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Department of Kinesiology, Université de Montréal, Montreal, Quebec, Canada H3C 3J7
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The mechanism by which mechanical forces acting through skeletal muscle cells generate intracellular signaling, known as mechanotransduction, and the details of how gene expression and cell size are regulated by this signaling are poorly understood. Mitogen-activated protein kinases (MAPKs) are known to be involved in mechanically induced signaling in various cell types, including skeletal muscle where MAPK activation has been reported in response to contraction and passive stretch. Therefore, the investigation of MAPK activation in response to mechanical stress in skeletal muscle may yield important information about the mechanotransduction process. With the use of a rat plantaris in situ preparation, a wide range of peak tensions was generated through passive stretch and concentric, isometric, and eccentric contractile protocols, and the resulting phosphorylation of c-Jun NH2-terminal kinase (JNK), extracellular regulated kinase (ERK), and p38 MAPKs was assessed. Isoforms of JNK and ERK MAPKs were found to be phosphorylated in a tension-dependent manner, such that eccentric > isometric > concentric > passive stretch. Peak tension was found to be a better predictor of MAPK phosphorylation than time-tension integral or rate of tension development. Differences in maximal response amplitude and sensitivity between JNK and ERK MAPKs suggest different roles for these two kinase families in mechanically induced signaling. A strong linear relationship between p54 JNK phosphorylation and peak tension over a 15-fold range in tension (r2 = 0.89, n = 32) was observed, supporting the fact that contraction-type differences can be explained in terms of tension and demonstrating that MAPK activation is a quantitative reflection of the magnitude of mechanical stress applied to muscle. Thus the measurement of MAPK activation, as an assay of skeletal muscle mechanotransduction, may help elucidate mechanically induced hypertrophy.
intracellular signaling; mitogen-activated protein kinases; c-Jun NH2-terminal kinase; eccentric contraction
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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MECHANOTRANSDUCTION IS THE fundamental mechanism by which mechanical stress acting through a cell initiates intracellular signaling. Through this mechanism, forces promote cellular growth and survival (10, 27), influence metabolic responses (17), and govern tissue architecture in various cell types (5, 32, 34, 39). Whereas all adhesion-dependent cells appear to be sensitive to mechanical forces (27, 35), this is especially evident in mechanocytes or cells routinely subjected to mechanical forces, such as vascular endothelial (21) and smooth muscle cells (25), airway smooth muscle cells (33), chondrocytes (16, 37), osteocytes (24), cardiomyocytes (29, 32, 38), and skeletal muscle cells (13, 34). Striated muscle cells are particularly responsive to mechanical stress, as evidenced by the fact that cellular size is in large part dictated by physical forces: loading elicits hypertrophy whereas unloading elicits atrophy (26).
Despite a great deal of research in this area, mechanotransduction remains poorly understood. A number of mechanisms have been proposed to explain the mechanochemical coupling on a molecular level, but none is generally accepted. Only slightly better understood are the details of the downstream signaling pathways through which mechanical stress affects gene expression.
Whereas numerous pathways have been implicated, the involvement of the mitogen-activated protein kinase (MAPK) cascades in mechanically induced signaling from the cytosol to the nucleus is consistent across various cell types (3, 15, 16, 18-20, 29-31, 38, 40). This function is compatible with the well-known role of the MAPKs as points of convergence for various signaling cascades regulating gene expression (11), including signaling cascades triggered by many soluble growth factors and their cell-surface receptors and cascades triggered by physical stresses through unknown transduction mechanisms. The MAPKs, classified into the c-Jun NH2-terminal kinase (JNK) family (also known as stress-activated protein kinases), the extracellular-regulated kinase (ERK) family, and the p38 family, translocate to the nucleus on activation through tyrosine and threonine phosphorylation by upstream MAPK kinases. There they phosphorylate a number of transcription factors such as Elk-1, activator protein-1 (AP-1), and serum response factor (SRF), thereby regulating gene expression (11).
Only a limited number of studies have examined mechanisms of mechanically induced hypertrophy in skeletal muscle, despite the well-documented adaptability of this tissue in response to the forces acting through it. These studies have employed models of passive stretch, perhaps the simplest and most easily studied form of mechanical stress, to stimulate skeletal muscle hypertrophy and have tended to focus on the role of autocrine secretion of growth factors. The events involved in the initiation of muscle hypertrophy are not known. Knowledge of early events related to mechanical stimulation is limited to findings that passive stretch induces, in a stretch-amplitude-dependent manner, immediate-early genes (6) and MAPK phosphorylation (22), the later which was recently demonstrated by our laboratory.
