellular energetics of muscle (for reviews see Jacobus
(1985a), Wallimann et al.(1992), and Saks et
al.(1994)). This enzyme catalyzes the reversible transfer of the
phosphoryl group between phosphocreatine (PCr) and ATP in the reaction:
MgADP
+ PCr
+
H
MgATP
+ creatine.
Muscle cells express three subunit isoforms of CK: M-, B-, and the mitochondrial one. M- and B-subunits form dimeric MM, MB, and BB isoenzymes. CK isoforms are subcellularly compartmentalized; part of them are soluble, whereas a significant amount of MM-CK is bound to intracellular structures, close to the sites of energy utilization where cellular ATPases reside. MM-isoform has been found to be attached to the myofibrillar M-band (Turner et al., 1973; Wallimann et al., 1977), to the sarcoplasmic reticulum (Baskin and Deamer, 1970; Levitsky et al., 1978), and to the sarcolemma (Sharov et al., 1977; Jockers-Wretou et al., 1977).
CK isoenzymes are localized also at the sites of energy production. Part of cytosolic MM-CK, predominantly in skeletal muscle, is associated with glycolytic enzymes. The second point of interaction of CK with ATP generation is at the outer surface of the inner mitochondrial membrane, where mitochondrial CK (mi-CK) isoenzyme is situated close to the adenine nucleotide translocase.
The distribution of CK isoenzymes in muscles depends on the type of metabolism. In cardiac and slow-twitch skeletal muscle with a permanent high level of energy production and utilization, there is an elevated relative proportion of mitochondrial CK (for review see Wyss et al.(1992)) and a relative low proportion of cytosolic subunits (Yamashita and Yoshioka, 1991). Fast-twitch skeletal muscles, having their metabolism based mainly on glycolytic activity, have a low proportion of mitochondrial isoform and a high proportion of cytosolic CK. These differences in CK isoform distribution have given rise to hypothesis of different roles of CK system in ``oxidative'' and ``glycolytic'' tissues.
According to this hypothesis, in oxidative tissues, the major role of CK operating with PCr is to provide the energy transport system. Mi-CK is functionally coupled to the adenine nucleotide translocase so that ATP generated by oxidative phosphorylation, after transport through the inner mitochondrial membrane, is transphosphorylated to PCr. This takes place at the expense of creatine diffusing from the sites of PCr utilization, i.e. sites where cytosolic CK is localized. Therefore, creatine and PCr are considered to be the main molecular species participating in the energy turnover between mitochondria and cellular ATPases.
High activity of mi-CK in the oxidative tissues ensures a
rapid phosphorylation of creatine in the mitochondrial compartment.
This is thought to keep local ADP concentration high in the vicinity of
adenine nucleotide translocase and, in such a way, to decrease the
apparent K for ADP. Obviously, such a
property of mi-CK seems to be very important because, as has been shown
recently (Saks et al., 1991, 1993), in ventricular muscle
(oxidative tissue) the K
of mitochondrial
respiration for ADP in situ is quite high, being considerably
higher than calculated values for free cytoplasmic [ADP].
In contrast, in glycolytic muscles, CK is supposed to provide mostly the ``energy buffering'' function. High cytosolic activity of CK localized near the sites of glycolytic production of ATP and, perhaps, functionally coupled to glycolysis, serves as a temporal buffer for high energy phosphates. This keeps ATP and ADP concentrations steady during short periods of elevated muscular contractile activity, i.e. during increased ATP splitting. Furthermore, under these conditions, CK reaction consumes protons so that ATP hydrolysis and the activation of glycolysis in working skeletal muscle are not followed by an intracellular acidification.
Inhibition of various sites in the intracellular CK system would
give much information about the role of this system in different
tissues. Unfortunately, so far there is no specific inhibitor of CK
that could be applied in living animals or even in isolated tissue
preparation in vitro. Another approach for studying the CK
system is to use animals fed by a creatine analog
-guanidinopropionic acid (
-GPA) (Fitch et al., 1974)
which acts as a competitive inhibitor of trans-sarcolemmal
creatine transport (Fitch et al., 1974; Chevli and Fitch,
1979). However, findings with this model of substrate deficiency should
be interpreted with caution. Most importantly, in spite of a
considerable decrease in total creatine content in muscles of
-GPA-fed rats, PCr levels are still in the K
range of CK and sufficient to support high energy fluxes
from mitochondria to myofibrils (Jacobus, 1985b).
