J Appl Physiol 114: 490–497, 2013.
First published November 29, 2012; doi:10.1152/japplphysiol.00789.2012.
Interaction between myoglobin and mitochondria in rat skeletal muscle
Tatsuya Yamada,1 Yasuro Furuichi,1 Hisashi Takakura,1 Takeshi Hashimoto,2 Yoshiteru Hanai,3
Thomas Jue,4 and Kazumi Masuda1
1
Faculty of Human Sciences, Kanazawa University, Kanazawa, Japan; 2Faculty of Sports Sciences, Ritsumeikan University,
Kusatsu, Japan; 3Department of Frontier Materials, Nagoya Institute of Technology, Nagoya, Japan; and 4Department of
Biochemistry and Molecular Medicine, University of California Davis, Davis, California
Submitted 26 June 2012; accepted in final form 19 November 2012
cytochrome c oxidase-subunit IV; oxygen transport; mitochondrial
respiration
OXYGEN TRANSPORT TO THE SKELETAL muscle is regulated by a
wide range of factors, including blood flow, hematocrit, capillary density, and oxygen diffusivity. In the myocytes, the
oxygen binding protein myoglobin (Mb) should presumably
play an important role. However, the Kroghian model of
oxygen transport does not include any functional role for Mb,
because the intracellular concentration and localization of Mb
have been unclear (21).
From a conventional physiology viewpoint, Mb with its high
affinity to oxygen can serve as an oxygen depot. The stored
oxygen is released when the intracellular PO2 drops to the
critical level. Moreover, Mb can facilitate intracellular oxygen
transport (3), which is believed to support the oxygen flux from
the sarcolemma to the mitochondrial surface (33).
Although in vitro studies have shown Mb diffusivity can
increase oxygen influx from capillary to myocyte, in vivo
experiments have not provided substantiating evidence (3). Mb
in respiring muscle appears to diffuse too slowly to influence
oxygen flux, except in cases of exceedingly low intracellular
PO2 (17, 18). These observations suggest that the role of Mb to
facilitate O2 transport is limited to times when the PO2 is low.
The role of Mb as a facilitator of O2 diffusion remains then
controversial.
Address for reprint requests and other correspondence: K. Masuda, Faculty
of Human Sciences, Kanazawa Univ., Kakuma-machi, Kanazawa-city, Ishikawa
920-1192, Japan (e-mail: [email protected]).
490
Whether respiration increases immediately after the start of
muscle contraction remains a highly debated topic (25, 31).
Some investigators have posited a delay in pulmonary O2
uptake and extraction and have interpreted the data to indicate
an impedance to oxygen (8, 10, 16). Others disagree with the
data interpretation. NMR studies show clearly that Mb releases
immediately its O2 at the start of muscle contraction, consistent
with a rapid rise in O2 consumption (V̇O2) (4). Recent NIRS
experiments on buffer perfused hindlimb also show Mb desaturating immediately (4, 26).
Why Mb desaturates so quickly, given the generally accepted inertia ascribed to oxidative phosphorylation, remains
puzzling (9). However, the sudden increase in V̇O2 at the onset
of contraction does not appear to depend on muscle ADP
concentration and implicates a direct role for Mb-mediated O2
delivery (4, 26). Indeed, previous experiments have suggested
that a direct Mb-mediated oxygen delivery might contribute to
mitochondrial respiration. When the oxygen-binding function
of Mb was blocked by CO in the presence of sufficient free O2
to support oxidative phosphorylation, mitochondrial respiration was still suppressed by ⬃70% (32).
The present study shows that Mb can interact directly with
mitochondria and can co-localize with complexes in mitochondria. This mitochondrial Mb interaction could have a potentially significant role in respiration at the start of muscle
contraction.
MATERIAL AND METHODS
Animals. Male Wistar rats weighing 282–375 g (n ⫽ 12) were used
for the experiments. Animals were housed in an air-conditioned room
and exposed to a 12-h light-dark photoperiod. A standard diet (Oriental Yeast, Tokyo, Japan) and water were provided ad libitum. All
procedures performed in the present study were approved by the
Ethics Committee on Animal Experimentation of Kanazawa University (protocol AP-10187).
