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Am J Physiol Endocrinol Metab 295: E195-E204, 2008. First published May 20, 2008; doi:10.1152/ajpendo.90276.2008
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Kinase-specific responsiveness to incremental contractile activity in skeletal muscle with low and high mitochondrial content

Vladimir Ljubicic and David A. Hood

Muscle Health Research Centre, School of Kinesiology and Health Science, York University, Toronto, Ontario, Canada

Submitted 6 March 2008 ; accepted in final form 9 May 2008


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Muscle contractions activate protein kinases, leading to signal transduction. We hypothesized that kinase activation would be influenced by mitochondrial content, as well as by contractile activity-induced increases in muscle O2 consumption (VO2). Kinase phosphorylation in high-oxidative red and low-oxidative white tibialis anterior (TA) muscle (RTA and WTA, respectively) with 2.5-fold differences in mitochondrial content were compared. Stimulation of the TA muscle elicited large increases in VO2 (3- to 6-fold and 4- to 60-fold above resting levels in WTA and RTA, respectively). At rest, AMP-activated protein kinase (AMPK), p38, p42, and p44 activation were nearly twofold greater in WTA than in RTA, suggesting an inverse relationship between mitochondrial content and kinase activation in resting muscle. During contractions, similar degrees of phosphorylation in RTA and WTA were evident as a function of VO2 for p38 and p42. During increases in VO2 up to sixfold above rest, greater responses were observed in RTA than in WTA for AMPK and p44, whereas Akt activation was greater in WTA. In RTA, elevations in VO2 elicited increases in AMPK and p44 activation, whereas Akt, p38, and p42 were less sensitive to increments in VO2. Reactive oxygen species (ROS) production was greater in mitochondria from white muscle, but when it was calculated in the context of the whole muscle, ROS production was twofold greater in red than in white myofibers. Thus mitochondrial content influences ROS production and is inversely related to kinase activation in resting muscle. During contractions, kinases are differentially sensitive to contraction-induced increments in VO2, suggesting that muscle mitochondrial content is important, but it is not the sole determinant of kinase activation during exercise of different intensities.

muscle oxygen consumption; kinase activation; muscle plasticity; signal transduction; mitochondrial biogenesis

WITHIN SECONDS OF THE ONSET of contractile activity in skeletal muscle, a number of rapid events occur that form part of the initial signaling process leading to downstream protein and lipid synthesis. These changes include Ca2+ flux, ATP turnover, and stimulation of O2 consumption (VO2) within the contracting muscle cells. Accumulating evidence supports the link between chronic alterations in these conditions and distinctive programs of gene expression that establish phenotypic diversity among skeletal myofibers, such as hypertrophy or mitochondrial biogenesis. For example, raising cytosolic Ca2+ by exposing skeletal muscle cells to caffeine or the Ca2+ ionophore A-23187 induced increases in proteins involved in mitochondrial biogenesis. These adaptations include increases in nuclear respiratory factor (NRF)-2 (NRF-2) binding to DNA, cytochrome c and malate dehydrogenase mRNA expression, and mitochondrial transcription factor A (Tfam) and peroxisome proliferator-activated receptor-{gamma} coactivator-1{alpha} (PGC-1{alpha}) protein content (27, 28, 61). PGC-1{alpha} is an important adaptive protein in skeletal muscle: it plays a key role in fiber type determination and mitochondrial gene expression (34, 52). In addition to a signaling role for Ca2+, chronic reductions in the ATP-to-ADP ratio, elevations in AMP levels, or administration of the creatine analog β-guanidinopropionic acid to rodents also elicit changes in muscle phenotype. The energy stress induced by these conditions has been shown to lead to alterations such as augmented NRF-1 DNA binding, {delta}-aminolevulinate synthase mRNA expression, PGC-1{alpha}, and cytochrome c protein content, as well as increased skeletal muscle mitochondrial density (10, 42, 79). Thus the adaptive response of skeletal muscle to an increase in cytosolic Ca2+ and ATP turnover mimics the stimulation of mitochondrial biogenesis by exercise (35).

Within mitochondria, a small fraction of the O2 consumed undergoes a one-electron reduction, resulting in production of reactive oxygen species (ROS). ROS production increases in muscle during contractile activity (57, 63) and with acute exercise (19). Recent studies have indicated the chemically induced ROS production can lead to mitochondrial reticulum elongation and branching complexity in fibroblast cells (49). Furthermore, patients with mitochondrial complex I deficiency exhibit elevated rates of ROS production and similar adaptive changes in mitochondrial morphology (50). Thus it has become clear that ROS are important signals involved in mitochondrial adaptations and that they likely represent an additional stimulus involved in the mitochondrial response to exercise.

The alterations in cellular homeostasis brought about by contractile activity, as noted above, affect the activation of kinases and phosphatases, resulting in the posttranslational modification of proteins (59, 68). Multiple kinases, including AMP-activated protein kinase (AMPK), protein kinase B (Akt), and the mitogen-activated protein kinases (MAPKs) p38, p42, and p44, are involved in the regulation of DNA transcription through phosphorylation of nuclear transcription factors. This enhances or inhibits the ability of transcription factors to bind DNA, affecting target gene transcription (10, 68, 70). Chronic activation of these signaling cascades by muscle contractions during intermittent bouts of acute physical activity can result in phenotypic adaptations such as mitochondrial biogenesis (35) and improved endurance performance (53) or myofiber hypertrophy and augmented force development (8). Therefore, the initiation of muscle plasticity begins with the early signals associated with contracting skeletal muscle leading to downstream kinase activation and gene expression.

