J Appl Physiol 100: 399 – 405, 2006.
First published September 15, 2005; doi:10.1152/japplphysiol.00873.2005.
Calcium-independent phospholipase A2 modulates cytosolic oxidant activity
and contractile function in murine skeletal muscle cells
Ming C. Gong, Sandrine Arbogast, Zhenheng Guo, Jeremy Mathenia, Wen Su, and Michael B. Reid
Department of Physiology, University of Kentucky, Lexington, Kentucky
Submitted 19 July 2005; accepted in final form 10 September 2005
reactive oxygen species; excitation-contraction species; antioxidants;
exercise; signal transduction
resting muscles, Zuo and colleagues (42) showed that basal
rates of superoxide anion release into the extracellular space
are also PLA2 dependent.
Muscle-derived ROS have a physiological role, promoting
contractile function and force production by unfatigued muscle
(26). To the extent that PLA2 supports endogenous ROS
production, PLA2 would also be expected to promote contractile function of skeletal muscle. This expectation is supported
by data from vascular smooth muscle. Studies by Guo et al.
(10) indicate that PLA2 mediates the increased force of contraction stimulated by phenylephrine or serotonin. Specifically,
the increase in force appears to be mediated by the iPLA2
isoform. Guo and associates found that iPLA2 is constitutively
expressed by vascular smooth muscle and that agonist-induced
increases in the force of contraction could be inhibited by
selective iPLA2 inhibition.
The biological importance of iPLA2 in skeletal muscle cells
is largely unstudied. Existing data imply that iPLA2 might
modulate oxidant production by muscle cells and thereby
influence contractile function. The present study evaluated this
postulate by testing three hypotheses. 1) iPLA2 is ubiquitously
expressed by murine skeletal muscles and muscle-derived cell
lines. Differentiated C2C12 myotubes and a panel of striated
muscles (limb, respiratory, cardiac) from adult mice were
tested for iPLA2 mRNA and protein levels. 2) iPLA2 contributes to intracellular oxidant activity in resting muscle fibers.
The 2⬘,7⬘-dichlorofluorescein (DCFH) oxidation assay was
used to measure oxidant activity in the cytosol of differentiated
muscle cells with and without iPLA2 inhibition. 3) iPLA2
promotes force production by skeletal muscle. Contractile
properties and fatigue characteristics of mouse limb muscle
were measured in vitro with and without iPLA2 blockade.
A2 (PLA2) superfamily catalyze
hydrolysis of the sn-2 ester bond on phospholipids to produce
arachidonic acid and other fatty acids (31). The PLA2 enzymes
are categorized into as many as 11 groups based on catalytic
activity, amino acid sequence, sequence homologies, and
splice variance (31). Among these enzymes, calcium-independent PLA2 (iPLA2), also known as group VI PLA2, is located
within the cell and does not require calcium for enzymatic
activity (38).
In skeletal muscle, several reports indicate that constitutive
PLA2 activity supports the production of reactive oxygen
species (ROS) by mitochondria (23) and 5-lipoxygenase (42).
Nethery and associates (24) have shown that PLA2 function is
essential for the rise in intracellular ROS that occurs during
repetitive, fatiguing contractions. In subsequent studies of
Reagents. 2⬘,7⬘-Dichlorodihydrofluorescein diacetate (DCFH-DA;
Molecular Probes, Eugene, OR) was dissolved in 100% ethyl alcohol,
diluted in Krebs-Ringer solution, and stored at ⫺80°C for later use.
Bromoenol lactone (BEL; Biomol International, Plymouth Meeting,
PA) was dissolved in 100% dimethyl sulfoxide (DMSO). Cu-Zn
superoxide dismutase (SOD; Oxis International, Portland, OR) was
dissolved in Krebs-Ringer solution. All other chemicals were obtained
from Sigma-Aldrich (St. Louis, MO).
