From the Departments of Molecular and Human Genetics,
§ Pathology, and ¶ Pediatrics, Baylor College of
Medicine, Houston, Texas 77030
Received for publication, July 24, 2000, and in revised form, October 20, 2000
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ABSTRACT |
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Voltage-dependent anion channels
(VDACs) form the main pathway for metabolites across the mitochondrial
outer membrane. The mouse vdac1 gene has been disrupted by
gene targeting, and the resulting mutant mice have been examined for
defects in muscle physiology. To test the hypothesis that VDAC1
constitutes a pathway for ADP translocation into mitochondria, the
apparent mitochondrial sensitivity for ADP
(Km(ADP)) and the calculated rate of
respiration in the presence of the maximal ADP concentration (Vmax) have been assessed using skinned fibers
prepared from two oxidative muscles (ventricle and soleus) and a
glycolytic muscle (gastrocnemius) in control and
vdac1 The mitochondrial outer membrane
(MOM),1 like the outer
membrane of Gram-negative bacteria, contains a major protein that
constitutes an aqueous pore, porin, also known as the
voltage-dependent anion channel (VDAC). When reconstituted
in artificial bilayers, VDAC conductance is high at low voltages,
whereas the channel reverts to a low conductance state when the voltage
is increased (1). Three VDAC isoforms have been characterized in
mammals: VDAC1, VDAC2, and VDAC3 (2-6). These isoforms constitute a
gene family that arose by gene duplication and divergence (7). Although the exact in vivo roles of VDACs are not yet known, it has
been speculated they are involved in the coupling of cellular energy demand to mitochondrial energy production since they constitute the
main pathway for small metabolites across the MOM (8-10).
Compartmentation of creatine kinase isoforms in the cytosol and
mitochondria of tissues with high and fluctuating energy demands has
led to the phosphocreatine circuit hypothesis as a mechanism linking
sites of ATP consumption and production (11, 12). Bessman and Fonyo
(13) were the first to demonstrate that creatine could exert acceptor
control of respiration by production of ADP from mitochondrial ATP. The
functional coupling of mitochondrial creatine kinase (miCK), located in
the intermembrane space, to respiration has been attributed to the
direct channeling of ATP and ADP between miCK and the
adenine-nucleotide translocase (ANT), located in the inner
mitochondrial membrane (14-17). In support of this hypothesis,
formation of a complex between the octameric form of miCK and VDAC has
been observed in vitro (18), and a complex formed by
octameric miCK, VDAC, and ANT has been purified from rat brain
mitochondria (19). These complexes may constitute the structural basis
for the functional coupling of miCK to respiration. It is believed that
the functional coupling of miCK to respiration increases the ADP
concentration in the vicinity of the ANT and compensates for the
barrier to ADP diffusion exerted by the MOM. Evidence that the MOM may
restrict ADP diffusion has been provided by experiments in
reconstituted systems showing that mitochondria and pyruvate kinase
compete for ADP generated by kinases present at different locations
with respect to the MOM (17, 20, 21). The functional coupling of miCK
to respiration has more recently been studied in skinned fibers
prepared from cardiac ventricle (22, 23). In these preparations, where
mitochondria preserve their in vivo structure, miCK coupling
to respiration decreases the apparent mitochondrial
Km for ADP (Km(ADP)). Unlike isolated mitochondria devoid of their intracellular
interactions, the in situ apparent
Km(ADP) is relatively high. This high
apparent Km(ADP) is presumed to reflect
the retarded diffusion of ADP across the MOM (23, 24). It has been
speculated that some unknown factors related to the cytoskeleton and
interacting with VDACs may control the permeability of the MOM in
oxidative muscles (24). In glycolytic skeletal muscle fibers, the
apparent Km(ADP) is low, similar to that
in isolated mitochondria (25, 26).
Since VDACs constitute a multigenic family with multiple isoforms, it
is reasonable to think that the different functions assigned to VDACs
may be carried out by distinct isoforms. The fact that expression of
each mouse VDAC isoform in yeast leads to different permeability
characteristics (27), as well as the differing ability of each isoform
to complement yvdac1-deficient yeast (7), supports this
hypothesis. In an attempt to further elucidate the roles of VDAC1 in
cell metabolism, VDAC1-deficient mice have been
generated.2 The mice are
viable and thus provide a means for addressing VDAC1 function in
vivo. As VDACs are believed to be responsible for MOM permeability
for small metabolites, including ADP, we studied the apparent
Km(ADP) in skinned fiber preparations
from two oxidative striated muscle types (ventricle and soleus) and from a glycolytic skeletal muscle (gastrocnemius). To examine the role
of VDAC1 in creatine coupling, we also studied the effect of creatine
on mitochondrial respiration.