As MAPK activation can be induced by tetanic muscle contraction (1, 28) as well as by passive stretch alone (22), it was hypothesized that activation is sensitive to tension either imposed passively on, or developed actively by, the muscle and that MAPK activation is a reflection of the mechanotransduction process. To demonstrate tension-dependent activation of MAPK, we compared contractile protocols that differed in their developed tension but not in their activation or fiber recruitment profiles.
The purpose of this paper was, therefore, to characterize the relationship between tension and MAPK activation and to demonstrate that the measurement of MAPK activation can be used as a tool for gaining insight into the mechanotransduction process and perhaps the initiation of hypertrophic signaling. We report a quantitative relationship between peak tension and MAPK phosphorylation, specifically the p54 JNK isoform. The relationships between phosphorylation and other tension-related parameters, such as rate of force development and aggregate tension, as measured by tension-time integral (TTI), were analyzed. Whereas speed appears to have little influence on MAPK activation, time of tension application in addition to peak tension should be factored into future models of mechanotransduction in skeletal muscle. Finally, a dose-response activation of MAPKs to mechanical stimulation supports a role of MAPKs in mechanically induced gene regulation in skeletal muscle.
An abstract of this work has been presented elsewhere (23).
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Physiology
In situ nerve-muscle preparation. Forty female Sprague-Dawley rats (Charles River), weighing 195 ± 5 g, underwent an in situ nerve-muscle preparation of the sciatic nerve and plantaris muscle. Animals were anesthetized with an intraperitoneal injection of ketamine and xylazine (61.5 mg/kg ketamine and 7.7 mg/kg xylazine) and maintained under anesthesia by hourly injections of 25% of the initial dose. After the experiment, animals were killed by anesthetic overdose. Treatment of animals was certified by the animal ethics committee of the Université de Montréal and conformed to the regulations of the Canadian Council of Animal Care.
The plantaris muscle of the left leg was surgically isolated from the other ankle extensors. Care was taken not to disrupt its vasculature, innervation, or tendon and not to apply any tension on the muscle during isolation. The other ankle extensors were denervated and tenotomized at the proximal end of their distal tendon to avoid separating the common tendon of the extensors and possibly damaging the plantaris tendon. The calcaneus was clipped, leaving a bone chip attached to the common tendon, and a silk ligature was firmly placed around the bone-tendon interface. After surgery, the animal was secured in the prone position within a stereotaxic frame. The left foot was immobilized with a clamp, and the left knee was pinned to the stereotaxic frame in a slightly flexed position. The silk ligature around the common tendon was attached to the lever arm of a muscle puller servomotor (Cambridge LR 350), without putting the isolated muscle under tension. The skin of the hindlimb was pulled into a bath that was filled with heated mineral oil maintained at 36-37°C. Core temperature was monitored by rectal probe and maintained at 35-36°C using a heating pad. To determine optimal length for muscle twitch tension development (Lo), the isolated muscle was indirectly stimulated through a platinum bipolar electrode placed on the sciatic nerve, and developed tension was simultaneously visualized on an oscilloscope and recorded with a microcomputer. Always beginning from a completely relaxed length, the muscle was slowly lengthened while supramaximal (5-V) single square pulses of 0.05 ms in duration were delivered once every 3 s by a Grass S88 stimulator. Lo determination to less than ±1 mm required, on average, 60 twitches. After determination of Lo, electrical stimulation was ceased, and the muscle was held at this length for 5 min before the onset of the contractile protocol.Contractile protocols.
effect of different types of contractions on mapk
phosphorylation.
Isometric, eccentric, and concentric contractions generated using
identical stimulation parameters were compared in terms of their
efficacy for activating MAPKs. Electrical stimulation in the form of
150 ms of supramaximal single square pulses, 0.05 ms in duration,
delivered at a frequency of 100 Hz, was applied to the sciatic nerve
once every 1 s for exactly 5 min (i.e., 300 tetanic contractions).
Muscle length was either kept constant at Lo
(isometric contraction) or varied from Lo to
Lo + 3 mm (eccentric contraction with
passive return to Lo) or
Lo + 3 mm to Lo
(concentric contraction with passive stretch return to
Lo + 3 mm) over 150 ms synchronously with
the electrical stimulation, by using a computer-controlled servomotor.
A 3-mm length excursion corresponds to ~10% of muscle length. A ramp
function was employed to keep velocity constant at 20 mm/s or
~
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3 mm and at
Lo + 3 mm. In addition to the eccentric
contractions performed between Lo and +3 mm,
eccentric contractions were performed between
Lo 3 mm and Lo.