Myogenic cell cultures. Myotubes were cultured from the murine
skeletal muscle-derived C2C12 myoblast line (American Type Culture
Collection, Rockville, MD) as described previously (16). Briefly,
C2C12 cells were cultured in DMEM supplemented with 10% fetal
calf serum and gentamycin at 37°C in the presence of 5% CO2.
Myoblast differentiation was initiated by replacing the growth me-
Address for reprint requests and other correspondence: M. B. Reid, Dept. of
Physiology, Univ. of Kentucky Medical Center, 800 Rose St., Lexington, KY
40536 (e-mail: [email protected]).
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
ENZYMES OF THE PHOSPHOLIPASE
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MATERIALS AND METHODS
8750-7587/06 $8.00 Copyright © 2006 the American Physiological Society
399
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Gong, Ming C., Sandrine Arbogast, Zhenheng Guo, Jeremy
Mathenia, Wen Su, and Michael B. Reid. Calcium-independent
phospholipase A2 modulates cytosolic oxidant activity and contractile function in murine skeletal muscle cells. J Appl Physiol
100: 399 – 405, 2006. First published September 15, 2005;
doi:10.1152/japplphysiol.00873.2005.—Phospholipase A2 (PLA2) activity supports production of reactive oxygen species (ROS) by
mammalian cells. In skeletal muscle, endogenous ROS modulate the
force of muscle contraction. We tested the hypothesis that skeletal
muscle cells constitutively express the calcium-independent PLA2
(iPLA2) isoform and that iPLA2 modulates both cytosolic oxidant
activity and contractile function. Experiments utilized differentiated
C2C12 myotubes and a panel of striated muscles isolated from adult
mice. Muscle preparations were processed for measurement of mRNA
by real-time PCR, protein by immunoblot, cytosolic oxidant activity
by the dichlorofluorescein oxidation assay, and contractile function by
in vitro testing. We found that iPLA2 was constitutively expressed by
all muscles tested (myotubes, diaphragm, soleus, extensor digitorum
longus, gastrocnemius, heart) and that mRNA and protein levels were
generally similar among muscles. Selective iPLA2 blockade by use of
bromoenol lactone (10 ␮M) decreased cytosolic oxidant activity in
myotubes and intact soleus muscle fibers. iPLA2 blockade also inhibited contractile function of unfatigued soleus muscles, shifting the
force-frequency relationship rightward and depressing force production during acute fatigue. Each of these changes could be reproduced
by selective depletion of superoxide anions using superoxide dismutase (1 kU/ml). These findings suggest that constitutively expressed iPLA2 modulates oxidant activity in skeletal muscle fibers by
supporting ROS production, thereby influencing contractile properties
and fatigue characteristics.
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J Appl Physiol • VOL
dized DCF was measured from representative areas of the fiber bundle
surface (0.27 mm2) by use of an epifluorescence microscope (Labophot-2, Nikon Instruments, Tokyo, Japan) and charge-coupled device
camera (Series 72, Dage-MTI, Michigan City, IN). A mechanical
shutter in the excitation light pathway was controlled by computer
using commercial data acquisition and analysis software (Optimas
4.02; Bioscan, Edmonds, WA) to standardize excitation time (33 ms).
Images were acquired in real time and stored in a desktop computer
for later analysis of mean emission intensity. Photooxidation artifact
was controlled by conducting experiments in a darkened laboratory
and by standardizing excitation parameters to minimize the cumulative delivered energy; residual error was corrected according to
Arbogast and Reid (2). Data were collected in paired comparisons of
treated vs. untreated solei from individual animals. Muscles were
mounted in separate chambers containing Krebs-Ringer solution aerated with 95% O2 and 5% CO2 at 37°C. Each was pretreated with 10
␮M BEL, 180 nM DMSO, 1 kU/ml SOD, or buffer (control) for 20
min and loaded with DCFH. After 60-min passive incubation, fluorescence emissions were measured to assess oxidant activity. Separate
experiments using a muscle-free system tested for chemical interactions between DCFH and each drug; none altered fluorescence.