Chemicals--
Chemical products were purchased from Sigma.
Animals--
The control and VDAC1-deficient
(vdac1 Preparation of Skinned Fibers--
Control and
vdac1 In Situ Mitochondrial Respiratory Studies--
Respiratory rates
in the presence of increasing concentrations of ADP were assessed using
an oxygraph (biological oxygen monitor, YSI Model 5300) and a
Clark-type electrode (oxygen probe, YSI Model 5331). Skinned fibers
prepared from different striated muscles were incubated at 22 °C in
4 ml of buffer A containing 2 mg/ml bovine serum albumin. The rate of
oxygen consumption was recorded by a MacLab/200 system (AD
Instruments). The solubility of oxygen was taken as 230 nmol of
oxygen/ml. The ADP concentrations used were 0.05-2 mM for
ventricle and soleus fibers and 0.005-1 mM for
gastrocnemius fibers. The functional coupling of miCK to respiration was assessed in the presence of 25 mM creatine, and the ADP
concentration used was 0.025-2 mM. At the end of the
experiments, fibers were removed, dried, and weighed. Rates of
respiration are given in µmol of oxygen/min/g (dry weight) (Fig.
1). The integrity of the MOM was assessed
in the presence of 8 µM cytochrome c (31,
32).
Citrate Synthase Activity--
Fresh heart, soleus, and
gastrocnemius were homogenized in 5 mM Hepes buffer (pH
8.0) containing 1 mM EDTA and 1 mM
dithiothreitol. Protein concentrations were determined using the BCA
protein assay (Pierce). Citrate synthase activity was measured in the
presence of 0.1 mM 5,5'dithio-bis-(2-nitrobenzoic
acid), 0.3 mM acetyl-CoA, and 0.5 mM
oxalacetate using 30 µg of protein from heart and soleus homogenates
and 100 µg of protein from gastrocnemius homogenate. The initial rate
of reaction of liberated CoA-SH was followed at 412 nm for 3 min.
Western Blotting--
To quantify the amount of each VDAC
isoform in different striated muscles, a commercial monoclonal antibody
(mAb1, Calbiochem) specific for VDAC1 (33) and two polyclonal
antibodies specific for VDAC2 (similarly generated as described (34))
and VDAC3 (34) were used. To control for variation in protein content, a cytochrome c-specific polyclonal antibody (H-104, Santa
Cruz Biotechnology) was used. Different tissues from control and
vdac1 Electron Microscopy--
Following dissection, small pieces of
the left ventricle, soleus muscle, and gastrocnemius muscle from
control and vdac1 Statistical Analysis--
A two-tailed, unpaired t
test (GraphPAD PRISM Version 2.0C) was employed.
In Situ Mitochondrial Sensitivity for ADP--
In skinned muscle
fibers, the apparent Km(ADP) reflects
the in situ mitochondrial sensitivity for ADP. It is
"apparent" because the exact concentration of ADP in the
mitochondrial intermembrane space is not determined. This value varies
significantly between muscle types (25, 26). To determine whether the
absence of VDAC1 increases the barrier to ADP diffusion across the MOM,
we determined the apparent Km(ADP) in
skinned fibers prepared from different striated muscle types in control
and vdac1
In the ventricle, there was a 30% increase in the apparent
Km(ADP) in
vdac1
In soleus fibers, surprisingly, the apparent
Km(ADP) decreased significantly in
vdac1
In gastrocnemius fibers, the apparent
Km(ADP) increased significantly in
vdac1 Citrate Synthase Activity--
Ventricle homogenates from
vdac1 Western Blot Analysis--
It is possible that loss of VDAC1
function leads to an alteration in the expression level of the
remaining VDACs. Using antibodies specific for each VDAC isoform, we
determined that the striated muscles surveyed in this study express all
three VDAC isoforms (Fig. 6). After
normalizing the expression level to the mitochondrial protein
cytochrome c, we did not detect any significant alteration in the expression of VDAC2 or VDAC3 in any of the striated muscles surveyed in vdac1 Ultrastructural Examination--
To examine whether the loss of
VDAC1 results in structural alterations of mitochondria, the different
striated muscles surveyed in control and
vdac1 For adequate mitochondrial energy production, a variety of small
metabolites such as pyruvate, ADP, and ATP must be transported across
the two mitochondrial membranes. VDACs form the main pathway for small
metabolites across the MOM and potentially offer another mechanism for
integrating cytosolic energy demand with mitochondrial energy
production. In this report, we describe the characteristics of in
situ mitochondria from different striated muscles lacking VDAC1.
