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Departments of 1 Physical Therapy & Rehabilitation Science2 Physiology, University of Maryland-Baltimore School of Medicine, Baltimore, MD 21201, USA
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(Received 24 April 2006;
accepted after revision 17 July 2006; first published online 20 July 2006)
Corresponding author D. W. Russ: Department of Physical Therapy & Rehabilitation Science, University of Maryland-Baltimore, School of Medicine, 100 Penn Street, Baltimore, MD 21201, USA. Email: druss{at}som.umaryland.edu
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Introduction |
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Several studies indicate that various forms of exercise increase MAPK phosphorylation in both rodent (Boppart et al. 2001; Martineau & Gardiner, 2001, 2002; Nader & Esser, 2001; Wretman et al. 2001) and human muscles (Widegren et al. 1998; Boppart et al. 1999; Thompson et al. 2003). Forced lengthening of muscle during active contractions (typically referred to as ‘eccentric’ contractions) and high-strain, passive stretch appear to produce greater MAPK phosphorylation than isometric or shortening (concentric) contractions, suggesting that mechanical tension is a key factor in the activation of MAPK cascades. Other factors have also been implicated in the MAPK phosphorylation, including metabolic changes (Wretman et al. 2001), hormones (Sherwood et al. 1999) and glycogen depletion (Dufresne et al. 2001). Moreover, the different stimuli appear to have variable effects on the different pathways. For example, ERK has been shown to be more responsive to metabolic factors than p38 (Widegren et al. 1998; Wretman et al. 2001), while JNK appears to be more responsive to tensile loading than ERK (Martineau & Gardiner, 2001).
One aspect of exercise that has been largely overlooked in studies of MAPKs is the activation frequency of the muscle during contraction, not to be confused with the contraction frequency, or duty cycle, which describes the time spent contracting relative to the time spent at rest. Given the results of studies demonstrating that electrical activity is a significant regulator of developing muscle cells (Eftimie et al. 1991; De Deyne, 2000), the role of activation frequency as a factor in MAPK activation merits further attention. Furthermore, Dentel et al. (2005) have recently demonstrated that phosphorylation of p38, one of the ‘stress-activated’ MAPKs, is similar in control and N-benzyl-p-toluene sulphonamide(BTS)-treated muscles, following identical stimulation protocols. Since BTS inhibits force production without affecting muscle depolarization or calcium transients, their data suggest a mechanism by which the number or frequency of activation pulses might regulate the activation of some MAPK cascades, because changes in membrane potential and calcium levels would be expected to occur with each pulse. Increased stimulation frequency has also been associated with increased fatigue (Garland et al. 1988), which some investigators suggest is an important stimulus for the muscle adaptation to activity (Schott et al. 1995).
Studying the effects of activation frequency independent of force production can be quite problematical, however, since changes in frequency along the linear portion of the force–frequency relationship (FFR) also produce changes in force. One potential approach is to use low frequencies of stimulation, at which no summation of force occurs. In such instances, however, changes in frequency (e.g. from 1 to 3 Hz) alter the duty cycle of contractions. Another solution is to compare high stimulation frequencies on the far right of the FFR, but the non-physiological rates used may saturate the non-contractile ATP demand and not reflect changes that occur at lower frequencies.
The present study used both the FFR and length–tension relationship to compare the effects of stimulation frequencies within a physiological range (15 versus 30 Hz), while maintaining the same active contractile force. Phosphorylation of MAPK was determined by immunoblotting with antibodies to the total and phosphorylated forms of three commonly studied MAPKs: ERK, JNK and p38. Glycogen depletion was measured as a marker of metabolic demand during the stimulation protocols. Based on previous work by others, we hypothesized that higher frequency stimulation would be associated with greater p38 phosphorylation (Dentel et al. 2005), that JNK phosphorylation would more closely follow total tension (Martineau & Gardiner, 2001) and that ERK phosphorylation would be related to fatigue and glycogen depletion (Dufresne et al. 2001). We further hypothesized that greater fatigue and glycogen depletion would be associated with increased stimulation frequency, regardless of total tension.
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Methods |
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Animal use was approved by the University of Maryland Institutional Animal Care and Use Committee. Male Sprague–Dawley rats (n = 24, Charles River Laboratories, Wilmington, MA, USA) were randomly distributed into one of three groups: 15 Hz stimulation at optimal length (15 Hz, n = 8), 30 Hz stimulation at suboptimal length (30 Hzsub, n = 8) and 30 Hz stimulation at supraoptimal length (30 Hzsupra, n = 8). Additional animals were used in experiments to determine the effects of the surgical procedure, independent of stimulation (n = 5), to evaluate EMG responses (n = 9) and to assay possible membrane damage (n = 3).