In the context of exercise, these rapid responses are related to the type, intensity, and duration of the contractile activity, as well as the muscle fiber type (12, 59, 68). A number of studies have examined the influence of these parameters on the extent of kinase sensitivity and responsiveness (7, 39, 59, 62, 68, 69, 74). Nader and Esser (59) demonstrated that acute low- or high-frequency electrical stimulation-induced contractile activity of fast- or slow-twitch muscle in situ differentially activated a number of signaling molecules such as p70S6K, Akt, and p38. Although the signaling kinase response to a multiplicity of acute contractile activity paradigms has been documented, the nature of the phosphorylation events characteristic of contractile activity representing different energy costs of contraction in high-oxidative red and low-oxidative white skeletal muscle remains poorly understood. Thus the purposes of this work were to investigate the phosphorylation of signaling kinases that are relevant to phenotypic adaptations in fast-twitch muscle with low or high mitochondrial content and the relationship between contractile activity-induced increases in VO2 and kinase activation. We hypothesized that activation of the kinase signaling network would be increased in response to elevated VO2 to a greater extent in low- than in high-oxidative muscle.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Adult male Sprague-Dawley rats (Charles River; 373.8 ± 3.8 g body wt, n = 35) were housed two per cage in a temperature-controlled room (20–21°C) with a 12:12-h light-dark cycle. Purina Rat Chow and water were provided ad libitum.

In situ acute stimulation procedure. All experiments involving animals were evaluated and approved by the York University Animal Care Committee in accordance with the regulations of the Canadian Council on Animal Care. Animals were prepared essentially as described previously (73). They were anesthetized with pentobarbital sodium (60 mg/kg), and the left common carotid artery was catheterized (PE-50) to allow us to monitor hemodynamic parameters with the use of a pressure transducer (model RS 3200, Gould, Cleveland, OH). The left tibialis anterior (TA) muscle was exposed and prepared for in situ direct muscle stimulation. The muscle of the other limb was also exposed to facilitate tissue sampling; it was wrapped in plastic to prevent dehydration. The distal tendon of the TA muscle was isolated, and a hooked pin was affixed to the tendon and attached to a strain gauge. Intramuscular stimulating electrodes were placed in the belly of the muscle, parallel to the fibers. The temperature of the stimulated muscle was maintained at 37°C with heat lamps and monitored continuously with a surface thermometer (Yellow Springs Instruments, Yellow Springs, OH). O2 (100%) was provided to the animal throughout the procedure. Direct, unilateral stimulation of the TA muscle was subsequently recorded over a 5-min period during one of four acute contractile activity protocols: 1) mild [100 Hz, 100-ms duration, 0.1 trains/s (TPS)] or 2) intense (1 TPS) tetanic (TET) contractile activity or 3) mild (1 Hz) or 4) intense (10 Hz) twitch (TW) contractions of the TA. As such, the total number of electrical impulses of the mild (300 stimuli) or intense (3,000 stimuli) TW and TET stimulation bouts were equated. Force and pressure signals were sampled online (Powerlab 4/SP, ADInstruments, Colorado Springs, CO) and stored for analysis using Chart 5 software. While the tissues were perfused, the TA muscles of the stimulated and contralateral nonstimulated limbs were quickly removed. The deep, high-oxidative red TA (RTA) and the superficial, low-oxidative white TA (WTA) portions were freeze clamped with aluminum tongs precooled in liquid nitrogen and stored at –70°C. These muscle sections represent the most divergent mitochondrial oxidative capacities within skeletal muscle. Animals were killed by exsanguination after a medial thoracotomy.

Estimation of muscle VO2. Similar to previous reports (1, 76), VO2 values were calculated on the basis of the known O2 cost of TW and TET contractions of rat skeletal muscle (37). Resting and contractile activity-induced skeletal muscle VO2 values were estimated according to values reported previously under identical experimental conditions (37). Steady-state VO2 levels during the TW and TET protocols are based on a measured 0.037 and 0.26 µmol O2·g muscle–1·contraction–1, respectively, in the absence of fatigue (37). The O2 cost of contractions is contingent on 1) the performance decline of the whole muscle, 2) the fiber composition of the rat hindlimb musculature (6), and 3) the knowledge that energy costs of contraction are a function of force development (29, 65). We assume (37) that force development per unit mass is similar for RTA and WTA (17) and that the initial loss of maximal force-producing capacity is attributed to contractile failure in the low-oxidative WTA portion (9, 14, 23, 24, 55), comprising ~60% of TA myofibers (51). Because the objective of the present study was to establish the activation of kinases as a function of VO2 in fast-twitch red and fast-twitch white muscle, these previously validated assumptions and calculations were essential to achieve this goal with the in situ muscle preparation. Hepple et al. (31, 32) also reproduced nearly identical skeletal muscle VO2 values in rat hindlimb muscle under similar conditions of O2 delivery during in situ acute contractile activity.