Immunohistochemistry. For immunohistochemistry, rats were anesthetized and perfused with Krebs-Henseleit buffer (118 mM NaCl, 5.9
mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 1.8 mM CaCl2, 20 mM
NaHCO3, and 15 mM glucose) into the abdominal aorta for 15 min at
body temperature. The buffer was then switched to 4% paraformaldehyde (PFA) in phosphate buffer (PB; 0.1 M, pH 7.2). After 10 min
of perfusion of 4% PFA, the fixed muscles were dissected. The
muscles were then dipped in 4% PFA in PB overnight at 4°C. After
that, the buffer was changed to 10, 20, and 30% sucrose in 0.1 M PBS
one after another and incubated overnight at 4°C. Samples were stored
at ⫺80°C until histochemical analysis. Serial cross and longitudinal
sections (6 ␮m thick) of the muscles were cut at ⫺20°C in a
cryomicrotome (CMR1510; Leica, Wetzlar, Germany), and the sections were adhered to glass slides. The glass slides were soaked in the
antigen activator reagent at 92°C for 10 min and washed three times
with PBS. Samples were soaked in PBS-T (PBS with 1% Tween 20)
at room temperature for 30 min and then in methanol for 1 min and
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Yamada T, Furuichi Y, Takakura H, Hashimoto T, Hanai Y, Jue
T, Masuda K. Interaction between myoglobin and mitochondria in rat
skeletal muscle. J Appl Physiol 114: 490 – 497, 2013. First published
November 29, 2012; doi:10.1152/japplphysiol.00789.2012.—The mechanisms underlying subcellular oxygen transport mediated by myoglobin (Mb) remain unclear. Recent evidence suggests that, in the
myocardium, transverse diffusion of Mb is too slow to effectively
supply oxygen to meet the immediate mitochondrial oxygen demands
at the onset of muscle contractions. The cell may accommodate the
demand by maintaining the distribution of Mb to ensure a sufficient
O2 supply in the immediate vicinity of the mitochondria. The present
study has verified the co-localization of Mb with mitochondria by
using biochemical histological and electron microscopy analyses.
Immunohistochemical and electron microscopy analysis indicates a
co-localization of Mb with mitochondria. Western blotting confirms
the presence of Mb colocalizes with the mitochondrial fraction and
appears more prominently in slow-twitch oxidative than in fast-twitch
glycolytic muscle. In particular, Mb interacts with cytochrome c
oxidase-subunit IV. These results suggest that a direct Mb-mediated
O2 delivery to the mitochondria, which may play a potentially significant role for respiration.
Myoglobin Interaction with Mitochondria in Muscle
Yamada T et al.
491
fuged at 230,000 g for 120 min at 4°C. The supernatant from this
high-speed spin was used as the cytosolic fraction in the immunoblots.
The mitochondrial pellet was washed initially by resuspension in
buffer A and repelleted at 10,000 g for 30 min at 4°C. This pellet was
then washed twice in buffer C (1 mM EDTA and 10 mM Tris, pH 7.4)
and resuspended in buffer C with 1% NP-40. Then, it was centrifuged
at 1,100 g for 20 min at room temperature, and the supernatant
containing this mitochondrial fraction was used for immunoblots and
adjusted to contain equal protein amounts in all samples.
Western blot analysis was performed as previously described (7).
In brief, equal protein amounts (4.5 ␮g/lane) of samples were loaded
onto 15% SDS-PAGE gels, and proteins were transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was incubated
in blocking buffer and then with an anti-Mb antibody, an antiVDAC-I (Abcam, Cambridge, UK), and an anti-␤-actin (Abcam) at
4°C overnight. It was then reacted with the secondary antibody, and
the signals were visualized by enhanced chemiluminescence (ECL)
(GE Healthcare, Piscataway, NJ). The signal intensity was quantified
with ImageJ imaging software.
Immunoprecipitation. The mitochondrial fraction was obtained by
the same procedure as Western blotting. In this preparation, buffer D
[20 mM Tris·HCl (pH 7.4), 50 mM NaCl, 250 mM sucrose, 50 mM
NaF, 5 mM sodium pyrophosphate, 1 mM DTT, 4 mg/l leupeptin, 50
mg/l trypsin inhibitor, 0.1 mM benzamidine, and 0.5 mM PMSF] was
used instead of buffers A and C.
The mitochondrial fraction of protein was divided into 1-ml PBS
aliquots. Dynabeads (Invitrogen, Carlsbad, CA) were added to the
samples, and then the samples were incubated at 4°C for 1 h.