Surgical procedure and stimulation protocols
Animals were anaesthetized with intraperitoneal ketamine and xylazine (40 and 10 mg (kg body weight)–1, respectively). The surgical procedure for the in situ fatigue protocols was essentially the same as that previously used (Lovering & De Deyne, 2004). Briefly, the distal tendon of the tibialis anterior muscle (TA) was released and secured in a custom-made metal clamp attached to a load cell (FT03; Grass Instruments, Warwick, RI, USA) mounted on a micromanipulator (Kite Manipulator; World Precision Instruments, Sarasota, FL, USA) to allow adjustment of muscle length. The signal from the load cell was fed via a DC amplifier (model P122; Grass Instruments) to an analog-to-digital board using commercial acquisition software (PolyVIEW version 2.1; Grass Instruments). The tibia was stabilized with a 16 gauge needle, and the peroneal nerve was dissected free through a small incision and clamped with a subminiature electrode (Harvard Apparatus, Holliston, MA, USA) that was used to stimulate the TA with supramaximal stimulation pulses (120% of the stimulation intensity that produced a maximal twitch response at optimal length). All pulses were 200 µs in duration and delivered by a Grass S48 stimulator in series with an isolation unit (model PSIU6; Grass Instruments). Throughout the experiments, the TA was protected from cooling by a heat lamp and from dehydration by mineral oil.
Once the animal was securely positioned for muscle stimulation, muscle length was adjusted to find the length that produced maximum twitch force (L0). For the 15 Hz group, all subsequent testing took place at L0. For the 30 Hzsub group, the muscle was stimulated at L0 with a 330 ms, 15 Hz train, and the force was recorded. The muscle length was then shortened incrementally until a 330 ms, 30 Hz train produced the same force that the 15 train produced at L0 (Fig. 1). The same procedure was followed for the 30 Hzsupra group, except that the muscle was incrementally lengthened rather than shortened. The 15 and 30 Hz trains were chosen because the goal was to compare two different frequencies in the physiological range of motor unit discharge rates. At 15 Hz, some summation of force occurred, although the degree of summation was considerably less than that seen with 30 Hz stimulation. Using less than 15 Hz stimulation would probably have produced isolated twitches. A higher frequency could have been used at L0, but this would have produced higher forces and would have required even greater length changes in the 30 Hzsub and 30 Hzsupra conditions to equalize the active tension production across groups.
Once the experimental length was determined, eight 330 ms trains were delivered to the muscle (two each at 1, 15, 30 and 80 Hz), separated by 15 s. The fatigue test was begun after 2 min of rest. The fatigue test consisted of 2 min of stimulation at a rate of one 330 ms train per second. The 15 Hz group received 15 Hz trains, and the 30 Hzsub and 30 Hzsupra groups received 30 Hz trains. Following the fatigue test, muscles received two testing trains each of 1, 15, 30 and 80 Hz, separated by two of the respective fatiguing trains, to control for activation history and maintain a constant level of fatigue, as previously described (Russ et al. 2002a). Following the stimulation, muscles were immediately dissected free, blotted dry and weighed, divided and snap-frozen in liquid nitrogen. The non-stimulated TA muscles were processed in the same manner to serve as controls. Once the muscles were removed, the anaesthetized animals were killed with an overdose of anaesthetic (100 mg kg–1 (80 and 20 mg/kg body weight, ketamine and xylazine respectively)) followed by open aortic transection. To confirm that there was no effect of the surgical procedure on MAPK phosphorylation, four animals were subjected to the muscle isolation procedure, the muscles attached to the force transducer, set at L0 and held for the duration of the stimulation protocol, but not stimulated (sham), and compared to the contralateral muscles, which were simply dissected and processed, as were the control muscles for the experimental groups.
Protein extraction
Muscle portions were brought to 4°C, weighed and maintained at 0–4°C throughout the protein extraction. Each portion was minced on cold glass, homogenized with a motorized pellet pestle (Kontes, Vineland, NJ, USA) in 15 volumes (w/v) high ionic strength buffer containing several protease inhibitors (Thompson et al. 2001) and centrifuged at 15 000g for 30 min. Two volumes of 100% HPLC-grade acetone were added to the supernatant to precipitate protein, followed by resuspension in 1 volume of dH2O and a second acetone precipitation. Both pellet and supernatant from the extract were resuspended in SDS sample buffer (Laemmli, 1970) and stored at –80°C. Protein concentrations were estimated (Lowry et al. 1951) using a standard curve of twice recrystallized bovine serum albumin (BSA).
