(Received for publication, April 18, 1995; and in revised form, October 6, 1995)
From the
The brindled mottled mouse (Mo), an animal model of
the Menkes' copper deficiency syndrome, was used for the
investigation of changes in respiratory flux control associated with
cytochrome c oxidase deficiency in muscle. Enzymatic analysis
of cardiac and skeletal muscles showed an approximately 2-fold decrease
in cytochrome c oxidase activity of brindled mutants in both
types of muscles as compared with controls. The activities of
NADH-cytochrome c oxidoreductase (respiratory chain segment
I-III) and succinate-cytochrome c oxidoreductase
(segment II-III) were normal. Assessment of mitochondrial
respiratory function was performed using chemically skinned musculus
quadriceps or heart muscle fibers isolated from control and brindled
mottled mice. In skeletal muscle, there was no difference found in
maximal rates of respiration. In the Mo
hearts, this
parameter was slightly lower than control. Alternately, the
determination of flux control coefficients of cytochrome c oxidase performed by a step by step inhibition of respiration with
increasing concentrations of azide or cyanide revealed significantly
sharper inhibition curves for brindled mice than for control,
indicating more than 2-fold elevated flux control coefficients of
cytochrome c oxidase. This investigation proved essential in
characterizing the metabolic effect of a cytochrome c oxidase
deficiency. We conclude, therefore, that application of metabolic
control analysis can be a valuable approach to study defects of
mitochondrial oxidative phosphorylation.
The brindled mouse is a variant of the X-linked mottled mutants
(Mo) with severe copper deficiency and is considered to be
an animal model of Menkes' syndrome (Menkes' kinky hair
disease)(1, 2, 3, 4, 5) .
The copper homeostasis disorders in humans, in mottled mice, or in
copper-deficient rats are all associated with distinct mitochondrial
alteration in various tissues. The brain and other regions of the
central nervous system are particularly affected (6, 7) . Among mitochondrial abnormalities are various
ultrastructural and biochemical changes, such as the well documented
depressed activity of cytochrome c oxidase (a mitochondrial
cuproenzyme) (7, 8, 9, 10) . Thus,
it was suggested that the lower level of energy metabolism caused by
the decrease in both copper concentration and cytochrome c oxidase activity may be responsible for brain degeneration
associated with the Menkes' disease(6, 11) .
However, there is little information available concerning mitochondrial
function or cytochrome c oxidase activity in cardiac and
skeletal muscles of animals with copper deficiency.
Measurements of maximal rates of mitochondrial respiration are often used for the functional determination of the different mitochondrial defects(12, 13, 14) . It has been shown that assessment of respiratory activities of saponin-skinned muscle fibers can be especially applicable for the study of small human biopsy specimens(15, 16, 17) . However, in cases where the defect is located at a non-rate-limiting step of oxidative phosphorylation, simply determining the maximal rate of respiration will not reveal the mitochondrial defect. Alterations in individual respiratory complexes, for example in cytochrome c oxidase activity, can therefore be hard to find or even missed. Recently it was suggested that metabolic control analysis of oxidative phosphorylation can be a successful approach for quantifying the enzymatic defect in certain mitochondrial diseases(18, 19, 20) . However, direct evidence is still absent, and acceptable pathological models were not used.
In this work we applied metabolic control
analysis (21, 22) to study the consequences of
cytochrome c oxidase deficiency in Mo mice. For
this we used incremental inhibition of the mitochondrial respiration
with increasing concentration of azide and cyanide. Due to the limited
amount of tissue, and in order to avoid possible artifacts of the
preparation, we used chemically skinned muscle fibers, which allowed us
to investigate mitochondrial functions in situ, without
isolation of mitochondria(15, 17, 23) .
Table 1and Table 2summarize the data of
enzymatic analysis of cardiac and skeletal muscles from control and
Mo mice. It can be seen that cytochrome c oxidase
activity of copper-deficient animals was 50% of normal activity in both
cardiac and skeletal muscles. On the other hand, the activities of
other mitochondrial enzymes, NADH-cytochrome c oxidoreductase
and succinate-cytochrome c oxidoreductase, were not
significantly different from the control values. The activity of the
mitochondrial marker enzyme, citrate synthase, was slightly higher in
the muscles of brindled mice with significant differences in the heart.
