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SKELETAL MUSCLE AND EXERCISE |
1 Copenhagen Muscle Research Centre, Department of Exercise and Sport Sciences, Section of Human Physiology, University of Copenhagen, Universitetsparken 13, Copenhagen, Denmark, 2100
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Abstract |
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(Received 23 December 2006;
accepted after revision 1 February 2007;
first published online 1 February 2007)
Corresponding author A. J. Rose: Copenhagen Muscle Research Centre, Department of Exercise and Sport Sciences, Section of Human Physiology, University of Copenhagen, Universitetsparken 13, Copenhagen, Denmark, 2100. Email: arose{at}ifi.ku.dk
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Introduction |
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Along with the unifunctional phosphorylase kinase, myosin light chain kinase and eukaryotic elongation factor 2 kinase, the multifunctional calmodulin-dependent protein kinases (i.e. CaMKI, II and IV) are likely to play functional roles in skeletal muscle (Chin, 2004). Of these, CaMKII is the best described (for a review see Sacchetto et al. 2005a), and was shown to be expressed in skeletal muscle more than 20 years ago (Campbell & MacLennan, 1982; Chiesi & Carafoli, 1982; Woodgett et al. 1982, 1983, 1984), and is likely to regulate gene transcription (Ojuka et al. 2002; Liu et al. 2005), ion homeostasis (Hawkins et al. 1994; Tavi et al. 2003) and metabolism (Watt et al. 2003; Wright et al. 2004; Singh et al. 2004; Sacchetto et al. 2005b). More recently, studies have demonstrated CaMKII expression in skeletal muscle of humans (Damiani et al. 1996; Margreth et al. 2000; Rose & Hargreaves, 2003; Rose et al. 2006).
The functional properties and regulation of skeletal muscle CaMKII have been reviewed in detail recently by Damiani and colleagues (Sacchetto et al. 2005a). Of note, despite different isoforms of CaMKII being expressed in skeletal muscle (Bayer et al. 1998; Rose & Hargreaves, 2003; Rose et al. 2006), the CaMKII of skeletal muscle displays properties similar to that of neural CaMKII (Woodgett et al. 1984; Pelosi & Donella-Deana, 2000; Rose et al. 2006). In particular, skeletal muscle CaMKII exists as a multimeric complex of 10–12 individual CaMKII enzymes (Woodgett et al. 1983), and upon rises in intracellular Ca2+ and Ca2+–CaM binding, undergoes a conformational change which relieves autoinhibition to increase catalytic activity (Colbran et al. 1989; Lengyel et al. 2001). In addition, skeletal muscle CaMKII can undergo autophosphorylation in response to prolonged exposure to Ca2+, which enables the kinase to be partially active in the absence of Ca2+–CaM (Pelosi & Donella-Deana, 2000; Rose et al. 2006).
Recently, it was shown that skeletal muscle CaMKII of humans was activated during exercise as indexed by higher autophosphorylation and autonomous activity as well as higher phosphorylation of a protein substrate phospholamban (Rose & Hargreaves, 2003; Rose et al. 2006). In particular, the activation of CaMKII in working skeletal muscle during exercise was rapid and sustained (Rose et al. 2006). However, in these studies, whether the activation was a result of local versus humoral factors during exercise could not be determined. Thus, the primary aims of the present study were to examine the time effect of contractions on skeletal muscle CaMKII and to gain further insight into the mechanisms as well as functional consequences of CaMKII activation in skeletal muscle.
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Methods |
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All materials were from Sigma-Aldrich (USA) unless stated otherwise.
Animals
Male Sprague–Dawley rats were used for experimentation and were fed standard laboratory chow and consumed water ad libitum, and kept on a constant 12: 12 h light–dark cycle. All experiments were approved by the Danish Animal Experimental Inspectorate and complied with the European Convention for the Protection of Vertebrate Animals used for Experiments and other Scientific Purposes (council of Europe no. 123, Strasbourg, France, 1985).