In addition to the concentric contractions performed between
Lo + 3 mm and Lo,
concentric contractions were performed between
Lo and Lo 3 mm.
All groups conformed to the 50:50 duty cycle design described in the
preceding section. This symmetry allowed time spent reaching
Lo and at Lo to be kept
constant across all groups, regardless of starting length. Refer to
Fig. 1 for a schematic representation of the various contractile
protocols. The Passive Stretch group described above was also included
in the analysis, for a total of eight groups or 32 points for
regression analysis. Refer to Fig. 2 for representative tension
tracings for the eight groups.
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Muscle excision.
Muscle excision was performed immediately after the contractile
protocol. Excision was performed in <10 s, and muscles were immediately frozen in liquid nitrogen and stored at 80°C for subsequent biochemical analysis.
Western Immunoblots Using Phospho-specific MAPK Antibodies
MAPK activation was assessed by measurement of dual phosphorylation using phospho-specific antibodies. As MAPK activation is controlled through this phosphorylation, measurements of phosphorylation should adequately reflect activity. A high degree of agreement between phosphorylation measurements and kinase assays has been demonstrated.Homogenization and sample preparation.
Frozen muscles were powdered in liquid nitrogen. Approximately 90 mg of
muscle powder were transferred to a cooled microcentrifuge tube and
kept in liquid nitrogen until the addition of 1 ml of ice-cold modified
RIPA buffer (50 mM HEPES, 150 mM NaCl, 5% glycerol, 5 mM EGTA, 2 mM
MgCl2, pH 7.4, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) containing a cocktail of protease inhibitors (Mini-Protease, Boehringer; 2 mM phenylmethylsulfonyl fluoride) and phosphatase inhibitors (100 µM sodium orthovanadate, 1 mM sodium pyrophosphate, 10 mM sodium fluoride). The sample was vortexed for 30 s and
placed on ice for 1 h. Throughout this 1-h period, samples were
frequently vortexed. Samples were then centrifuged at 4,500 g, 4°C, for 1 h to remove insoluble material. The
supernatants were decanted, and pellets were discarded. Protein
concentration of the supernatants was determined by Bradford protein
assay (Bio-Rad). Samples of equal total protein concentration were then
prepared for SDS-PAGE by diluting an appropriate amount of the
supernatant in reducing sample buffer (60 mM Tris, 10% glycerol, 2%
SDS, 5% 
-mercaptoethanol, pH 6.8) and boiling for 1 min.
Electrophoresis and Western blot.
Forty samples, each containing 200 µg of total protein, were loaded
onto 16-cm-long 9% acrylamide gels and separated overnight at 10°C.
The 40 separated samples were simultaneously electrotransferred to a
single piece of polyvinylidene difluoride membrane (Millipore) and
visualized by Ponceau S stain to confirm successful transfer and equal
sample loading of all lanes. Membranes were blocked with 3% BSA in
Tris-buffered saline + Triton (TBST; 50 mM Tris, 150 mM NaCl, pH
7.4, 0.5% Triton X-100), bathed overnight at 4°C in primary antibody
solution, and bathed for 1.5 h at room temperature in secondary
antibody solution. All washes were done in TBST. Monoclonal antibodies
directed against phosphorylated (p) JNK (reactive with p-JNK1, p-JNK2,
and p-JNK3), ERK (reactive with p-ERK1 and p-ERK2), and p38 (reactive
with p-p38 and p-p38) (Santa Cruz Biotechnology) were prepared at a
concentration of 1:2,000 in TBST plus 1% BSA and 0.5%
NaN3, whereas horseradish peroxidase-conjugated anti-mouse
IgG secondary antibodies (Jackson Immunoresearch) were prepared at
1:10,000 in TBST plus BSA. Two hundred micrograms of whole cell lysate
of ultraviolet-treated HeLa cells or heat-shocked NIH/3T3 cells (Santa
Cruz Biotechnology) were used as a positive control for p38
phosphorylation. Membranes were then bathed in chemiluminescence
substrate (ECL, Amersham) and exposed to blue-light-sensitive film (ECL
Film, Amersham) for 5-20 min. Membranes were stripped, reblocked,
and reprobed to obtain ERK, JNK, and p38 phosphorylation values from
every sample. Films were quantified by densitometry using a flatbed
scanner and NIH Image software. In cases where the range in signal
amplitude exceeded the dynamic range of film (~16-fold resolution),
short and long exposures of the same blots were quantified. A ratio was
calculated from the results of samples that were within the linear
range on both films and used as a conversion factor for the pooling of
data from two films of the same blot.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Effects of Contraction Type on MAPK Phosphorylation
The phosphorylation of both JNK and ERK MAPKs was observed to be increased above unstimulated and unstretched control levels immediately after the three 5-min contractile protocols that were compared: eccentric (Lo to Lo + 3 mm), isometric (Lo), and concentric (Lo + 3 mm to Lo) (Fig. 3). Refer to Table 1 for group descriptive statistics. For both JNK and ERK, eccentric contractions resulted in significantly more phosphorylation than isometric contractions, which in turn resulted in significantly more phosphorylation than concentric contractions.
