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Am J Physiol Cell Physiol 300: C1280–C1290, 2011.
First published February 2, 2011; doi:10.1152/ajpcell.00496.2010.
Protein composition and function of red and white skeletal
muscle mitochondria
Brian Glancy and Robert S. Balaban
Laboratory of Cardiac Energetics, National Heart, Lung, and Blood Institute, National Insitutes of Health,
Bethesda, Maryland
Submitted 6 December 2010; accepted in final form 1 February 2011
proteomics; fast and slow-twitch muscle; iTRAQ; energetics; oxidative phosphorylation
SKELETAL MUSCLE-specific oxygen consumption can increase
over 100-fold from rest to maximal oxygen uptake (V̇O2max)
(52). An additional threefold increase in power output can be
achieved during maximal anaerobic exercise (43). Thus skeletal muscle is faced with a wide range of energetic demands
dependent on both exercise intensity and duration. Slow and
sustained activities such as maintaining posture or low-intensity exercise result in the recruitment of red, oxidative muscle
fibers. Conversely, shorter bouts of high-intensity exercise are
accomplished by the activation of white, glycolytic myocytes.
Accordingly, protein expression in red and white fibers is tuned
to meet their respective energetic demands. White muscle is
characterized by a predominance of glycolytic enzymes and the
Address for reprint requests and other correspondence: B. Glancy, NHLBI,
NIH, 10 Center Dr., Rm. B1D416, Bethesda, MD 20892 (e-mail: glancybp
@nhlbi.nih.gov).
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fast isoforms of contractile proteins, whereas red muscle has
greater abundance of contractile protein slow isoforms and
higher oxidative enzyme content (16, 38, 51). The larger
oxidative capacity in red muscle is due, at least in part, to a
two- to threefold greater mitochondrial content compared with
white muscle (19, 24). However, how these mitochondria in
different muscle types are poised to perform different tasks by
their relative mitochondrial protein expression is unclear.
Despite the large difference in mitochondrial content, red
and white myocytes rest at the same tissue-specific oxygen
consumption rate (32, 39) and, surprisingly, white muscle
fibers maintain a higher resting energetic state (32–34). These
results suggest qualitative differences in the regulation of
oxidative phosphorylation between red and white muscle. Indeed, Jackman and Willis (24) found that mitochondria isolated from rabbit soleus had greater electron transport chain
(ETC) activity and maximal (State 3) respiration with pyruvate ⫹
malate (P⫹M) or palmitoyl carnitine ⫹ malate (PC⫹M) as
oxidative substrates than did mitochondria from gracilis muscle. However, Schwerzmann and colleagues (47) found no
difference in State 3 respiration with P⫹M, glutamate ⫹
malate (G⫹M), or succinate in mitochondria isolated from cat
soleus and gracilis. Furthermore, while Pande and Blanchaer
(40) found no difference with P⫹M, they did find higher State
3 respiration with PC⫹M in mitochondria from red muscle
compared with white. These conflicting results necessitate a
deeper look into potential differences between mitochondria
from red and white skeletal muscle.
The goal of this study was to evaluate the nuclear programming of mitochondrial protein expression in red and white
skeletal muscle and to relate compositional differences with
functional outcomes. We hypothesized that mitochondria from
red muscle would have a greater capacity for fat oxidation and
higher ETC activity. To test these predictions we examined the
mitochondrial proteome using two-dimensional (2D) differential in gel electrophoresis (DIGE) and isobaric tag for relative
and absolute quantitation (iTRAQ) labeling with mass spectrometry, assessed ETC complex content, composition, and
activity by blue native (BN)-PAGE and optical spectroscopy,
and related protein content to function by measuring State 3
respiration with different fuels as well as isocitrate dehydrogenase activity.
MATERIALS AND METHODS
Mitochondrial isolation. All procedures were approved by the
National Heart, Lung, and Blood Institute ACUC and performed in
accordance with the guidelines described in the Animal Care and
Welfare Act (7 USC 2142-13). Porcine vastus intermedius [red, 70%
type I fibers (48)] and gracilis [white, 70% type IIb fibers (53)]
muscles were excised upon death and immediately placed in ice-cold
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Glancy B, Balaban RS. Protein composition and function of red
and white skeletal muscle mitochondria. Am J Physiol Cell Physiol 300: C1280 –C1290, 2011. First published February 2, 2011;
doi:10.1152/ajpcell.00496.2010.—Red and white muscles are faced
with very different energetic demands. However, it is unclear whether
relative mitochondrial protein expression is different between muscle
types. Mitochondria from red and white porcine skeletal muscle were
isolated with a Percoll gradient. Differences in protein composition
were determined using blue native (BN)-PAGE, two-dimensional
differential in gel electrophoresis (2D DIGE), optical spectroscopy,
and isobaric tag for relative and absolute quantitation (iTRAQ).
Complex IV and V activities were compared using BN-PAGE in-gel
activity assays, and maximal mitochondrial respiration rates were
assessed using pyruvate (P) ⫹ malate (M), glutamate (G) ⫹ M, and
palmitoyl-carnitine (PC) ⫹ M. Without the Percoll step, major cytosolic protein contamination was noted for white mitochondria. Upon
removal of contamination, very few protein differences were observed
between red and white mitochondria. BN-PAGE showed no differences in the subunit composition of Complexes I–V or the activities of
Complexes IV and V. iTRAQ analysis detected 358 mitochondrial
proteins, 69 statistically different. Physiological significance may be
lower: at a 25% difference, 48 proteins were detected; at 50%, 14
proteins were detected; and 3 proteins were detected at a 100%. Thus
any changes could be argued to be physiologically modest. One area
of difference was fat metabolism where four ␤-oxidation enzymes
were ⬃25% higher in red mitochondria. This was correlated with a
40% higher rate of PC⫹M oxidation in red mitochondria compared
with white mitochondria with no differences in P⫹M and G⫹M
oxidation. These data suggest that metabolic demand differences
between red and white muscle fibers are primarily matched by the
number of mitochondria and not by significant alterations in the
mitochondria themselves.
MITOCHONDRIAL PROTEIN COMPOSITION BY MUSCLE FIBER TYPE
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mM glycine, 14 mM MgSO4, pH 7.8) for 30 min followed by 30 min
in incubation buffer [preincubation buffer plus 0.2% wt/vol Pb(NO3)2
and 8 mM ATP, pH 7.8]. Gels were placed in water and then imaged
against a CB background with infrared lights. Images shown below
were converted to grayscale. Complex IV activity was measured by
incubating gel lanes in 50 mM KPO4, pH 7.2, 0.05% wt/vol diaminobenzidine, and 0.0001% wt/vol bovine heart cytochrome c and imaging against a white light background. Activities per mole enzyme were
quantified by comparing the optical density of the activity band to the
respective protein band for both red and white mitochondria using
ImageJ software (National Institutes of Health, Bethesda, MD).