Dynabeads were collected with magnets, and normal mouse immunoglobulin G (nIgG; Santa Cruz Biotechnology) was added to the
supernatants and incubated at 4°C overnight. Samples were centrifuged at 7,000 g at 4°C for 30 s, and aliquots of this supernatant were
reacted with primary antibodies against Mb, cytochrome c oxidasesubunit IV (COX-IV), and mouse nIgG at 4°C overnight. Dynabeads
were then added and incubated at 4°C for 2 h. The pellet was collected
with magnets and washed with PBS. The final pellet from immunoprecipitation (IP) was analyzed by SDS-PAGE and immunoblotting.
The antibody used for Mb immunoprecipitation and detection was the
same as used for Western blotting. Mouse anti-COX-IV (Abcam) was
used for immunoprecipitation and detection. Mouse anti-GAPDH
(Abcam) and mouse anti-cytochrome c (Santa Cruz Biotechnology)
were used for detection of each protein.
Statistical analysis. All data are expressed as means ⫾ SD. Variables among groups were compared using one-way ANOVA, and a
Tukey-Kramer post hoc test was conducted if the ANOVA indicated
a significant difference. The level of significance was set at P ⬍ 0.05.
RESULTS
Mitochondrial myoglobin detected by immunohistochemistry and electron microscopic observations. Each muscle tissue
was well stained with the Alexa fluor 488-conjugated anti-Mb
(green) and Alexa fluor 568-conjugated anti-VDAC-I [red;
Figs. 1 and 2 show the immunofluorescent images of Mb and
VDAC-I in the cross sections of rat soleus, gastrocnemius
deep, plantaris, and gastrocnemius surface (Sol, GasD, Pla, and
GasS, respectively)]. Mb (green) was detected in almost all
muscle cells (Fig. 1, A, D, G, and J). VDAC-I (red), a specific
mitochondrial protein (Fig. 1, B, E, H, and K), was observed
throughout the stained tissue in all muscle groups. Superposition of the Mb and for VDAC-I images clearly showed colocalization of Mb with mitochondria (Fig. 1, C, F, I, and L). Sol
contains more Mb than GasD or Pla. In GasS, signals of Mb
and VDAC-I (especially Mb) were quite weak (Fig. 1, J and
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subsequently washed for 10 min in PBS. The samples were then
incubated in blocking solution (1% BSA in PBS-T) for 30 min. After
washing, samples were incubated overnight at 4°C with mouse
anti-myoglobin and rabbit anti-voltage-dependent anion channel
(VDAC)-I (Abcam, Cambridge, UK). The secondary antibodies for
Mb and VDAC-I were anti-rabbit antibodies conjugated to Alexa
Fluor 488 (Invitrogen, Carlsbad, CA) and anti-mouse antibodies
conjugated to Alexa Fluor 568 (Invitrogen), respectively. The secondary antibodies were incubated with the slides for 1 h at room
temperature. After being washed, a cover glass with the nuclear
counterstain reagent Dapi-Fluoromount-G (Southern Biotech, Birmingham, AL) was applied to the samples.
The images of the sections were imported with a resolution of
1,360 ⫻ 1,024 pixels, and the exposure time was adjusted depending
on the negative control. The colocalization areas were calculated with
JACoP [plugin software of Image J: version 1.33, NIH, Bethesda, MD
(2)]. Single fibers were distinguished along the lines of the plasma
membrane from the cross-section image. Thresholds were set visually,
according to the intensity of the yellow aspect of each fiber. Fifty
fibers were identified from each muscle, and the regions of colocalization (yellow regions) and total Mb (yellow plus green parts) were
calculated.
Electron microscopic imaging. Isolated gastrocnemius muscle was
fixed with 4% paraformaldehyde (PFA), 0.05% glutaraldehyde (GA)
(distilled EM grade; Electron Microscopy Sciences, Hatfield, PA) in
0.1 M PB, pH 7.4, at 4°C for 90 min, and then the sample was rinsed
three times with 0.1 M PB for 15 min each. Then belly muscle was cut
out into forms of some block. The muscles were dehydrated through
a series of graded ethanol (50%, 70%) at 4°C for 20 min each. The
samples were infiltrated with a 50:50 mixture of ethanol and resin (LR
White; London Resin, Berkshire, UK) three times for 20 min each.