Preparation of skeletal muscle tissue lysates and immunoblotting. We investigated the responsiveness of multiple signaling kinases to contraction-induced increases in VO2. Frozen RTA and WTA sections were pulverized to a fine powder with a stainless steel mortar that was cooled to the temperature of liquid nitrogen. The protein extraction was performed as previously described (36) with modifications (69). Briefly, powdered tissues were diluted 1:20 (wt/vol) in buffer B [20 mM HEPES (pH 7.4), 2 mM EGTA, 50 mM β-glycerophosphate, 1 mM dithiothreitol, 1 mM Na3VO4, 10% glycerol, 1% Triton X-100, 10 µM leupeptin, 5 µM pepstatin A, 10 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride]. Homogenates were rotated for 1 h at 4°C, sonicated (3 times, each for 5 s) on ice, and then centrifuged at 14,000 g for 10 min at 4°C. The protein concentrations of the supernates were determined by the Bradford method, with BSA as the standard (13). Proteins (50 µg) extracted from the muscle homogenates were resolved by SDS-PAGE (10–12% polyacrylamide) and subsequently electroblotted to nitrocellulose membranes (Amersham, Baie D'Urfé, PQ, Canada). The membranes were blocked (1 h) with 5% skim milk in 1x Tris-buffered saline-Tween 20 [TBST: 25 mM Tris·HCl (pH 7.5), 1 mM NaCl, and 0.1% Tween 20] solution and then incubated overnight at 4°C with phosphorylated (Thr180/Tyr182) p38 MAPK (1:250 dilution), p38 MAPK (1:1,000 dilution), phosphorylated (Thr172) AMPK{alpha} (1:250 dilution), AMPK{alpha} (1:1,000 dilution), phosphorylated (Thr202/Tyr204) p42/p44 (1:500 diluted), total p42/p44 (1:1,000 diluted), phosphorylated (Ser473) Akt (1:750 dilution), or total Akt (1:1,000 dilution). All antibodies were purchased from New England Biolabs (Mississauga, ON, Canada). For overnight incubations, antibodies were diluted in 5% BSA-TBST, with the exception of phosphorylated p42/p44, which was suspended in 5% skim milk-TBST. After three 5-min washes with TBST, blots were incubated for 1 h at room temperature with the appropriate secondary antibody coupled to horseradish peroxidase. Blots were then washed (3 times, each for 5 min) with TBST and visualized with enhanced chemiluminescence (ECL). Hyperfilm ECL (Amersham) exposure time was optimized so that blot intensities were within the linear range of detection. Films were then scanned and analyzed by using SigmaScan Pro 5 software (Jandel Scientific, San Rafael, CA). Kinase activation status was determined as the ratio of the phosphorylated form of the protein to the total protein content for each sample.

Cytochrome c oxidase activity. Cytochrome c oxidase (COX) activity in RTA and WTA sections from the nonstimulated leg was evaluated as described previously (30). Enzyme activity was determined as the maximal rate of oxidation of fully reduced cytochrome c measured by the change in absorbance at 550 nm in a spectrophotometer (model DU-64, Beckman).

Mitochondrial isolation from red and white muscle. Another group of male Sprague-Dawley rats (234.9 ± 2.9 g body wt, n = 8) was used for the mitochondrial experiments. Pooling of RTA and WTA with sections of red and white gastrocnemius, respectively, provided sufficient material (0.8–1 g muscle tissue) for the isolation protocol. Muscle portions were placed into chilled buffer 1, finely minced, and then homogenized. Differential centrifugation and mechanical and protease digestions were employed to fractionate subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria, as described previously (54). SS and IMF subfractions were resuspended in resuspension medium, and an aliquot of the suspension was taken for measurements of protein content (13).

Mitochondrial respiration. Samples of isolated SS and IMF mitochondrial subfractions were incubated with VO2 buffer at 30°C in a water-jacketed respiratory chamber with continuous stirring. VO2 (natoms O2·mg–1·min–1) was assessed in the presence of 11 mM glutamate (state 4 respiration) or glutamate + 0.4 mM ADP (state 3 respiration) using a Clark O2 electrode (Yellow Springs Instruments), as described elsewhere (3, 54).

Mitochondrial ROS production. ROS were measured as described previously (3). Briefly, SS and IMF mitochondria (50 µg) from red and white muscle were incubated with VO2 buffer in a 96-well plate. ROS production was assessed at 37°C for 30 min during state 4 and state 3 respiration by addition of 11 mM glutamate and glutamate + 0.4 mM ADP, respectively, immediately before the addition of 50 µM dichlorodihydrofluorescein diacetate. Fluorescence emission at 480–520 nm measured with a multidetection microplate reader (Synergy HT, Biotek Instruments, Winooski, VT) is directly related to ROS production. Data were recorded and interpreted using KC4 (version 3.0) software. ROS production measured in absolute fluorescence units was linear over the entire measurement period. ROS levels are expressed per nanoatom of O2 consumed, measured during the mitochondrial respiration assay.