Differential gel electrophoresis. 2D DIGE was performed on whole
tissue homogenates and isolated mitochondria from red and white
skeletal muscle. Protein (1 mg) was suspended in 250 ␮l lysis buffer
[15 mM Tris·HCl, 7 M urea, 2 M thiourea, and 4% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) (wt/vol)]
and kept on ice with frequent vortexing for 5 min. Samples were then
spun at 16,000 g for 10 min at 4°C, and the supernatant was
transferred to a fresh microcentrifuge tube. This was repeated two
more times. Red (25 ␮g), white (25 ␮g), and red ⫹ white (12.5 ␮g
each) samples were mixed with 0.3 nmol Cy5, Cy3, or Cy2 dyes,
respectively (GE Healthcare, Piscataway, NJ) in 1 ␮l dimethylformamide and incubated in the dark for 30 min on ice. To quench protein
binding to the CyDyes, 1 ␮l of 10 mM lysine was then added to each
sample and incubated on ice for at least 15 min. Samples were
combined and added to rehydration solution [7 M urea, 2 M thiourea,
4% CHAPS (wt/vol), 13 mM DTT, 1% pH 3–11NL Pharmalyte
(vol/vol), and 2 ␮l of Destreak reagent] to a final volume of 210 ␮l
and placed on ice for 5 min before being loaded onto 11 cm
Immobiline DryStrip gels (pH 3–11NL, Sigma-Aldrich, St. Louis,
MO). Isoelectric focusing (IEF) was achieved by active rehydration
for 12 h at 30 V followed by stepwise application of 500 V (1 h),
1,000 V (1 h), a gradient to 6,000 V (2 h), and a final step at 6,000 V
(1.2 h) for a total of ⬃15,000 volt hours (Ettan IPG Phor2). After IEF,
gel strips were incubated in SDS equilibration solution plus 0.05 g
DTT for 10 min. The gel strip was then placed atop an 8 –16%
Tris·HCl gel and run as described above for 2D BN-PAGE. Upon
completion of SDS-PAGE, gels were imaged on a Typhoon variable
mode imager at a resolution of 100 ␮m.
iTRAQ labeling, mass spectrometry, and protein identification.
Samples were labeled using the iTRAQ Reagents 8plex Kit (Applied
Biosystems, Foster City, CA). Mitochondria, 200 ␮g lysed proteins
(see above), were added to six volumes of cold acetone (⫺20°C) and
incubated for 1 h at ⫺20°C. After being spun at 13,000 g for 10 min
and the supernatant discarded, 20 ␮l dissolution buffer, 2 ␮l reducing
reagent, and 1 ␮l denaturant were added. The samples were then
incubated in a shaking water bath at 60°C for 1 h. Cysteine block (1
␮l) was added, and the samples were incubated in a room temperature
shaking water bath for 10 min followed by the addition of 20 ␮g
trypsin. The trypsinized samples were incubated overnight at 37°C.
Isopropanol (50 ␮l) was added to iTRAQ reagents 113–119 and 121,
and each reagent was added to a different sample. Tetraethylammonium bicarbonate was added to bring the pH above 7.5, and the
samples were incubated for 2 h at room temperature before combining
all samples and drying with a Speedvac. After being reconstituted
with 0.1% formic acid (FA), the digest was desalted on a Waters Oasis
HLB column and eluted with 60% acetonitrile (ACN) ⫹ 0.1% FA.
The eluted peptide mixture was then dried by Speedvac.
The sample was reconstituted with 100 ␮l strong cation exchange
buffer A (10 mM KH2PO4, 20% ACN, pH 2.7) and separated on a
PolyLC PolySULFOETHYL A column (200 ⫻ 2.1 m, 5 ␮m, 200 Å)
with a linear 200 ␮l/min gradient of 0 –70% buffer B (10 mM
KH2PO4, 20% ACN, 500 mM KCl, pH 2.7) in 45 min on an Agilent
1200 LC device with Chemstation B.02.01 control software. Fractions
were collected each minute and eventually pooled into 24 fractions.
After being dried by Speedvac, the fractions were desalted and eluted
on a Waters Oasis HLB column as described above. Samples were
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isolation buffer (in mM: 280 sucrose, 10 HEPES, 1 K2EDTA, 1
EGTA, pH 7.1). After fat and connective tissue were trimmed off, 60
g of muscle were weighed out, cut into small pieces on a glass Petri
dish on ice, placed into 270 ml isolation buffer, and homogenized with
a commercial food processor covered by ice for 2 min at 30% power.
Protease (50 mg Subtilisin A) was added and the mixture continually
stirred for 15 min when 50 ml of isolation buffer plus 1% wt/vol fatty
acid-free BSA was added. The solution was centrifuged at 800 g for
10 min at 4°C to pellet down contractile protein and cellular debris.
The supernatant was decanted through a double layer of cheesecloth,
phenylmethylsulfonyl fluoride (3 mg/40 ml) was added, and the
solution was centrifuged at 8,000 g for 10 min to pellet down the
mitochondrial fraction. The supernatant was discarded, and 80 ml
isolation buffer was added to the pellet. After gently being removed
from the side of the centrifuge tube, the pellet was resuspended slowly
with four to five passes of a tight-fitting pestle in a dounce homogenizer and centrifuged again at 8,000 g for 10 min. The supernatant was
discarded and the pellet was resuspended in 3.4 ml of wash solution
(250 mM sucrose, 10 mM HEPES, 0.1% wt/vol fatty acid-free BSA,
pH 7.4). Four milliliters of the crude mitochondrial suspension were
placed atop 32 ml of Percoll solution (30% vol/vol Percoll, 250 mM
sucrose, 10 mM HEPES, 0.1% wt/vol fatty acid-free BSA, pH 7.4)
and centrifuged at 68,000 g for 40 min. Typically, two layers formed
during the Percoll gradient, a top layer containing cytosolic contaminants and a bottom layer containing the purified mitochondrial
fraction. Occasionally, a small layer of blood would form below the
mitochondrial layer. The mitochondrial layer was removed using a
plastic transfer pipet, and wash solution was added until the volume
was 40 ml. After being centrifuged for 10 min at 10,000 g, the
supernatant was discarded and the pellet resuspended in 40 ml wash
solution followed by a 10-min spin at 10,000 g. The final mitochondrial pellet was suspended in 500 ␮l solution B (in mM: 137 KCl, 10
HEPES, 2.5 MgCl2, 0.5 K2EDTA, pH 7.1) yielding a protein concentration of 20 –35 mg/ml measured using a Bradford Assay (USB
Quant Kit, Cleveland, OH).
Optical spectroscopy. ETC cytochrome content was measured by
adding 50 ␮l mitochondria to 950 ␮l of a 2% Triton, 100 mM
phosphate buffer, pH 7.0, and recording absorbance from 300 to 800
nm (oxidized). Sodium hydrosulfite was then added and absorbance
again recorded from 300 to 800 nm (reduced). Cytochrome contents
were calculated using the optical absorbance differences between the
oxidized and reduced spectra (6) using a custom-written IDL program
(Research Systems, Boulder, CO) and millimolar extinction coefficients of 12, 14, and 21 for cytochromes a,a3, b, and c⫹c1, respectively. Cytochrome b and c spectra were separated using oxidized and
reduced spectra from purified cytochrome c.
Native gel electrophoresis. BN-PAGE was performed using the
NativePAGE Novex Bis-Tris System (Invitrogen, Carlsbad, CA) with
4 –16% 1 mm bis-Tris gels. Solubilized mitochondria (30 ␮g) were
added to each lane and run at 4°C for 1 h at 150 V and 1.3 h at 250
V. For 2D BN-PAGE, gel lanes were cut and incubated in SDS
equilibration solution [50 mM Tris·HCl (pH 8.8), 6 M urea, 30%
glycerol, and 2% SDS] plus 0.1 g DTT for 10 min. After equilibration
entire lanes or individual complex bands were applied to SDS-PAGE
gels. Two whole lanes were placed atop an 8 –16% Tris·HCl gel,
sealed with 0.5% agarose plus bromophenol blue, and run in TGS
buffer (25 mM Tris, 192 mM glycine, 0.1% wt/vol SDS, pH 8.3) at
room temperature for 10 min at 50 V and 70 min at 180 V. Individual
bands were placed in 16.5% Tris-Tricine gel lanes and sealed and run
in TGS buffer as described for the whole gel lanes. After SDS-PAGE,
gels were stained in Coomassie blue (CB) (0.75% wt/vol CB, 30%
methanol, 3% phosphoric acid) overnight. The following morning,
gels were destained in 30% methanol and 3% phosphoric acid and
imaged.