Cytochrome c reduction assay. Superoxide anion activity was
measured using an adaptation of the spectrophotometric technique
described by McCord and Fridovich (20). In brief, cytochrome c
(Sigma-Aldrich) was added to oxygenated, buffered Krebs-Ringer
solution to achieve 10 ␮M final concentration. A spectrophotometer
(Biomate 3, Thermo Spectronic, Rochester, NY) was used to measure
absorbance at 540 nm (A540), 550 nm (A550), and 560 nm (A560). The
cytochrome c solution was supplemented with a superoxide-generating system (1.25 ␮M hypoxanthine, 20 mU/ml xanthine oxidase) plus
10 ␮M BEL, 180 nM DMSO (diluent control), 1 kU/ml SOD, or
nothing (buffer control). After 30 min, absorbance was remeasured at
each wavelength. A550 values were normalized [A550 normalized ⫽
A550/0.5(A540 ⫹ A560)] and used to compute net cytochrome c
reduction (in pmol) from a standard curve. In follow-up studies using
prereduced cytochrome c, we tested the capacity of 10 ␮M BEL to
inhibit changes in normalized A550 caused by addition of 250 ␮M
hydrogen peroxide (Sigma-Aldrich) or 1 mM NOC-7 (nitric oxide
donor; Calbiochem, La Jolla, CA).
Contractile studies. Using established methods (28), soleus muscles were excised and transferred to a dissecting chamber containing
Krebs-Ringer solution at room temperature (composition in mmol:
137 sodium chloride, 4 potassium chloride, 1 magnesium chloride, 1
potassium phosphate, 12 sodium bicarbonate, and 12 calcium chloride) equilibrated with 5% CO2-95% O2 (pH 7.30). The tendons were
isolated using a dissecting microscope; 2-0 silk sutures were used to
fix the proximal tendon to a glass rod and the distal tendon to a force
transducer (model BG200G, Kulite Industries, Leonia, NJ) mounted
on a micrometer. The muscle was positioned between platinum
stimulating electrodes in a temperature-controlled muscle bath containing oxygenated, buffered Krebs-Ringer solution at room temperature. Muscles were stimulated to contract isometrically using electrical field stimulation (supramaximal voltage, 0.2-ms pulse duration).
Force transducer output was recorded using an oscilloscope (model
546601B, Hewlett-Packard, Palo Alto, CA) and a chart recorder
(BD-11E; Kipp and Zonen, Delft, The Netherlands). In each experiment, muscle length was adjusted to optimize twitch force [optimal
length (Lo)], and bath temperature was increased to 37°C. BEL was
then added to achieve a bath concentration of 10 ␮M, or SOD was
added to achieve 1 kU/ml; DMSO diluent was added in BEL control
experiments. Thirty minutes were allowed for thermal and drug
equilibration. The force-frequency relationship was determined using
contractions evoked at 2-min intervals using stimulus frequencies of
1, 15, 30, 50, 80, 120, 160, 250, and 300 Hz and a tetanic train
duration of 500 ms. One minute later, we induced acute fatigue by
stimulating repetitive submaximal tetanic contractions (40 Hz, 0.5
train/s, 500-ms train duration) for 5 min. After the experiment, Lo was
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dium with differentiation medium: DMEM supplemented with 2%
horse serum. Differentiation was allowed to continue for 96 h before
experimentation (changing to fresh medium at 48 h).
Animal use. Animal procedures were conducted in accordance with
the National Institutes of Health Guide for the Care and Use of
Animals and were approved by the Institutional Review Board of the
University of Kentucky. Adult male HSD-ICR mice (Harlan Sprague
Dawley, Indianapolis, IN) were deeply anesthetized and killed by
exsanguination after which the diaphragm, soleus, extensor digitorum
longus (EDL), gastrocnemius, and heart muscles were surgically
isolated for analysis.