In situ experiments
Rats (190–230 g) were anaesthetized by intraperitoneal injection of sodium pentobarbital (5 mg per 100 g body wt). With some animals (n
= 8–12 per time-point), an in situ stimulation protocol was applied as a model of exercise, as previously described (Richter et al. 1984), to examine the time effect of contractions on signalling proteins. This protocol of muscle contraction was used rather than exercise as it results in recruitment of the entire fibre population of the stimulated muscles, the effect of local versus humoral factors can be accounted for by comparing the stimulated versus the resting contralateral hindlimb muscles, and it allows rapid collection of muscle tissue during the stimulation. In brief, the gastrocnemius muscles of both hindlimbs were exposed by surgically removing skin and connective tissues around these muscles. In addition, the sciatic nerve of the right hindlimb was carefully exposed. Afterwards, the rats were placed in a standard position by fixing the knee in a set position by inserting a needle under the patella tendon. A hook was then placed under the Achilles tendon of the same leg, and this was connected to a force transducer as previously described (Wojtaszewski et al. 1996). An electrode was placed around the sciatic nerve, and the hindlimb was stretched to a standard basal tension. The animals rested in this position for 10 min after which the gastrocnemius muscle was either freeze-clamped and dissected immediately (representing time 0), or was stimulated to contract isometrically via the sciatic nerve (train rate: 0.5 Hz; train duration: 200 ms; pulse rate: 100 Hz, pulse duration: 0.1 ms; 5–20 V; DISA Impulse Generator) for 10 s, 1, 3, 10 or 30 min and muscles were freeze-clamped during stimulation and dissected free. To ensure that all muscle fibres were recruited, the voltage was adjusted to a level where no further increase in tension was produced during the first few seconds of stimulation. Using this protocol, there was an initial 5–10% reduction in tension development during the initial 2–3 min of stimulation, with a further 
5% fall after 10 min, after which tension development was stable during the remaining 20 min (data not shown). For the animals stimulated for 10 and 30 min, the gastrocnemius muscle from the contralateral leg was also sampled (30–45 s after sampling the other limb) to examine the possible effects of humoral stimuli resulting from the stimulation or surgery. In some animals, the gastrocnemius muscle was freeze-clamped immediately after surgery and dissected free. To control for the time effect of surgery and the small amount of tension placed on the hindlimb induced by the experimental set-up, some (n
= 4) animals were placed in the set-up with the electrode on the nerve without stimulation for 40 min after which the muscle was freeze-clamped and dissected free. All muscle samples were placed in liquid nitrogen after dissection and later stored at –80°C until required. After experimentation, the animals were killed by cervical dislocation while unconscious.
Ex vivo experiments
Rats (45–55 g) were anaesthetized by intraperitoneal injection of sodium pentobarbital (5 mg per 100 g body wt). Extensor digitorum longus (EDL) muscles from both hindlimbs were carefully excised and placed in incubation chambers (multi myograph system organ bath 700MO; Danish Myo Technology A/S, Aarhus, Denmark) and suspended from the tendons by ligatures at resting tension (4–5 mN). After muscle excision, the rats were killed by cervical dislocation while unconscious and muscles were constantly bathed in Krebs–Ringer–Henseleit buffer (118.5 mM NaCl, 24.7 mM NaHCO3, 4.74 mM KCl, 1.18 mM MgSO3, 1.18 mM KH2PO4, and 2.5 mM CaCl2, pH 7.4) containing 2 mM pyruvate and 0.01% (w/v) BSA at 30°C which was gassed with 95% O2 and 5% CO2.
In one experiment (n = 6 per treatment), muscles were preincubated with 25 µM 1-[N,O-bis-(5-isoquinolinesulphonyl)-N-methyl-L-tyrosyl]-4-phenyl-piperazine (KN62; LC Laboratories, MA, USA) + 0.1% DMSO or 0.1% DMSO only for 1 h. In another experiment (n = 8 per treatment), muscles were preincubated for 2 h with 75 µM myristoylated autocamtide-2-related inhibitory peptide 2 (myr-AIP2; N-myr-KKKLRRQEAFDAL-OH; KJ Ross-Petersen ApS, Denmark). KN62 and AIP2 are allosteric (Hidaka & Yokokura, 1996) and competitive (Ishida et al. 1998) inhibitors of CaMKII, respectively. KN62 inhibits CaMKI, CaMKII and CaMKIV but presumably not other kinases (Hidaka & Yokokura, 1996; Davies et al. 2000) and has been used to inhibit CaMKII in rat skeletal muscle previously (Wright et al. 2004). AIP is a specific CaMKII inhibitor when compared with other multifunctional CaM kinases, cAMP-dependent protein kinase and protein kinase C (Ishida et al. 1995, 1998) and has been used in vitro previously (De Santiago et al. 2002). One muscle from the same animal served as the basal control while the other was electrically stimulated, via platinum electrodes in contact with the incubation buffer, to contract for 60 s following the preincubation period, with the same pattern described above except that the voltage was 50 V. Tension development was recorded at 0.2, 4.2, 20.2 and 58.2 s, and muscles were frozen at 60 s. A representative tension–time profile is shown in Fig. 1. Basal muscles were frozen within several minutes of stimulating the corresponding muscle, and samples were stored at –80°C until required. EDL was chosen as fast-twitch EDL muscles have higher expression of trisk95 than slow-twitch soleus muscle (Vassilopoulos et al. 2005; A. J. Rose & E. A. Richter, data not shown), and has similar fibre-type distribution as the gastrocnemius in that they are comprised mainly of fast-twitch muscle fibres (Armstrong & Phelps, 1984) and that there were no differences in CaMKII and trisk95 expression when comparing gastrocnemius and EDL muscles (n = 8; data not shown).