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Western blots reveal that both p54 and p46 isoforms of the JNK family are phosphorylated in response to tension. However, whereas p54 phosphorylation was observed over a wide range of tension, p46 phosphorylation was only observed in conjunction with a saturating p-p54 signal. Only p54 phosphorylation was quantified and reported here. As a result of the eccentric contractile protocol, p54 JNK phosphorylation surpassed 80-fold control levels, 10-fold isometric levels, or 40-fold concentric levels.
The p44 ERK (ERK1) isoform of the ERK family was observed to be phosphorylated in response to tension, and this phosphorylation was quantified. The eccentric protocol resulted in ERK1 phosphorylation of fivefold control levels, the isometric protocol in fourfold control levels, and the concentric protocol in threefold control levels.
The amplitude of phosphorylation reported here is in line with typically reported levels of MAPK activation in response to mechanical stress (2, 15).
The Static Lo + 3 mm group or the Passive Stretch Lo to Lo + 3 mm control group did not result in more JNK phosphorylation than did the Static Lo baseline control group, indicating that the JNK phosphorylation resulting from the anisometric contractile groups cannot be attributed to the 3-mm length excursion imposed on the muscle. These same control groups resulted in ERK phosphorylation that was twofold greater than control levels. Thus part of the ERK phosphorylation induced by the anisometric protocols may be attributed to the length excursion. These findings can also be interpreted as indicating that the ERK system is more sensitive to low tensions than is the JNK system.
Passive stretch or contractile activity in any form were not observed to induce phosphorylation of p38 MAPK above control values, as measured immediately after the 5-min protocol.
See Fig. 4 for representative p-JNK,
p-ERK, and p-p38 Western blots.
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Relationship Between Tension and MAPK Activation
Seven contractile protocols were used to generate maximal peak tensions ranging from 226 to 655 g or 3.3- to 11.2-fold greater than twitch tension (Table 1). An unstimulated cyclic stretch group, the Passive Stretch Lo to Lo + 3 mm group, generated on average peak tensions of 44 g or 0.8-fold greater than twitch tension. When taken together, the eight groups produced a 15-fold range in peak tension. Group descriptive data are summarized in Table 1. Representative tension tracings are illustrated in Fig. 2.Phosphorylation values for p54 JNK (p-JNK densitometry values
linearized by log10 transformation) and p44 ERK (p-ERK raw densitometry values) were subjected to regression and correlational analyses against
the parameters of peak tension, TTI, and dT/dt. Raw ERK densitometry values and JNK densitometry values linearized by log10
transformation were used for this analysis. Positive
tension-phosphorylation relationships were observed for both JNK and
ERK. Results of linear regression analyses are summarized in Table
2. For both JNK and ERK, the relationship
between normalized peak tension and phosphorylation was stronger than
between TTI or dT/dt and phosphorylation. For all parameters
measured, the correlation with JNK phosphorylation was consistently
stronger than the correlation with ERK phosphorylation.
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The strongest relationships observed, as assessed by correlation
coefficient, were between peak tension measurements and JNK phosphorylation (r2 > 0.85). Of these,
normalized average peak tension over the first 60 s had the
highest correlation coefficient (r2 = 0.89). When group average raw densitometry data were plotted against
average peak tension over the first 60 s, a simple power function (y = 0.3 x4.67)
produced a near-perfect fit (r2 = 0.998).
These relationships are illustrated in Fig.
5.