Very recently, mice with a homozygous null mutation for the gene-encoding M-subunit of CK were created (van Deursen and Wieringa, 1992; van Deursen et al., 1993). Being completely deprived of MM- and MB-isoforms of CK, muscles of these animals express normal levels of mi-CK and have normal concentrations of free ATP, PCr, and inorganic phosphate. One can suggest that certain mechanisms of compensation for the lack of M-CK are involved leading to rearrangements of cellular energy pathways. If CK plays different roles in oxidative and glycolytic muscles, these mechanisms should be tissue-specific. Therefore, we decided to investigate the intrinsic properties of mitochondria (this study) and myofibrils (Ventura-Clapier et al., 1995) in situ in skinned fibers taken from various muscles as well as the functional ability of bound CK of these mice. The data described in the companion paper (Ventura-Clapier et al., 1995) have demonstrated that no fundamental remodelling occurs in myofibrils. The results obtained in the present study show that oxidative and glycolytic muscles have different patterns of regulation of mitochondrial respiration by ADP and different types of adaptation to the M-CK deficiency, thus giving support to the hypothesis of different roles of the CK system in these muscles.
Six control C57BL/6 and 5 transgenic
mice were weighed and anesthetized with an intraperitoneal injection of
ethyl carbamate according to the recommendations of the Institutional
Animal Care Committee (INSERM, Paris, France). Heart, soleus
(predominantly slow-twitch), gastrocnemius (predominantly fast-twitch)
muscles, brain, lung, kidneys, and liver were isolated, weighed, and
placed in a modified Krebs solution containing (mM): NaCl,
118; KCl, 4.7; NaHCO, 25; KH
PO
,
1.2; and MgSO
, 1.2.
CK activity was determined using the coupled enzyme
assay of glucose-6-phosphate dehydrogenase and hexokinase producing
NADPH. NADPH production was measured spectrophotometrically at 340 nm
(Gilford Spectrophotometer, Corning, NY). The CK activity was assayed
in a solution containing (mM): HEPES, 30; MgCl, 5;
dithiothreitol, 0.5; ADP, 1.2; PCr, 20; glucose, 20; NADP, 0.6; P
,P
-di(adenosine-5`)
pentaphosphate (to inhibit myokinase), 0.01; and 2 IU/ml
glucose-6-phosphate dehydrogenase and hexokinase at a pH of 7.1 and 30
°C. Determination of citrate synthase activity was performed
according to Srere(1969). Each determination was carried out in
duplicate.
Respiratory rates were determined by a Clark electrode (Yellow Spring Instruments) in an oxygraphic cell containing 10-15 fiber bundles in 3 ml of solution R at 22 °C with continuous stirring. The solubility of oxygen was taken to be 460 ng atoms of O/ml. After measurement, the bundles were removed and dried. Respiration rates were expressed as ng atoms of O/min/mg dry weight.
Solutions S and R contained 10 mM EGTA-CaEGTA buffer (free Ca concentration, 100
nM), 3 mM free Mg
, 20 mM taurine, 0.5 mM dithiothreitol, and 20 mM imidazole. Ionic strength was adjusted to 0.16 M by
addition of potassium methanesulfonate. Solution S (pH 7.0) also
contained 5 mM MgATP and 15 mM PCr. Solution R (pH
7.1) contained 5 mM glutamate, 2 mM malate, 3 mM phosphate, and 2 mg/ml fatty acid free bovine serum albumin,
instead of high energy phosphates.
Functional activity of mi-CK in skinned fibers was assessed by enhancement in the rate of oxygen consumption after creatine addition in the presence of submaximal ADP concentrations (Veksler et al., 1987). Myokinase functional activity was evaluated by an increase in the respiration rate after addition of 1 mM AMP in the presence of 0.2 mM ATP.
To obtain ADP kinetic parameters, muscle fibers were exposed to
increasing [ADP] in the presence or in the absence of
creatine (20 mM). The ADP-stimulated respiration above basal
oxygen consumption was plotted in order to determine the apparent K for ADP and V
.
Table 2also shows CK isoenzyme distribution in various tissues. Agarose electrophoresis followed by staining for CK activity demonstrated that ventricular and skeletal muscles (as well as brain tissue) of mutant animals were completely devoid of isoenzymes containing the M-CK subunit. M-CK activity in brain of control mice was also below the threshold of detection. Very low levels of MB- and BB-isoenzymes in control muscular tissues prevented their precise quantification. However, mutant murine muscles in some cases showed the clear appearance of BB-CK activity so that it was possible to estimate the percent representation of this isoenzyme.
There were no significant differences between control and transgenic tissues in mi-CK-specific activity. Thus, M-CK deficiency was followed by neither a compensatory expression of mitochondrial isoenzyme nor a significant appearance of B-CK.
