Molecular Imaging Research Center, Departments of 1Physiology and 2Radiology, Michigan State University, East Lansing, Michigan
Submitted 13 October 2004 ; accepted in final form 22 November 2004
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ABSTRACT |
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calcium; energetics; mechanical stress; metabolism
Several signaling cascades involving families of mitogen-activated protein kinases (MAPKs) are responsive to mechanical stress in smooth, skeletal, and myocardial muscle (1, 9, 17, 23, 24, 26, 30, 36, 38, 39, 42, 43). These include extracellular signal-regulated kinase (ERK)1/2, c-Jun NH2-terminal kinase (JNK), and p38 MAPK and are known to be involved in cell proliferation, differentiation, apoptosis, and adaptations to stress (7, 17). Several transcription factors key to muscle phenotype transition are downstream of p38 MAPK, including cyclic AMP response element-binding protein, myocyte enhancer factor 2C, and peroxisome proliferator-activated receptor- coactivator-1
, with the latter having been shown to stimulate mitochondrial biogenesis and fast-to-slow phenotypic transition in skeletal muscle (41, 45). Thus MAPK are putative activators of genes important in both hypertrophic and phenotypic transformations in muscle in response to changes in mechanical stress.
The relationship between skeletal muscle force production and MAPK activation has been studied extensively in rodent (11, 17, 26, 3840) and human tissues (1, 30, 36). Phosphorylation of MAPKs increases as a result of increased active (11, 17, 26, 34, 3840) and passive tension (5, 11, 26, 40). A positive correlation of peak tension with phosphorylation has been demonstrated for JNK, ERK1/2, and p38 MAPK when eccentric > isometric > concentric > passive tension (listed in decreasing order of activation), with regard to the type of mechanical stress used and the extent of MAPK phosphorylation (11, 15, 22, 26, 34, 38, 40) . However, these studies failed to separate the effects of force production from other factors involved in muscle contraction.
To determine whether the force of contraction is mechanistically linked to MAPK phosphorylation, isolated superfused muscles from adult mice were electrically stimulated in the presence and absence of a specific inhibitor of actomyosin ATPase [N-benzyl-p-toluene sulfonamide (BTS)] to eliminate force development (10). In the absence of BTS, the phosphorylation of p38 MAPK in stimulated muscle was 2.5- to threefold that in nonstimulated muscle. In the presence of BTS, Ca2+ handling appeared relatively unchanged, but force production was inhibited by nearly 95%. However, there was still a two- to threefold increase in p38 MAPK phosphorylation. In contrast, electrical stimulation did not change phosphorylation of ERK1/2 relative to nonstimulated muscles. Ca2+ handling was not inhibited by BTS in these preparations. These observations demonstrate that it is not cross-bridge formation per se but a process associated with actively contracting muscle that is the signal for the mechanosensitivity of p38 MAPK. Interestingly, isometric contractions capable of activating p38 MAPK had no effect on ERK1/2, suggesting that normal bouts of physiological activity are not sufficient to activate this kinase.
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MATERIALS AND METHODS |
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Animals and isolated muscle preparations. All mouse care and experimental protocols were approved by the All University Committee on Animal Use and Care at Michigan State University. Adult male Swiss Webster mice (Harlan, Indianapolis, IN) were maintained in a controlled environment with a 12:12-h light-dark cycle and food and water administered ad libitum. Harvesting of intact muscles was performed on mice anesthetized to a deep plane of surgical anesthesia with an intraperitoneal injection of pentobarbital sodium (50 mg/kg). The extensor digitorum longus (EDL) muscles were ligated at the proximal and distal tendons with 5-0 silk sutures, removed from the hindlimbs, and immediately placed in organ baths to recover. Muscles were incubated in modified mouse Ringer solution containing (in M) 0.9712 NaCl, 0.0046 KCl, 0.025 NaHCO3, 0.0025 CaCl2, and 0.0116 MgSO4, and 10 mg/l gentamicin and then equilibrated with 95% O2-5% CO2. The pH was 7.4 at 37°C. Superfusate temperature was measured in a subset of experiments using a K-type thermocouple (Omega Engineering, Stamford, CT) adjacent to the muscle and maintained at the required temperatures ±0.2°C by circulating water through a glass-jacketed organ bath (Radnoti Glass Technology, Monrovia, CA).