Mitochondrial ROS production within the context of the whole muscle was estimated on the basis of our results from the mitochondrial ROS assay in conjunction with previous data on mitochondrial content in skeletal muscle (38). We calculated ROS production per milligram of red and white muscle by assuming that 1) SS and IMF mitochondria constitute ~20% and 80% of the mitochondrial volume within muscle, respectively, and 2) the mitochondrial content in white and red muscle is ~2% and 6% by volume, respectively (38).

Statistical analyses. The data were analyzed using paired and unpaired Student's t-tests and ANOVA, as appropriate. Bonferroni's post hoc test was used to test significant differences revealed by ANOVA. Statistically significant distinctions between fiber types (fold differences) were computed using the raw data sets before conversion to the fold differences. Significance was accepted at P < 0.05. Values are means ± SE.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Mitochondrial content and kinase phosphorylation in RTA and WTA. We estimated mitochondrial content in the low- and high-oxidative fiber types by measuring COX activity (77). COX enzyme activity was 4.7 ± 0.3 and 11.5 ± 1.1 U/g muscle in WTA and RTA, respectively (Fig. 1A). This disparity in mitochondrial enzyme activity between low- and high-oxidative skeletal muscles is in agreement with previous work (9, 66).


Figure 1
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  		Fig. 1. Phosphorylation of signaling kinases in low- and high-oxidative portions of resting, nonstimulated tibialis anterior (TA) muscle. TA was sectioned into red (RTA) and white (WTA) muscle and assayed for cytochrome c oxidase (COX) activity (A) and Akt (B), AMP-activated protein kinase (AMPK, C), p38 MAPK (D), p42 MAPK (E), and p44 MAPK (F) activation by Western blotting. Kinase activation is expressed as the ratio of the phosphorylated form of the protein to total protein expression. Values are means ± SE of repeated experiments (n = 7–9). Top blot: phosphorylated (P) form of the protein; bottom blot: total protein content. *P < 0.05 vs. RTA.

 
The phosphorylation state of kinases known to be responsive to acute contractile activity in skeletal muscle, including AMPK, Akt, and p38, p42, and p44 MAPKs, was measured in RTA and WTA at rest. Basal phosphorylation status (i.e., the phosphorylated form of the protein relative to the total protein content of the kinase) of Akt was similar between the two fiber types (Fig. 1B); this finding coincides with earlier observations made in red and white gastrocnemius (69). In contrast, AMPK and p38, p42, and p44 MAPK activation was 1.6- to 2.3-fold greater in WTA than in RTA (Fig. 1, C–F).

Muscle performance and calculated VO2 during acute contractile activity. To investigate acute contractile activity-induced VO2 and intracellular signaling in low- and high-oxidative fiber types, unilateral electrical stimulation of the TA was employed. Endurance performance of the TA during the four 5-min acute contractile activity paradigms is shown in Fig. 2A. Initial TW and TET tension outputs were 1,470 ± 84 and 8,640 ± 400 mN, respectively, a 5.9-fold difference (P < 0.05). Muscle force was potentiated throughout the 1-Hz TW protocol, attaining 117.4 ± 10% of initial tension after 5 min of contractile activity. Muscle fatigue was modest, but significant, during the 0.1-TPS TET contractions, with force dropping to 83.1 ± 3% of initial force. Despite the sevenfold difference in the O2 cost of maximal TW and TET contractions (37), more intense 10-Hz TW and 1-TPS TET contractile activity elicited similar decrements in maximal force output to 45.6 ± 4% and 35.4 ± 3% of initial tension, respectively. Blood pressure was well maintained throughout the experimental protocol, averaging 115.6 ± 1.5 mmHg for all animals.


Figure 2
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  		Fig. 2. Muscle tension and estimated O2 consumption (VO2) during acute in situ contractile activity. A: force production was recorded over 5 min of direct muscle stimulation of TA eliciting twitch (TW) or tetanic (TET) contractions. TW contractions were induced at a frequency of 1 or 10 Hz. TET contractions were evoked at a frequency of 0.1 or 1 train per second (TPS). Values are means ± SE (n = 8–9). *P < 0.05 vs. initial tension. B: estimated VO2 in RTA and WTA at rest and in response to contractile activity based on muscle performance during TW and TET protocols. Tissue-specific differences in VO2 during 10-Hz TW and TET procedures are due to contraction failure in low-oxidative WTA.