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Analysis performed according to contraction type revealed that the positive tension-phosphorylation relationship holds within the eccentric groups and within the isometric groups, as illustrated in Fig. 5. This supports the fact that MAPK phosphorylation is independent of contraction type.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The present study suggests that MAPKs may be involved in mechanically induced signaling in skeletal muscle. A strong relationship between peak tension, developed actively or passively, and p54 JNK MAPK phosphorylation demonstrates that MAPK phosphorylation can be used as a quantitative marker of the magnitude of mechanical stress applied to a muscle. This study also supports the fact that differences in MAPK phosphorylation resulting from controlled in situ eccentric, isometric, and concentric contractile protocols are due to differences in magnitude of developed tension between the different contractile protocols, independent of contraction type. Finally, the results of this study indicate that peak tension is a better predictor of MAPK phosphorylation than are other tension-related parameters, such as TTI and peak dT/dt.
The relationship between peak tension and phosphorylation is demonstrated to hold over a 15-fold range in tension produced with passive stretch and contractile protocols employing different types of contraction (i.e., concentric, isometric, eccentric). The strength of this relationship, and the fact that the positive relationship holds even within the range of peak tensions generated by protocols employing the same type of contraction, both support the fact that contraction-type differences are due to differences in tension and not contraction type per se.
The study of the mechanical sensitivity of MAPKs in skeletal muscle, as presented here, does not address directly the mechanism of mechanotransduction or the proximity of the MAPKs to the initial mechanochemical coupling event. Rather, the purpose of the study is to demonstrate that MAPK activation can be used as a quantitative measurement of the magnitude of mechanical stress applied to a muscle. This in turn may be used to elucidate early mechanosensitive events upstream of the MAPKs, including the mechanotransduction process itself. Mechanotransduction and the events leading to mechanically induced MAPK activation are unclear and may occur through any of a number of mechanisms, such as the release of growth factors stored in the extracellular matrix, influx of ions through stretch-sensitive channels, or direct activation of kinases through mechanical deformation. The measurement of mechanically induced MAPK activation, therefore, represents a simple and reliable assay to investigate the effects of disrupting various cellular components and signaling pathways suspected of participating in mechanotransduction and mechanical regulation of gene expression.
Furthermore, by assessing the efficacy of different types of stimulation on MAPK phosphorylation, the measurement of mechanically induced MAPK activation can be used to elucidate the nature of the mechanical stress to which muscle is most sensitive. Determination of the sensitivity of MAPKs to amplitude, frequency, velocity, and duration of mechanical stimulation can provide new insight into the design of protocols for the maintenance of muscle mass in situations of decreased loading and also represents another approach for elucidating the mechanism of mechanotransduction. In the present study, the assessment of the relationship between various tension-related parameters (peak tension, TTI, peak dT/dt) and MAPK activation represents an application of this strategy. For example, the finding of a low correlation between peak dT/dt and phosphorylation suggests that mechanically induced signaling is largely independent of contraction velocity. This, combined with a fair correlation between TTI and phosphorylation, would suggest that two protocols developing identical peak tension at different frequencies but with identical TTI would induce similar levels of signaling. Furthermore, a high correlation between peak tension and MAPK phosphorylation and a low correlation between peak dT/dt and phosphorylation are both consistent with a mechanotransduction mechanism based on elastic deformation. The relative importance of these parameters in the modeling of mechanical sensitivity needs to be addressed directly with studies designed to vary TTI and dT/dt.
Delay kinetics, time course, and time-dependent changes in
sensitivity of mechanically induced MAPK activation are other
parameters that need to be addressed to fully model the sensitivity of
muscle to mechanical stimulation. Although these issues are beyond the scope of this study, the complexity of the delay kinetics can, nevertheless, be appreciated from the data presented here. The finding
that average peak tension over the first minute is a slightly better
predictor of MAPK phosphorylation than maximal peak tension or average
peak tension over the first 10 or 30 s, combined with the
interesting observation that a dramatically different rate of fatigue
between contraction types is apparent between the 30th and 60th second
of the protocol (Fig. 6), suggests that
phosphorylation measured immediately after the 5-min protocols is
determined by more than 30 s of stimulation.
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Although this study does not address directly the role of MAPKs in mechanically induced gene regulation and muscle hypertrophy, the dose-response behavior of p54 JNK MAPK to mechanical stimulation, as presented here and as previously described in response to passive stretch (22), certainly supports a role for MAPKs in relaying a hypertrophic stimulus to the nucleus and possibly triggering a program of events necessary for hypertrophy. This is further supported by the report of a rapid stretch-amplitude-dependent induction of c-jun and c-fos in response to passive stretch (6). A similar magnitude-dependent activation of JNK and induction of immediate early genes by mechanical stress have been reported in smooth muscle cells (15).