Experimental procedures. Isolated EDL muscles were set at a fixed length by tying one end of the muscle ligature to a glass hook and the other to an isometric force transducer (Astro-Med, West Warwick, RI) fitted with a positioning micrometer. Muscles were fixed at their in vivo resting length and measured between ligatures in situ (typically 1618 mm) before being adjusted to their optimal resting length (Lo) using the length-tension relationship. Electrical stimulation was delivered via two platinum plate electrodes adjacent to the muscle and generated using a Grass S88 Stimulator (Grass Instruments, Quincy, MA). Force was recorded using an analog-to-digital converter (ADC) (model AT MIO16E; National Instruments, Austin, TX) controlled using commercially available software (LabScribe/NI; iWorx, Dover, NH). Analysis of mechanical transients was performed using a custom algorithm for physiological data developed in our laboratory using a MatLab programming environment (Mathworks, Natick, MA). To test the effects of BTS on force production, 25, 50, 75, or 150 µM BTS was added to the bath immediately after a short series of five control twitches at 1 Hz. At 10-min intervals, a short series of five twitches were obtained to determine the degree of inhibition by BTS at each concentration level.
To test the effects of force generation on MAPK phosphorylation, isometric contractions were induced at 10 Hz for a period of 15 min. The contralateral EDL muscles were used as controls by fixing them at resting length and incubating them in Ringer solution in a separate organ bath for an equivalent period of time. The degree of MAPK phosphorylation in the absence of force production was tested as follows. Forty min before the stimulation protocol, 75 µM BTS was added to the bath and brief test twitches confirming 9597% inhibition of initial force were performed, followed by the same 15-min stimulation protocol used in control experiments. After these stimulation protocols, the muscles were removed from their tendons, weighed, flash frozen, and stored at 80°C until further analysis.
Ca2+ measurements. The relative changes in Ca2+ handling in the presence of BTS were measured using fluorescence microscopy. Measurements were performed using a Nikon TE2000U inverted microscope outfitted with a dual channel model 814 photomultiplier detection system (Photon Technology International, Lawrenceville, NJ) coupled to a model D-104 grating photometer (DeltaRAM; Photon Technology International). Excitation was set at 550 nm and emission was set at 570 nm using a model C3286 filter cube (Chroma Technology, Rockingham, VT) specific to rhod-2 and a long working distance lens objective (x10 Plan Fluor). Measurements were obtained for isolated EDL muscles fixed horizontally at resting length and positioned with a micrometer as done in the organ bath experiments. A custom bath was constructed of Perspex with provisions for perfusion using a Bioptechs microcirculation pump (Bioptechs, Butler, PA), with stimuli delivered via bath-applied platinum electrodes using a Grass model s48 stimulator (Astro-Med). Muscles were mounted in the bath at both ends, one on a stainless steel post fixed to a micromanipulator and the other on an isometric force transducer (Kent Scientific, Torrington, CT). Force was recorded digitally using an analog channel on the DeltaRAM ADC so that force and fluorescence measurements were synchronized. The analog signal was recorded on an Astro-Med DASH II thermal array recorder. Temperature was controlled using a model TS-4 LPD stage heater and a model PTU-3 controller (Physitemp, Clifton, NJ). Muscles were loaded with rhod-2 AM for 30 min at 3035°C, and, once this step was completed, muscles were stimulated and Ca2+ and force were recorded concurrently. Muscles were subsequently treated with 75 µM BTS and Ca2+, and force was assayed again after 30 min.
Tissue preparation and Western blot analysis.
Muscles were homogenized in isotonic saline buffer (25 mM Tris·HCl, pH 7.6, and 138 mM NaCl) containing 10 mM dithiothreitol (DTT); 10% (vol/vol) glycerol; phosphatase inhibitors 20 mM sodium pyrophosphate (NaPPi), 50 mM NaF, 25 mM -glycerophosphate, and 1 mM sodium orthovanadate (Na3VO4); protease inhibitors 0.01% leupeptin, 0.05% PMSF, and 0.01% pepstatin; and detergent [1% (wt/vol) sodium dodecyl sulfate (SDS)]. The protein concentration of the homogenates was determined using the Bio-Rad DC protein reagent kit. Equal amounts (40 µg) of homogenate proteins were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) through an 8% (wt/vol) polyacrylamide gel, and then they were electrophoretically transferred onto a nitrocellulose membrane.
Western blot analysis was accomplished by blocking the membrane for 1 h with isotonic saline buffer containing 3% (wt/vol) bovine serum albumin, 5% (wt/vol) nonfat dry milk, and 50 mM NaF (blocking buffer), followed by an overnight incubation at 4°C with a rabbit antibody that specifically detects either the phosphorylated isoforms of p38 (Thr180/Tyr182) or total endogenous isoforms of p38. Primary antibodies were diluted (1:1,000) in blocking buffer. Goat anti-rabbit antibody conjugated to horseradish peroxidase (HRP) was used to detect bound primary antibody. The antibody-HRP conjugate was diluted (1:2,000) with isotonic saline buffer containing 5% (wt/vol) nonfat dried milk. Bound second antibody was detected using the Phototope-HRP Western blot chemiluminescence kit (Cell Signaling Technology). Chemiluminescent signals were captured on Amersham ECL Hyperfilm.