Figure 1: Oxygraph traces of respiratory activities of mitochondria in saponin-skinned ventricular fibers taken from control and mutant mice. The arrows indicate time of additions of fibers (the quantities are expressed in mg of dry weight), ADP, and creatine. Note a weaker augmentation of the respiration rate after creatine addition in the transgenic preparation.
Fig. 2shows the averaged values of the index of CK functional coupling in two oxidative tissues. In both ventricular and soleus muscles of wild-type mice, at 100 µM ADP, creatine induced a great stimulation in the oxygen consumption rate. In gastrocnemius muscle, the stimulation of respiration by creatine was negligible (not shown). Transgenic oxidative muscles demonstrated a significantly lower extent of respiratory rate enhancement by creatine, thus indicating an alteration in the relationship between supply of mitochondria with ADP by mi-CK and by diffusion from the extramitochondrial space.
Figure 2: Functional activity of mi-CK and myokinase expressed as percentage of the respiration rate increase after addition of creatine or AMP respectively. *, p < 0.05;**, p < 0.01;***, p < 0.001 versus respective controls.
The experimental protocol described in Fig. 1allowed us also to evaluate the absolute values of mitochondrial respiration in a given tissue at high [ADP] (1 mM) in the presence of creatine. In ventricular muscle of mutant mice, this value (81.3 ± 4.3 ng atoms/min/mg dry weight, n = 5) was found to be significantly (p < 0.01) higher than in control muscle (60.6 ± 2.3 ng atoms/min/mg dry weight, n = 5). In contrast, respiration rates at high [ADP] in soleus muscle, being lower than in ventricular tissue, were similar in M-CK-deficient (35.1 ± 4.2 ng atoms/min/mg dry weight, n = 4) and wild-type (34.5 ± 3.6 ng atoms/min/mg dry weight, n = 4) preparations.
We have also tested the ability of myokinase compartmentalized in saponin-skinned fibers to produce ADP for mitochondrial respiration in oxidative (ventricle) and glycolytic (gastrocnemius) muscles. (Unfortunately, myokinase functional activity was not measured in soleus muscle because not enough tissue was available). Addition of 0.2 mM ATP to the medium without adenine nucleotides induced a marked rate of oxygen consumption by skinned fibers due to ADP generated by various cellular ATPases. Further addition of 1 mM AMP leads to a production of ADP in the myokinase reaction and stimulation of the mitochondrial respiration. The degree of this stimulation was the same in control and transgenic ventricles (Fig. 2). In control gastrocnemius muscle, AMP addition only slightly increased the oxygen consumption rate, the effect being approximately 5 times smaller than in ventricular tissue. However, in transgenic gastrocnemius muscle, the percentage of AMP-stimulated respiration was significantly higher than in control, being close to the values observed in ventricular muscle.
To check this possibility, we studied the dependence of the mitochondrial respiration rate in saponin-skinned fibers on ADP concentration. Although the fibers possessed high ATPase activity, to keep [ADP] constant during the measurement of respiration rate at any given [ADP] in the medium, in some experiments we added an ADP-regenerating system: 20 mM glucose and 5 IU/ml hexokinase. The results were similar in the presence and in the absence of this system.
Fig. 3a shows the dependence of respiration on ADP
concentration in representative control ventricular fibers in the
presence and in the absence of creatine. The preparations were taken
from the same animal. The dependence is very well fitted by the
Michaelis-Menten equation. From this figure and from the
double-reciprocal plot of the data (Fig. 3b), one can
see that creatine substantially decreased the apparent K for ADP and, at the same time,
increased the maximal respiration rate. These effects reflect the
effective coupling between mi-CK and oxidative phosphorylation. The
difference between the [ADP]/respiration rate curves explains
the stimulation of the oxygen consumption rate by creatine at
submaximal ADP concentrations.
Figure 3:
O consumption rates of skinned
ventricular fibers plotted as a function of ADP concentration.
Respiration of fibers taken from control (a and b)
and mutant (c and d) mice was measured in the absence (squares, solid lines) or in the presence (circles, broken lines) of 20 mM creatine.
Experimental data were fitted by a Michaelis-Menten equation (a and c); b and d, double reciprocal
plots of 1/O
consumption rates versus 1/[ADP] (data were derived from a and c, respectively).
As Fig. 3, c and d, shows, in transgenic ventricular muscle preparations (also
taken from the same animal) the dependence of respiration rate on
[ADP] in the absence of creatine was greatly altered.
However, in the presence of creatine, the curves were very similar; as
a result, apparent K values for ADP in
the presence and in the absence of creatine are much closer than in the
case of control.