The MAPKs, in particular the JNK MAPKs, and calcineurin, an alternate pathway to the nucleus, are believed to be essential components of the hypertrophic response in cardiac muscle (7, 9). Recent evidence suggests that calcineurin may act through JNK (7). Inhibition of calcineurin in skeletal muscle has recently been shown to abolish the hypertrophy typically induced by synergist-ablation muscle overload (8). It should not be surprising to find that chronic inhibition of the JNK pathway will also abolish the potential for hypertrophy. This is a reasonable hypothesis, not only in light of the rapid and transient activation of MAPKs in response to mechanical stress, but also in light of the longer contribution of autocrine growth factor action in the hypertrophic response (12).
MAPK-family-specific differences in response amplitude and sensitivity to tension suggest that, whereas the JNK and ERK MAPKs may transduce a mechanically induced signal in parallel, they may serve different functions in the regulation of muscle plasticity. JNK phosphorylation is not observed below a peak tension threshold that is on the order of two- to fourfold greater than twitch tension. However, above this threshold, phosphorylation increases exponentially with tension to as much as 80-fold baseline values at around 10-fold twitch tension. By contrast, the observed signal amplitude of ERK phosphorylation is only fivefold. However, an ERK response at the very low tensions produced by the passive stretch protocols employed here suggests a higher sensitivity to tension. The lower response amplitude of ERK may be partly explained by a higher baseline level of activity because of its higher sensitivity. The finding of a tighter relationship, as assessed by correlation coefficient, between JNK phosphorylation and peak tension than between ERK and tension suggests that p54 JNK may have a more important role in mechanically induced gene regulation than does p44 ERK.
In contrast to the JNK and ERK families of MAPKs, the p38 family was not found to be activated above control levels by 5 min of contractile activity. This suggests that p38 is either not involved in skeletal muscle mechanotransduction or follows a different time course of activation. The latter appears to be the case as p38 activation resulting from 5 min of contractile activity can be observed at later time points (unpublished data). This is in accord with other reports of contractile activity-induced MAPK activation. Goodyear et al. (14) showed very low p38 activation (less than twofold greater than control values) occurring significantly later than JNK activation in exercised rat muscle, and Widegren et al. (36) showed p38 activation to be at least an order of magnitude lower than ERK activation in exercised human muscle.
In summary, the present study demonstrates a possible involvement of MAPKs in mechanically induced signaling in skeletal muscle. A quantitative relationship between p54 JNK phosphorylation and peak tension supports the fact that JNK activation is a reflection of mechanical stimulation applied to a muscle and may be used as an assay of the mechanotransduction process. Such an assay represents a useful tool for the elucidation of early signaling events involved in mechanically induced muscle hypertrophy and for the understanding of the nature of the mechanical stimuli to which muscle is sensitive.
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ACKNOWLEDGEMENTS |
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This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada.
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FOOTNOTES |
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Address for reprint requests and other correspondence: P. F. Gardiner, Université de Montréal, Dept. of Kinesiology, P. O. Box 6128 Centre-Ville, Montreal, Quebec, Canada H3C 3J7 (E-mail: Phillip.Gardiner{at}umontreal.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.
Received 11 July 2000; accepted in final form 29 March 2001.
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REFERENCES |
|---|
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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1.
Aronson, D,
Dufresne SD,
and
Goodyear LJ.
Contractile activity stimulates the c-Jun NH2-terminal kinase pathway in rat skeletal muscle.
J Biol Chem
272:
25636-25640,
1997
2.
Aronson, D,
Violan MA,
Dufresne SD,
Zangen D,
Fielding RA,
and
Goodyear LJ.
Exercise stimulates the mitogen-activated protein kinase pathway in human skeletal muscle.
J Clin Invest
99:
1251-1257,
1997[Web of Science][Medline].
3.
Berk, BC,
Corson MA,
Peterson TE,
and
Tseng H.
Protein kinases as mediators of fluid shear stress stimulated signal transduction in endothelial cells: a hypothesis for calcium-dependent and calcium-independent events activated by flow.
J Biomech
28:
1439-1450,
1995[Web of Science][Medline].
4.
Boppart, MD,
Aronson D,
Gibson L,
Roubenoff R,
Abad LW,
Bean J,
Goodyear LJ,
and
Fielding RA.
Eccentric exercise markedly increases c-Jun NH2-terminal kinase activity in human skeletal muscle.
J Appl Physiol
87:
1668-1673,
1999
5.
Chicurel, ME,
Chen CS,
and
Ingber DE.
Cellular control lies in the balance of forces.
Curr Opin Cell Biol
10:
232-239,
1998[Web of Science][Medline].
6.