In-gel activity assays were performed on BN-PAGE gels immediately after electrophoresis. Complex V activity was measured by
incubating gel lanes in preincubation buffer (35 mM Tris·HCl, 270
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MITOCHONDRIAL PROTEIN COMPOSITION BY MUSCLE FIBER TYPE
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RESULTS
White muscle has lower mitochondrial content but similar
mitochondrial composition. The large difference in protein
content between red and white skeletal muscle is depicted in
Fig. 1A as most protein spots are red or green, signifying higher
protein content in red or white muscle, respectively. ATP
synthase ␤-subunit, the most abundant mitochondrial protein in
red and white muscle, is highlighted in Fig. 1, B and C, to
demonstrate the twofold difference in mitochondrial content
between muscle types. In contrast to the whole muscle DIGE,
Fig. 1D demonstrates that mitochondria isolated from red and
white skeletal muscle have very similar protein composition
because nearly all the protein spots are yellow or the same
between mitochondrial types. Proteins listed in Fig. 1, A and D,
are those that have been identified many times by our laboratory (4, 5, 8, 20, 27, 41, 45) as well as by Kim et al. (30).
Mitochondria from red and white skeletal muscle have
similar oxidative phosphorylation components. Native protein
complexes were evaluated in red and white muscle mitochondria using BN-PAGE. The similarity between red and white
mitochondrial protein complexes is shown in Fig. 2A. In
addition to similar complex contents, the activities of ETC
complexes IV and V were also no different between mitochondrial types (Fig. 2, B–D). Separation of whole BN-PAGE gel
lanes by molecular weight (Fig. 3) suggests little difference in
the composition of each native complex between red and white
skeletal muscle mitochondria. Examining the composition of
ETC Complexes I, III, IV, and V individually also yields no
differences (Fig. 4).
Contents of ETC cytochromes a,a3, b, and c⫹c1 per milligram of mitochondrial protein were quantified based on optical
density. Table 1 shows that both red and white skeletal muscle
mitochondria have about 1 nmol cytochrome a,a3/mg of mitochondria and that there is no difference in cytochrome b or
c⫹c1 contents.
iTRAQ quantification suggests modest protein differences
between red and white skeletal muscle mitochondria. There
were 485 unique proteins identified and quantified with iTRAQ
labeling and mass spectrometry. Of those proteins, 481 were
identified in all six samples yielding three red-to-white protein
ratios. Two ratios were obtained for the remaining 4 proteins.
Of the 485 proteins, 358 (74%) were considered mitochondrial
proteins. Though mass spectrometry was sensitive enough to
detect 127 nonmitochondrial proteins, the abundance of most
was low enough to not be seen in 2D DIGE gels (Fig. 1B).
Indeed, 88% of the spectra originated from mitochondrial
proteins. The largest sources of nonmitochondrial spectra were
trypsin (2.4%) and myosin isoforms (1.6%). Nonmitochondrial
contamination was no different between mitochondrial types.
There were significant differences in 69 proteins between
red and white mitochondria. Of those 69, 48 proteins were at
least 25% different and are shown in Table 2. The only
apparent whole pathway difference between mitochondrial
types, whether employing the 25% threshold or not, was in fat
metabolism. Proteins associated with all four ␤-oxidation enzymes were at least 25% higher in red muscle mitochondria. A
full list of identified proteins, ratios, and P values can be found
in Supplemental Materials online at the AJP-Cell Physiol
website.
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dried again and reconstituted with 0.1% FA. Liquid chromatography
was performed on an Eksigent nanoLC-Ultra 1D plus system (Dublin,
CA). Peptide digest was first loaded on a Zorbax 300SB-C18 trap
(Agilent, Palo Alto, CA) at 6 ␮l/min for 5 min and then separated on
a PicoFrit analytical column (100 mm long, ID 75 ␮m, tip ID 10 ␮m,
packed with BetaBasic 5-␮m 300 Å particles, New Objective,
Woburn, MA) using a 40-min linear gradient of 5–35% ACN in 0.1%
FA at a flow rate of 250 nl/min. Mass analysis was carried out on an
LTQ Orbitrap Velos (Thermo Fisher Scientific, San Jose, CA) with
data-dependent analysis mode, where MS1 scanned full MS mass
range from m/z 300 to 2,000 at 30,000 mass resolution, and six HCD
MS2 scans were sequentially carried out at resolution of 7500 with
45% collision energy, both in the Orbitrap.
MS/MS spectra from 24 fractions were searched against the Swiss
Prot (Swiss Institute of Bioinformatics, updated August 10, 2010,
21241 entries) database, taxonomy Mammalia using a six-processor
Mascot (Matrix Science, London, UK; version 2.3) cluster at NIH
(http://biospec.nih.gov), with precursor mass tolerance at 20 ppm,
fragment ion mass tolerance at 0.05 Da, trypsin enzyme with 2
miscleavages, methyl methanethiosulfonate of cysteine and iTRAQ
8plex of lysine and the NH2-terminus as fixed modifications, and
deamidation of asparagine and glutamine, oxidation of methionine,
and iTRAQ 8plex of tyrosine as variable modifications. The resulting .dat file was loaded into Scaffold Q⫹ (version Scaffold_3_00_04,
Proteome Software, Portland, OR) to filter and quantify peptides and
proteins. Peptide identifications were accepted at 80.0% or higher
probability as specified by the Peptide Prophet algorithm (28) and an
false discovery rate (FDR) of ⬍1%. Protein identifications were
accepted at 90.0% or higher probability and contained at least two
identified peptides with FDR ⬍1%. Protein probabilities were assigned by the Protein Prophet algorithm (37). Proteins that contained
similar peptides and could not be differentiated based on MS/MS
analysis alone were grouped to satisfy the principles of parsimony.
Peptides were quantified using the centroided reporter ion peak
intensity, with minimum of 5% of the highest peak in the spectrum.
Intrasample channels were normalized based on the median ratio for
each channel across all proteins. The isobaric-tagged samples were
normalized by comparing the median protein ratios for the reference
channel. Quantitative protein values were derived from only uniquely
assigned peptides. Protein quantitative ratios were calculated as the
median of all peptide ratios.
Mitochondrial respiration. Mitochondrial oxygen consumption (Jo)
was measured using a Clark-type electrode in a water-jacketed chamber maintained at 37°C. Incubations were carried out in a 1.5-ml final
volume of respiration medium containing (in mM) 100 KCl, 50
MOPS, 20 glucose, 10 K2PO4, 10 MgCl2, 1 EGTA, and 0.2% wt/vol
BSA, pH 7.00. Mitochondria, 375 ␮g mitochondrial protein were
added to the chamber with either pyruvate ⫹ malate (P⫹M, 1 mM
each), glutamate (G) ⫹ M (10 mM ⫹ 1 mM), or palmitoyl-carnitine
(PC) ⫹ M (10 ␮M ⫹ 1 mM) as oxidative substrates. Preliminary
experiments concluded that 1.3 mM ADP was necessary to elicit
maximal State 3 Jo. This amount of ADP results in the consumption
of all the oxygen in the chamber while still at State 3. Thus State 4
(resting) respiration was determined before State 3 upon the phosphorylation of a 0.13 mM addition of ADP. The respiratory control
ratio (RCR) was determined as the ratio of State 3/State 4 (13).
Isocitrate dehydrogenase activity. NAD-linked isocitrate dehydrogenase (IDH) activity in red and white skeletal muscle mitochondria
was measured by following the appearance of NADH at 340 nm in
0.05% Triton buffer containing (in mM): 50 Tris acetate, 8 isocitrate,
1.3 MnCl2, 0.7 ADP, and 0.33 NAD, pH 7.4 (11). Measurements were
made on the same samples used for iTRAQ analysis.