The different types of mammalian muscle fibers differ in such aspects
as myosin heavy chain composition and mitochondrial content, conferring
relatively distinct physiological and biochemical properties (36).
Skeletal muscle fibers have generally been categorized into three
types, identified as slow oxidative, fast oxidative glycolytic, and
fast glycolytic (37). Cardiac muscle is composed of slow oxidative
fibers. In this study, we have shown that the different striated
muscles surveyed express all three VDAC isoforms in wild-type mice. The
absence of VDAC1 did not affect the expression of the other VDAC
isoforms, so we can assume that the results described in this report
are related directly to the absence of VDAC1. The different striated
muscles from mice lacking VDAC1 exhibited different types of in
situ mitochondrial adaptation. In ventricle and gastrocnemius
fibers, we measured a 30% increase in the apparent
Km(ADP) in the absence of VDAC1, which
indicates that the barrier to ADP diffusion across the MOM has
increased. This result supports the hypothesis that VDAC1 constitutes a
pathway for ADP across the MOM and is consistent with recent results of
Vander Heiden et al. (38), who showed a disruption of
ATP/ADP exchange across the MOM upon growth factor withdrawal and
induction of apoptosis. This defect results from a loss of MOM
permeability to metabolic anions and correlates with the changes in
conductance properties that accompany closure of VDACs. The observation
that the Vmax did not change in the absence of
VDAC1 may mean that, in these two striated muscles, the absence of
VDAC1 alters the properties of only the MOM, without affecting the
mitochondrial inner membrane.
In contrast to the ventricle and gastrocnemius, the soleus exhibited a
decrease in both the apparent Km(ADP)
and Vmax, which suggests that, in this oxidative
skeletal muscle, the absence of VDAC1 affects the properties of both
the MOM and the mitochondrial inner membrane. In mammals, three ANT
isoforms have been characterized, and their tissue specificity
determined (40-42). In ANT1-deficient mice, a severe reduction in the
state III respiration rate has been reported in skeletal muscle (39). It is possible that, in the
vdac1 The decreased apparent Km(ADP) in the
vdac1 The apparent Km(ADP) represents an
adaptable mechanism for mitochondrial regulation. Indeed, a 3-fold
decrease in the apparent Km(ADP) in
skinned cardiac fibers in rats fed a creatine analog that competitively
inhibits creatine transport across the plasmalemma has been reported
(44). This decreased apparent Km(ADP) is
thought to compensate for the reduced cellular level of creatine and
phosphocreatine that would presumably affect the
phosphocreatine/creatine shuttle system. It has also been reported that
a decrease in the apparent Km(ADP) in
skinned cardiac and oxidative skeletal muscle fibers occurs in mice
deficient in microtubule-associated protein (25). As an
adaptation to the absence of VDAC1, soleus fibers may take on the
characteristics of glycolytic muscle, accounting for the decrease in
the apparent Km(ADP). Xu et
al. (27) have reported that, when reconstituted into liposomes,
VDAC2 appears to exist in two forms differing with respect to
conductance and selectivity. It is plausible that, in the soleus of
vdac1 There is a tissue specificity of mitochondria with respect to
morphology, structural organization, and oxidative capacity (45-47).