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The gastrocnemius muscle samples were mechanically powdered under liquid nitrogen to make each aliquot taken from a sample representative of the whole sample. After powdering, visible connective tissue was removed and separate aliquots were taken for protein extraction. For extraction, samples were homogenized while in an ice slurry (i.e. 0°C) in a buffer (15 µl per mg tissue) containing 50 mM Tris (pH 7.4), 150 mM NaCl, 10% (v/v) glycerol, 1 mM EDTA, 1 mM EGTA, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 1 mM PMSF, 1 mM dithiothreitol, 1 mM benzamidine, 0.5% (v/v) protease inhibitor cocktail, and 1% (v/v) Nonidet P-40 using a polytron homogenizer (PT 1200, Kinematica) until no visible particles remained. The homogenates were mixed thoroughly by end-over-end rotation at 4°C for 30 min, and then spun at 6000 g for 10 min at 4°C. The clarified supernatant was taken and stored at –80°C until required. A small aliquot of each lysate was taken and diluted for protein concentration determination prior to storage.
Analytical techniques
Protein concentration of tissue extracts was determined in triplicate using the bicinchoninic acid (BCA) method using bovine serum albumin standards (Pierce, USA) and BCA assay reagents (Pierce, USA). A maximal coefficient of variance of 5% was accepted between replicates. Samples were immunoblotted for protein expression and phosphorylation was according to Rose et al. (2005). The primary antibodies used were anti-CaMKII (BD Biosciences-Pharmingen, USA; 612624), anti-phospho-Thr287-CaMKII (Cell Signalling Technology, Inc., USA; 3361), anti-phospho-Thr (Cell Signalling Technology, Inc., USA; 9381) and anti-triadin/trisk95 (Affinity Bioreagents, USA; MA3-927). Secondary antibodies were from DakoCytomation (Denmark). Band intensity was quantified by Kodak imaging software (Kodak 1D 3.5, USA). Preliminary experiments demonstrated that the amounts of protein loaded were within the dynamic range for the conditions used and the results obtained (data not shown). To measure kinase activity muscle extracts were analysed according to Rose & Hargreaves (2003). In brief, CaMKII activity of muscle extracts was measured in the presence (maximal activity) or absence (autonomous activity) of Ca2+–calmodulin with autocamtide-2 (Upstate Biotechnology, USA) as the peptide substrate. This assay has been shown to be specific for CaMKII as using a specific inhibitor for CaMKII reduces the majority of phosphotransfer activity by lysate proteins in the presence or absence of calmodulin (Rose & Hargreaves, 2003). To measure trisk95/triadin phosphorylation, 10 µl of anti-triadin antibody was mixed with 1 mg (gastrocnemius) or 0.5 mg (EDL) of tissue lysate protein with 40 µl 30% (v/v) protein-G-sepharose (Amersham Biosciences, Sweden) in a final volume of 500 µl and mixed overnight at 4°C. Immunocomplex pellets were washed several times and resuspended with 30 µl of 1 x sample buffer. Samples were denatured and immunoblotted using phospho-Thr antibodies. Following capture, membranes were stripped (Restore Western Blot Stripping Buffer, Pierce Biotech. Inc., USA) and re-probed with anti-trisk95 antibodies (Affinity Bioreagents, USA; MA3-927).