SDS-PAGE and immunoblotting
Muscle extracts (20 µg total protein per lane) were electrophoresed on discontinuous (5% stacking, 10% separating) SDS gels. Bands were visualized with Coomassie Blue R250 and analysed densitometrically. The myosin heavy chain band in every lane of each gel served as an internal loading control and was shown to be within the linear range of the stain. A phosphorylase b standard was electrophoresed and scanned on every gel to control for any variation in gel staining.
Immunoblotting was performed (Towbin et al. 1979) using polyvinylidene fluoride (PVDF) membranes (Millipore Corp., Bedford, MA, USA) with the gels transferred at 4°C. The membranes were dried overnight before use, then blocked for 2 h in 5% milk (50 mM NaF was included for phosphorylated blots) in 150 mM NaCl, 10 mM Tris, pH 7.5 (TBS), incubated overnight at 4°C with monoclonal antibodies to the phosphorylated forms of JNK (pJNK), ERK1/2 (pERK) and p38 (pp38) (Santa Cruz Biological, Santa Cruz, CA, USA: sc-6254, sc-7383 and sc-7973, respectively). The blots were rinsed, and then incubated for 2 h at room temperature with an antimouse IgG/M, horseradish peroxidase-conjugated secondary antibody (Sigma). The blots were then developed in tetramethyl benzidine (TMB) solution (KP Laboratories, Gaithersburg, MD, USA), dried overnight, scanned densitometrically (Liu et al. 1999) and analysed using Scion Image (Scion Corp., Frederick, MD, USA). Once the blots were scanned, they were stripped in erasing buffer (2% SDS (w/v), 62.5 mM Tris-HCl (pH 6.8) and 100 mM β-mercaptoethanol) as previously described (Thompson et al. 2003), and then re-probed with antibodies for the total form of the MAPK measured on the initial blot (Santa Cruz Biological: JNK (sc-571), ERK1/2 (sc-94), p38 (sc-7972)). The same secondary antibody was used for the pan-p38 blots, but an antirabbit IgG (Sigma) was used for the pan-ERK and pan-JNK blots. Scanning and densitometric analysis were performed as above. The optical density of the phospho-MAPK bands was normalized to the optical density of the pan-MAPK bands for the corresponding sample, and the stimulated values were compared to the control values, as previously described (Thompson et al. 2003). Primary antibody dilutions were made in 1% BSA/TBS with 0.05% Tween (TBS-T) at 1:2000. Secondary dilutions were at 1:10 000.
Glycogen determination
One portion of the stimulated and control muscles was lyophillized and homogenized, and glycogen content was determined spectrophotometrically from the absorbance at 490 nm, as described by Ortmeyer (2001). The difference in glycogen content between the stimulated and control muscles was used as an index of metabolic demand. Although other metabolite assays could have been used, glycogen seemed an appropriate marker, since others have shown that glycogen depletion can affect MAPK pathway activation (Dufresne et al. 2001). Furthermore, it has been suggested that the sarcolemmal and sarcoplasmic reticulum pumps of skeletal muscle are selectively glycolytic (Han et al. 1992; Xu et al. 1995), and we hypothesized that it would be the activity of these pumps that would increase metabolic demand with increased stimulation frequency in our protocols.
Evans Blue dye assay
Since we were concerned that our 30 Hzsupra protocol might cause fibre damage, we conducted a commonly used assay to assess muscle fibre damage on muscles from three animals that underwent the 30 Hzsupra protocol. As previously described (Lovering & De Deyne, 2004), animals received an intraperitoneal injection of 1% (w/v) Evans Blue dye (EBD; Sigma) in phosphate-buffered saline (PBS, pH 7.4) at a volume of 1% body mass (1 mg EBD (0.1 ml PBS)–1 (10 g body weight)–1). The solution was sterilized by passage through a Millex-GP 0.22 µm filter (Millipore, Bedford, MA, USA) and administered 24 h before the stimulation protocol to ensure a good signal (Hamer et al. 2002). Evans Blue dye binds serum albumin and is detected by fluorescence microscopy (at 568 nm) in the extracellular space. Presence of the protein-bound dye inside the muscle fibre indicates muscle fibre damage. Portions of the frozen muscle samples were cryosectioned (10 µm thickness) and subsequently evaluated for the presence of intracellular EBD using fluorescence microscopy by an investigator blinded to the origin of the sections.