 
Estimated VO2 levels of the high- and low-oxidative sections of the TA during contractile activity, as well as in the nonstimulated contralateral control muscle, were based on previously reported results (37). Resting, or noncontracting, skeletal muscle VO2 is 0.37 µmol·g–1·min–1 (Fig. 2B). With contractile activity, VO2 calculations take into account the extent of contraction-induced fatigue exhibited by the muscle, as well as the proportion of low- and high-oxidative fibers in the tissue. For example, the 0.1-TPS TET protocol reduced force output of the TA to 83.1% of initial tension (Fig. 2B). We made the assumptions that the loss of maximal force production of the muscle is due to contractile failure in the low-oxidative WTA portion (9, 14, 23, 24, 55), accounting for 60% of TA myofibers (51), and that force development per unit mass is similar for RTA and WTA (17). The O2 cost of one maximal TET contraction was reported to be 0.26 µmol·g–1·contraction–1 in the absence of fatigue (37). Therefore, VO2 in the RTA during 0.1-TPS TET was calculated as follows: 0.26 µmol·g–1·contraction–1 x 6 contractions/min = 1.56 µmol·g–1·min–1. In the WTA, where contractile failure was responsible for the 16.9% decrement in force production in the muscle, VO2 was determined as follows: 0.26 µmol·g–1·contraction–1 x 6 contractions/min x 43.1/60 = 1.12 µmol·g–1·min–1.

During the 1-TPS TET contractile activity protocol, the added O2 cost of each tetanic contraction of 0.26 µmol/g for full force output would decrease to zero for the fast-twitch WTA as tension development of the entire muscle decreases to 35.4% of initial. Thus we estimate that VO2 of completely failed muscle would revert rapidly to resting levels as contraction failure envelopes 100% of the WTA muscle mass (37).

The multiple acute contractile activity protocols account for a wide degree of induced skeletal muscle VO2 estimates. In WTA, the calculated VO2 estimates (Fig. 2B) represent 3.0-, 5.6-, and 6.0-fold elevations in VO2 above resting levels. For the RTA, the VO2 levels equate to 4.2-, 6.0-, 37-, and 60-fold increases in VO2 over the resting value.

Fiber type-specific kinase phosphorylation during contractile activity-induced increases in VO2. To compare intracellular signaling responsiveness in low- and high-oxidative fiber types, we assessed protein kinase phosphorylation in the estimated VO2 range of 0.37 µmol·g–1·min–1 (i.e., resting WTA and RTA and noncontracting WTA) to 2.22 µmol·g–1·min–1 (i.e., 1-Hz TW contractions), a 6-fold increase in VO2. Akt activation in WTA was increased twofold at VO2 of 2.08 µmol·g–1·min–1 compared with rest and 2.22 µmol·g–1·min–1 (Fig. 3A). Furthermore, Akt remained activated twofold above nonstimulated WTA during the 1-TPS TET protocol, when contractions of these fibers would presumably have ceased due to fatigue (37). Akt phosphorylation in RTA was unchanged during VO2 increments in this range.


Figure 3
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  		Fig. 3. Signaling kinase response with increasing contractile activity-induced VO2. Activation of Akt (A), AMPK (B), p38 MAPK (C), p42 MAPK (D), and p44 MAPK (E) is expressed as fold change compared with resting, nonstimulated values in high-oxidative RTA and low-oxidative WTA. Estimated VO2 values are indicative of RTA and WTA VO2 at rest and during mild 1-Hz TW and 0.1-TPS TET contractile activity, WTA VO2 at 10-Hz TW, and WTA VO2 at 1-TPS TET, as described in Fig. 2B legend. Values are means ± SE (n = 7–9). *P < 0.05 vs. resting, nonstimulated muscle (0.37 µmol O2·g–1·min–1). {dagger}P < 0.05 vs. 2.22 µmol O2·g–1·min–1 in the same fiber type.

 
A stepwise elevation in AMPK activation in RTA was evident with stimulation-induced increases in VO2 (Fig. 3B). AMPK phosphorylation was 2.1-fold higher at VO2 of 1.56 µmol·g–1·min–1 than at rest and further increased by 1.5-fold at VO2 of 2.22 µmol·g–1·min–1. In contrast, acute contractile activity-associated increments in VO2 did not alter AMPK activation in the low-oxidative WTA, similar to previous results (25).

The increase in p38 phosphorylation within this sixfold range of VO2 was modest for WTA and RTA. Compared with resting, nonstimulated levels, p38 activation in WTA was 1.4-fold higher at VO2 of 1.12 µmol·g–1·min–1 and 1.9-fold higher after stimulation, when fatigue returned VO2 to 0.37 µmol·g–1·min–1 (Fig. 3C). In RTA, p38 activation was 1.5-fold greater at VO2 of 2.22 µmol·g–1·min–1 in RTA than at rest (Fig. 3C).

Across this range of incremental VO2, p42 activation was unchanged in RTA and WTA (Fig. 3D). In contrast, the p44 response pattern in both fiber types was comparable to that observed with AMPK and was divergent in red, compared with white, muscle. In RTA, levels of p44 activation were 2.3- and 3.5-fold greater at contractile activity-induced VO2 of 1.56 and 2.22 µmol·g–1·min–1, respectively, than at rest (Fig. 3E). During contractile activity, p44 phosphorylation was less marked in WTA than in RTA, increasing to a level twofold higher at VO2 of 2.08 µmol·g–1·min–1 than at rest.