Averaged values of mitochondrial kinetics are
presented in Table 3. In control ventricular muscle, K for ADP in the absence of creatine was
found to be 137 ± 16 µM, a value much higher than
those reported for isolated mitochondria (Chance and Williams, 1955;
Bygrave and Lehninger, 1967).
Addition of creatine switches on
coupled mi-CK, resulting in a more than a two times decrease of
apparent K in control ventricular muscle (Table 3). Calculated V
of oxygen
consumption in this muscle was higher in the presence of creatine,
probably due to an accelerated production of ADP at the expense of ATP
by mi-CK in the vicinity of adenine nucleotide translocase.
Mitochondria in transgenic ventricular tissue showed altered
sensitivity to ADP. In the absence of creatine, Kof oxidative phosphorylation for ADP
was about two times lower than that in control. This means that in
mutant myocardium, access of extramitochondrial ADP to adenine
nucleotide translocase and, possibly, export of ATP from the
mitochondrial compartment were facilitated. This could contribute to
the significantly higher maximal respiration rate observed in the
absence of creatine, in transgenic ventricular fibers, as compared to
control fibers. Creatine addition only slightly decreased the apparent K
. Interestingly, in the presence of
creatine, neither K
for ADP nor V
was different from control values in the same
conditions.
These data clearly show that in M-CK-deficient
ventricular muscle, the properties of the mitochondrial compartment are
essentially changed so that the importance of coupled mi-CK for
facilitating the ADP/ATP exchange between the mitochondrial and
cytosolic compartments is diminished. The adapted mitochondria
themselves or, more precisely, contact structures between the inner
mitochondrial membrane and cytosol appear to be able to facilitate
adenine nucleotide transportation. Qualitatively, the same data were
obtained for another oxidative muscle, soleus (Table 3). In the
absence of creatine, skinned fibers from wild-type mice showed quite
high K values for ADP, even higher than
in ventricular muscle (209 ± 10 µM). Mi-CK was well
coupled to oxidative phosphorylation because creatine significantly
decreased the K
for ADP. However, in
transgenic soleus muscle, the K
was
already significantly lower in the absence of creatine, like in
ventricular muscle. Thus, these results demonstrate that two types of
oxidative muscles have similar low mitochondrial sensitivity to ADP in
control, and the M-CK deficiency seems to be followed by similar
adaptive changes with regard to K
.
In
transgenic soleus muscle, in contrast to ventricular muscle, there was
no increase in the maximal oxygen consumption rate in the absence of
creatine, as compared to control. In general, this muscle was
characterized by lower oxidative capacity, so that the V was about 50% of the respective value for
ventricular tissue.
Properties of total mitochondrial population in
glycolytic, predominantly fast-twitch white muscle (gastrocnemius) were
completely different from those in oxidative tissues (Table 3).
In control muscle, mitochondrial sensitivity to ADP was extremely high,
the K for ADP being as low as 7.5
± 0.5 µM, that is
20-25 times smaller
than in oxidative muscles. Despite the presence of mi-CK, creatine did
not change kinetic parameters of respiration, thus indicating either
the absence of coupling between mi-CK and oxidative phosphorylation or
the absence of a role for this coupling in regulation of oxidative
phosphorylation in fast-twitch muscle. As expected, oxidative capacity
of this muscle estimated by the maximal oxygen consumption rate was
considerably lower than in oxidative muscles.
In contrast to
ventricular and soleus muscles, in gastrocnemius muscle, M-CK
deficiency induced a decrease rather than an increase in mitochondrial
sensitivity to ADP. The apparent Kvalues
for ADP both in the absence and in the presence of creatine were
approximately 2.5 times higher than those for control tissue. At the
same time, oxidative capacity of the fast-twitch transgenic muscle was
significantly increased (Table 3).
Obviously, the increase in K for ADP as well as the rise in the
maximal rate of oxygen consumption could be the result of either
uniform changes in properties of total mitochondrial population or the
appearance of a new population of mitochondria in the muscle. In the
latter case, the total mitochondrial population would show a
heterogeneity in their properties. Analysis of the dependence of
respiration rate on ADP concentration gives evidences in favor of the
existence of a different mitochondrial population in transgenic
gastrocnemius muscle. It can be seen in Fig. 4that the
respiration of skinned gastrocnemius fibers taken from control mice is
rather well-fitted by a single Michaelis-Menten curve, whereas
respiration of transgenic fibers show clear deviations from hyperbolic
dependence. Two groups of mitochondria seem to exist, one having a low K
for ADP, as in control, and another one
with a high K
value. Unfortunately, due
to technical reasons, there was no possibility to obtain enough
experimental points to calculate these K
values with adequate accuracy. Thus, one should realize that
the apparent K
values obtained in our
experiments for transgenic gastrocnemius muscle seem to represent
averaged K
values for different existing
mitochondrial populations. Apparently, the ``new'' population
in M-CK-deficient white muscle having low sensitivity to ADP
contributes to the increased respiratory capacity in the tissue.