Real-time PCR determination of iPLA2 mRNA. Differentiated myotubes and individual muscles were placed in RNAlater solution
immediately after isolation. As described previously (10), RNA was
extracted using Tri Reagent (Molecular Research Center, Cincinnati,
OH). The cDNA was synthesized by Moloney murine leukemia virus
reverse transcriptase using random hexamers (Invitrogen, Carlsbad,
CA). The following real-time PCR primers were used: 5⬘-AGATGTCTTTCGTCCCAGCAA-3⬘ (forward), 5⬘-ACTGTTGCACAGATCCAGATGG-3⬘ (reverse), which include sequence from two adjacent
exons from mouse iPLA2; and 5⬘-AGAAACGGCTACCACATCCAA-3⬘ (forward), 5⬘-GGGTCGGGAGTGGGTAATTT-3⬘ (reverse)
for mouse 18S rRNA. The real-time PCR reactions were performed
using a QuantiTect SYBRgreen PCR kit (Qiagen, Valencia, CA) in an
ABI Prism 7000 Sequence Detection System. Specificity of the PCR
was verified by dissociation-curve analysis and agarose gel electrophoresis. Each sample was analyzed in triplicate in every experiment.
iPLA2 mRNA was normalized to the 18S rRNA signal; relative
quantification was by standard curve analysis. Template controls were
not included in each real-time PCR run.
Immunoblot of iPLA2. Isolated muscle preparations were immediately frozen in liquid nitrogen for processing by established methods
(10). In brief, after pulverization and lysis, protein concentrations
were determined by bicinchoninic acid protein assay kit (Pierce,
Rockford, IL). Equal amounts of protein were subjected to SDSpolyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride (PVDF) membrane (Fisher). Nonspecific binding sites
on the PVDF membrane were blocked by 4% nonfat milk in PBST
buffer (PBS plus 0.1% Tween 20). iPLA2 was detected by using
anti-iPLA2 antibodies from Cayman (Ann Arbor, MI; 1:7,500 dilution). The immunoreactive bands were blotted using horseradish
peroxidase-conjugated goat anti-rabbit antibodies (Jackson ImmunoResearch Laboratories) and detected by enhanced chemiluminescence. iPLA2 protein levels were quantified by Kodak 1D imageanalysis software (Eastman Kodak, Rochester, NY).
iPLA2 assay. iPLA2 activity was measured using the method of
Yang et al. (40) as adapted previously (10). In brief, the reaction was
conducted in Ca2⫹-free medium containing 4 mM EDTA, 1 mM ATP,
and 2 mM dithiothreitol. The membrane fraction was isolated from
muscle homogenates by 100,000-g centrifugation at 4°C; ⬃100 ␮g
membrane proteins were incubated with a mixture of unlabeled and
14
C-labeled 1,2-dipalmitoyl-sn-3-glycero phosphoryl choline for 90
min at 40°C. Free fatty acids were extracted using modified Dole
reagents; radiolabeled free fatty acids were quantified by liquid
scintillation counting. Specific iPLA2 activity was expressed as picomoles of free fatty acid released per milligram of protein in 1 min
(pmol 䡠 min⫺1 䡠 mg⫺1).
Intracellular oxidant activity. As described previously (2), oxidant
activity was measured by use of a diffusible fluorochrome probe,
DCFH-DA. The acetate group is cleaved by cytosolic esterases to
yield DCFH, a polar nonfluorescent molecule retained by the cell.
DCFH is converted by cytosolic oxidants, including ROS (15), to its
fluorescent derivative, 2⬘,7⬘-dichlorofluorescein (DCF; 480-nm excitation, 520-nm emissions). The rate of DCF accumulation is proportional to net cytosolic oxidant activity. Fiber bundles were loaded with
DCFH by in vitro incubation with DCFH-DA (50 ␮M) for 45– 60 min,
time periods that enable probe equilibration. Accumulation of oxi-
IPLA2
IN SKELETAL MUSCLE
401
measured using an electronic caliper, and the muscle was removed,
blotted dry, and weighed. Cross-sectional area was calculated according to Close (4).