Increased contraction-induced VO2 and kinase activation in the high-oxidative RTA. In the present study, calculated contraction-elicited increases in VO2 for the high-oxidative RTA ranged from 0.37 µmol·g–1·min–1 at rest to 22.2 µmol·g–1·min–1 during 10-Hz TW contractile activity (Fig. 4). Two distinct patterns of kinase activation were evident. The first, apparent from the Akt (Fig. 4A), p38 (Fig. 4C), and p42 (Fig. 4D) responses, involved progressive increases in activation with increases in VO2 to two- to threefold (Akt and p42) to three- to fourfold (p38) above resting muscle. The greatest increases occurred at 13.81 µmol·g–1·min–1, rather than at the highest VO2 attained. In contrast, AMPK (Fig. 4B) and p44 (Fig. 4E) displayed significant increases in activation (3- to 4-fold) that were initiated at much lower levels of VO2 and, thereafter, reached a plateau (p44) or were modestly reduced (AMPK) at higher workloads.


Figure 4
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  		Fig. 4. Kinase activation in high-oxidative section of TA muscle, with increasing contractile activity-induced VO2. Akt (A), AMPK (B), p38 MAPK (C), p42 MAPK (D), and p44 MAPK (E) activation is expressed as fold change compared with resting values. Calculated VO2 values correspond to RTA at rest and during the 4 contractile activity conditions corresponding to the VO2 values, as described in Fig. 2B legend. Values are means ± SE (n = 7–9). *P < 0.05 vs. resting activation.

 
Mitochondrial VO2 and ROS production in red and white skeletal muscle. In view of the important roles played by mitochondria in signal transduction pathways in skeletal muscle, we isolated SS and IMF mitochondrial subfractions from red and white muscles and measured rates of respiration and ROS production. As described in Table 1, the yield of SS and IMF mitochondrial protein recovered from the fractionation process was greater when the organelles were isolated from red than from white skeletal muscle. The greater mitochondrial yield from red muscle, indicative of organelle content, is also reflected by the higher COX enzyme activity of the RTA, as shown in Fig. 1A. Glutamate (state 4)- and glutamate + ADP (state 3)-stimulated respiration was 1.5- to 1.7-fold higher in SS mitochondria isolated from red muscle than in the SS subfraction recovered from white muscle (Table 1). In contrast, rates of IMF mitochondrial respiration were similar between fiber types. The mean respiratory control ratio of 7.2–9.4 for all samples was indicative of well-coupled and functional mitochondria. Consistent with previous results, ROS production from isolated mitochondria was greater during state 4 than during state 3 respiration and was higher in the SS than in the IMF mitochondrial subfraction (2, 3). In SS mitochondria, ROS production per nanoatom of O2 consumed during state 4 and state 3 respiration was 1.4- to 1.5-fold lower in mitochondria isolated from red than from white muscle. ROS production also tended to be lower in IMF mitochondria isolated from red muscles (–20%) than from white muscles.


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  		Table 1. Mitochondrial yield, O2 consumption, and ROS production in SS and IMF subfractions

 
We wished to assess the potential contribution of ROS signaling within the context of the whole muscle, as we have done for the signaling kinases. We assumed that 1) SS and IMF mitochondria constitute ~20% and 80% of the mitochondrial volume within muscle, respectively, and 2) the mitochondrial content in white and red muscle is ~2% and 6% by volume, respectively (38). We then calculated ROS production per milligram of tissue in red and white muscle sections. This analysis revealed that ROS production per milligram of muscle was about twofold greater (P < 0.05) in red than in white myofibers during state 4 and state 3 respiration (Fig. 5).


Figure 5
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  		Fig. 5. Calculated mitochondrial reactive oxygen species (ROS) production per milligram of red and white skeletal muscle. Subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondrial ROS production levels measured during state 4 and state 3 respiration (Table 1) in red (solid bars) and white (open bars) skeletal muscle were combined using a weighted average of 0.8 for IMF and 0.2 for SS mitochondria to provide ROS production per microgram of mitochondria. These values were multiplied by an estimate of mitochondrial volume fraction (2%, 20 µg/mg muscle for white; 6%, 60 µg/mg muscle for red) to provide an assessment of ROS expressed per milligram of muscle mass. AFU, arbitrary fluorescence units. Values are means ± SE (n = 8). *P < 0.05 vs. red.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The aim of the present study was to relate contraction-elicited increases in muscle VO2 with the phosphorylation of kinases involved in the initiation of muscle plasticity. We selected the TA muscle, because it is readily accessible for in situ electrical stimulation. In addition, the TA consists of fast-twitch, low- and high-oxidative fiber types, which allows for assessment of the influence of mitochondrial content on altered VO2-induced signal transduction. Our results suggest that mitochondrial content influences kinase phosphorylation in a kinase-specific fashion, at rest and during contractions. Furthermore, kinase activation is not strictly proportional to increases in skeletal muscle VO2. Indeed, the activation of individual kinases displays a differential sensitivity to increases in muscle VO2.