This may reflect a possible adaptational increase in mitochondrial
content due to depressed oxidative metabolism, since no difference in
fiber typing of m. quadriceps was observed. This skeletal muscle of
brindled mutants also has decreased activities of lactate
dehydrogenase, adenylate kinase, and creatine kinase, while in cardiac
muscle only adenylate kinase activity was reduced. The activities of
aspartate aminotransferase were normal in both heart and m. quadriceps (Table 1).
Next, we determined the maximal respiration activities of chemically skinned muscle fibers. Saponin is frequently used as a skinning agent to ensure mitochondrial intactness(15, 17) . In addition to this we adapted a procedure of plasma membrane permeabilization with glycerol as a chemical skinning agent previously used for mechanical experiments only(26) . As shown in Table 3and Fig. 1the mitochondrial function in these fibers is similarly preserved as reported for saponin-skinned fibers(15, 17) . When comparing the maximal rates of mitochondrial respiration in skinned m. quadriceps fibers isolated from normal and brindled mice (Table 3) almost no difference was observed. The same result was obtained using saponin-skinned fibers and glycerol-skinned fibers with two different substrate combinations: glutamate + malate, and glutamate + malate and succinate. In each case the maximal rate of respiration was achieved by the addition of 1 mM ADP. On the other hand, skinned fibers isolated from the cardiac muscle of copper-deficient animals demonstrated slightly lower respiratory parameters than control (Table 3).
Figure 1: Representative traces of respiration from glycerol-skinned fibers from control mouse heart. Skinned fibers (1.4 mg, dry weight) in the medium for oxygraphic measurement. Additions were as follows: 10 mM glutamate (GLU), 5 mM malate (MAL), 1 mM ADP, 10 µM carboxyatractyloside (CAT). The top line is the direct measure of oxygen concentration, and the bottom line is the traces differentiation.
To determine the flux
control coefficients for cytochrome c oxidase we used
titrations of the rate of respiration of skinned fibers with the
specific inhibitors of this enzyme: sodium azide (28) and
potassium cyanide(29) . To do this, the maximal rate of
respiration (in the presence of 1 mM ADP) was inhibited by
incrementally increasing sodium azide (0-800 µM) or
potassium cyanide (0-60 µM) concentrations. The
inhibition of respiration of skinned cardiac fibers from control (open circles) and Mo mice (filled
circles) by sodium azide is shown in Fig. 2(upper
part). It can be seen that the shapes of the curves are
different for control and brindled mice. The inhibition curve for
brindled mutants is characterized by a sharper slope and, therefore,
higher sensitivity to azide. A similar effect was found for skinned
fibers isolated from m. quadriceps of control and brindled mice (Fig. 2, lower part; compare open and filled circles). In terms of metabolic control analysis, this
means that the flux control coefficient of cytochrome c oxidase is much higher in copper-deficient brindled mice than in
control mice. To determine the flux control coefficient (C
) using this noncompetitive inhibitor, the
following equation can be used.
Figure 2: Inhibition of mitochondrial respiration of glycerol-skinned fibers by azide. Upper part, skinned fibers (1.5-2.0 mg, dry weight) were isolated from heart (left ventricle) of control (open circles) and brindled (filled circles) mice. The rate of respiration was measured in the presence of 10 mM glutamate, 5 mM malate, 10 mM succinate, and 1 mM ADP with subsequent additions of the azide concentrations indicated. The data points are averages of three experiments. Lower part, skinned fibers (3.0-3.5 mg, dry weight) were isolated from m. quadriceps of control (open circles) and brindled (filled circles) mice. The rate of respiration was measured as described above. The data points are averages of five experiments.
where J is the respiration flux, dJ is the
decrement of respiration flux caused by the increment of inhibitor
addition dI, and K is the dissociation
constant for sodium azide. To exclude possible changes of the K
value under the conditions of copper deficiency
it was necessary to determine the inhibition curves of cytochrome c oxidase activity by azide. The dependence of cytochrome c oxidase activity of heart muscle homogenates from control and
Mo
mice on azide concentration is shown in Fig. 3.
In the inset the data are transformed using the Dickson
linearization. Both curves were within experimental error
similar with the same K
value for azide (85.5
± 5.1 µM and 83.9 ± 9.2 µM for
control and brindled mice, respectively). Interestingly, in the m.
quadriceps homogenates we determined K
values of
73.5 ± 6.9 µM and 107.3 ± 6.7
µM for control and brindled mice, respectively. Thus, it
was possible to calculate the flux control coefficients using . As shown in Table 4, both heart and quadriceps
muscles of Mo
mice have significantly higher values of
flux coefficients of cytochrome c oxidase.