Dawes, NJ,
Cox VM,
Park KS,
Nga H,
and
Goldspink DF.
The induction of c-fos and c-jun in the stretched latissimus dorsi muscle of the rabbit: responses to duration, degree and re-application of the stretch stimulus.
Exp Physiol
81:
329-339,
1996[Abstract].
7.
DeWindt, LJ,
Lim HW,
Haq S,
Force T,
and
Molkentin JD.
Calcineurin promotes protein kinase C and c-Jun NH2-terminal kinase activation in the heart. Cross-talk between cardiac hypertrophic signaling pathways.
J Biol Chem
275:
13571-13579,
2000
8.
Dunn, SE,
Burns JL,
and
Michel RN.
Calcineurin is required for skeletal muscle hypertrophy.
J Biol Chem
274:
21908-21912,
1999
9.
Force, T,
Hajjar R,
Del MF,
Rosenzweig A,
and
Choukroun G.
Signaling pathways mediating the response to hypertrophic stress in the heart.
Gene Expr
7:
337-348,
1999[Web of Science][Medline].
10.
Frisch, SM,
Vuori K,
Ruoslahti E,
and
Chan HP.
Control of adhesion-dependent cell survival by focal adhesion kinase.
J Cell Biol
134:
793-799,
1996
11.
Garrington, TP,
and
Johnson GL.
Organization and regulation of mitogen-activated protein kinase signaling pathways.
Curr Opin Cell Biol
11:
211-218,
1999[Web of Science][Medline].
12.
Goldspink, G.
Changes in muscle mass and phenotype and the expression of autocrine and systemic growth factors by muscle in response to stretch and overload.
J Anat
194:
323-334,
1999.
13.
Goldspink, G,
Scutt A,
Loughna PT,
Wells DJ,
Jaenicke T,
and
Gerlach GF.
Gene expression in skeletal muscle in response to stretch and force generation.
Am J Physiol Regulatory Integrative Comp Physiol
262:
R356-R363,
1992
14.
Goodyear, LJ,
Chang PY,
Sherwood DJ,
Dufresne SD,
and
Moller DE.
Effects of exercise and insulin on mitogen-activated protein kinase signaling pathways in rat skeletal muscle.
Am J Physiol Endocrinol Metab
271:
E403-E408,
1996
15.
Hamada, K,
Takuwa N,
Yokoyama K,
and
Takuwa Y.
Stretch activates Jun N-terminal kinase/stress-activated protein kinase in vascular smooth muscle cells through mechanisms involving autocrine ATP stimulation of purinoceptors.
J Biol Chem
273:
6334-6340,
1998
16.
Hung, CT,
Henshaw DR,
Wang CC,
Mauck RL,
Raia F,
Palmer G,
Chao PH,
Mow VC,
Ratcliffe A,
and
Valhmu WB.
Mitogen-activated protein kinase signaling in bovine articular chondrocytes in response to fluid flow does not require calcium mobilization.
J Biomech
33:
73-80,
2000[Web of Science][Medline].
17.
Ihlemann, J,
Ploug T,
Hellsten Y,
and
Galbo H.
Effect of tension on contraction-induced glucose transport in rat skeletal muscle.
Am J Physiol Endocrinol Metab
277:
E208-E214,
1999
18.
Ikeda, M,
Takei T,
Mills I,
and
Sumpio BE.
Calcium-independent activation of extracellular signal-regulated kinases 1 and 2 by cyclic strain.
Biochem Biophys Res Commun
247:
462-465,
1998[Web of Science][Medline].
19.
Komuro, I,
Kudo S,
Yamazaki T,
Zou Y,
Shiojima I,
and
Yazaki Y.
Mechanical stretch activates the stress-activated protein kinases in cardiac myocytes.
FASEB J
10:
631-636,
1996[Abstract].
20.
Li, S,
Kim M,
Hu YL,
Jalali S,
Schlaepfer DD,
Hunter T,
Chien S,
and
Shyy JY.
Fluid shear stress activation of focal adhesion kinase. Linking to mitogen-activated protein kinases.
J Biol Chem
272:
30455-30462,
1997
21.
Malek, AM,
and
Izumo S.
Control of endothelial cell gene expression by flow.
J Biomech
28:
1515-1528,
1995[Web of Science][Medline].
22.
Martineau, LC,
and
Gardiner PF.
Static stretch induces MAPK activation in skeletal muscle (Abstract).
FASEB J
13:
A410,
1999.
23.
Martineau, LC,
and
Gardiner PF.
Eccentric contractions cause rapid and massive activation of MAPK in rat skeletal muscle in situ (Abstract).