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RESULTS |
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The potential that relatively small changes in force production were sufficient to activate p38 MAPK could not be excluded by the BTS experiments because of the residual amount (<5%) of force over the time course. If p38 MAPK activation were exquisitely sensitive to contraction-induced mechanical stress, even the small amounts of force generated by BTS-treated muscles might be sufficient to stimulate phosphorylation. To test this hypothesis, untreated muscles were stimulated at 10 Hz to generate an amount of force equivalent to that generated by BTS-treated muscles. Because a single twitch generated 9-g force, nontreated muscles were stimulated for
150 contractions in the short-stimulation protocol. Figure 2 (inset) shows that nontreated muscles stimulated for a short duration generated slightly more force than BTS-treated EDL muscles within 15 min. Despite the greater force produced with the short-stimulation protocol, there was not a significant increase in p38 MAPK phosphorylation. This was true whether muscles were frozen immediately after the last contraction (15 s) or were allowed to rest for the entire 15-min time course and then frozen (data not shown). If p38 MAPK signaling were as sensitive as hypothesized, the degree of phosphorylation in the nontreated muscles subjected to the short-term stimulation would be identical to that in the BTS-treated muscles under the same experimental conditions. In fact, the phosphorylation ratio was not significantly different from that of resting muscles (Fig. 5), demonstrating that force production was not the physiological signal that activated p38 MAPK in response to muscle contractile activity.
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DISCUSSION |
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These studies relied on the use of BTS as a specific inhibitor of force. BTS is an aryl sulfonamide that has been shown to weaken S1 subfragments of myosin with F-actin in in vitro motility assays. Kinetic analysis of the action of BTS using skinned fiber preparations has shown that it decreases the rate of Pi release as well as the dissociation of S1 and ADP (35). The concomitant ATPase activity is directly inhibited by the decreased release of Pi (35). The molecular action of BTS is highly specific and has been shown to minimally affect Ca2+ handling [sarcoplasmic reticulum (SR) ATPase activity] in intact frog fibers (10) or saponin-treated fish fibers (44). Therefore, the use of BTS allows the putative signal of force production to be separated from other signals generated by muscle stimulation.
MAPK phosphorylation and mechanical signaling. Mechanical stress is a change in force per cross-sectional area, whereas mechanical strain is a change in force per change in length. Muscles contract in vivo, with small changes in muscle length (low component of mechanical strain) depending on pennation, but force per cross-sectional area increases substantially during contraction (high component of mechanical stress) (12, 18, 20). Hence, in the isometric contraction model used in the present study, mechanical stress was the dominant physical force. In contrast, contractions with concurrent increases in length (eccentric contractions) have a larger strain component than isometric contractions do and can cause substantial damage to the sarcolemmal membrane and the SR (14, 21). Thus, for studies of mechanical signaling, increases in the proportion of mechanical strain to overall muscle loading may result in substantial muscle damage (2, 25). On the basis of these facts, interpretation of the intracellular signaling by MAPKs in exercising muscle may be confounded by underlying membrane and organelle damage. By using isometric contractions, we designed the present study to minimize mechanical strain (or any damage) under physiological conditions.
The results of the present study show two important findings with respect to mechanical stress in isometric contractions. First, Fig. 4 shows that p38 MAPK responded to contractile activity because there was robust phosphorylation in stimulated muscles relative to the contralateral resting controls. However, the inhibition of force production through a loss in cross-bridge formation from BTS administration (Fig. 3) did not attenuate the phosphorylation response (Fig. 4). This supports the argument that p38 MAPK, while activated as a consequence of electrical stimulation, does not respond to mechanical loading and that some other aspect of physiological activity associated with contraction, perhaps metabolic in nature, must be responsible. This result conflicts with a previous study of isolated superfused rat EDL muscles in which the investigators concluded that ERK1/2 was sensitive to a metabolic signal, whereas p38 MAPK was sensitive to mechanical force (40). The disagreement of those previous results with those of the present study might be explained by the nature of the contractions performed. Wretman et al. (40) used tetanic contractions with changes in muscle length, eccentric and concentric, resulting in a strain component not present in the experiments described in the present study. Strain-dependent activation of p38 MAPK has been shown in rat EDL muscle. Boppart et al. (5) showed that passive stretch of rat EDL with forces as small as 0.06 N resulted in a fivefold increase in p38 MAPK phosphorylation. Their study also showed similar trends in slow-twitch soleus muscles; however, the relative increases in p38 MAPK phosphorylation were not as dramatic until mechanical strain was >0.24 N, suggesting fiber-specific effects. However, ERK1/2 phosphorylation with passive stretch was identical independent of muscle fiber-type content. Taken together, these results suggest that ERK1/2 responds to strain in the presence or absence of active force generation and is not fiber type dependent, while p38 MAPK responds to events associated with muscle activation but is not dependent on force production. Consistent with this conclusion is that isometric contractions did not activate ERK1/2 in the present study (Fig. 6).