Muscle excitation
Changes in the compound muscle action potential (CMAP) that occurred as a result of the three stimulation protocols (n = 3 per group) were measured as described elsewhere (Binder-Macleod & Barrish, 1992). Briefly, one needle electrode was inserted into the TA, and the reference electrode was inserted into the quadriceps muscle. The electromyographic signal was amplified (model P55 AC amplifier, Astro-Medical, Warwick, RI, USA) and then converted to a digital signal, recorded, and stored as described for the load cell signal.
Data analysis
For the animal and muscle characteristics (Table 1), one-way analyses of variance (ANOVA) was used to test for differences across the experimental protocols, with post hoc comparisons made using the Student–Newman–Keuls test. Differences in the decline in force produced by the four testing trains were evaluated using a two-way (stimulation protocol x testing train frequency) ANOVA, with the Student–Newman–Keuls test used for post hoc comparisons. Planned, a priori contrasts were performed to assess within-group differences in MAPK phosphorylation between control and stimulated conditions using Student's paired t tests. One-way ANOVAs were used to test for effects of stimulation protocol on the differences in phosphorylation, glycogen depletion and CMAP peak-to-peak amplitude, with post hoc comparisons made using the Student–Newman–Keuls test.
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Results |
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No differences in animal age, body weight or muscle wet weight were present across the three experimental groups. The combination of altering muscle length and stimulation frequency achieved the desired goal of producing comparable initial forces for each protocol, despite the twofold difference in frequency between the 15 and the two 30 Hz groups (see values in bold in Table 1), although there were differences in experimental muscle length and passive resting tension. The force–time integrals (FTI) of initial trains in the fatiguing protocols were also not significantly different (30 Hzsupra, 0.705 ± 0.113 N s; 15 Hz, 0.815 ± 0.131 N s; and 30 Hzsupra, 0.917 ± 0.129 N s) Despite the high passive tension in the 30 Hzsupra protocol, the strain in this protocol (
15%) was less than that previously reported (20–25%) in studies of the effects of passive stretch on MAPKs (Boppart et al. 2001; Martineau & Gardiner, 2001, 2002; Wretman et al. 2001).
Phosphorylation of MAPK
No differences in the relative amounts of the total MAPKs were found across the three protocols. However, the phosphorylation of the three MAPKs studied differed in response to the different stimulation protocols. There were significant effects of stimulation protocol on the increase in phosphorylated/total content in the stimulated versus control muscles for JNK (F = 6.29, P < 0.001) and p38 (F = 3.96, P = 0.03), but not ERK (F = 0.13, P = 0.80). The degree of JNK phosphorylation followed the pattern of total (active plus passive) tension differences across the three stimulation protocols, with the greatest changes seen in the 30 Hzsupra group, followed by the 15 Hz and then the 30 Hzsub group, with both the 30 Hzsupra and 15 Hz groups exhibiting significant changes relative to the control muscles (Fig. 2). In contrast, significant p38 phosphorylation (increase versus control) was observed in the 30 Hzsub and 30 Hzsupra groups, but not in the 15 Hz group. None of the protocols produced a significant change in ERK1/2 phosphorylation relative to control values. No changes in the phosphorylation of any of the MAPKs were detected in the sham versus control muscles. No differences in the total content of the three MAPKs across groups were present.
Fatigue
Muscle fatigue was assessed as both the decline in peak force during the 2 min of the stimulation protocols (Table 1) and as the decline in the force production of the four testing trains (1, 15, 30 and 80 Hz) delivered before and after each protocol (Fig. 3A). There was a significant effect of stimulation protocol on the decline within the fatigue protocol (F = 10.6, P < 0.01). Over the course of the fatigue protocols, the total FTI was greatest in the 15 Hz protocol, followed by the 30 Hzsupra, then the 30 Hzsupra group, although these differences were not significant (Table 1). This finding is consistent with the different degrees of fatigue observed during the protocols. While we cannot rule out a possible effect of the differences in total FTI, we believe that any effects of FTI would have biased against finding the results we did (see Discussion). For the declines in peak forces produced by the testing trains, there were significant effects of both stimulation protocol and testing train frequency (F = 7.67, P < 0.01 and F = 5.19, P < 0.01, respectively). The 15 Hz protocol produced the least fatigue, regardless of whether fatigue was assessed as the decline within the fatigue protocol (Table 1) or as the decline in the forces of the different testing trains (Fig. 3). There were trends for greater fatigue in the 30 Hzsupra group versus the 30 Hzsub group, but these differences did not achieve statistical significance.