Figure 3:
Inhibition of cytochrome c oxidase activity of cardiac homogenates by azide. The homogenate
(50 mg, wet weight/ml) was obtained from hearts of control mice (open circles) and of Mo mice (filled
circles). The cytochrome c oxidase activity was measured
as described under ``Experimental Procedures.'' Inset, Dickson plot of azide inhibition of cytochrome c oxidase activity.
To prove the result of these titration experiments, another specific inhibitor of cytochrome c oxidase, potassium cyanide, was applied. The inhibition curves of oxygen consumption of skinned muscle fibers with KCN are shown on Fig. 4. Once again, it can be seen that KCN titrations for control and brindled mice are strikingly different for both types of skinned fibers from heart and m. quadriceps with less pronounced sigmoidal behavior in copper-deficient mutants. The control coefficients obtained from KCN titrations are summarized in Table 4. In this case of irreversible enzyme inhibition the flux control coefficients were calculated using the following equation.
Figure 4:
Inhibition of mitochondrial respiration of
glycerol-skinned fibers by cyanide. Upper part, skinned fibers
(1.5-2 mg, dry weight) of control (open circles) and
brindled (filled circles) mice heart. The respiration was
measured in the presence of 10 mM glutamate, 5 mM malate, 10 mM succinate, and 1 mM ADP. Curves
with the following parameters (cf. (27) ) were fitted
to the titration points. Open circles: flux control
coefficient (C) = 0.11; maximal amount of
inhibitor (I
) = 5.8 µM;
dissociation constant of the inhibitor (K
) = 0.55 µM;
initial rate of respiration (J) = 57.1 ng atoms of
oxygen/min/mg, dry weight. Filled circles: C
= 0.54; I
= 5.6
µM; K
= 0.41
µM; J = 53 ng atoms of oxygen/min/mg, dry
weight. Lower part, skinned fibers (3-3.5 mg, dry
weight) from m. quadriceps of control (open circles) and
brindled (filled circles) mice. The respiration was measured
as described above. Curves with the following parameters were fitted to
the titration points. Open circles: C
= 0.09; I
= 7.1
µM; K
= 0.35
µM; J = 16.1 ng atoms of oxygen/min/mg
dwt. Filled circles: C
= 0.38; I
= 8.9 µM; K
= 0.9 µM; J = 15.4 ng atoms of oxygen/min/mg, dry
weight.
where I is the maximal amount of inhibitor. To
avoid overestimation of flux control coefficients, we performed
nonlinear regression analysis of the entire inhibitor titration
curves(27) . Interestingly, the values determined by titrations
with cyanide are substantially smaller than the values with azide.
Nevertheless, as seen in Table 4, the values of flux control
coefficients obtained from copper-deficient brindled mice are
approximately 2-fold elevated, compared with the control values for
both cardiac and skeletal muscles.
To examine the cause for the
reduced cytochrome c oxidase activities in copper-deficient
mice, we determined the cytochrome content in Triton X-100 solubilized
membrane pellets of homogenates from heart and skeletal muscles from
difference spectra in the -band of cytochromes. Reduced minus
oxidized spectra of these membrane pellets are shown in Fig. 5, A and B. It was found that the cytochrome aa
content is in cardiac homogenates of Mo
mice about 1.4-fold lower in comparison with control homogenates (cf. quantitative data in Table 1), whereas no
significant difference was observed for homogenates from skeletal
muscle (cf. quantitative data in Table 2).
Figure 5:
Difference spectra of cytochromes in
homogenates of control and Mo mice. The membrane fractions
of homogenates from heart (containing 50 mg, wet weight, of tissue/ml)
and m. quadriceps (containing 100 mg, wet weight, tissue/ml) were
prepared as described under ``Experimental Procedures.'' Upper spectrum of each part, dithionite reduced minus oxidized
difference spectrum of membrane fraction from controls; lower
spectrum of each part, dithionite reduced minus oxidized
difference spectrum of membrane fraction from Mo
mice.
In the present study mottled brindled copper-deficient mice
were used as a model of the more common cytochrome c oxidase
deficiency(30, 31, 32, 33, 34, 35) .