MAPK phosphorylation and other physiological signaling. During contractile activity, depolarization of the sarcolemmal membrane results in activation of the ryanodine receptor, thereby initiating Ca2+ release from the SR. The rise in cytosolic Ca2+ immediately activates SR ATPases to pump Ca2+ back into the SR as well as causes a conformational change in troponin, permitting the attachment of myosin to actin and initiating the myosin power stroke (19). Active SR ATPases and actomyosin ATPases consume ATP, liberating ADP and Pi (8). Therefore, within the normal contractile cycle, three tenable signals are generated: force, altered Ca2+ homeostasis, and altered ATP-free energy homeostasis. The present study shows directly that inhibition of force by BTS had no effect on p38 MAPK phosphorylation (Fig. 4), thereby eliminating force production as the signaling mechanism. This implies that either altered Ca2+ homeostasis or altered ATP-free energy homeostasis should be considered a signal.
In this study, Ca2+ homeostasis in isolated superfused fast-twitch muscles was investigated using rhod-2 AM as the Ca2+ indicator. Incubation with BTS resulted in the massive loss of force production (Fig. 1) while having no apparent deleterious effects on Ca2+ handling (Fig. 2). However, p38 MAPK phosphorylation was still elevated by electrical stimulation. Interestingly, the short-term stimulation protocol (Fig. 3) resulted in no significant increase in p38 MAPK phosphorylation relative to unstimulated controls (Fig. 5). This is consistent with the expectation that the amount of Ca2+ released as a function of time will scale with the number of twitches produced. Therefore, the rise in intracellular Ca2+ cannot be eliminated as a signal for contraction-induced phosphorylation of p38 MAPK.
Because there is an energetic cost associated with Ca2+ handling and force production, it is possible that perturbations in ATP homeostasis could also signal for the phosphorylation of p38 MAPK. In fast-twitch muscles, the estimated cost of force production (actomyosin ATPase activity) is 70% of the overall ATP use, with the balance being consumed by Ca2+ handling (SR ATPase activity >20%) and membrane ion pumps (<5%) (32). Even though energetic costs were not directly measured in the present study, the use of BTS to inhibit actomyosin ATPase activity implies a reduction in the overall ATP consumption proportionate to the measured decrease in force generation (Fig. 1). However, the energetic cost of Ca2+ handling and meager force production is still above resting ATPase activities. Our observation that the extent of p38 MAPK phosphorylation was similar in stimulated muscle in the presence and absence of BTS (Fig. 4) implies that in energetic signal-mediated p38 MAPK phosphorylation, either the response to the signal was saturated at very low perturbations of ATP free energy or the signal in both tissues was of similar strength and was not energetically mediated.
One possible signaling mechanism not necessarily accounted for in the present study was the effect of reactive oxygen species (ROS). ROS are generated during intensive contractile activity (33) and stimulate the phosphorylation of ERK1/2 in response to repeated concentric contractions (40). However, Wretman et al. (40) observed that p38 MAPK was not phosphorylated in response to concentric contractions; thus the production of ROS appears to have no role in activating p38 MAPK signaling. Our study showed that p38 MAPK phosphorylation increased 2.5- to threefold in response to chronic isometric contractions (Fig. 4), whereas the phosphorylation of ERK1/2 did not change relative to nonstimulated muscle (Fig. 6). Although we cannot definitively rule out that isometric contractions generate ROS, our observation that ERK1/2 was not phosphorylated, but that p38 MAPK was, most likely rules out ROS as a potential signaling mechanism in isometric contractions.
Concluding remarks. On the basis of the results of the present study, we conclude that p38 MAPK is significantly activated in isolated mouse fast-twitch muscle in response to chronic isometric contractions. The observation that the fold increases in p38 MAPK phosphorylation were similar in stimulated muscle in the absence and presence of BTS indicates that mechanical stress is not the signaling mechanism for activation of p38 MAPK. Furthermore, the observation that ERK1/2 was not phosphorylated in response to chronic isometric contractions suggests that ERK1/2 responds to strain in the presence or absence of active force generation, while p38 MAPK responds to events associated with muscle activation but not force production. These putative signaling mechanisms could include changes in intracellular Ca2+ and/or changes in ATP-free energy homeostasis.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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