Glycogen depletion
All of the protocols produced marked and significant declines in glycogen (all P < 0.02). There were, however, no significant differences in depletion across groups (Fig. 3B).
Myofibre damage
We were concerned that isometric contractions of lengthened TAs might cause damage, so we administered EBD 24 h before this protocol to assess myofibre damage (Hamer et al. 2002; Lovering & De Deyne, 2004). Approximately 25 sections were evaluated, with an average of 45 ± 8 muscle fibres per field. Thus, > 1100 fibres were assessed. Although isolated EBD-positive fibres were detected sporadically, there was no evidence of damage in the three additional animals subjected to the 30 Hzsupra protocol following Evans Blue dye injection compared to the contralateral control TAs.
Muscle excitation
The peak-to-peak CMAP amplitude was preserved well during the 15 Hz protocol (5.7 ± 11.1% decline), but marked declines were observed in both 30 Hz groups, with greater declines in the 30 Hzsupra (40.3 ± 15.0%) versus the 30 Hzsub group (25.6 ± 8.2%; Fig. 4). Although only a small number of animals were tested for CMAP changes (n = 3 per group), the differences between the 15 Hz group and the other two groups were significant (P = 0.02 in each case).
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Discussion |
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The pattern of p38 phosphorylation observed with the in situ preparation in the present study (increased after the 30 Hzsub and 30 Hzsupra protocols, but not after the 15 Hz protocol) is consistent with, although smaller in magnitude than, the in vitro findings of Dentel et al. (2005). Differences in species (mouse versus rat) and muscle (EDL versus TA) and preparation (in vitro versus in situ) may have contributed to the difference in the magnitude of the responses observed in the two studies, but the difference in stimulation protocol is probably the main factor. The 30 Hz protocols in the present study delivered 1400 total stimulation pulses over the fatiguing protocol and administration of the testing trains, whereas the protocol used by Dentel et al. (2005) involved 10 Hz stimulation for 15 min, for a total of 9000 stimulation pulses.
The JNK phosphorylation resulting from the stimulation protocols used in the present study followed the total (active plus passive) tension of the different protocols, a pattern similar to that observed by other investigators (Boppart et al. 1999, 2001; Martineau & Gardiner, 2001). Like the p38 phosphorylation, the JNK response seen in the present study was more moderate than that in many previous investigations (Boppart et al. 2001; Martineau & Gardiner, 2001; Wretman et al. 2001), possibly because the earlier studies evaluated maximal forced lengthening during maximal contractions. In addition to very high forces, such contractions often produce disruptions of muscle fibre membranes (Hamer et al. 2002; Lovering & De Deyne, 2004). The submaximal isometric contractions in the present study did not produce maximum active tension, and the results of the EBD assays suggest that the even the 30 Hzsupra protocol did not produce muscle fibre damage, despite high passive tension.
Given the wide variety of exercise and stimulation protocols that have been shown to increase ERK1/2 phosphorylation, it was surprising that we did not observe any significant changes in phosphorylation of this MAPK. Again, the short duration of the protocols and submaximal contractions utilized may have been insufficient to activate that pathway. This lack of a change is not without precedent, however, since Dentel et al. (2005) also reported no changes in pERK following their longer stimulation protocol.
Regarding fatigue, the two 30 Hz protocols produced greater declines in muscle force than the 15 Hz protocol, supporting the hypothesis that increased stimulation frequency increases fatigue. Our working hypothesis was that this increase in fatigue with increasing frequency was the result of an added metabolic demand owing to the greater ion-pump activity associated with the increased number of activation pulses (Homsher, 1987; Stienen et al. 1995). No differences in our assay of metabolic cost (glycogen depletion) were observed across protocols, however. It is possible that a different metabolite assay (e.g. phosphocreatine or inorganic phosphate) might have shown some relationship to fatigue, but glycogen seemed an appropriate marker, because others have suggested that the sarcolemmal and sarcoplasmic reticulum pumps of skeletal muscle are preferentially glycolytic (Han et al. 1992; Xu et al. 1995). It is possible that the reduced summation of force with the 15 Hz protocol contributed to the lack of a difference in glycogen depletion and fatigue. It has been suggested that force generation is more metabolically demanding than force maintenance during electrically stimulated contractions (Hogan et al. 1998; Russ et al. 2002b). The reduced summation at 15 versus 30 Hz stimulation required that force be regenerated after relaxation to a greater degree, and may thereby have decreased the differences in metabolic demand and fatigue between the 15 and 30 Hz protocols.