We first examined the relationship between mitochondrial content and kinase phosphorylation in resting skeletal muscle. The disparity in organelle volume between the TA sections was confirmed by the nearly twofold greater mitochondrial COX enzyme activity measured in the RTA than the WTA. With the exception of Akt, basal kinase activation was inversely related to mitochondrial content: it was higher in white than in red muscle. This suggests that kinases in red muscle have a greater potential for activation in response to stimuli such as contractile activity. Our findings are similar to previous assessments of basal kinase activation between skeletal muscles with low (e.g., epitroclearis and TA) and higher (e.g., soleus) mitochondrial content (40, 45, 72, 78). These data suggest that mitochondrial concentration has an influence on the extent of kinase activation in resting muscle. However, other characteristics unique to red or white muscle fiber types, including rates of ROS production, intracellular Ca2+ concentration, and protein phosphatase activity, could influence basal kinase activation. It is known that resting Ca2+ concentrations are higher in red than in white skeletal muscle (15). This higher Ca2+ level could promote a greater activation of Ca2+-activated phosphatases, which would lower basal kinase phosphorylation in red muscle. Data describing muscle protein phosphatase activity appear to support this idea, since the expression of the Ca2+-activated protein phosphatase-2B was greater in red than in white muscle (11). The results of these studies suggest that a lower protein phosphatase content in white muscle may be partly responsible for the higher basal kinase phosphorylation in this tissue.

Signaling kinases in skeletal muscle are sensitive to a diverse array of stimuli associated with contractions, including the mode of contractile activity (59), the frequency of muscle activation (67) and contraction (7), the magnitude of passive and active tension development (18, 56), Ca2+ cycling within the myofibers (16, 21), and contraction-associated metabolites (26). In the present study, we sought to relate the activation of kinases to a common denominator of various modes of contractile activity: contraction-induced increases in aerobic energy demand, represented by VO2. Similar to previous reports (1, 76), the VO2 values in the present investigation were calculated on the basis of data obtained on the O2 cost of twitch and tetanic contractions of rat skeletal muscle (37). More recently, nearly identical muscle VO2 values have been reported in rat hindlimb muscle under similar conditions of O2 delivery during in situ acute contractile activity (31, 32). Three of the four contractile activity protocols employed in the present study induced fatigue, presumably because of the initial contraction failure of low-oxidative, white fibers (37). Reduced contractile activity of the WTA would, in turn, attenuate VO2 in those fibers (37) and result in whole muscle VO2 values between those for pure red and white muscle.

In response to increments in skeletal muscle VO2 evoked by electrical stimulation, we assessed kinase activation in high- and low-oxidative muscle (Fig. 3). The responses displayed a striking heterogeneity, which might be expected, since white fibers are likely working at a higher percentage of their maximum VO2 than red fibers at any given level of VO2. However, it is evident that this higher degree of metabolic stress in white fibers is not the only factor involved in the activation of kinases during contractile activity. For example, similar degrees of p38 and p42 phosphorylation were evident in white and red muscle as a function of VO2. This suggests that, as VO2 increases, common intracellular events that are unrelated to metabolic stress occur to invoke p38 and p42 activation during contractile activity. In contrast, markedly divergent responses between high- and low-oxidative muscle were evident for AMPK and p44. The activation of these kinases was greater in red than in white muscle. Thus, despite similar increases in absolute energy cost, the intracellular milieu within white and red muscle must differ significantly with respect to the activation of these two kinases. This is further exemplified by the response of Akt, which exhibited the opposite response: a greater activation was evident in white than in red muscle. It is interesting to note that Akt appears to have a critical role in signaling the phenotypic adaptations related to skeletal muscle hypertrophy and opposing atrophy (7, 12). In WTA, Akt and p38 remained activated during stimulation, even when the muscle was apparently no longer producing contractile force, consistent with the findings of others after periods of inactivity or recovery (7, 59, 62). Thus our results suggest that, during increases in muscle VO2, the activation of intracellular signaling is, in part, dependent on the oxidative capacity of the muscle, but it is specific for each kinase.

In addition to activation by phosphorylation, the activity of these kinases may be affected allosterically by cofactors intrinsic to the muscle. For example, AMP binds AMPK, thereby rendering the protein more receptive to phosphorylation (26, 68). Interestingly, Dudley et al. (22) showed lower free AMP (AMPf) levels in skeletal muscle with higher mitochondrial contents within a range of contraction-induced VO2 values. Our results, which indicate a greater activation of AMPK in RTA than in WTA (Fig. 3B), suggest that factors other than AMPf are important for AMPK activation during contractile activity. These include the status of upstream mediators of AMPK, such as LKB1, which may be activated by increasing VO2 to a greater extent in the RTA muscle. Therefore, future investigations of kinase signaling in skeletal muscle should account for protein phosphorylation levels and should also include other potential markers of activation, such as upstream kinase status and allosteric influences.