Statistical analyses. All data were analyzed using a paired Student’s t-test. A P value of 0.05 was used to determine significant
differences.
MITOCHONDRIAL PROTEIN COMPOSITION BY MUSCLE FIBER TYPE
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Mitochondrial function correlates with protein abundance.
For red skeletal muscle mitochondria, there was no difference
in maximal respiration or RCR between G⫹M (491.7 ⫾ 73.2
and 13.1 ⫾ 1.9), P⫹M (510.5 ⫾ 77.7 and 12.1 ⫾ 2.3), or
PC⫹M (448.2 ⫾ 30.9 and 10.4 ⫾ 2.4). Conversely, white
muscle mitochondria respiring on PC⫹M had lower State 3
Jo and RCR (321.2 ⫾ 12.4 and 4.7 ⫾ 0.9) than with G⫹M
(470.0 ⫾ 19.5 and 10.3 ⫾ 2.6). Figure 5 also shows that white
muscle mitochondria had a 40% lower State 3 Jo with PC⫹M
compared with red muscle mitochondria. Differences in RCR
between red and white mitochondria did not reach statistical
significance.
Previous reports showed higher NAD-linked IDH activity in
white mitochondria compared with red (21, 24). iTRAQ analAJP-Cell Physiol • VOL
ysis here showed a less than 12% difference in NAD-IDH
subunits ␣ and ␤, however, the ␥ subunit was 85 ⫾ 24% higher
in white mitochondria. We measured NAD-IDH activity on the
same samples used for iTRAQ analysis and found that white
muscle mitochondria had 57 ⫾ 10% higher activity than red
muscle mitochondria (Fig. 6).
DISCUSSION
The current study demonstrates that protein content and
function are similar between mitochondria from red and white
porcine skeletal muscle. Red and white skeletal muscle mitochondria contain ⬃1 nmol of cytochrome a,a3 per milligram of
protein (Table 1), similar to that value found in porcine heart
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Fig. 1. Two-dimensional (2D) differential in
gel electrophoresis (DIGE) of whole muscle
homogenates (A) and isolated mitochondria
(D) from red and white porcine skeletal muscle. Red spots signify higher protein content
in red muscle, green spots are higher in white
muscle, and yellow spots mean equal protein
content. Whole muscle ATP synthase, ␤ subunit is highlighted (B is red, C is white) to
represent the difference in mitochondrial content between fiber types.
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MITOCHONDRIAL PROTEIN COMPOSITION BY MUSCLE FIBER TYPE
(22) and are capable of nearly identical maximal respiration
rates (Fig. 5). The primary difference found between red and
white muscle mitochondria was in fat oxidation. Protein expression of the entire ␤-oxidation pathway was upregulated in
red mitochondria (Table 2), which resulted in a higher rate of
maximal respiration with a fat fuel source compared with white
mitochondria (Fig. 5).
A key feature of this study was the use of a Percoll gradient
during the mitochondrial isolation process. Without the Percoll
gradient step, major cytosolic protein contamination was noted
in the 2D DIGE of the white but not red muscle mitochondria
(results not shown). The disproportionate contamination between mitochondrial types before using the Percoll gradient
may explain some of the discrepancies in the literature. Previous studies looking at differences in maximal respiration between mitochondria from red and white muscle have found
State 3 respiration with P⫹M higher in red (24, 36) or no
different (2, 7, 40, 47, 54); with G⫹M, higher in white (10) or
no different (47); with succinate, higher in red (7) or no
different (47); and with PC⫹M, higher in red (24, 36, 40). Of
these studies, only Schwerzmann et al. (47) reported using a
Percoll gradient, and, similar to our results (Fig. 5), they found
no difference in State 3 respiration between red and white cat
muscle mitochondria with P⫹M or G⫹M. These results suggest that great care must be taken when comparing skeletal
muscle mitochondrial samples as results normalized to milligram of protein will always be skewed toward a lesser contaminated sample. Marker enzyme activities, such as citrate
synthase, are often used in an attempt to avoid this problem;
AJP-Cell Physiol • VOL
however, citrate synthase activity is reportedly different between red and white muscle mitochondria (24) making it a
potentially unreliable marker for these types of studies. As
such, the use of a purification step such as a Percoll gradient or
a protein screen such as a 2D DIGE is recommended when
comparing mitochondrial samples from muscles that may differ in fiber type composition.
Increased expression of glycolytic and fast isoforms of
contractile proteins and decreased expression of oxidative
enzymes in white compared with red skeletal muscle has been
well-characterized previously (9, 16, 23, 29, 38, 51), a profile
similar to which was found between the red and white porcine
muscles used in this study (Fig. 1A). However, because mitochondria can comprise ⬍3% of skeletal muscle volume density
(19), it is difficult to discern relative mitochondrial protein
expression differences from mitochondrial content differences
in the whole muscle studies. Using our isolated mitochondrial
preparation, we were able to compare and quantify the relative
expression of 358 mitochondrial proteins from red and white
skeletal muscle. Only 3 of 89 detected proteins associated with
ETC Complexes I–V were significantly and 25% different
between red and white mitochondria (Table 2). Further support
for these results is shown by Figs. 1D and 2– 4 in which no
apparent differences in the abundance of oxidative phosphorylation complexes or their respective subunits are found. Moreover, the contents of cytochromes a, b, and c were no different
between red and white muscle mitochondria (Table 1).
It is important to point out that this protein content data is
only one of the elements in the complex control of mitochon-
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Fig. 2. Native protein complexes in red and white muscle mitochondria were resolved by blue native (BN)-PAGE (A). Whole gel lane density traces for both
mitochondrial types are shown to the right of the gel. Activities of Complexes IV (C) and V (B) were assessed with BN-PAGE in-gel activity assays. Activities
of Complex IV and Complex V per mole of enzyme relative to red mitochondria are shown (D).
MITOCHONDRIAL PROTEIN COMPOSITION BY MUSCLE FIBER TYPE
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dria function. Though the protein contents might be very
similar, the regulatory mechanisms including allosteric factors,
substrates, and posttranslational modifications can completely
change the flux patterns. Thus this analysis of protein content
can only provide information on the potential flux of a given
pathway. The actual physiological flux is likely also influenced
by a host of other factors not fully understood in this complex
network. One approach to look for posttranslational modifications is to assay extracted activity of the enzymes involved. To
begin this process, we assayed the activity of Complex IV and
V activity as well as maximal respiration of the isolated
mitochondria. With the use of BN-PAGE in-gel activity assays,
Complex IV and V activities per mole of enzyme were no
different between mitochondria from red and white skeletal
muscle (Fig. 2, B–D). Given that both Complex IV and V have
been suggested to be sites of flux control (18, 55), perhaps it is
not surprising then that State 3 respiration with both P⫹M and
G⫹M were no different between red and white mitochondria
(Fig. 5). It should be stressed that these assays were conducted
on mitochondria extracted from anesthetized resting muscle.
Thus any persistent posttranslational modifications of the complexes related to work load will be in the resting muscle state.
As previously shown in the heart (46), extracted complex V
activity increases with workload consistent with a posttranslational modification. Whether this occurs in skeletal muscle has
yet to be established. These functional results combined with
the proteomic data above and previous findings that the Km
values for both ADP and Pi are no different between mitochondria isolated from red and white muscle (36, 44) suggest that
many of the acute regulatory mechanisms within these mitochondria are also similarly poised when isolated from resting
muscle. This similarity in protein content and activity level per
mole enzyme, for those we queried, suggest that the configuAJP-Cell Physiol • VOL
ration of mitochondria in these two muscle types is nearly
identical to produce ATP as a function of mitochondrial volume. This might represent an “optimal” ratio of mitochondrial
volume to ATP production to optimize the use of cell volume
for energy support of fiber contraction. The use of a minimal
volume of the “optimal” mitochondria would reserve the area
within the cell to optimize the cross-sectional area of fibers for
contractile power, independent of the muscle action.