Figure 4:
O consumption rates of skinned
fibers from gastrocnemius muscle plotted as a function of ADP
concentrations. Respiration of fibers taken from control (squares) and mutant (circles) mice was measured in
the absence of creatine. Experimental data were fitted by a
Michaelis-Menten equation for control (solid line) and
transgenic (broken line)
preparations.
Table 3shows also values of acceptor control ratio (ratio of
the V to basal respiration rate in the absence
of ADP) determined for skinned fibers of various muscles. It can be
seen that there were no statistically significant differences in this
parameter between control and transgenic muscles. Thus, alterations in
mitochondrial sensitivity to ADP found in muscles of mutant mice were
not accompanied with changes in coupling between oxygen consumption and
phosphorylation.
To check if the adaptation to M-CK deficiency in oxidative and glycolytic muscles is followed by alterations in quantity of respiratory units, the specific activity of citrate synthase, a marker mitochondrial matrix enzyme, was determined in extracts of ventricular and gastrocnemius muscles. Citrate synthase activity in transgenic ventricular tissue (74 ± 18 IU/g wet weight, n = 4) had a tendency to be higher than in control (39 ± 5 IU/g wet weight, n = 3) although the difference was not statistically significant. However, citrate synthase activity in mutant gastrocnemius muscle (21.3 ± 2.9 IU/g wet weight, n = 5) was significantly higher than in control muscle (6.3 ± 0.6 IU/g wet weight, n = 3), thus implying a prominent rise in oxidative potential in genetically glycolytic muscle under conditions of M-CK deficiency.
M-CK-deficient mice represent a very useful model for
studying the role of the CK system in muscle tissue. In a previous
article, van Deursen et al.(1993) have reported that resting
skeletal muscles (a complex of fast- and slow-twitch muscles,
gastrocnemius-plantaris-soleus) of M-CK knock out mice have normal
levels of ATP and PCr. PCr could be utilized because its content
declines during muscle exercise. Mutant mice show no alterations in
absolute muscle force, but the performance of this muscle complex is
changed. In contrast to control muscles, M-CK-deficient muscles
demonstrate a drop in force after a few twitches. Furthermore, P NMR magnetization transfer methods have shown the
absence of significant fluxes through the CK reaction in mutant
skeletal muscles at rest in spite of the presence of mi-CK and the
ability of muscles to utilize PCr during activity.
The present study has confirmed that mutant mice are anatomically similar to the control animals. Electrophoretic analysis of mutant tissues has shown a complete absence of M-subunit of CK. However, this defect was not followed by marked alterations in internal organ macrostructure. Examination of M-CK-deficient animals has revealed no signs of cardiac hypertrophy, congestive heart failure, or alteration in muscle mass.
Knocking out the gene for M-CK subunit does not result in marked changes in other subunit expression. We have not found significant differences in mi-CK-specific activity in muscle and brain tissues between mutant and control animals. Interestingly, muscles of wild-type mice contained very low levels of BB- and MB-isoenzymes, so that quantification of these forms was not possible by electrophoresis followed by CK-specific staining. Zymograms of ventricular and gastrocnemius homogenates of murine muscles obtained by van Deursen et al.(1993) also show the absence of clear traces of BB-isoenzyme. In contrast, ventricular (Popovich et al., 1989; Awaji et al., 1990; Savabi and Kirsch, 1991; Field et al., 1994), as well as skeletal (Yamashita and Yoshioka, 1991), muscles of another rodent species, rat, contain marked activities of BB- and MB-isoforms. Thus, the distribution of the CK isoenzymes varies even between close mammalian species.
Some preparations from mutant muscles in our study revealed measurable quantities of the BB-form. Accordingly, a slight increase of BB-CK in ventricular tissue of M-CK-deficient mice was reported earlier (van Deursen et al., 1993). However, these changes were too weak to speculate on their significance.
The most important findings of this study are that
mitochondrial sensitivities to ADP are completely different between
oxidative and glycolytic muscles of wild-type mice and change in
opposite directions under conditions of M-CK deficiency. The K values of oxidative phosphorylation in situ for ADP in control ventricular and soleus muscles are
much higher than K
values obtained in
isolated mitochondrial preparations. Other authors (Seppet et
al., 1991; Saks et al., 1991, 1993; Clark et
al., 1994) using skinned ventricular fibers or cardiomyocytes to
study respiration of mitochondria also found rather high K
values for ADP (100-300
µM). In contrast, isolated cardiac mitochondria showed
severalfold lower apparent K
values,
about 20-30 µM (Chance and Williams, 1955; Bygrave
and Lehninger, 1967). It is very important to understand the reason for
this discrepancy, because the apparent K
of oxidative phosphorylation for ADP is one of the basic
parameters in the theory of regulation of cellular energy metabolism.