Data analyses. Statistical analyses were performed using commercial software (Sigma Stat, SPSS, Chicago, IL). Averaged data are
reported as means ⫾ SE. Overall differences in a single end point
among two or more groups were tested by one-way ANOVA and post
hoc comparison by Tukey’s test (13). For recurrent measurements, a
two-way, repeated-measures ANOVA was used (13). Paired comparisons between two conditions were evaluated by Student’s paired
t-test (41). Differences were considered significant at P ⬍ 0.05.
RESULTS
Fig. 1. Calcium-independent phospholipase A2 (iPLA2) mRNA levels in
murine striated muscle. Data depict mean iPLA2/18S values (⫾SE) in diaphragm (Diaph), heart, gastrocnemius (Gastroc), soleus, and extensor digitorum longus (EDL) as measured using real-time PCR; n ⫽ 3– 6/group. *P ⬍
0.05 vs. diaphragm.
J Appl Physiol • VOL
Fig. 2. iPLA2 protein in muscle. A: representative autoradiograph depicts
85-kDa bands detected in striated muscles. B: mean protein levels in individual
muscles (⫾SE) as measured by densitometry. *P ⬍ 0.05 vs. gastrocnemius.
[77 ⫾ 3 (SE) %DMSO control; P ⬍ 0.05] and myotubes [74 ⫾
8 (SE) %DMSO control; P ⬍ 0.05]. Similar decrements in
oxidant activity were produced by selective ROS depletion. As
shown in Fig. 3B, exposure to SOD (degrades superoxide
anions) decreased oxidant activity in soleus fibers (P ⬍ 0.05)
and in myotubes (P ⬍ 0.05).
The similarity of BEL and SOD actions does not reflect
direct scavenging of superoxide anions by BEL. We tested the
capacity of each compound to degrade superoxide anions
generated by a superoxide-generating system in the absence of
muscle cells. As shown in Fig. 4, the superoxide signal was
blocked by SOD. The signal was unaffected by BEL or its
diluent, DMSO, at the concentrations used. Follow-up studies
using a similar protocol determined that BEL does not inhibit
changes in cytochrome c redox state caused by exposure to
hydrogen peroxide (P ⬍ 0.36) or a nitric oxide donor (P ⬍
0.72). In combination, these data suggest that BEL inhibits
oxidant activity via iPLA2 blockade and not by direct antioxidant actions.
iPLA2 blockade and muscle function. iPLA2 effects on
contractile function were tested using excised soleus muscles
pretreated with 10 ␮M BEL. Specific force was depressed by
15–23% across a range of submaximal stimulus frequencies
(30 – 80 Hz; P ⬍ 0.05). There were no changes in maximal
tetanic force (32.8 ⫾ 2.0 N/cm2 treated vs. 34.6 ⫾ 1.8 N/cm2
control), twitch force (4.0 ⫾ 0.4 vs. 4.5 ⫾ 0.4 N/cm2), time to
peak twitch force (24 ⫾ 1 vs. 23 ⫾ 2 ms), or twitch half
relaxation time (30 ⫾ 2 vs. 33 ⫾ 1 ms). Normalized for
variations in maximal force, the relative force-frequency curve
(Fig. 5A) demonstrates the contractile depression caused by
iPLA2 inhibition. As with oxidant activity, SOD pretreatment
mimicked the effect of iPLA2 inhibition, shifting the relative
force-frequency curve rightward (Fig. 5B).
Acute fatigue characteristics were also affected by iPLA2
inhibition. During repetitive, submaximal tetanic (40 Hz) stimulation, developed force was systematically depressed in BELtreated muscles (Fig. 6A), a difference that was evident at time
0 and throughout most of the 300-s protocol. SOD-treated
muscles exhibited a similar response during acute fatigue (Fig.
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iPLA2 expression patterns. iPLA2 gene expression was a
robust finding among widely divergent murine muscle preparations. iPLA2 mRNA was clearly detectable by real-time PCR
in each of five striated muscles (Fig. 1). Constitutive iPLA2
mRNA levels are comparable among diaphragm, gastrocnemius, and EDL; relative to diaphragm, levels are lower in
soleus (P ⬍ 0.05) and heart (P ⬍ 0.02). For comparison, we
also screened differentiated myotubes from the stable, mouse
muscle-derived C2C12 cell line and confirmed measurable
levels of iPLA2 mRNA [mean iPLA2/18S RNA: 3.7 ⫾ 1.2
(SE)]. The iPLA2 mRNA content in myotubes was higher
overall than in excised muscles (P ⬍ 0.05).