In vitro experiments
All phospho-specific antibodies used were tested for phosphospecificity as described by Rose et al. (2006). Extraction of rat muscle samples without phosphatase inhibitors and incubation with phosphatase completely removed or greatly reduced signal obtained when using the phospho-specific antibodies used in this study (data not shown).
To further examine the relationship between CaMKII autophosphorylation and autonomous activity, an in vitro experiment was performed to manipulate CaMKII autophosphorylation levels. This was achieved by taking aliquots of a rat skeletal muscle extract (final concentration: 2.4 µg µl–1) and incubating them with differing amounts of Ca2+ and CaM (Upstate Biotechnology, USA). All samples were reacted in quadruplicate in a buffer containing 10 mM Hepes (pH 7.2), 1 mM EGTA, 0.5 mM Na3VO4 and 400 µM ATP under four different conditions. One condition was without added Ca2+ or CaM, another was with 2 mM CaCl2, another with 2 mM CaCl2 + 0.12 µM CaM, and another with 2 mM CaCl2 + 1.2 µM CaM. These reaction mixes were preheated to 30°C, and the reaction was initiated by the addition of the sample. The reaction proceeded for 1 min at 30°C and was stopped by placing in an ice slurry (0°C) and the addition of 5 mM EGTA (final concentration). An aliquot of each sample was then taken for immunoblot analysis of CaMKII autophosphorylation. Aliquots of the remaining sample were taken and CaMKII was immunoprecipitated in duplicate as described above and these immunopreciptates were analysed for CaMKII activity (Rose & Hargreaves, 2003).
Calculations and statistics
Statistical analyses were performed using SPSS 13.0 for Windows. Initially, descriptive analyses were performed, and as all data was normally distributed, the data were analysed using parametric statistical tests. To test whether the data displayed equal variances, Levene's test for homogeneity of variance was used, and when variances were not equal, the data were transformed appropriately (Montgomery, 2001). For the time-course samples, an unpaired t test was performed initially to examine whether there was an effect of placing the muscle in the experimental set-up for 10 min and then sampling versus sampling immediately after surgery. One-way ANOVA was conducted to evaluate whether there were differences between time-points during stimulation. When the ANOVA indicated significance, Tukey's post hoc testing was used. To test for effects between the data from stimulated versus the non-stimulated muscle of the same animal, paired t tests were conducted. To test for the combined effect of surgery and placing in the experimental set-up for 40 min, data from samples taken immediately after surgery as well as after 40 min in the set-up without stimulation were compared using unpaired t tests. The strength of association between variables was analysed by Pearson correlations. For the ex vivo work, two-way ANOVA was conducted with post hoc testing using SigmaStat v. 3.1. Differences were considered to be significant when P was less than 0.05.
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Results |
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There was no effect of in situ contractions on maximal CaMKII activity or CaMKII expression (Figs 2 and 3). In contrast, there was a transient increase in Ca2+–calmodulin-independent (i.e. autonomous) CaMKII activity in response to contractions. More specifically, autonomous CaMKII activity was 10 ± 1% of maximal at time 0, and increased to 20–25% of maximal at 1–3 min of contractions (Fig. 2). Autonomous CaMKII activity was 18 ± 2% and 17 ± 3% of maximal at 10 and 30 min of contractions, respectively, but only 10 min was different from time 0 (Fig. 2). There were no differences in autonomous CaMKII activity between time 0, 10 and 30 min when compared with the unstimulated contralateral muscles (Fig. 2). Importantly, there were no significant differences in CaMKII expression and phosphorylation between samples taken at basal, time 0 or 40 min of placement in the experimental set-up, indicating that the effect of electrical stimulation was a real and not simply due to placement in the experimental set up (data not shown).
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M and 
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isoforms (r2
= 0.73; P < 0.05; data no shown), the optical densities of both of these bands were summed. Similar to autonomous CaMKII activity, there was a rapid and transient increase in skeletal muscle CaMKII phosphorylation at Thr287 in response to contractions (Fig. 3). Specifically, there was a 1.5-fold increase in pThr287-CaMKII after 10 s of contractions compared with time 0, and was even higher (3- to 5-fold) after 3 min of contractions (Fig. 3). When expressed relative to CaMKII expression, CaMKII phosphorylation was also higher at 10 min of contractions when compared with time 0 or the resting contralateral muscle at this time (Fig. 3). At 30 min of contractions, there were no differences in CaMKII phosphorylation when compared with time 0 or the resting contralateral muscle at this time (Fig. 3). There was a positive-linear correlation between CaMKII phosphorylation at Thr287 and autonomous CaMKII activity (r2 = 0.43, P < 0.05; Fig. 4A). Furthermore, when the level of autophosphorylation was manipulated by prior incubation of skeletal muscle proteins with varying concentrations of free Ca2+ and CaM, there was also a positive-linear correlation between CaMKII phosphorylation and activity (r2 = 0.73, P < 0.05; Fig. 4B).