It was found that disruption of the outer mitochondrial membrane in
skinned cardiac fibers by an osmotic shock dramatically decreased the K
(Saks et al., 1993). Analyzing
these results, Saks et al.(1993) have suggested that in
mitochondria in situ, the outer mitochondrial membrane
restricts free ADP diffusion from the extramitochondrial space and thus
leads to an increase in the apparent K
for ADP. The authors have proposed that some unknown factor
controls the permeability of the outer membrane to ADP, and a procedure
of mitochondrial isolation eliminates the regulatory role of this
factor. It is possible also that the reduced ADP diffusion in
nonisolated mitochondria of oxidative muscles is the consequence of a
high local concentration of macromolecules in the vicinity of the
mitochondrial compartment in situ. In agreement with this
latter view are results of Gellerich et al.(1993) showing an
increase in resistance of outer membrane to adenine nucleotides due to
elevated oncotic pressure.
Whatever the cause for the low
sensitivity of in situ mitochondria to ADP, at physiological
ADP concentration in ventricular and slow-twitch muscles, the
functional coupling of mi-CK with oxidative phosphorylation is a
powerful and important mechanism of regulation of the ADP/ATP ratio in
the vicinity of the adenine nucleotide translocase. Our data show that
working mi-CK is able to significantly decrease the K values for ADP making them close to the
cytosolic range of [ADP]. However, this function of mi-CK may
be disturbed if the PCr/creatine transport system is impaired. If
cytosolic M-CK is switched off, as is the case in M-CK knocking out,
one end of the ``PCr shuttle'' is eliminated and the
transport system does not work. Under these conditions, ADP rather than
creatine should play the main role of diffusible phosphate acceptor in
the cells. However, ADP would be able to play this role only if the
sensitivity of mitochondria to ADP is higher than in normal cells. This
is exactly what we have observed in oxidative fibers of M-CK-deficient
mice, where the apparent K
values for ADP
were substantially lower than those in wild-type mice. Such an
adaptation, at least in the heart, does not seem to involve alterations
in translocase properties because the mitochondrial sensitivity to ADP
in the presence of creatine is the same in mutant and control mice.
One should point out that as far as a high K value is a prerequisite for evidencing the coupling between
the translocase and mi-CK, it is difficult to make a clear conclusion
about the degree of this coupling in mutant mitochondria. It might be
possible that the coupling is weaker in mutant oxidative muscles as
compared to control ones.
Our data are in very good accord with
those recently obtained by Clark et al.(1994) in rats fed by a
creatine analogue, -GPA. Such a diet reduces the cellular levels
of creatine and PCr, affecting the PCr/creatine transport system at its
substrate site. These authors have found a significant (3-fold)
decrease in apparent K
for ADP in skinned
ventricular fibers of
-GPA-fed rats, as compared with controls.
It seems likely that in cells with an impaired PCr/creatine transport system the diffusion barrier between adenine nucleotide translocase and the extramitochondrial space becomes more permeable not only for import of ADP but also for export of ATP and, probably, for PCr as well (van Deursen et al., 1993; Wallimann, 1994). M-CK-deficient muscles are able to utilize PCr during exercise, obviously, in the reaction catalyzed either by CK or some unknown enzyme which is able to metabolize PCr. Both MM- and MB-isoforms are absent, BB-isoform activity is extremely low. At least in the myofibrillar compartment of mutant muscle cells, there is no enzyme capable of using PCr (Ventura-Clapier et al., 1995). Therefore, the only enzyme that can catalyze PCr degradation is mi-CK. Thus, one has to hypothesize that communications between mi-CK and its cytosolic substrates and products are somehow facilitated.
The
underlying mechanism responsible for the regulation of adenine
nucleotide exchange in situ between translocase and the
extramitochondrial space in oxidative muscles is currently unclear. The
data obtained in the present study and by Clark et al. (1994)
indicate that in M-CK-deficient mice and in -GPA-fed rats, the
hypothetical factor restricting the adenine nucleotide exchange is
either absent or switched off. Thus, interesting studies can be
conducted by comparing the structural and functional properties of the
mitochondrial compartment in these experimental models and in normal
animals to identify this factor and to provide insight into the problem
of adenine nucleotide exchange between the mitochondrial compartment
and the cytosol.