Tissue-specific differences in function are generally met
with upregulation of protein expression of entire pathways
(26). The increased expression of all ␤-oxidation enzymes in
red compared with white muscle mitochondria shown here falls
in line with this theory. Only 2 of 22 detected tricarboxylic acid
cycle and pyruvate dehydrogenase proteins were significantly
and 25% different between mitochondrial types. These results
combined with the similarities in oxidative phosphorylation
machinery and State 3 respiration with P⫹M make the greater
content and activity of NAD-linked IDH in white mitochondria
a curious finding. Jackman and Willis (24) also found increased IDH activity in white muscle mitochondria, suggesting
that regulation of NADH supply to the ETC may be different
between red and white muscle mitochondria. Higher IDH
activity would predict a greater NADH level and may explain
why reactive oxygen species production is higher in white
muscle mitochondria (3, 10, 42, 49). Flux through IDH also
results in formation of ␣-ketoglutarate (AKG), which plays an
important role in amino acid metabolism as the ␣-keto acid pair
for glutamate in transaminase reactions occurring in both
mitochondria and the cytosol. Hutson (22) showed that white
skeletal muscle has greater relative activity of branched chain
aminotransferase in the cytosol than in mitochondria, whereas
red muscle has branched chain aminotransferase activity exclusively in the mitochondria. Hutson (22) also suggests that
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Fig. 3. Composition of native complex components by 2D BN-PAGE. Gel lanes as shown in Fig. 2 were separated in the second dimension by molecular weight
(MW) with an SDS buffer. A: red skeletal muscle mitochondria. B: white skeletal muscle mitochondria.
C1286
MITOCHONDRIAL PROTEIN COMPOSITION BY MUSCLE FIBER TYPE
muscles with lower mitochondrial content may release more
keto acids than those with higher mitochondrial content. Thus
the higher IDH activity in white muscle mitochondria may be
a mechanism for increased AKG production to be used by
cytosolic transaminase reactions.
These studies were conducted using porcine vastus intermedius [70% type I (48)] and gracilis [70% type IIb (53)] muscles.
We initially attempted to use mitochondria isolated from rabbit
soleus and gracilis [98% and 99% type I and IIb, respectively
(39)], but the yield after the mitochondrial purification process
was too small to proceed with the full proteomic analysis. We
were, however, able to measure the cytochrome a content in
rabbit soleus and gracilis homogenates and isolated mitochondria. Just as with porcine muscle, cytochrome a content per
milligram of mitochondria was similar in both muscle types in
the rabbit. Cytochrome a content per gram wet weight, on the
other hand, was 6.5-fold higher in rabbit soleus versus gracilis
compared with a twofold difference between red and white
Table 1. Cytochrome-to-protein ratios in
isolated mitochondria
Red muscle
White muscle
Cytochrome a,a3,
nmol/mg
Cytochrome b,
nmol/mg
Cytochrome c⫹c1,
nmol/mg
1.15 ⫾ 0.07
1.05 ⫾ 0.05
0.80 ⫾ 0.07
0.73 ⫾ 0.04
0.62 ⫾ 0.05
0.54 ⫾ 0.03
Values are means ⫾ SE. n ⫽ 19 and 20 for white and red muscle
mitochondria, respectively.
AJP-Cell Physiol • VOL
porcine muscle. Thus it is likely that the differences detected
here with regard to substrate oxidation would be three to four
times as large in rabbit muscle mitochondria, though we would
still predict similar content and activities of oxidative phosphorylation proteins between muscle types. In fact, in our
initial studies on rabbit muscle, there were no differences in the
stoichiometry between ETC complexes or in the activities of
Complexes IV or V per mole of enzyme (as in Fig. 2) between
soleus and gracilis mitochondria (results not shown). Furthermore, Leary et al. (35) reported no differences in oxidative
phosphorylation enzyme activities or P⫹M State 3 respiration
between red and white muscle mitochondria from rainbow
trout as well as increased fat oxidation enzyme activity in red
mitochondria when normalized to cytochrome a. The collective findings from the pig and rabbit here, and fish (35) and cat
(47) previously, suggest that the similarity in oxidative phosphorylation components between red and white muscle mitochondria may be found across a wide range of species, though
it is possible that smaller species, such as rats or mice, may
hold larger differences.
This primary focus of this study was to assess differences in
the nuclear programming of mitochondrial protein expression
between red and white skeletal muscle. The similarities in
protein composition found here suggest there is little difference
in metabolic capacity between red and white mitochondria.
However, differences in metabolic control may still exist. For
example, it is fully possible, and perhaps likely, that there are
differences in posttranslational modifications such as phos-
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Fig. 4. Composition of oxidative phosphorylation components using 2D BN-PAGE. Individual bands corresponding to electron transport chain (ETC) Complexes
I (A), III (B), IV (C), and V (D) were cut from gel lanes as shown in Fig. 2 and separated by molecular weight using an SDS buffer. Whole gel lane density
traces for both mitochondrial types are shown to the right of each gel.
C1287
MITOCHONDRIAL PROTEIN COMPOSITION BY MUSCLE FIBER TYPE
Table 2. iTRAQ ratios for significantly and ⫾25% different proteins in mitochondria from red and white muscle
UniProtKB Entry
Name
Identified Proteins
Molecular Mass,
kDa
White/Red
Ratio
P
ACS2L_HUMAN
MCAT_HUMAN
Homo sapiens
Homo sapiens
75
33
0.60 ⫾ 0.08
1.29 ⫾ 0.10
0.038
0.035
ACADV_BOVIN
ACADL_PIG
ACADS_PIG
Bos taurus
Sus scrofa
Sus scrofa
71
48
45
0.74 ⫾ 0.04
0.67 ⫾ 0.02
0.79 ⫾ 0.05
0.000
0.004
0.011
ECHD2_BOVIN
HCDH_PIG
THIM_BOVIN
PECI_MOUSE
ECHA_PIG
Bos taurus
Sus scrofa
Bos taurus
Mus musculus
Sus scrofa
32
34
42
43
83
0.71 ⫾ 0.08
0.80 ⫾ 0.04
0.79 ⫾ 0.04
0.73 ⫾ 0.02
0.76 ⫾ 0.04
0.012
0.031
0.002
0.007
0.002
NU2M_PIG
COXAM_BOVIN
ATPF2_BOVIN
Sus scrofa
Bos taurus
Bos taurus
39
13
33
1.30 ⫾ 0.01
1.99 ⫾ 0.17
0.77 ⫾ 0.02
0.002
0.028
0.007
DHRS4_PIG
IDHP_PIG
MAON_HUMAN
Sus scrofa
Sus scrofa
Homo sapiens
28
48
67
0.75 ⫾ 0.04
0.79 ⫾ 0.05
0.80 ⫾ 0.01
0.021
0.008
0.001
ODPX_BOVIN
Bos taurus
54
0.80 ⫾ 0.06
0.020
IDH3G_PIG
Sus scrofa
11
1.85 ⫾ 0.24
0.016
BCKD_HUMAN
Homo sapiens
46
0.80 ⫾ 0.04
0.031
ODBB_BOVIN
ALAT1_BOVIN
HIBCH_BOVIN
MCCB_MOUSE