After this infiltration, they require three changes of 100% LR white at
4°C for 1 h each. The samples were transferred to a fresh 100% resin
and were polymerized at 50°C overnight. The blocks were ultra-thin
sectioned at 80 nm with a diamond knife using an ultramicrotome
(Ultracut UCT; Leica), and sections were placed on nickel grids. The
grids were then placed into the rabbit anti-Mb antibody (Santa Cruz
Biotechnology, Santa Cruz, CA) in 1% BSA at 1:1,000 dilution, PBS
for 90 min at room temperature followed by rinsing with 1% BSA,
and PBS three times for 1 min. And then they were floated on drops
of the secondary antibody attached with 10 nm-colloidal gold particles
(Anti Rabbit IgG; British BioCell International) for 1 h at room
temperature. After rinsing with PBS, the grids were placed in 2% GA
in 0.1 M PB. Afterward, the grids were dried and stained with 2%
uranylacetate for 15 min, and then rinsed with distilled water followed
by being secondary-stained with Lead stain solution (Sigma-Aldrich)
at room temperature for 3 min. The grids were observed by a
transmission electron microscope (JEM-1200EX; JEOL) at an acceleration voltage of 80 kV. Digital images (2,048 ⫻ 2,048 pixels) were
taken with a charge-coupled device (CCD) camera (VELETA, Olympus Soft Imaging Solutions).
Western blotting. Animals were anesthetized (50 mg pentobarbital
sodium/100 g body wt). For biochemical studies, four calf muscles
[the soleus (Sol), plantaris (Pla), and gastrocnemius surface (GasS)
and deep portion (GasD)] were quickly isolated; the tissues were
washed in ice-cold saline, removed from the connective tissues and
nerves, and then frozen in liquid nitrogen. Tissues were stored at
⫺80°C. Muscles (⬃300 mg) were homogenized in lysis buffer A (in
mM: 250 sucrose, 5 NaN3, 2 EGTA, 100 PMSF, 1 pepstatin A, 10
leupeptin, 20 HEPES-Na, pH 7.4) with the use of a loose-fitting
Dunce (Teflon-glass) homogenizer (Asahi Techno Glass, Tokyo,
Japan), and then centrifuged at 600 g for 10 min at 4°C to remove
nuclei and debris. One fraction of the supernatant was used as the total
tissue fraction in the immunoblots, whereas the other was centrifuged
at 10,000 g for 30 min at 4°C to precipitate mitochondrial fragments.
The result of the supernatant was diluted with 0.75 vol of buffer B
(1.167 M KCl and 58.3 mM Na4P2O7·10 H2O, pH 7.4), and centri-
•
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Myoglobin Interaction with Mitochondria in Muscle
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Yamada T et al.
Mb
VDAC-I
Merge
B
C
D
E
F
G
H
I
J
K
L
Pla
GasS
K). The colocalization of the Mb and VDAC-I signals were
also quite weak in this muscle group (Fig. 1L).
The longitudinal-sections of GasD have both Mb-rich and
Mb-poor fibers (Fig. 2A), and a colocalization of VDAC-I with
Mb was observed in the Mb-rich fibers (Fig. 2C). However,
GasS has few fibers that express large amounts of Mb (Fig.
2G), and therefore the merged VDAC-I and Mb immunofluorescent images show very few colocalized regions (Figs. 1L
Mb
VDAC-I
Merge
B
C
D
E
F
G
H
I
GasS
Fig. 2. Histochemical images of Mb and mitochondria in
muscle longitudinal sections. Immunofluorescent imaging of
Mb and VDAC-I in the longitudinal sections of GasD and GasS
muscles. Mb is green (A, D, G), VDAC-I (mitochondrial marker) is
red (B, E, H), and colocalization of Mb with mitochondria is
yellow in the merged images (C, F, I). Magnified images of
GasD are the squared areas in A–C. Scale bars are 50 ␮m and
25 ␮m in the magnified images of GasD. Colocalization was
widely observed in Mb-rich GasD fibers, whereas GasS fibers
showed little colocalization.
GasD
GasD
A
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Fig. 1. Histochemical images of myoglobin and mitochondria
in muscle cross sections. Immunofluorescent imaging of myoglobin (Mb) and voltage-dependent anion channel (VDAC)-I in
the cross sections from four rat calf muscles [soleus (Sol),
gastrocnemius deep portion (GasD), plantaris (Pla), and gastrocnemius surface (GasS)]. Mb is green (A, D, G, J); VDAC-I
(mitochondrial marker) is red (B, E, H, K); colocalization of
Mb with mitochondria is yellow in the merged images (C, F, I,
L). Scale bars are 50 ␮m. Signals of colocalization are widely
observed in the oxidative muscles (Sol and GasD), whereas the
glycolytic muscles show minimal colocalization (GasS).