Previous findings provided direct evidence for decreased activities of
copper-containing enzymes (particularly of cytochrome c oxidase), being most probably responsible for mitochondrial
abnormalities and brain degeneration associated with Menkes'
disease(11) . Histochemical investigations of Menkes'
mutants showed elevated copper concentration in organelle-free
cytoplasm as compared with nuclei, mitochondria, or lysosomes,
suggesting the disturbed copper transport from the cytosol to the
organelles in the cell(36) . Using P NMR
spectroscopy, it was shown that in the brain the observed decreased
energy metabolism (decline in ATP content, PCr:Cr ratio, and
mitochondrial respiration) can be a pathophysiological mechanism of
disturbed nervous function in copper deficiency and Menkes'
diseases(37) .
In our study an approximately 2-fold lower cytochrome c oxidase activity is seen in cardiac and skeletal muscles of brindled mice ( Table 1and Table 2). As has been pointed out(38) , this decline cannot be related exclusively to the role of copper as a metal center of cytochrome c oxidase, but also may be due to a decreased synthesis of nuclear encoded subunits. Reduced expression of cytochrome c oxidase subunits in the cerebellum, spinal cord, and other regions of central nervous system in Menkes' disease was shown using specific antibodies against subunits II and IV of cytochrome oxidase (10) . This reflects also a decrease of synthesis of mtDNA-encoded subunits.
The K values for azide (70-100 µM)
obtained in this study are close to the value recently reported for
isolated cytochrome c oxidase (28) . As has been shown
previously, the Cu
and cytochrome a
sites are involved in the binding of cytochrome c oxidase inhibitors like azide and cyanide(29) . It is
therefore noteworthy that the K
values for azide
inhibition obtained in heart homogenates were not significantly
different for control and mottled brindled mice. This indicates an
absence of changes in cytochrome c oxidase binding sites for
azide in this copper deficiency. Therefore, similarly as in
brain(10, 38) , the decreased activity of this enzyme
in cardiac muscle of Mo
mice seems to be related (at least
in part) to a decreased expression of the enzyme and is visible from
the decreased levels of cytochrome aa
obtained in
heart homogenates. In skeletal muscle, these changes are small (and due
to the higher experimental error not significant) even if the elevated
amount of mitochondria per mg, wet weight, is taken into consideration (cf. citrate synthase levels in Table 2). On the other
hand, different K
values for azide inhibition were
observed in quadriceps homogenates, pointing to possible alterations in
enzymatic function of cytochrome c oxidase in this muscle.
To date little is known concerning changes in other enzymatic patterns of cardiac and skeletal muscles associated with, or caused by, copper deficiency. As has been suggested recently, copper deficiency may cause compensatory mechanisms to maintain cardiac ATP levels(39) . In our study we estimated these possible adaptational alterations in brindled mutants by assaying the activities of certain key enzymes. The most remarkable changes were revealed in m. quadriceps (Table 2). We found a significant decrease in the activities of lactate dehydrogenase, adenylate kinase, and creatine kinase in muscle homogenates of brindled mottled mice. We obtained, however, an elevation in the activity of citrate synthase, which was even more pronounced in heart muscle. This is most probably indicative of the mentioned adaptational response. Increasing the mitochondrial volume and/or density may allow the tissue to compensate for the lack of cytochrome c oxidase. This observation is in accordance with increased mitochondrial:myofibrillar ratios and an expanded mitochondrial area reported for copper-deficient rat hearts(40, 41) .
The apparent conflicting results of mitochondrial respiration and oxidative phosphorylation in various models of copper deficiency (39, 42, 43) may be explained by different procedures of mitochondrial isolation and examination in those studies. To avoid possible artifacts of the mitochondrial preparation the respiratory parameters of cardiac and skeletal muscle mitochondria were determined using chemically skinned fibers. As has been shown in our previous studies, this approach has a number of advantages(15, 17) . Selective permeabilization of plasma membrane essentially results in unobstructed access to the mitochondria in skinned fibers for substrates or ADP. In this way, the mitochondrial parameters can be estimated inside thin muscle fibers. It allows one to investigate the total mitochondrial population, in situ, without isolation of these organelles and using very small samples of tissue.
The significantly lower values of flux control coefficients for cytochrome oxidase observed with KCN titration as compared with azide titration can be explained by the more specific inhibition of cytochrome c oxidase by cyanide. Due to the fact that in some conditions azide may also inhibit ATP splitting and ATP synthesis activities of mitochondrial ATPase(44) , the application of this inhibitor may have resulted in an overestimation of the value of flux control coefficient for cytochrome c oxidase (Table 4).
Summarizing, this report demonstrates that determination of flux control coefficients can be a valuable approach to study defects of mitochondrial oxidative phosphorylation.