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In response to contractions in situ, trisk95 phosphorylation at Thr residues was 20- to 30-fold higher at 1 and 30 min, when compared with time 0 (Fig. 5). Furthermore, trisk95 phosphorylation was higher in the contracting compared with the non-stimulated muscles at 30 min of stimulation, and there was no difference between trisk95 phosphorylation when comparing non-stimulated and time 0 muscles (Fig. 5). When EDL muscles were contracted ex vivo, there was a 15- to 20-fold increase in trisk95 Thr phosphorylation (Fig. 6). Taken together, these data indicate that the increase in trisk95 phosphorylation at Thr residues is due to local stimuli within the contracting muscle. Indeed, the increase in trisk95 phosphorylation with contraction was largely or completely blunted when muscles were preincubated with CaMKII inhibitors myr-AIP2 and KN62, respectively (Fig. 6). Importantly, the increase in skeletal muscle CaMKII phosphorylation by contraction was also blocked by CaMKII inhibition (Fig. 6). Furthermore, the magnitude of fatigue was exacerbated when muscles were preincubated with both CaMKII inhibitors (Fig. 7).
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Discussion |
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As shown in Figs 2 and 3, there is a similar pattern between the changes in CaMKII autonomous activity and CaMKII phosphorylation at Thr287 during contractions and there was a significant positive correlation between these two variables (Fig. 4A). In addition, when CaMKII Thr287 phosphorylation level was artificially manipulated by incubation of skeletal muscles lysates in vitro with variable amounts of Ca2+ and CaM, there was a stronger positive correlation (Fig. 4B). Taken together, these findings suggest that the increases in CaMKII autonomous activity during contractile activity are mediated by the increases in CaMKII phosphorylation at Thr287. Indeed, expression of CaMKII that has a non-phosphorylatable residue at this site displays a lack of increase in autonomous activity when exposed to stimuli that increase Ca2+ (Hanson et al. 1989). However, only 43% of variance of CaMKII autonomous activity could be accounted for by Thr287 phosphorylation (Fig. 5A), suggesting that there may be additional factors involved. These factors might include phosphorylation of CaMKII on other sites (Colbran, 1993; Migues et al. 2006) or localization and binding to substrate proteins (Bayer & Schulman, 2001).
The mechanism by which skeletal muscle CaMKII becomes phosphorylated during exercise probably occurs via autophosphorylation. Indeed, similar to neuronal CaMKII (Hudmon & Schulman, 2002), skeletal muscle CaMKII probably exists as a multimeric complex of 12 individual CaMKII subunits (Woodgett et al. 1983, 1984), and upon rises in cytosolic [Ca2+] and Ca2+–CaM binding, the CaMKII multimeric complex undergoes intersubunit phosphorylation at Thr287 (Fig. 4B; Hudmon & Schulman, 2002; Rose et al. 2006). In terms of the functional consequences of CaMKII autophosphorylation, the affinity for calmodulin is greatly enhanced (Meyer et al. 1992), and thus the rapid autophosphorylation may be a mechanism to accelerate the activation of CaMKII with contractions. In addition, autophosphorylation may allow CaMKII to remain partially active between Ca2+ transients by disrupting autoinhibition (Hudmon & Schulman, 2002), thereby allowing persistent phosphorylation of downstream substrates. However, it should be noted that the increase in autonomous activity was only 15–25% of Ca2+–CaM-stimulated activity during the majority of contraction time (Fig. 2), meaning that the rise in activity during the Ca2+ spike may have been quantitatively more important in the overall increase in CaMKII activity. Indeed, the patterns of CaMKII phosphorylation (Fig. 3) and phosphorylation of the putative CaMKII substrate trisk95 (Fig. 5), which may be indicative of in vivo CaMKII activity during contraction (discussed below), were remarkably different. The transient nature of the increase in autophosphorylation with contraction (Fig. 3) is difficult to explain, but may be related to differences in Ca2+ uptake and release kinetics by the sarcoplasmic reticulum and thus Ca2+–CaM binding, or a differential action of CaMKII phosphatases on CaMKII over time. Indeed, it has been observed that there is a transient increase in stimulus-induced intracellular Ca2+ during repeated tetanic contraction of single muscle fibres (Westerblad & Allen, 1991). Also, skeletal muscle CaMKII may indirectly regulate its own dephosphorylation by increasing PP1c activity through phosphorylation of the PP1c-targeting subunit GM (Sacchetto et al. 2005b). This could be the case as dephosphorylation of glycogen synthase was higher in contracting rat skeletal muscle in situ using the same protocol (J. N. Nielsen & J. F. P. Wojtaszewski, unpublished observations), and PP1c-GM mediates glycogen synthase dephosphorylation by contraction (Aschenbach et al. 2001).