Since our marker of metabolic demand failed to explain the observed differences in fatigue, we conducted a supplementary set of experiments on a small number of animals to assess whether changes in neuromuscular excitation could have contributed to our findings. Given that the highest activation frequency used during the fatiguing protocol was 30 Hz, we did not expect to find any impairment. To our surprise, the EMG data indicated that the greater fatigue at higher frequencies might be the result of some form of depolarization impairment. The m-wave changes could be due to several factors (Keenan et al. 2006), and our measurements did not distinguish among impairments at the level of the peripheral nerve, the neuromuscular junction or sarcolemma. Nevertheless, our findings suggest that increased depolarization failure, possibly as a result of alterations in potassium gradients (Cairns et al. 2003), contributed to some extent to the increased fatigue seen with increased stimulation frequency. The greater fatigue seen in the 30 Hzsupra versus the 30 Hzsub protocol could result from an added effect of stretch-activated ion channels in the 30 Hzsupra condition (Allen, 2004). A loss of excitation might have limited the glycogen depletion we observed in the 30 Hz protocols, since inactive fibres would not consume glycogen. However, a loss of fibre activation would bias against the greater p38 phosphorylation in the 30 Hz protocols, such that we may have underestimated the effect of stimulation frequency on that response.
Initial force production was the same across the three experimental protocols, but the greater fatigue observed with 30 Hz stimulation led to lower total FTI over the course of the three protocols, although these differences in FTI were not significant (Table 1). If the total FTI were a factor in the MAPK responses we observed, it would probably have biased against our results. For example, FTI was lowest in the 30 Hzsupra group, yet it showed greater JNK phosphorylation than the other groups. Likewise, the 15 Hz group exhibited the lowest p38 phosphorylation, despite having the greatest FTI. No ERK changes were observed across any protocols, suggesting that FTI did not play a role.
The pattern of fatigue resembled that of p38 phosphorylation, with the two 30 Hz protocols producing greater fatigue than the 15 Hz protocol. If p38 signalling is involved in the adaptive response of skeletal muscle to exercise, this observation might offer some support for the hypothesis that fatigue itself is a factor in inducing a training response, possibly due to the changes in high-energy phosphates or pH that typically accompany fatigue (Rooney et al. 1994; Schott et al. 1995). Another possibility might be changes in resting intramuscular calcium that have been observed with fatigue (Chin & Allen, 1996). Regardless, it seems much more likely that differences in muscle activation account for our results, rather than differences in fatigue, given that p38 phosphorylation has been observed with repetitive activation when contractile force is eliminated (Dentel et al. 2005).
Although muscle activation frequency, independent of force production, has received little attention, the results of this study indicate that changes in muscle activation frequency can influence certain physiological responses of skeletal muscle during repetitive contraction, even when the contractile force is kept the same. We observed increased fatigue associated with greater activation frequencies, a finding that appears to be associated with a failure of depolarization. Furthermore, it appears that at least one MAPK (p38) is frequency sensitive, possibly responding to the ion fluxes associated with muscle stimulation (e.g. Na+–K+; Ca2+). Functionally, the frequency-sensitive nature of p38 is in line with previous studies that have suggested that p38 phosphorylation is sufficient to induce mitochondrial biogenesis (Akimoto et al. 2005) and possibly fast-to-slow myosin heavy chain conversion (Gredinger et al. 1998) in skeletal muscle. Each of these responses is associated with frequent-activation, low-load contractile protocols, such as chronic low-frequency stimulation (Reichmann et al. 1985). Speculating further, reduced activation of frequency-sensitive signalling pathways might account for some of the blunted response to voluntary exercise seen in aged muscle, since voluntary motor unit discharge rates are known to decline with age (Kamen et al. 1995; Rubinstein & Kamen, 2005). Further studies of the effects of activation frequency on skeletal muscle are needed to address these issues in greater depth.
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0.05) from control value for the same protocol). Post hoc testing indicated significant differences between the phosphorylation of JNK in response to the 30 Hzsupra protocol and that associated with the other 2 protocols, and between the phosphorylation of p38 in response to the 30 Hzsub protocol and that in response to the 15 Hz protocol (# significant difference between protocols).
significant difference (P