GasD
Sol
A
Myoglobin Interaction with Mitochondria in Muscle
Mb overlapping VDAC-I (%)
100
80
*†
*
60
*
40
20
0
Sol
GasD
Pla
GasS
Fig. 3. Comparison of the colocalization areas. The areas of colocalization
of Mb and mitochondria are significantly higher in Sol and GasD than in
GasS. Moreover, the colocalization area of Sol is higher than that of Pla.
The data are presented as means ⫾ SD of 4 rats and represent 50 muscle
fibers. *Significant difference vs. GasS (P ⬍ 0.05). †Significant difference
vs. Pla (P ⬍ 0.05).
Yamada T et al.
493
E), Pla (Fig. 7C), and GasS (Fig. 7D)]. Mb co-precipitated with
COX-IV from the mitochondrial fraction of each muscle tissue
homogenate (Fig. 7, A–D). Also, COX-IV co-precipitated with
Mb of GasD homogenate (Fig. 7E). The specificity of the
complex composition was further demonstrated by the absence
of COX-IV co-precipitation with IgG HC and IgG LC in Sol
(Fig. 7A), GasD (Fig. 7B), Pla (Fig. 7C), and GasS (Fig. 7D),
and the absence of GAPDH co-precipitations with Mb, nIgG,
and COX-IV (Fig. 7F). GAPDH localizes mainly on cytosol,
whereas a previous study shows it also localizes on mitochondria (27). We also detected GAPDH from mitochondrial fraction (Fig. 7F, lane of mito). The results of GAPDH detection
can be used as a negative control of immunoprecipitation (cf,
DISCUSSION). Cytochrome c was not detected in the immunoprecipitates of either Mb or COX-IV (Fig. 8), an indication that
neither Mb nor COX-IV has interaction with cytochrome c.
DISCUSSION
Mb localization with mitochondria. In the present biochemistry view, Mb exists as a cytosolic protein. In contrast, the
present study shows that Mb can localize in both the cytosol
and the mitochondria. Detection of the colocalization of Mb
with the mitochondrial marker protein VDAC-I (20) demonstrates the presence of Mb in the mitochondria. The mitochondrial fractions did not show any immunoreactivity to the
cytosolic marker protein ␤-actin (5). These observations reveal
that the mitochondrial fraction was well precipitated, and the
cytosolic fraction, including cytosolic Mb, did not contaminate the mitochondrial fraction. Therefore, the Mb detected
in the mitochondrial fraction by Western blotting did not
include cytosolic Mb.
A fraction of Mb in the myocytes was localized with the
mitochondria, as evidenced by immunohistochemical and
Western blotting analysis (Figs. 1, 2, 5). Electron microscopic
images corroborated the colocalization. The immunohistochemical analysis of longitudinal sections showed striped pattern in the areas of colocalization (Fig. 2F). Previous work
revealed that both Mb and mitochondria are abundant in the
I-band of the cardiac cells (15). Thus the striped pattern noted
in the longitudinal section would indicate that Mb and mitochondria interact on the I-bands in the skeletal muscles. The
sarcoplasmic reticulum (SR) localizes around the I-band in the skeletal muscle cells, and, therefore, these data suggest that the
mitochondria could potentially associate physically and functionally with the SR (6) on the I-band region. The electron
microscopic images clearly show that Mb localize in both the
cytosol and the mitochondria (Fig. 4).
The human heart mitochondrial proteome study has detected
Mb in the mitochondria by osmotic shock methods (28). The
results suggested that Mb is present in the mitochondrial outer
membrane. However, the investigators could not confidently
determine whether the detected Mb originated only from the
mitochondria without any cytosolic contamination. Our study
shows clearly that Mb can interact with the mitochondria.
Mechanism of Mb colocalization. How Mb localizes to the
mitochondria remains moot. Most mitochondrial proteins are encoded by nuclear genes and are, therefore, imported from the
cytosol. The protein import machineries of mitochondria are
the translocase of the outer membrane (TOM) complex and the
translocase of the inner membrane (TIM) complex. TOM
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and 2I). The magnified VDAC-I and Mb merged images of
GasD show the stripe pattern indicative of colocalization of Mb
with mitochondria (Fig. 2F). The colocalization area of Mb and
VDAC-1 in Sol, GasD, and Pla was significantly higher than in
GasS. Furthermore, the colocalization area of Sol was higher
than in Pla (Fig. 3).