Immunoblot analysis identified a single major iPLA2 protein
band at the expected molecular mass of ⬃85 kDa in each of the
excised muscles tested (Fig. 2A) and in C2C12 myotubes (data
not shown). Averaged densitometry data (Fig. 2B) illustrate the
similarity of iPLA2 protein contents across individual mouse
muscles. We detected no differences among skeletal muscles,
but iPLA2 protein content was lower in heart than gastrocnemius (P ⬍ 0.02).
iPLA2 and intracellular oxidant activity. Cytosolic oxidant
activity was measured in intact muscle cells by use of the
DCFH oxidation assay. Low levels of basal activity were
detectable in soleus muscle fibers and in C2C12 myotubes (data
not shown). The iPLA2 contribution to this signal was tested by
use of BEL, a selective iPLA2 inhibitor (11).
Enzymatic iPLA2 activity in skeletal muscle is decreased
by 85% by treatment with 10 ␮M BEL [34.1 ⫾ 2.2 (SE)
pmol䡠 min⫺1 䡠mg⫺1 basal vs. 5.0 ⫾ 0.5 pmol䡠 min⫺1 䡠mg⫺1
treated; n ⫽ 3/group; P ⬍ 0.01]. As shown in Fig. 3A, 10 ␮M
BEL also decreases oxidant activity in intact muscle fibers
402
IPLA2
IN SKELETAL MUSCLE
Fig. 3. iPLA2 blockade decreases cytosolic
oxidant activity in intact muscle cells. A:
paired comparisons of cytosolic oxidant activity in C2C12 myotubes or intact fibers of
soleus muscles incubated with 10 ␮M bromoenol lactone (BEL) or 180 nM dimethyl
sulfoxide (DMSO), the diluent control; BEL
lowered oxidant activity in both cell types
(P ⬍ 0.05); n ⫽ 9 comparisons/cell type. B:
paired comparisons of myotubes and muscle
fibers incubated with 1 kU/ml superoxide dismutase (SOD) or buffer; SOD lowered oxidant activity in both cell types (P ⬍ 0.05);
n ⫽ 6 (myotubes) or 3 (fibers).
DISCUSSION
iPLA2 expression patterns among skeletal muscles have not
been examined at either the message or protein levels. Nor has
a functional role for iPLA2 been identified in this cell type.
These are the first data to indicate that iPLA2 modulates
oxidant activity and contractile properties of muscle. Our
findings help bridge gaps among prior reports on this subject,
Fig. 4. BEL does not scavenge superoxide anions. Values depict mean
superoxide anion activities (⫾SE) measured using the cytochrome c reduction
assay. Experiments compared 4 cell-free Krebs-Ringer solutions containing a
superoxide-generating system (1.25 ␮M hypoxanthine, 20 mU/ml xanthine
oxidase) plus either 1) 10 ␮M BEL, 2) 180 nM DMSO (diluent control), 3) 1
kU/ml SOD, or 4) no additive (buffer control). Superoxide activity was
unaffected by DMSO or BEL but was largely abolished by SOD (⫺96%
inhibition vs. buffer control; *P ⬍ 0.001).
J Appl Physiol • VOL
broadening our understanding of phospholipase function in
skeletal muscle.
iPLA2 expression in striated muscle. Fukusawa and Serrero
(9) originally reported that iPLA2 activity was less in skeletal
muscle and heart than in nine other rat tissues; in particular, it
was two to three orders of magnitude lower than intestine.