Our data have provided evidence that regulation of
mitochondrial respiration by ADP in a fast-twitch, glycolytic
gastrocnemius muscle has properties completely different from those in
oxidative muscles. The apparent K of
respiration for ADP is 20-25 times lower in gastrocnemius muscles
than in oxidative tissues, thus indicating the absence of a diffusion
barrier for adenine nucleotides between translocase and the
extramitochondrial space. The difference in K
values could not result from higher diffusional distance
between the extracellular space and mitochondria in soleus because slow
oxidative muscles have been shown to have a much greater mitochondrial
volume density in the subsarcolemmal area, in contrast to glycolytic
muscles (Philippi and Sillau, 1994). Moreover, our data show that the K
for ADP measured in skinned
preparations does not depend on cell diameters because fibers in mouse
soleus muscle are apparently not larger than those in gastrocnemius
(see Fig. 2in van Deursen et al.(1993)). This means
that the diffusion barrier for adenine nucleotides in oxidative cells
seems to be located near the boundary of mitochondrial compartment
rather than in the bulk of cytosol.
Mi-CK has no influence on the ADP-stimulated respiration in gastrocnemius muscle. Therefore, functional coupling between mi-CK and translocase in fast-twitch muscle mitochondria appears to be absent. Obviously, these mitochondria do not need coupling because their sensitivity to ADP is already very high. Possibly, in this type of muscles, mi-CK plays the same role as cytosolic MM-CK does, to balance the PCr/ATP ratio, thus providing an intracellular temporal energy buffer.
Different patterns of mitochondrial sensitivity to ADP in oxidative and glycolytic muscles found in the present study are in good agreement with the hypothesis of muscle type specificity of the control of cellular respiration. In a recent work, Kushmerick et al. (1992) have reported the dependence of oxygen consumption rate on calculated [ADP] in slow- and fast-twitch muscle. The authors have concluded that in fast-twitch muscle (where, as the data of our work imply, the compartmentation of adenine nucleotides is either absent or negligible) but not in slow-twitch muscle, oxygen consumption rates could be explained by a feedback control of cellular respiration by cytosolic [ADP]. Evidently, in fast-twitch muscle, actual [ADP] near translocase appears to be close to that in cytosol calculated from data of NMR experiments.
Under conditions of M-CK
deficiency, the properties of mitochondria in gastrocnemius muscle are
altered. The apparent Kfor ADP
increases, but a detailed analysis of the [ADP]-respiration
rate relationship shows that this relationship is not well fitted by
the Michaelis-Menten equation so that the determined value of the K
probably reflects an averaged K
value for different mitochondrial
populations having different properties. One may suggest the appearance
of a mitochondrial population with a high K
for ADP, like in oxidative muscle. Furthermore,
M-CK-deficient glycolytic gastrocnemius muscle increases its oxidative
capacity substantially. The maximal oxygen consumption rate per unit of
tissue weight rises by 50%. This could be explained by an increase in
mitochondrial content. Earlier, the electron microscopy revealed an
expanded mitochondrial compartment in this muscle (van Deursen et
al., 1993). Besides, the specific activity of citrate synthase, a
marker mitochondrial enzyme, increases significantly in gastrocnemius
muscle (van Deursen et al.(1993) and the present study).
It is interesting to note that the increase in mitochondrial content in mutant fast-twitch muscle is not accompanied by an enhancement of mi-CK activity. However, we have found alterations in mitochondrial myokinase functional activity; being very weak in control gastrocnemius muscle, the functional activity of this enzyme increased in M-CK deficiency and became close to that in oxidative ventricular fibers.
Our findings
show that the adaptive strategy in white muscle seems to be directed
toward appearance of some properties of red, oxidative fibers. Similar
alterations were observed in fast-twitch muscle of -GPA-fed rats
where activities of aerobic enzymes (Shoubridge et al., 1985)
as well as mitochondrial ATP synthesis rate (Freyssenet et
al., 1994) were found to increase by 30-40%. Taken together,
these observations suggest that in fast-twitch muscle, elimination of
buffer function of CK/PCr system induces a compensatory augmentation of
the oxidative potential. In contrast, slow-twitch skeletal muscle
reveals another type of adaptation. We did not notice changes in the
maximal respiratory capacity of skinned fibers from soleus muscle of
mutant mice. Accordingly, the activities of aerobic enzymes were
reported to be unchanged in soleus muscle of
-GPA-fed rats
(Shoubridge et al., 1985).