In striated muscles, there are subsarcolemmal and intermyofibrillar mitochondria. Abnormal subsarcolemmal and intermyofibrillar
mitochondria associated with defects of components of the mitochondrial
electron transport chain have also been described (48-50). By
analyzing muscle sections by electron microscopy, we observed an
abnormal configuration of the cristae of subsarcolemmal mitochondria in the skeletal muscles of vdac1 The functional coupling of miCK to mitochondrial respiration decreases
the apparent Km(ADP), a finding that
supports the hypothesis that, in vivo, the stimulation of
oxidative phosphorylation depends on the activity of peripheral kinases
(52). Creatine-stimulated respiration occurs when miCK is functionally
coupled to oxidative phosphorylation, and it has been suggested that
the octameric form of miCK located in the intermembrane space connects
the MOM via VDACs to ANT. It has been theorized that creatine diffuses through VDACs and is converted by miCK in the presence of ATP to
phosphocreatine and ADP. Phosphocreatine then leaves the mitochondria and is used at ATP-consuming sites, whereas ADP returns to the matrix
via ANT to generate ATP (11, 12). In this study, we did not observe an
alteration of the effect of creatine on mitochondrial respiration,
which could mean either that VDAC1 is not required for the diffusion of
creatine through the MOM or that VDAC2 and VDAC3 (or some alternate
means) are sufficient for diffusion. From this study, we can also
conclude either that the structural basis for the functional coupling
of miCK to respiration does not require VDAC1 or that VDAC2 and VDAC3
are sufficient to account for the connection between the MOM and ANT
via miCK.
In conclusion, our results suggest that VDAC1 is involved in the
transport of ADP across the MOM. We cannot draw the same conclusion for
creatine. The absence of VDAC1 has differing effects on the properties
of in situ mitochondria from different striated muscles.
Similar studies on mice lacking VDAC2, VDAC3, or some combination of
VDAC isoforms will be necessary to completely elucidate the roles of
VDAC isoforms in cellular metabolism and their possible involvement in
mitochondrial diseases.
/
mice. We observed a
significant increase in the apparent
Km(ADP) in heart and gastrocnemius,
whereas the Vmax remained unchanged in both
muscles. In contrast, a significant decrease in both the apparent
Km(ADP) and
Vmax was observed in soleus. To test whether
VDAC1 is required for creatine stimulation of mitochondrial respiration
in oxidative muscles, the apparent
Km(ADP) and
Vmax were determined in the presence of 25 mM creatine. The creatine effect on mitochondrial
respiration was unchanged in both heart and soleus. These data,
together with the significant increase in citrate synthase activity in
heart, but not in soleus and gastrocnemius, suggest that distinct
metabolic responses to altered mitochondrial outer membrane
permeability occur in these different striated muscle types.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
) mice used in this study
were of a mixed genetic background (C57BL6/129SvEv). Unless otherwise
noted, in each set of experiments, at least five littermate control and five vdac1
/
mice
were used. All mice were ~3 months of age.
/
mice were anesthetized
with Metofane, and the hearts were quickly removed and placed in a
cooled, well oxygenated (95% O2 + 5% CO2),
modified Krebs solution containing 118 mM NaCl, 4.7 mM KCl, 25 mM NaHCO3, 1.2 mM KH2PO4, and 1.2 mM
MgSO4 (25). To study the in situ properties of
mitochondria, we used the method of skinned fiber preparation described
by Veksler et al. (29). For heart skinned fibers, the left
ventricle was opened, and muscle strips from the endocardium were cut
lengthwise to avoid mechanical damage of the cells. Muscle fiber
bundles from the left ventricle, soleus (slow-twitch skeletal muscle),
and gastrocnemius (fast-twitch skeletal muscle) were placed in a cooled
solution containing 2.77 mM CaK2EGTA, 7.23 mM K2EGTA, 6.56 mM
MgCl2, 5.7 mM Na2ATP, 15 mM phosphocreatine, 0.5 mM
dithiothreitol, 50 mM potassium methanesulfonate, 20 mM imidazole, and 20 mM taurine (pH 7.1)
containing 50 µg/ml saponin. The fibers were incubated with gentle
stirring at 4 °C for 30 min to solubilize the sarcolemma.
Permeabilized (skinned) fibers were then placed in buffer A (2.77 mM CaK2EGTA, 7.23 mM K2EGTA, 1.38 mM MgCl2, 0.5 mM dithiothreitol, 3 mM
K2HPO4, 2 mM malic acid, 5 mM pyruvic acid, 100 mM potassium
methanesulfonate, 20 mM imidazole, and 20 mM
taurine (pH 7.1)) for 10 min with gentle stirring at 4 °C to wash
out soluble metabolites, in particular ADP (30). The wash was repeated twice.