Tang et al. (35) measured iPLA2 mRNA in a mouse-rat
multiple-tissue Northern blot. They detected a 3 kb iPLA2
transcript that was abundant in muscle and barely detectable in
heart. Forsell and associates (8) detected four iPLA2 transcripts
of 1.8, 2.0, 3.2, and 4.2 kb in a human multiple-tissue Northern
blot. All four transcripts were detectable in skeletal muscle and
in heart. More recently, Mancuso et al. (18) and Tanaka et al.
(34) have also reported that skeletal muscle and heart express
a membrane-associated iPLA2 with an mRNA of ⬃3.4 kb.
Note that iPLA2 expression by muscle was not the focus of
these prior studies. Separate skeletal muscles were not evaluated individually, and protein levels were not measured.
To define iPLA2 distribution among muscles, we measured
iPLA2 mRNA and protein levels in a panel of striated muscle
preparations selected for adaptational divergence. Murine skeletal muscles included an endurance-adapted respiratory muscle
(diaphragm), an endurance-adapted limb muscle (soleus), a
sprint-adapted limb muscle (EDL), and a limb muscle of
intermediate contractile and metabolic properties (gastrocnemius). Highly aerobic cardiac muscle was also included for
comparison, as were differentiated myotubes from the immortalized C2C12 cell line derived from mouse skeletal muscle.
iPLA2 mRNA and protein levels were detectable in all of these
preparations and were of similar magnitude among the various
excised muscles. This finding suggests that constitutive iPLA2
expression is a robust property of striated muscle, at least in the
mouse, and that expression patterns are not strongly adaptation
dependent.
iPLA2 and oxidant activity. Several prior reports indicate
that PLA2 activity supports ROS production by skeletal muscle. Nethery and coworkers (24) studied intracellular ROS
levels in isolated, perfused rat hemidiaphragms. They showed
that ROS levels increase during repetitive, fatiguing contrac-
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6B). As muscles fatigue, relaxation between tetanic contractions becomes incomplete such that residual force persists in
the absence of electrical stimulation (37). In the present protocol, residual force increased monotonically as control muscles fatigued; the rise in residual force was unaffected by either
BEL or SOD pretreatment (data not shown).
IPLA2
IN SKELETAL MUSCLE
403
Fig. 5. iPLA2 blockade inhibits force production by unfatigued muscle. Values depict mean
forces (⫾SE) developed by soleus muscles
stimulated electrically in vitro. A: compared
with muscles incubated with DMSO alone,
pretreatment with 10 ␮M BEL depressed force
at stimulus frequencies of 30 –120 Hz (*P ⬍
0.05). B: pretreatment with 1 kU/ml SOD
depressed force relative to control over the
stimulation range of 30 – 80 Hz (*P ⬍ 0.05).
Po, maximal tetanic force.
that elevate cytoplasmic calcium, e.g., repetitive muscle contraction (7, 19, 24, 27), heat stress (42), or direct mitochondrial
exposure to calcium (23). Elevated calcium levels activate the
14-kDa calcium-dependent PLA2 isoform (cPLA2), which
stimulates ROS production at supranormal rates (24, 42).
Integrating these data, the calcium-independent iPLA2 isoform
appears to play a housekeeping role, supporting low rates of
ROS production under resting conditions, whereas the cPLA2
isoform provides a calcium-sensitive mechanism of stimulating
additional ROS production when cytosolic calcium levels rise.
Our present data may underestimate iPLA2 contributions to
oxidant activity. It is possible that iPLA2 inhibition by BEL
was incomplete. This error, if present, is likely to be small,
because the concentration we used (10 ␮M) exceeds the EC50
by more than threefold (11). Another potential source of
underestimation is the DMSO used in diluent control studies.
DMSO has antioxidant effects (12). To the extent DMSO
buffered muscle-derived oxidants, BEL effects would be obscured. This would lessen the apparent contribution of iPLA2
to oxidant activity. One final limitation of our present study,
Fig. 6. Effect of iPLA2 blockade on acute
fatigue. Values depict mean forces (⫾SE)
developed by soleus muscles during 5 min of
repetitive 40-Hz stimulation. Forces were depressed by pretreatment with either 10 ␮M
BEL (A) or 1 kU/ml SOD (B) relative to the
corresponding control group (P ⬍ 0.05 for
each comparison).