Positive signals of Mb in electron microscopic imaging were
detected in ultra-thin section of GasD tissue. Most of all Mb in
images were present in cytosol, especially nearby I-bands (Fig. 4),
which were in concordance with our immunofluorescent results
(Fig. 2). Besides, it is noted that some Mb was close to or
localized inside of mitochondria (Fig. 4). These results indicate
that Mb was observed as points localized around or in the
mitochondria.
Mitochondrial myoglobin detected by Western blotting.
Western blot of ␤-actin assessed the cytosolic contamination in
the mitochondrial fraction. The cytosolic protein marker ␤-actin was only found in the cytosolic fractions of Sol (Fig. 5A),
GasD (Fig. 5B), Pla (Fig. 5C), and GasS (Fig. 5D). It showed
no immunoreactivity to the corresponding mitochondrial fraction, which showed the mitochondrial fraction contained no
significant cytosolic contamination (Fig. 5). Moreover, the
mitochondrial outer membrane marker VDAC-I was detected
only in the mitochondrial fraction. There was no immunoreactivity to VDAC-I in the cytosolic fractions of the muscles (Fig. 5).
Western blot revealed the presence of Mb in both the
cytosolic and mitochondrial fractions. Comparison of the mitochondrial Mb levels of the four calf muscles as a fraction of
VDAC-I revealed that slow-twitch oxidative muscles (Sol and
GasD) have more mitochondrial Mb than fast-twitch glycolytic
muscles (GasS; Fig. 6).
Interaction between Mb and COX-IV detected by immunoblotting after immunoprecipitation. Immunoprecipitation experiments on the solubilized mitochondrial fractions determined the subcellular localization and the physical association
between Mb and mitochondrial components. Protein complexes in the mitochondrial fractions were precipitated with
antibodies against Mb and COX-IV, and then probed for
COX-IV, Mb, or GAPDH [Sol (Fig. 7A), GasD (Fig. 7, B and
•
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Myoglobin Interaction with Mitochondria in Muscle
A
•
Yamada T et al.
C
MF
M
Z
M
B
F
Z
M
MF
M
M
E
M
M
Fig. 4. Electron microscopic imaging of Mb in mitochondria. Mb immunogold labeling in GasD tissue shows mitochondrial localization of Mb. A: the wide view
around sarcolemma (bar ⫽ 2 ␮m). B and C: magnified views of subsarcolemmal mitochondria (bar ⫽ 50 nm). D: the wide view of myofibril (bar ⫽ 2 ␮m) and
its magnified view of intermyofibril mitochondria (bar ⫽ 500 nm). Sections are 80 nm thin.
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D
Myoglobin Interaction with Mitochondria in Muscle
total
cyt
mito
B
total
cyt
mito
Mb
VDAC-I
β-actin
C
D
Mb
VDAC-I
β-actin
recognizes and imports the signal sequences of precursor
proteins (24); however, almost all studies on mitochondrial
protein import systems have been conducted with yeast or
bacterial cell models. In the mammalian cell, especially the
skeletal muscle cell, the presence and function of any mitochondrial protein import system still require experimental
verification. In addition, since mitochondria are dynamic organelles that constantly fuse and divide (30), Mb may be
imported in the fusion process.
Previous studies (28, 29) have indicated that Mb might
localize to the mitochondrial outer membrane via heme uptake
by apomyoglobin, which occurs at the mitochondrial outer
membrane (29). Other research has proposed that Mb binds to
the mitochondrial outer membrane via an electrical charge
effect (22). However, our data at present cannot determine the
mechanisms by which the mitochondria interact with Mb.
Nevertheless, the results show an interaction between the
mitochondria and Mb, which may play an important role in
influencing mitochondrial respiration and intracellular oxygen
transport.
Mb interaction with COX-IV. The electron microscopic
images do show some Mb located inside the mitochondria and
suggest a potential Mb interaction with complex IV. The
mitochondria locating under sarcolemma, fairly large mitochondria have more Mb than those locating intermyofibril.