FASEB J
14:
A318,
2000.
24.
Mikuni-Takagaki, Y.
Mechanical responses and signal transduction pathways in stretched osteocytes.
J Bone Miner Metab
17:
57-60,
1999[Web of Science][Medline].
25.
Osol, G.
Mechanotransduction by vascular smooth muscle.
J Vasc Res
32:
275-292,
1995[Web of Science][Medline].
26.
Roy, RR,
Baldwin KM,
and
Edgerton VR.
The plasticity of skeletal muscle: effects of neuromuscular activity.
Exerc Sport Sci Rev
19:
269-312,
1991[Medline].
27.
Ruoslahti, E.
Stretching is good for a cell.
Science
276:
1345-1346,
1997
28.
Ryder, JW,
Fahlman R,
Wallberg HH,
Alessi DR,
Krook A,
and
Zierath JR.
Effect of contraction on mitogen-activated protein kinase signal transduction in skeletal muscle. Involvement Of the mitogen- and stress-activated protein kinase 1.
J Biol Chem
275:
1457-1462,
2000
29.
Sadoshima, J,
and
Izumo S.
Mechanical stretch rapidly activates multiple signal transduction pathways in cardiac myocytes: potential involvement of an autocrine/paracrine mechanism.
EMBO J
12:
1681-1692,
1993[Web of Science][Medline].
30.
Schmidt, C,
Pommerenke H,
Durr F,
Nebe B,
and
Rychly J.
Mechanical stressing of integrin receptors induces enhanced tyrosine phosphorylation of cytoskeletally anchored proteins.
J Biol Chem
273:
5081-5085,
1998
31.
Seko, Y,
Takahashi N,
Tobe K,
Kadowaki T,
and
Yazaki Y.
Pulsatile stretch activates mitogen-activated protein kinase (MAPK) family members and focal adhesion kinase (p125FAK) in cultured rat cardiac myocytes.
Biochem Biophys Res Commun
259:
8-14,
1999[Web of Science][Medline].
32.
Sharp, WW,
Simpson DG,
Borg TK,
Samarel AM,
and
Terracio L.
Mechanical forces regulate focal adhesion and costamere assembly in cardiac myocytes.
Am J Physiol Heart Circ Physiol
273:
H546-H556,
1997
33.
Smith, PG,
Garcia R,
and
Kogerman L.
Strain reorganizes focal adhesions and cytoskeleton in cultured airway smooth muscle cells.
Exp Cell Res
232:
127-136,
1997[Web of Science][Medline].
34.
Vandenburgh, HH,
Solerssi R,
Shansky J,
Adams JW,
and
Henderson SA.
Mechanical stimulation of organogenic cardiomyocyte growth in vitro.
Am J Physiol Cell Physiol
270:
C1284-C1292,
1996
35.
Watson, PA.
Function follows form: generation of intracellular signals by cell deformation.
FASEB J
5:
2013-2019,
1991[Abstract].
36.
Widegren, U,
Jiang XJ,
Krook A,
Chibalin AV,
Bjornholm M,
Tally M,
Roth RA,
Henriksson J,
Wallberg HH,
and
Zierath JR.
Divergent effects of exercise on metabolic and mitogenic signaling pathways in human skeletal muscle.
FASEB J
12:
1379-1389,
1998
37.
Wright, MO,
Nishida K,
Bavington C,
Godolphin JL,
Dunne E,
Walmsley S,
Jobanputra P,
Nuki G,
and
Salter DM.
Hyperpolarisation of cultured human chondrocytes following cyclical pressure-induced strain: evidence of a role for alpha 5 beta 1 integrin as a chondrocyte mechanoreceptor.
J Orthop Res
15:
742-747,
1997[Web of Science][Medline].
38.
Yamazaki, T,
Komuro I,
and
Yazaki Y.
Molecular aspects of mechanical stress-induced cardiac hypertrophy.
Mol Cell Biochem
164:
197-201,
1996.
39.
Yano, Y,
Geibel J,
and
Sumpio BE.
Cyclic strain induces reorganization of integrin alpha 5 beta 1 and alpha 2 beta 1 in human umbilical vein endothelial cells.
J Cell Biochem
64:
505-513,
1997[Web of Science][Medline].
40.
Zou, Y,
Hu Y,
Metzler B,
and
Xu Q.
Signal transduction in arteriosclerosis: mechanical stress-activated MAP kinases in vascular smooth muscle cells.
Int J Mol Med
1:
827-834,
1998[Web of Science][Medline].
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