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Fig. 1.
Oxygraph trace obtained with ventricle
skinned fibers. Oxygen consumption is plotted as a function of ADP
concentration. The Michaelis-Menten equation was used to obtain the
apparent Km(ADP) and
Vmax. In the presence of 25 mM
creatine, there was a decrease in the apparent
Km(ADP). gdw, g (dry
weight).
/
mice were homogenized in
a Polytron in 5 mM Hepes buffer (pH 8.0) containing 1 mM phenylmethylsulfonyl fluoride and anti-proteases (2 µg/ml aprotinin, 2 µg/ml leupeptin, 1 µg/ml pepstatin A, and 2 µg/ml antipain). 20-30 µg of total protein were separated on duplicate 4-12% gradient SDS-polyacrylamide gels. The separated proteins were transferred to polyvinylidene difluoride membranes (Roche
Molecular Biochemicals) using a Bio-Rad Trans-Blot system at 380 mA for 1 h in buffer containing 50 mM Tris, 40 mM glycine, 0.05% SDS, and 20% ethanol. Membranes were
blocked with 10% skimmed milk in buffer containing 25 mM
Tris, 3 mM potassium chloride, 140 mM sodium
chloride, and 0.05% Tween 20 (pH 7.4) overnight at 4 °C and then
incubated with mAb1 (5 µg/ml), anti-VDAC2 antibody (1:50 dilution),
anti-VDAC3 antibody (1:500 dilution), or anti-cytochrome c
antibody (1:500 dilution) for 1 h at room temperature. The
secondary antibody used was peroxidase-conjugated anti-mouse IgG
(1:10,000 dilution) for mAb1, anti-chicken IgY (1:10,000 dilution) for
the two polyclonal antibodies specific for VDAC2 and VDAC3, and
anti-rabbit IgG (1:5000 dilution) for the polyclonal antibody specific
for cytochrome c. The membranes were developed using an ECL
reaction and exposed to Kodak X-Omat films for 1-10 min.
/
mice were
fixed immediately in 3% phosphate-buffered glutaraldehyde. Samples
were post-fixed in 1% buffered osmium tetroxide and then dehydrated
through graded concentrations of alcohol, followed by embedding in
Araldite. Thin and thick sections were made, and the tissue was
examined on thin sections using a Joel 1210 transmission electron
microscopy. Photographs were made of selected regions.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
mice.
/
mice (309.4 ± 21.9 µM in vdac1
/
versus 220.6 ± 22.2 µM in control mice;
p = 0.020) (Fig.
2A). In the presence of 25 mM creatine, the apparent
Km(ADP) decreased in both control
(74.40 ± 5.39 µM) and
vdac1
/
(78.86 ± 4.25 µM) mice, these two values being comparable to each other
(Fig. 2A). The Vmax (calculated rate
of oxygen consumption in the presence of the maximal ADP concentration)
was not significantly different between control and
vdac1
/
mice in the absence or
presence of 25 mM creatine (Fig. 2B and Table
I). In comparison with wild-type mice,
basal respiration (no added ADP) was unchanged in
vdac1
/
mice in the absence or
presence of 25 mM creatine (Table I).
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Fig. 2.
A, apparent
Km(ADP) in saponin-skinned fibers
prepared from wild-type (black bars) and
vdac1 /
(white bars)
ventricles in the absence and presence of 25 mM creatine
(Cr). *, p < 0.05; N.S., no
significant difference. B, Vmax in
saponin-skinned fibers prepared from wild-type (black bars)
and vdac1
/
(white
bars) ventricles in the absence and presence of 25 mM
creatine. gdw, g (dry weight).
Respiratory parameters of in situ mitochondria from wild-type and
vdac1/
striated muscles in the absence and presence of 25 mM creatine
/
mice (212.0 ± 11.3 µM) in comparison with control mice (304.7 ± 2.7 µM; p < 0.0001) (Fig.
3A). In the presence of 25 mM creatine, the apparent
Km(ADP) decreased in both control
(106.0 ± 2.4 µM) and
vdac1
/
(71.40 ± 8.90 µM) mice, these two values being significantly different
(p = 0.0074) (Fig. 3A). In comparison with
wild-type fibers, there was a significant decrease in the
Vmax in
vdac1
/
mice, and this was
unchanged in the presence of 25 mM creatine (Fig.