Indeed, COX-IV was detected in the immunoprecipitates of
Mb and COX-IV, whereas it was not detected in the immunoprecipitates of nIgG (Fig. 7, A–D). Comparatively, GAPDH,
which localizes mainly to the cytosol but also to the mitochondria [Fig. 7E, lane of mito (27)], was not detected in the
immunoprecipitates of Mb (Fig. 7E). Since GAPDH localizes
either the cytosol or the mitochondria, the results of GAPDH
detection would be used as a negative control of immunoprecipitation. Undetection of GAPDH in the immunoprecipitates
of Mb indicated that COX-IV detected from the same sample
is not the mitochondrial contaminants. Additionally, undetection of GAPDH in the immunoprecipitates of COX-IV re-
VDAC-I−
Mb−
2.0
*
*
1.6
1.2
0.8
0.4
0
Sol
GasD
Pla
GasS
Fig. 6. Comparison of the mitochondrial myoglobin content of the four rat
calf muscles. The amounts of myoglobin and VDAC-I were determined,
and all calculated data were normalized by the heart muscle values. The
data from the four rats are presented as means ⫾ SD. *Significant
difference vs. GasS (P ⬍ 0.05).
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Fig. 5. Detection of myoglobin in the mitochondrial fraction of rat skeletal
muscles. Mitochondrial marker protein (VDAC-I), cytosolic marker protein
(␤-actin), and Mb in the entire tissue (total), cytosolic fractions (cyt), and
mitochondrial fractions (mito) from each muscle [Sol (A), GasD (B), Pla (C),
and GasS (D)] were determined by Western blotting. The results obtained for
VDAC-I and ␤-actin indicate that the mitochondrial fraction was not contaminated by the cytosolic fraction. Mb was detected in the mitochondrial fractions
of each muscle.
495
Yamada T et al.
vealed that Mb detected from the same sample is not the
cytosolic contaminants. Therefore, GAPDH can be considered
as the negative control for the immunoprecipitation experiment. These results suggest that COX-IV specifically interacts
with the Mb in the electron transfer system (ETS).
Mb-mediated O2 delivery. A previous study has hypothesized a direct Mb-mediated oxygen delivery to accompany
mitochondrial electron flow (32). The observation of Mb
bound to COX-IV appears consistent with a Mb direct role in
affecting the ETS. Furthermore, the presence of mitochondrial
Mb would enable the mitochondria to respire immediately and
to stimulate ATP synthesis at the onset of contractions. Mb
could then support mitochondrial electron transfer and facilitate O2 consumption. Thus the direct interaction of Mb with
COX-IV may have a basic role in intracellular oxygen dynamics.
Mb colocalization in muscle groups. Immunohistochemical
analysis revealed that the area of colocalization in the slowtwitch oxidative muscles was greater than that in the fasttwitch glycolytic muscles (Fig. 3), which was supported by the
results of Western blotting. The Mb content in the mitochondrial fraction was significantly higher in slow-twitch oxidative
muscles (Sol, GasD) than in the fast-twitch glycolytic muscles
(GasS, Pla; Fig. 6). Previous work has demonstrated that the
slow-twitch oxidative fibers possess large amounts of Mb and
mitochondria, whereas fast-twitch glycolytic muscles possess
significantly lesser amounts (23).
Gueguen et al. (12) reported a higher respiration rate in
permeabilized fibers dissected from type I fiber-dominant muscle than those from type II-dominant muscle. Isolated mitochondria from type I muscle fibers also showed faster respiration than those from type II fibers (13). Type I fibers have more
Mb than type II fibers. Our experiments show a larger Mbmitochondria colocalization area in the oxidative muscles relative to the glycolytic muscles, which suggests that the interaction between the mitochondria and Mb may contribute to the
enhanced oxidative capacity and respiration rate of type I
slow-twitch oxidative fibers over type II glycolytic fibers.
Mb / VDAC-I (AU)
A
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Myoglobin Interaction with Mitochondria in Muscle
A
E
B
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Yamada T et al.
IgG HC
IgG HC
IgG LC
IgG LC
COX-IV
Mb
C
D
CON
Mb IgG COX-IV
IgG HC
IP
F
IgG LC
IB : Mb
IgG HC
GAPDH
COX-IV
IP
IgG COX-IV
IP
IB : COX-IV
IB : COX-IV
IgG LC
mito CON
Mb
IgG COX-IV
IP
IB : GAPDH
Fig. 7. Detection of COX-IV from immunoprecipitates of mitochondrial myoglobin. Mb, COX-IV, immunoglobin G heavy chain (IgG HC), and immunoglobin
G light chain (IgG LC) were immunoprecipitated from detergent-solubilized mitochondrial fractions. COX-IV was detected in the immunoprecipitates of
COX-IV and Mb but not in those from nIgG. Co-immunoprecipitation of Mb and COX-IV was confirmed in each muscle [Sol (A), GasD (B), Pla (C), and GasS
(D)]. Mb was detected in the immunoprecipitates of Mb and COX-IV but not in those from nIgG (E). Therefore, COX-IV specifically binds Mb. GAPDH was
detected from mitochondrial fraction, which shows GAPDH localizes on mitochondria. GAPDH was not detected in the immunoprecipitates (F). CON is the
control sample (entire heart tissue sample).