KAT1_HUMAN
Bos taurus
Bos taurus
Bos taurus
Mus musculus
Homo sapiens
43
55
43
61
48
1.77 ⫾ 0.13
0.72 ⫾ 0.06
1.54 ⫾ 0.12
1.27 ⫾ 0.03
0.70 ⫾ 0.06
0.030
0.049
0.045
0.012
0.041
RT06_BOVIN
RT16_HUMAN
RM10_BOVIN
RM49_BOVIN
Bos taurus
Homo sapiens
Bos taurus
Bos taurus
14
15
29
19
0.73 ⫾ 0.05
0.74 ⫾ 0.07
2.03 ⫾ 0.18
1.30 ⫾ 0.06
0.001
0.001
0.029
0.037
ABCB8_MOUSE
ABCBA_HUMAN
MRP2_MOUSE
CMC2_HUMAN
SCMC1_BOVIN
MTX2_PIG
MOSC2_BOVIN
OXA1L_BOVIN
Mus musculus
Homo sapiens
Mus musculus
Homo sapiens
Bos taurus
Sus scrofa
Bos taurus
Bos taurus
78
79
174
74
53
30
37
49
1.68 ⫾ 0.09
0.68 ⫾ 0.04
0.80 ⫾ 0.03
0.61 ⫾ 0.03
1.98 ⫾ 0.10
1.33 ⫾ 0.04
1.53 ⫾ 0.19
1.33 ⫾ 0.02
0.018
0.018
0.019
0.005
0.010
0.015
0.037
0.003
GPDM_BOVIN
Bos taurus
81
1.62 ⫾ 0.15
0.009
MMSA_RAT
Rattus norvegicus
58
0.72 ⫾ 0.07
0.010
GPX4_HUMAN
GLRX5_HUMAN
Homo sapiens
Homo sapiens
22
17
0.47 ⫾ 0.01
1.36 ⫾ 0.02
0.001
0.004
COQ4_HUMAN
HINT2_BOVIN
ES1_HUMAN
IPYR2_MOUSE
Homo sapiens
Bos taurus
Homo sapiens
Mus musculus
30
17
28
38
1.27 ⫾ 0.03
0.79 ⫾ 0.05
0.80 ⫾ 0.04
0.77 ⫾ 0.05
0.012
0.049
0.033
0.045
C1QBP_BOVIN
Bos taurus
31
1.40 ⫾ 0.04
0.012
CHC10_HUMAN
PTRF_HUMAN
BR44_HUMAN
Homo sapiens
Homo sapiens
Homo sapiens
14
43
14
1.40 ⫾ 0.09
2.49 ⫾ 0.14
0.80 ⫾ 0.04
0.049
0.008
0.031
Red and white muscle mitochondrial samples (n ⫽ 3 pairs) were isobaric tag for relative and absolute quantitation (iTRAQ) labeled and analyzed by mass
spectrometry. Shown above are the 48 mitochondrial proteins that were significantly and at least 25% different between red and white skeletal muscle mitochondria.
The full list of proteins and ratios can be found in Supplemental Materials online at the AJP-Cell Physiol website. White/red values are means ⫾ SE.
AJP-Cell Physiol • VOL
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Fat Metabolism
Acetyl-coenzyme A synthetase 2-like, mitochondrial
Mitochondrial carnitine/acylcarnitine carrier protein
Very long-chain specific acyl-CoA dehydrogenase,
mitochondrial
Long-chain specific acyl-CoA dehydrogenase, mitochondrial
Short-chain specific acyl-CoA dehydrogenase, mitochondrial
Enoyl-CoA hydratase domain-containing protein 2,
mitochondrial
Hydroxyacyl-coenzyme A dehydrogenase, mitochondrial
3-ketoacyl-CoA thiolase, mitochondrial
Peroxisomal 3,2-trans-enoyl-CoA isomerase
Trifunctional enzyme subunit alpha, mitochondrial
Oxidative Phosphorylation
NADH-ubiquinone oxidoreductase chain 2
COX assembly mitochondrial protein homolog
ATP synthase mitochondrial F1 complex assembly factor 2
NADP-Linked Reactions
NADPH-dependent carbonyl reductase/NADP-retinol
dehydrogenase
Isocitrate dehydrogenase [NADP], mitochondrial
NADP-dependent malic enzyme, mitochondrial
TCA Cycle
Pyruvate dehydrogenase protein X component
Isocitrate dehydrogenase [NAD] subunit gamma,
mitochondrial
Amino Acid Metabolism
Branched-chain alpha-ketoacid dehydrogenase kinase
Branched-chain alpha-ketoacid dehydrogenase E1 component
beta chain
Alanine aminotransferase 1
3-hydroxyisobutyryl-CoA hydrolase, mitochondrial
Methylcrotonoyl-CoA carboxylase beta chain, mitochondrial
Kynurenine–oxoglutarate transaminase 1
Ribosomal Proteins
28S ribosomal protein S6, mitochondrial
28S ribosomal protein S16, mitochondrial
39S ribosomal protein L10, mitochondrial
39S ribosomal protein L49, mitochondrial
Membrane/Transport
ATP-binding cassette sub-family B member 8, mitochondrial
ATP-binding cassette sub-family B member 10, mitochondrial
ATP-binding cassette sub-family C member 2
Calcium-binding mitochondrial carrier protein Aralar2
Calcium-binding mitochondrial carrier protein SCaMC-1
Metaxin-2
MOSC domain-containing protein 2, mitochondrial
Mitochondrial inner membrane protein OXA1L
Other
Glycerol-3-phosphate dehydrogenase, mitochondrial
Methylmalonate-semialdehyde dehydrogenase [acylating],
mitochondrial
Phospholipid hydroperoxide glutathione peroxidase,
mitochondrial
Glutaredoxin-related protein 5, mitochondrial
Ubiquinone biosynthesis protein COQ4 homolog,
mitochondrial
Histidine triad nucleotide-binding protein 2, mitochondrial
ES1 protein homolog, mitochondrial
Inorganic pyrophosphatase 2, mitochondrial
Complement component 1 Q subcomponent-binding protein,
mitochondrial
Coiled-coil-helix-coiled-coil-helix domain-containing protein
10, mitochondrial
Polymerase I and transcript release factor
Brain protein 44
Taxonomy
C1288
MITOCHONDRIAL PROTEIN COMPOSITION BY MUSCLE FIBER TYPE
phorylation, acetylation, or S-nitrosylation between mitochondrial types. Indeed, Feng et al. (14) reported twice as many
carbonylated proteins in white versus red muscle mitochondria.
However, as the functional significance of any of these posttranslational modifications is still largely unclear, they were
left outside the scope of this project. The metabolic poise of red
and white skeletal muscle, respectively, may also contribute to
differences in metabolic control, which has been shown to
change as a function of rate, both within mitochondria (17, 31),
as well as for the mitochondrial contribution to cellular ATP
free energy (25). White muscle fibers are recruited for shorter
durations and thus result in mitochondria spending more time
near State 4 where the proton leak exerts greater control over
respiration (17, 31). In fact, white muscle mitochondria are
reported to have a higher rate of proton leak (35). Despite these
probable differences in metabolic control in vivo, the similarity
in metabolic capacity between red and white muscle mitochondria suggests that nuclear programming of mitochondrial protein expression may be configured to meet maximal energy
demands per unit volume rather than the specific patterns of
muscle activation.
AJP-Cell Physiol • VOL
Fig. 6. NAD-linked isocitrate dehydrogenase (IDH) activity in red and white
skeletal muscle mitochondria. NADH appearance was measured spectrophotometrically on the same samples used for isobaric tag for relative and absolute
quantitation (iTRAQ) analysis. *Significantly different from red muscle mitochondria.
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Fig. 5. Mitochondrial respiration in the presence of different oxidative substrates. Mitochondria (0.25– 0.5 mg mito protein/ml) were incubated with 10
mM Pi and either glutamate ⫹ malate (G⫹M), pyruvate ⫹ malate (P⫹M), or
palmitoyl-carnitine ⫹ malate (PC⫹M). A: maximal (State 3) respiration of red
and white skeletal muscle mitochondria. B: respiratory control ratio (State
3/State 4) of red and white skeletal muscle mitochondria. *Significantly
different from red muscle mitochondria.