3B and Table I). Basal respiration remained unchanged
between control and vdac1
/
mice
in the absence or presence of 25 mM creatine (Table I). This reduction in the apparent Km(ADP)
led us to consider the possibility that the VDAC1-deficient MOM
of soleus muscle is rendered more fragile and therefore is damaged by
saponin treatment. However, the percentage of stimulation of
respiration by cytochrome c was not significantly different
between control (7.27 ± 4.94%, n = 3) and
vdac1
/
(9.58 ± 1.86%,
n = 5) soleus muscles. This result suggests that the
integrity of the MOM in vdac1
/
soleus fibers is similar to that in control soleus fibers.
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Fig. 3.
A, apparent
Km(ADP) in saponin-skinned fibers
prepared from oxidative skeletal muscles of wild-type (black
bars) and vdac1 /
(white bars) mice in the absence and presence of 25 mM creatine (Cr). ***, p < 0.0001; **, p < 0.01. B,
Vmax in saponin-skinned fibers prepared from
oxidative skeletal muscles of wild-type (black bars) and
vdac1
/
(white bars)
mice in the absence and presence of 25 mM creatine. *,
p < 0.05. gdw, g (dry weight).
/
mice (16.13 ± 2.37 µM) in comparison with control mice (7.77 ± 0.42 µM; p = 0.0085) (Fig.
4A). In the presence of 25 mM creatine, the apparent
Km(ADP) increased in both control
(13.52 ± 2.36 µM) and
vdac1
/
(25.37 ± 3.88 µM) mice, these two values being significantly different
(p = 0.0395) (Fig. 4A). In gastrocnemius
muscle, miCK is not functionally coupled to respiration; the apparent
Km(ADP) is relatively low and is
unchanged by the addition of creatine (25). The increased apparent
Km(ADP) in the gastrocnemius in the
presence of creatine in this study is similar to what has previously
been measured in rat fibers prepared from the gastrocnemius (26) and
atria (35). The Vmax remained unchanged between
control and VDAC1-deficient fibers and was unaffected by the addition of 25 mM creatine (Fig. 4B and Table I). Basal
respiration also remained unchanged between control and VDAC1-deficient
fibers and was unaffected by the addition of 25 mM creatine
(Table I).
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Fig. 4.
A, apparent
Km(ADP) in saponin-skinned fibers
prepared from glycolytic skeletal muscles of wild-type (black
bars) and vdac1 /
(white bars) mice in the absence and presence of 25 mM creatine (Cr). **, p < 0.01;
*, p < 0.05. B, Vmax
in saponin-skinned fibers prepared from glycolytic skeletal muscles of
wild-type (black bars) and
vdac1
/
(white bars)
mice in the absence and presence of 25 mM creatine.
N.S., no significant difference; gdw, g
(dry weight).
/
mice had a higher citrate
synthase activity (348 ± 33 mIU/mg of protein) in comparison with control mice (174 ± 14 mIU/mg of protein; p = 0.0030) (Fig. 5). There was no
significant change in citrate synthase activity between control and
vdac1
/
mice in both the soleus
(193 ± 8 mIU/mg of protein in control versus 220 ± 46 mIU/mg of protein in
vdac1
/
mice) and gastrocnemius
(64 ± 9 mIU/mg of protein in control versus 72 ± 13 mIU/mg of protein in vdac1
/
mice) (Fig. 5).
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Fig. 5.
Citrate synthase activity in total
homogenates from different striated muscle types. **,
p < 0.01; N.S., no significant
difference.
/
mice (Fig.
6).
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Fig. 6.
Western blot showing the distribution of VDAC
isoforms in different striated muscle types from wild-type (+) and
vdac1 /
(
) mice. Cytochrome c was used
as a control signal to normalize the protein load. As determined by
densitometry, the relative values corresponding to this representative
figure (VDAC signal/cytochrome c signal) are as follows.