Moreover, endurance training increases both CS activity and
Mb concentration, implying that Mb may play a role in enhancing both O2 storage and Mb-mediated O2 delivery. The
Mb concentration is also increased by endurance training in rat
skeletal muscle (1, 19), implying an increase in O2 storage and
O2 flux mediated by Mb.
IgG HC
IgG LC
Cyto.C
CON
Mb IgG COX-IV
IP
IB : Cyto.C
Fig. 8. Detection of cytochrome c from immunoprecipitates of mitochondrial
myoglobin. Cytochrome c detection was also performed in the immunoprecipitates of Mb, COX-IV, and nIgG with GasD muscle tissue to confirm
specific protein-protein interaction of Mb and COX-IV. Cytochrome c was not
detected in either immunoprecipitate, which shows mitochondrial Mb does not
interact with cytochrome c.
Implication for respiratory control. The localization of Mb
to the mitochondria and to COX-IV could potentially facilitate
the rise in V̇O2 at the start of muscle contraction. Much debate
surrounds the V̇O2 on-kinetics at the start of muscle contraction
(25, 31). Proponents for a V̇O2 or metabolic impedance point to
delays observed in the pulmonary O2 uptake and extraction
data. Opponents point to an errant analysis in interpreting noisy
breath-to-breath O2 uptake data and in using changes in blood
flow and oxygen extraction to correlate precisely to any metabolic surge, especially in respiration, without considering the
role of Mb.
Indeed, the NMR and NIRS have observed the intracellular
Mb deoxygenating immediately at the onset of muscle contraction (4, 26). The rapid release of O2 from Mb implies that
respiration has suddenly stepped up with no intervening
delays. NIRS experiments on buffer perfused hindlimbs
have corroborated the NMR findings and have also detected
an immediate desaturation of Mb, consistent with a rapid
surge in respiration.
However, recent research also shows that the diffusivity of
Mb appears much slower than that previously expected (11, 14,
17, 18). These observations raise questions about how Mb can
respond quickly to the increase in the mitochondrial oxygen
demand. The presence of a myoglobin-mitochondria complex
would provide an alternative route of Mb-mediated O2 delivery
to the mitochondria. Mb colocalization with the mitochondria,
specifically with COX-IV, presents a new paradigm to understand the regulation of intracellular oxygen transport and mitochondrial respiration.
J Appl Physiol • doi:10.1152/japplphysiol.00789.2012 • www.jappl.org
Downloaded from http://jap.physiology.org/ by 10.220.32.246 on June 17, 2017
CON Mb IgG COX-IV CON Mb
Myoglobin Interaction with Mitochondria in Muscle
In conclusion, the present study presents experimental evidence that Mb localizes to the mitochondria and interacts with
COX-IV. In addition, the mitochondrial Mb content and the
area of colocalization are significantly higher in the slowtwitch oxidative muscles compared with the fast-twitch glycolytic muscles. Taken together, these findings suggest that Mb
can directly interact with the mitochondria. The specific interaction of Mb and COX-IV (complex IV) remains an open
question. Mb can bind to COX-IV from the matrix or intermembrane side and may be mediated by other proteins. Further
studies must then clarify the Mb-mitochondria and MbCOX-IV interaction, especially with respect to the impact on
respiration.
GRANTS
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: T.Y. and K.M. conception and design of research;
T.Y. and Y.F. performed experiments; T.Y. and H.T. analyzed data; T.Y.,
H.T., T.J., and K.M. interpreted results of experiments; T.Y. and K.M.
prepared figures; T.Y. and K.M. drafted manuscript; T.Y., T.H., Y.H., T.J., and
K.M. edited and revised manuscript; T.Y. and K.M. approved final version of
manuscript.
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J Appl Physiol • doi:10.1152/japplphysiol.00789.2012 • www.jappl.org
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This research was supported by a Grant-in-Aid for scientific research from
the Japanese Ministry of Education, Science, Sports, and Culture (20680032,
21650167; K. Masuda), with partial support from the Yamaha Motor Foundation for Sports (K. Masuda). The international collaboration was supported by Japan Society for the Promotion of Science, Bilateral Programs
(7301001471; K. Masuda).
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