Mitochondria do not exist only as isolated organelles in
vivo, but also as reticular networks located both near the
sarcolemma and within skeletal muscle fibers. The isolation
procedure used here is designed to recover both intermyofibrillar (IMF) and subsarcolemmal (SS) mitochondria, though
the percent contribution of each to the final yield is unknown.
IMF mitochondria are reported to comprise a greater proportion of the total mitochondrial population in white versus red
skeletal muscle (19). The lack of difference in protein composition between red and white mitochondria may indicate that
either a similar proportion of IMF and SS mitochondria were
isolated from both red and white mitochondria or that IMF and
SS mitochondria are not largely different themselves, though
the latter would contradict a number of previous reports (1, 12,
15, 50). In addition, while disruption of the mitochondrial
reticulum during the isolation process may remove any potential effect fission and fusion events have on mitochondrial
composition and/or function, the lack of difference in fission/
fusion proteins OPA1, FIS1, PARL, and MTP18 (Supplemental Materials) suggests these processes may also be similar
between red and white skeletal muscle mitochondria.
The remarkable similarity of the oxidative phosphorylation
complexes on a milligram of protein basis between the red and
white muscle mitochondria suggest that the volume packing of
these energy conversion components are nearly identical despite the large difference in peak aerobic ATP demands of the
muscles. Thus the differences in the ATP requirements of these
different muscles are accomplished by varying the number of
mitochondria with essentially identical ATP production capacities. This observation suggests that the volume packing of
mitochondrial oxidative phosphorylation elements in these
muscles is near optimal providing the maximum area for
muscle contraction elements for power with minimal volume
for energetic support.
In summary, protein content and function are similar between mitochondria from red and white skeletal muscle. Both
red and white muscle mitochondria contain about 1 nmol
cytochrome a per milligram of mitochondrial protein, though
there is a slight upregulation in fat oxidation in red muscle
MITOCHONDRIAL PROTEIN COMPOSITION BY MUSCLE FIBER TYPE
mitochondria. This suggests that differences in metabolic need
between fiber types are met by the number of mitochondria
rather than modifying the composition of individual mitochondria. There may be an optimal mitochondrial configuration to
support oxidative phosphorylation in muscle.
ACKNOWLEDGMENTS
We thank Stephanie French, Darci Phillips, and David Chess for technical
assistance and Yong Chen for work with the mass spectrometry samples.
GRANTS
This study was supported by Intramural Funding of the Division of
Intramural Research, National Heart, Lung, and Blood Institute.
DISCLOSURES
REFERENCES
1. Adhihetty PJ, Ljubicic V, Menzies KJ, Hood DA. Differential susceptibility of subsarcolemmal and intermyofibrillar mitochondria to apoptotic
stimuli. Am J Physiol Cell Physiol 289: C994 –C1001, 2005.
2. Alvarado Rigault MY, Blanchaer MC. Respiration and oxidative phosphorylation by mitochondria of red and white skeletal muscle. Can J
Biochem 48: 27–32, 1970.
3. Anderson EJ, Neufer PD. Type II skeletal myofibers possess unique
properties that potentiate mitochondrial H2O2 generation. Am J Physiol
Cell Physiol 290: C844 –C851, 2006.
4. Aponte AM, Phillips D, Harris RA, Blinova K, French S, Johnson DT,
Balaban RS. 32P labeling of protein phosphorylation and metabolite
association in the mitochondria matrix. Methods Enzymol 457: 63–80,
2009.
5. Aponte AM, Phillips D, Hopper RK, Johnson DT, Harris RA, Blinova
K, Boja ES, French S, Balaban RS. Use of (32)P to study dynamics of
the mitochondrial phosphoproteome. J Proteome Res 8: 2679 –2695, 2009.
6. Balaban RS, Mootha VK, Arai A. Spectroscopic determination of
cytochrome c oxidase content in tissues containing myoglobin or hemoglobin. Anal Biochem 237: 274 –278, 1996.
7. Blanchaer MC. Respiration of mitochondria of red and white skeletal
muscle. Am J Physiol 206: 1015–1020, 1964.
8. Boja ES, Phillips D, French SA, Harris RA, Balaban RS. Quantitative
mitochondrial phosphoproteomics using iTRAQ on an LTQ-Orbitrap with
high energy collision dissociation. J Proteome Res 8: 4665–4675, 2009.
9. Cai D, Li M, Lee K, Wong W, Chan K. Age-related changes of aqueous
protein profiles in rat fast and slow twitch skeletal muscles. Electrophoresis 21: 465–472, 2000.
10. Capel F, Buffiere C, Patureau Mirand P, Mosoni L. Differential
variation of mitochondrial H2O2 release during aging in oxidative and
glycolytic muscles in rats. Mech Ageing Dev 125: 367–373, 2004.
11. Chen RF, Plaut GW. Activation and inhibition of Dpn-linked isocitrate
dehydrogenase of heart by certain nucleotides. Biochemistry 2: 1023–
1032, 1963.
12. Cogswell AM, Stevens RJ, Hood DA. Properties of skeletal muscle
mitochondria isolated from subsarcolemmal and intermyofibrillar regions.
Am J Physiol Cell Physiol 264: C383–C389, 1993.
13. Estabrook R. Mitochondrial respiratory control and the polarographic
measurement of ADP/O ratios. Meth Enzymol 10: 41–47, 1967.
14. Feng J, Xie H, Meany DL, Thompson LV, Arriaga EA, Griffin TJ.
Quantitative proteomic profiling of muscle type-dependent and age-dependent protein carbonylation in rat skeletal muscle mitochondria. J
Gerontol A Biol Sci Med Sci 63: 1137–1152, 2008.
15. Ferreira R, Vitorino R, Alves RM, Appell HJ, Powers SK, Duarte JA,
Amado F. Subsarcolemmal and intermyofibrillar mitochondria proteome
differences disclose functional specializations in skeletal muscle. Proteomics 10: 3142–3154, 2010.
16. Gelfi C, Vigano A, De Palma S, Ripamonti M, Begum S, Cerretelli P,
Wait R. 2-D protein maps of rat gastrocnemius and soleus muscles: a tool
for muscle plasticity assessment. Proteomics 6: 321–340, 2006.
17. Hafner RP, Brown GC, Brand MD. Analysis of the control of respiration rate, phosphorylation rate, proton leak rate and protonmotive force in
isolated mitochondria using the “top-down” approach of metabolic control
theory. Eur J Biochem 188: 313–319, 1990.
AJP-Cell Physiol • VOL
18. Harris D, Das A. Control of mitochondrial ATP synthesis in the heart.
Biochem J 280: 561–573, 1991.
19. Hoppeler H, Hudlicka O, Uhlmann E. Relationship between mitochondria and oxygen consumption in isolated cat muscles. J Physiol 385:
661–675, 1987.
20. Hopper RK, Carroll S, Aponte AM, Johnson DT, French S, Shen RF,
Witzmann FA, Harris RA, Balaban RS. Mitochondrial matrix phosphoproteome: effect of extra mitochondrial calcium. Biochemistry 45: 2524 –
2536, 2006.
21. Howlett RA, Willis WT. Fiber-type-related differences in the enzymes of
a proposed substrate cycle. Biochim Biophys Acta 1363: 224 –230, 1998.
22. Hutson SM. Subcellular distribution of branched-chain aminotransferase
activity in rat tissues. J Nutr 118: 1475–1481, 1988.
23. Isfort RJ, Wang F, Greis KD, Sun Y, Keough TW, Bodine SC,
Anderson NL. Proteomic analysis of rat soleus and tibialis anterior
muscle following immobilization. J Chromatogr B Analyt Technol Biomed
Life Sci 769: 323–332, 2002.