Anti-VDAC2 antibody: wild-type and
vdac1
/
ventricles, 0.566 and
0.579, respectively; wild-type and
vdac1
/
soleus muscles, 2.034 and
1.691, respectively; and wild-type and
vdac1
/
gastrocnemius muscles,
0.454 and 0.285, respectively. Anti-VDAC3 antibody: wild-type and
vdac1
/
ventricles, 0.507 and
0.475, respectively; wild-type and
vdac1
/
soleus muscles, 4.684 and
2.831, respectively; and wild-type and
vdac1
/
gastrocnemius muscles,
0.608 and 0.305, respectively.
/
mice were studied by
electron microscopy. Although the three striated muscles showed
alterations in mitochondrial structure, the extent of these changes was
qualitatively different among the three muscles. In the control soleus
sample, the subsarcolemmal population of mitochondria were considerably
smaller than adjacent nuclei and had regular cristae (Fig.
7A). The same population of
mitochondria in the vdac1
/
soleus approached the size of the adjacent nuclei, and the cristae were
more compacted (Figs. 7B and
8). In the
vdac1
/
gastrocnemius, the
subsarcolemmal population of mitochondria were also increased in size
in comparison with the same population in the control mice (Fig.
9). The structural changes in heart mitochondria were less pronounced than in the two skeletal muscles. Although mitochondria from the
vdac1
/
ventricle were increased
in size relative to control mitochondria, they did not reach the size
of the nucleus (Fig. 10). Furthermore, the cristae were still visible and appeared less compacted in the
mutant mice (Fig. 10B).
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Fig. 7.
Electron microscopic study showing
subsarcolemmal population of mitochondria from wild-type
(A) and
vdac1 /
(B) soleus muscles. Magnification × 8000.
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Fig. 8.
Electron microscopic study showing
subsarcolemmal population of mitochondria in the
vdac1 /
soleus. Magnification × 3200 (A) and 9600 (B).
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Fig. 9.
Electron microscopic study showing
mitochondria from wild-type (A) and
vdac1 /
(B) gastrocnemius muscles. Magnification × 2500 (A) and 4000 (B).
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Fig. 10.
Electron microscopic study showing
intermyofibrillar population of mitochondria from wild-type
(A) and
vdac1 /
(B) ventricles. Magnification × 10,000.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
soleus, there is a
decrease in the expression of ANT1 and/or ANT3, thus accounting for the
reduction in the Vmax. However, a child with
deficiency of VDAC1 in skeletal muscle was reported to have only a
slight decrease in ANT at the protein level (43). Alternatively, a
defect in the respiratory chain may be present.
/
soleus could correspond
to a change in the biophysical properties of the MOM, e.g.
the MOM may become nonspecifically permeable. Cytochrome c
effect on respiration is typically used to determine the integrity of
the MOM (31, 32). Since we did not find a difference in the stimulation
of respiration by cytochrome c between control and
vdac1
/
mice, we conclude that
the decreased apparent Km(ADP) in the
vdac1
/
soleus is not due to the
disruption of the MOM by saponin treatment.
/
mice, VDAC2 exists
predominantly in its high conductance state, thus accounting for their
relatively low apparent Km(ADP).
/
mice. The morphologically abnormal mitochondria in the
vdac1
/
skeletal muscles may
result from an alteration in the interaction of cytoskeletal elements
with the MOM. In support of this notion, it has been reported that
VDACs bind the cytoskeletal element MAP2 in brain mitochondria
(51).
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ACKNOWLEDGEMENT |
---|
We thank Professor Valdur A. Saks for helpful discussions.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant R01 GM055713-02 (to W. J. C.) and the Baylor College of Medicine Mental Retardation Research Center.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Molecular
and Human Genetics, Baylor College of Medicine, Rm. S821, One Baylor
Plaza, Houston, TX 77030. Tel.: 713-798-8305; Fax: 713-798-8704; E-mail: wcraigen@bcm.tmc.edu.
Published, JBC Papers in Press, October 23, 2000, DOI 10.1074/jbc.M006587200
2 M. J. Sampson, E. Weeber, K. Anflous, M. Levy, W. K. Decker, J. D. Swealt, and W. J. Craigen, manuscript in preparation.
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ABBREVIATIONS |
---|
The abbreviations used are: MOM, mitochondrial outer membrane; VDAC, voltage-dependent anion channel; miCK, mitochondrial creatine kinase; ANT, adenine-nucleotide translocase; mAb, monoclonal antibody.
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