Differential kinase sensitivity to increments in VO2 is best observed over the broad 60-fold range exhibited by fast-twitch red muscle during contractile activity (Fig. 4). Our data indicate that relatively small increments in VO2 are required to elicit substantial elevations in the activation of p44 and AMPK. However, although p44 phosphorylation was maintained at high levels of VO2, AMPK phosphorylation returned toward that found in unstimulated muscle. The AMPK response may possibly be due to a leveling off, or decrease, of AMPf concentrations at higher levels of VO2 (22) or the kinase activity of its upstream activators. Nonetheless, these results demonstrate a high sensitivity of AMPK and p44 activation in response to initial increases in VO2. In contrast, Akt, p38, and p42 phosphorylation progressively increased to the maximum extent at higher VO2 levels. This broad range of energy demands at which different kinases are activated in high-oxidative red muscle would appear to be beneficial, in that they allow for the possibility that signaling events could occur at low to high levels of exercise. If cross talk, or redundancy, exists among kinase activation pathways, then this activation at various levels of VO2 permits phenotypic adaptations over all workload ranges. On the other hand, in the case of activated pathways that are mutually exclusive and affect separate downstream targets, specific adaptations are allowed when mild exercise, rather than more severe contractile activity, is invoked. In this interpretation, we recognize that our measures of kinase activation are reflective only of events at the onset of contractile activity, whereas typical phenotypic adaptations to exercise, such as mitochondrial biogenesis, may require longer exercise durations per bout (e.g., 20–30 min). In this context, it would also be useful in the future to examine the time course of kinase activation from the onset of contractile activity extending to more prolonged bouts of exercise.

The kinases that we have assessed ultimately activate downstream transcription factors and coactivators known to increase transcriptional activity. For example, AMPK phosphorylation leads to the activation of NRF-1 (10) and PGC-1{alpha} (44), both of which have important roles in the transcriptional activation of genes encoding mitochondrial proteins. Phosphorylation of p38 and Akt has been shown to activate the transcription factors activating transcription factor-2 (4) and Foxo (70), respectively, whereas p42/p44 signaling results in enhanced expression of nuclear factor of activated T cells and activator protein-1-dependent gene (71). Moreover, immediate early gene expression of c-fos, c-jun, serum response factor, and Egr-1 has been shown to increase in response to several distinct forms of contractile activity (20, 41, 58, 60), supporting the hypothesis that the kinases studied here are mediators of the stress response to exercise, including mitochondrial biogenesis (35). The principle of functional redundancy, exhibited here by the multiplicity of kinase activation in response to similar degrees of contraction-induced VO2, suggests that the downstream propagation of this metabolic signal is essential to the adaptive plasticity of muscle.

We further dissected the mechanism of VO2-induced signaling in skeletal muscle by isolating mitochondria and assessing organelle respiration and ROS production. ROS can act through several different pathways of signal transduction, making use of signaling cascades such as protein kinases, phosphatases, phospholipases, and Ca2+ (43). Indeed, ROS, such as H2O2, can strongly induce the activation of p42, p44, and p38 MAPK signaling in a dose- and time-dependent manner in skeletal muscle myoblasts (47). H2O2 also triggers the phosphorylation of p38 and AMPK in isolated skeletal muscle preparations (48, 75). We assessed the potential contribution of ROS signaling within the context of the whole muscle, as reported for the signaling kinases. These estimates were based on measured mitochondrial ROS production, as well as reasonable estimates of SS and IMF mitochondrial content in red and white muscle (38). The data reveal that mitochondria-derived ROS generation is greater in fast-twitch red than in fast-twitch white muscle. Of course, these results do not take into account the fact that ROS are also simultaneously neutralized by endogenous antioxidant molecules within the context of the whole muscle environment. In addition, alternative whole muscle sources of ROS production [e.g., xanthine oxidase enzymes and NAD(P)H oxidases (64)] contribute to the steady-state levels of ROS within the cell. Recently, results obtained using the permeabilized muscle fiber technique showed that ROS production was two- to threefold higher in white than in red myofibers (5). Thus these alternative contributing factors likely determine this higher rate of production in white fibers, since the mitochondrial content is substantially (2- to 3-fold) lower (Fig. 1A) in white than in red muscle. Measures of antioxidant protein content and activity in muscle homogenates, as well as H2O2 scavenging rate in permeabilized fibers, indicate a greater capacity for removal of ROS in high- than in low-oxidative fibers (5, 33). This may be due to a negative-feedback mechanism by which greater ROS-induced signaling leads to increased antioxidant gene expression (46). Thus, although mitochondria-derived ROS production is greater in red than in white muscle, the antioxidant capacity of red muscle must exceed that of white muscle, resulting in a greater overall steady-state ROS level in white muscle fibers (5). This may, in turn, contribute to the generally higher level of basal kinase activation in WTA muscle (Fig. 1).

In conclusion, our findings show that mitochondrial content influences the rate of ROS production and is inversely related to the activation of several kinases in resting muscle. During contractile activity, kinases are differentially sensitive to contraction-induced increments in skeletal muscle VO2, resulting in a broad range of exercise intensities in which kinases are active. This selective activation of intracellular signaling pathways and the divergent responses of red and white muscle likely play a part in determining the activation of downstream effectors (i.e., transcription factors) during exercise of varying intensities. This will lead to different muscle phenotypic adaptations, depending on 1) the exercise intensity and 2) the oxidative capacity of the fiber type that is recruited.


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This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC). V. Ljubicic was a recipient of NSERC Postgraduate Scholarships and is a Doctoral Research Award scholar of the Heart and Stroke Foundation of Canada. D. A. Hood holds a Canada Research Chair in Cell Physiology.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. A. Hood, School of Kinesiology and Health Science, York Univ., Toronto, ON, Canada M3J 1P3 (e-mail: dhood{at}yorku.ca)

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.


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90276.2008v1
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