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tions and that this increase is inhibited by PLA2 blockade. In a
follow-up study (23), they later identified muscle mitochondria
as the likely site of PLA2-dependent ROS production within
muscle fibers. Extracellular ROS release by muscle fibers also
appears to be modulated by PLA2. Zuo et al. (42) identified
5-lipoxygenase as the primary source of superoxide anions
released from rat diaphragm fiber bundles, both under basal
conditions and during heat stress. Their study suggested that
PLA2 supports superoxide anion production by generating
arachidonic acid for 5-lipoxygenase metabolism. Thus PLA2 is
implicated as an upstream modulator of ROS production via
two cellular sources, mitochondria and 5-lipoxygenase, that
affect the activity of muscle-derived ROS in both intracellular
and extracellular compartments.
PLA2 effects on muscle-derived ROS appear to be state and
isoform dependent. Our data identify the calcium-independent
iPLA2 isoform as a major determinant of ROS activity under
resting conditions. This is consistent with the facts that cytosolic calcium concentration is low in resting muscle fibers (36).
ROS production is increased above resting levels by conditions
404
IPLA2
IN SKELETAL MUSCLE
ACKNOWLEDGMENTS
We thank Andy Mead for assistance with the cytochrome c reduction assay.
GRANTS
This project was supported by National Heart, Lung, and Blood Institute
Grants HL-59878 and HL-45721 (M. B. Reid) and HL-67284 (M. C. Gong).
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and PLA2 studies in general, is the widespread reliance on
pharmacological probes to assess isoform specificity. BEL is a
potent, irreversible inhibitor of iPLA2 with specificity vs.
calcium-dependent PLA2 isoforms of ⬎1,000-fold (11) and is
the standard intervention for contemporary studies of iPLA2
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compliment existing drug studies.
iPLA2 and contractile function. iPLA2 blockade caused a
drop in the forces developed by unfatigued muscle at physiological activation frequencies. This is consistent with the decrease in cytosolic oxidant activity that we observed after
iPLA2 inhibition. Prior work by our laboratory (1, 14, 28, 29)
and by others (6, 21, 30, 39) has shown that antioxidants
decrease the force of contraction in unfatigued muscle. This
phenomenon is illustrated in our present experiments by the
actions of SOD, an antioxidant enzyme that selectively depletes muscle-derived superoxide anions. As with iPLA2
blockade, SOD exposure caused both cytosolic oxidant activity
and force production to fall. The most straightforward interpretation of these data is that constitutive iPLA2 activity
contributes to redox homeostasis in resting muscle, supporting
ROS production and redox-sensitive contractile function.
Our data contribute to the evidence that ROS effects on
muscle function are temperature dependent. Muscle-derived
ROS activity and ROS effects on muscle function are strongly
affected by temperature (2, 5, 43). To optimize resolution of
these processes, the present studies were conducted at 37°C. In
contrast, Nethery and associates (24) showed that intramuscular ROS levels are very low at 24°C and that iPLA2 inhibition
using 25 ␮M BEL has no discernable effect on contractile
function. Differences between our findings and those of Nethery et al. are consistent with reports that ROS activity is
diminished by cooling (2, 43) and that ROS effects are less
evident at subphysiological temperatures (24).
Conclusions. These findings identify iPLA2 as a constitutively expressed modulator of cytosolic oxidant activity and
contractile function in skeletal muscle cells. They contribute to
growing evidence that the PLA2 superfamily is a major regulator of ROS production by muscle. Pharmacological studies
indicate that PLA2 effects are isoform dependent, such that
iPLA2 regulates oxidant activity under basal conditions and
cPLA2 boosts ROS production during metabolic stress, i.e.,
fatiguing exercise or hyperthemia. This model has important
implications for redox-dependent signaling in muscle and highlights the importance of future experiments using genetic
interventions to define the roles of iPLA2 and cPLA2.
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34.
IN SKELETAL MUSCLE