In the presence of creatine, the maximal oxygen consumption rate calculated from the ADP kinetics experiments was similar in mutant and control ventricular fibers (67.6 and 64.2 ng atoms/min/mg dry weight, respectively). However, respiration in the presence of high [ADP] just after addition of a low quantity of ADP and creatine (Fig. 1) was significantly higher in M-CK-deficient than in control fibers (81.3 versus 60.6 ng atoms/min/mg dry weight). The only difference between these two protocols is that the second one is considerably shorter. It seems likely that the adapted mitochondrial populations of mutant ventricular muscle have increased oxidative capacity, probably due to the appearance of new mitochondria; however, they are not able to maintain high activity for a relatively long time. Some increase in citrate synthase-specific activity found in ventricular mutant muscle is of borderline significance, and this does not allow us to make any clear conclusion about mitochondrial content in M-CK-deficient ventricle.
In summary, our data show that knocking out the M-subunit of CK results in completely different patterns of adaptations in oxidative and glycolytic muscles (Fig. 5). The results obtained are consistent with the hypothesis of different roles of CK in different muscle types. Mitochondria in ventricular and soleus muscles normally have low sensitivity to ADP, and mi-CK functionally coupled with oxidative phosphorylation in the PCr/creatine pathway regulates adenine nucleotide levels in the mitochondrial compartment. It is tempting to speculate that M-CK deficiency and, consequently, an impairment of one end of the pathway result in alterations in ADP compartmentation, the role of ADP as transportable phosphate acceptor increases, and, to match these conditions, mitochondrial sensitivity to ADP rises. It appears that diffusion barriers between the mitochondrial compartment including mi-CK and extramitochondrial space become weaker, thus enabling mi-CK to serve the cytosolic compartment. This could explain the utilization of PCr by muscles of M-CK-deficient mice during exercise (van Deursen et al., 1993). In normal glycolytic muscle, adenine nucleotide compartmentation is negligible, mitochondrial sensitivity to ADP is very high, and respiration is controlled by cytosolic [ADP]. Fast-twitch muscle of mutant mice is deprived of enzyme controlling utilization of a powerful energy resource (PCr), and, to compensate this potential energy deficiency, the adaptation is directed toward enhancing the oxidative capacity of the muscle. Further studies might reveal the mechanisms responsible for such different patterns of adaptation as well as identify the factor(s) controlling mitochondrial accessibility for ADP.
Figure 5:
Model of main energy pathways in oxidative
and glycolytic muscles of control and mutant animals. In oxidative
muscle, mitochondrial CK is functionally coupled to the translocase (T) on the inner mitochondrial membrane (i.m.) so
that most of the ATP transported from the mitochondrial matrix enter
the CK reaction and is transphosphorylated into PCr leaving the
mitochondrial compartment. The permeability of the outer mitochondrial
membrane (o.m.) for adenine nucleotides is quite low, and this
results in high K of respiration for ADP.
Creatine (Cr) imported through the outer membrane decreases
the apparent K
by increasing the local
ADP concentration. In the cytosol, a relatively weak glycolytic (G) pathway produces ATP which is equilibrated with the PCr
pool by the soluble CK. At the sites of energy utilization (myofibrils), ATPase (A) preferentially uses ATP
produced at the expense of PCr in the reaction catalyzed by bound
MM-CK. In the mutant oxidative muscle, due to the absence of soluble
and bound MM-CK, the role of the adenylate pathway between the
mitochondria and energy-consuming sites is increased. The permeability
of the outer membrane for adenine nucleotides is elevated, and this is
followed by a decrease in K
of
respiration for ADP. Because the diffusion barrier for ATP and ADP
between extra- and intramitochondrial compartments is weaker, the
muscles are able to utilize ATP produced at the expense of PCr in the
CK reaction catalyzed by the mitochondrial isoenzyme. The absence of
the energy-buffering function mediated in normal muscles by the soluble
CK is compensated by some elevation in oxidative and glycolytic
capacities. In glycolytic muscle, the contribution of oxidative
phosphorylation to the ATP production is relatively small compared to
glycolysis. The respiration could be controlled by cytosolic
[ADP] because the adenine nucleotides are able to easily pass
the barrier between extra- and intramitochondrial compartments. A high
pool of PCr freely equilibrated with ATP by soluble CK can serve as an
energy buffer. M-CK deficiency limits the utilization of this buffer in
energy-consuming compartments. To compensate this, glycolytic and
predominantly oxidative capacities increase. The muscle acquires some
properties of slow-twitch skeletal muscle (and/or the proportion of
slow-twitch fibers increases). The augmentation of the oxidative
potential is followed by an elevation of K
for ADP, a characteristic feature of mitochondria in
oxidative muscle.