24. Jackman MR, Willis WT. Characteristics of mitochondria isolated from
type I and type IIb skeletal muscle. Am J Physiol Cell Physiol 270:
C673–C678, 1996.
25. Jeneson JA, Westerhoff HV, Kushmerick MJ. A metabolic control
analysis of kinetic controls in ATP free energy metabolism in contracting
skeletal muscle. Am J Physiol Cell Physiol 279: C813–C832, 2000.
26. Johnson DT, Harris RA, Blair PV, Balaban RS. Functional consequences of mitochondrial proteome heterogeneity. Am J Physiol Cell
Physiol 292: C698 –C707, 2007.
27. Johnson DT, Harris RA, French S, Aponte A, Balaban RS. Proteomic
changes associated with diabetes in the BB-DP rat. Am J Physiol Endocrinol Metab 296: E422–E432, 2009.
28. Keller A, Nesvizhskii AI, Kolker E, Aebersold R. Empirical statistical
model to estimate the accuracy of peptide identifications made by MS/MS
and database search. Anal Chem 74: 5383–5392, 2002.
29. Kim NK, Joh JH, Park HR, Kim OH, Park BY, Lee CS. Differential
expression profiling of the proteomes and their mRNAs in porcine white
and red skeletal muscles. Proteomics 4: 3422–3428, 2004.
30. Kim NK, Park HR, Lee HC, Yoon D, Son ES, Kim YS, Kim SR, Kim
OH, Lee CS. Comparative studies of skeletal muscle proteome and
transcriptome profilings between pig breeds. Mamm Genome 21: 307–319,
2010.
31. Korzeniewski B, Mazat JP. Theoretical studies on the control of oxidative phosphorylation in muscle mitochondria: application to mitochondrial
deficiencies. Biochem J 319: 143–148, 1996.
32. Kushmerick MJ, Meyer RA, Brown TR. Regulation of oxygen consumption in fast- and slow-twitch muscle. Am J Physiol Cell Physiol 263:
C598 –C606, 1992.
33. Kushmerick MJ, Moerland TS, Wiseman RW. Mammalian skeletal
muscle fibers distinguished by contents of phosphocreatine, ATP, and Pi.
Proc Natl Acad Sci USA 89: 7521–7525, 1992.
34. Kushmerick MJ, Moerland TS, Wiseman RW. Two classes of mammalian skeletal muscle fibers distinguished by metabolite content. Adv Exp
Med Biol 332: 749 –760, 1993.
35. Leary SC, Lyons CN, Rosenberger AG, Ballantyne JS, Stillman J,
Moyes CD. Fiber-type differences in muscle mitochondrial profiles. Am J
Physiol Regul Integr Comp Physiol 285: R817–R826, 2003.
36. Mogensen M, Sahlin K. Mitochondrial efficiency in rat skeletal muscle:
influence of respiration rate, substrate and muscle type. Acta Physiol
Scand 185: 229 –236, 2005.
37. Nesvizhskii AI, Keller A, Kolker E, Aebersold R. A statistical model for
identifying proteins by tandem mass spectrometry. Anal Chem 75: 4646 –
4658, 2003.
38. Okumura N, Hashida-Okumura A, Kita K, Matsubae M, Matsubara
T, Takao T, and Nagai K. Proteomic analysis of slow- and fast-twitch
skeletal muscles. Proteomics 5: 2896 –2906, 2005.
39. Pagliassotti MJ, Donovan CM. Influence of cell heterogeneity on skeletal muscle lactate kinetics. Am J Physiol Endocrinol Metab 258: E625–
E634, 1990.
40. Pande SV, Blanchaer MC. Carbohydrate and fat in energy metabolism of
red and white muscle. Am J Physiol 220: 549 –553, 1971.
41. Phillips D, Aponte AM, French SA, Chess DJ, Balaban RS. SuccinylCoA synthetase is a phosphate target for the activation of mitochondrial
metabolism. Biochemistry 48: 7140 –7149, 2009.
42. Picard M, Csukly K, Robillard ME, Godin R, Ascah A, BourcierLucas C, Burelle Y. Resistance to Ca2⫹-induced opening of the permeability transition pore differs in mitochondria from glycolytic and oxida-
300 • JUNE 2011 •
www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.32.247 on June 18, 2017
No conflicts of interest, financial or otherwise, are declared by the author(s).
C1289
C1290
43.
44.
45.
46.
47.
tive muscles. Am J Physiol Regul Integr Comp Physiol 295: R659 –R668,
2008.
Sargeant AJ, Hoinville E, Young A. Maximum leg force and power
output during short-term dynamic exercise. J Appl Physiol 51: 1175–1182,
1981.
Scheibye-Knudsen M, Quistorff B. Regulation of mitochondrial respiration by inorganic phosphate: comparing permeabilized muscle fibers and
isolated mitochondria prepared from type-1 and type-2 rat skeletal muscle.
Eur J Appl Physiol 105: 279 –287, 2009.
Schieke SM, Phillips D, McCoy JP, Jr, Aponte AM, Shen RF, Balaban
RS, Finkel T. The mammalian target of rapamycin (mTOR) pathway
regulates mitochondrial oxygen consumption and oxidative capacity. J
Biol Chem 281: 27643–27652, 2006.
Scholz TD, Balaban RS.. Mitochondrial F1-ATPase activity of canine
myocardium: effects of hypoxia and stimulation. Am J Physiol Heart Circ
Physiol 266: H2396 –H2403, 1994.
Schwerzmann K, Hoppeler H, Kayar SR, Weibel ER. Oxidative capacity of muscle and mitochondria: correlation of physiological, biochemical, and morphometric characteristics. Proc Natl Acad Sci USA 86:
1583–1587, 1989.
Sorensen MT, Oksbjerg N, Agergaard N, Petersen JS. Tissue deposition rates in relation to muscle fibre and fat cell characteristics in lean
AJP-Cell Physiol • VOL
49.
50.
51.
52.
53.
54.
55.
female pigs (Sus scrofa) following treatment with porcine growth hormone
(pGH). Comp Biochem Physiol A Physiol 113: 91–96, 1996.
Starkov AA, Fiskum G. Regulation of brain mitochondrial H2O2 production by membrane potential and NAD(P)H redox state. J Neurochem
86: 1101–1107, 2003.
Takahashi M, Hood DA. Protein import into subsarcolemmal and intermyofibrillar skeletal muscle mitochondria. Differential import regulation
in distinct subcellular regions. J Biol Chem 271: 27285–27291, 1996.
Vitorino R, Ferreira R, Neuparth M, Guedes S, Williams J, Tomer
KB, Domingues PM, Appell HJ, Duarte JA, Amado FM. Subcellular
proteomics of mice gastrocnemius and soleus muscles. Anal Biochem 366:
156 –169, 2007.
Weibel ER, Hoppeler H. Exercise-induced maximal metabolic rate scales
with muscle aerobic capacity. J Exp Biol 208: 1635–1644, 2005.
Weiler U, Appell HJ, Kremser M, Hofacker S, Claus R. Consequences
of selection on muscle composition. A comparative study on gracilis
muscle in wild and domestic pigs. Anat Histol Embryol 24: 77–80, 1995.
Willis WT, Jackman MR. Mitochondrial function during heavy exercise.
Med Sci Sports Exerc 26: 1347–1353, 1994.
Wilson DF, Owen CS, Holian A. Control of mitochondrial respiration: a
quantitative evaluation of the roles of cytochrome c and oxygen. Arch
Biochem Biophys 182: 749 –762, 1977.
300 • JUNE 2011 •
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Downloaded from http://ajpcell.physiology.org/ by 10.220.32.247 on June 18, 2017
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