To gain further insight into the regulation of skeletal muscle CaMKII activity with exercise as well as functional consequences of CaMKII activation, an examination of the phosphorylation of putative downstream substrates was conducted. Unlike humans and other mammals (Briggs et al. 1992; Damiani et al. 2000; Margreth et al. 2000; Rose et al. 2006), rat skeletal muscle does not express the CaMKII substrate phospholamban (A. J. Rose & E. A. Richter, unpublished observation; Vangheluwe et al. 2005), and thus it could not be examined in the present study. On the other hand, endogenously expressed CaMKII can phosphorylate another SR-associated protein trisk95, presumably at Thr37 (Damiani et al. 1995, 2003; Colpo et al. 2001), and trisk95 is expressed in rat (Carl et al. 1995; Vassilopoulos et al. 2005; Fig. 5) but not human (Thevenon et al. 2003) skeletal muscle. The majority of trisk95 is embedded within the lumen of the SR, but a small part of the C-terminus is cytoplasmic (Marty, 2004). CaMKII is anchored to the cytoplasmic side of junctional and longitundinal SR membranes (Sacchetto et al. 2005a), and can phosphorylate triadin at the cytoplasmic C-terminal domain (Colpo et al. 2001). In response to contractions in situ, trisk95 phosphorylation at Thr residues was rapidly increased, and was sustained during the 30 min stimulation period, with no change in the contralateral muscle during stimulation (Fig. 5). Furthermore, trisk95 phosphorylation was higher in EDL muscles electrically stimulated to contract ex vivo, a response that was greatly blunted by treatment of muscles with chemical inhibitors of CaMKII activity (Fig. 6), further indicating that trisk95 is a substrate of CaMKII. This is not inconceivable as CaMKII is localized to the junctional SR (Chu et al. 1990; Damiani et al. 2003), and endogenous CaMKII inhibition blunts Ca2+–CaM-induced trisk95 phosphorylation in SR vesicles from rabbit fast-twitch muscle (Damiani et al. 2003). Taken together, similar to exercising humans (Rose et al. 2006), these data show that the activation of skeletal muscle CaMKII is rapid and sustained during contractions, and occurs via a local mechanism within the contracting muscle, probably via allosteric activation by Ca2+–CaM.
Trisk95 is an integral membrane protein within the junctional SR in skeletal muscle and physically interacts with calsequestrin and the ryanodine receptor (RyR1; SR Ca2+ release channel; Ohkura et al. 1998; Groh et al. 1999) and inhibits excitation-induced Ca2+ release (Rezgui et al. 2005); probably by decreasing RyR1 open probability (Groh et al. 1999). It has been hypothesized (Sacchetto et al. 2005a) that phosphorylation of trisk95 (Figs 5 and 6) can relieve this inhibitory action on the RyR1 to potentiate Ca2+ release rate during repeated muscle contractions. Indeed, although controversial (Hain et al. 1994), CaMKII can increase RyR1 activity (Dulhunty et al. 2001), and inhibition of CaMKII leads to a blunting of force and Ca2+ release, but not Ca2+ uptake, during twitches of murine fast-twitch muscle (Tavi et al. 2003). In support of this, there was a greater magnitude of fatigue during stimulation of isolated whole rat EDL muscles when CaMKII was inhibited (Fig. 7). It is important to highlight that this effect was seen when CaMKII was inhibited by two structurally unrelated inhibitors which affect CaMKII activity via different mechanisms. This effect of CaMKII on the RyR1 and Ca2+ release is probably mediated by trisk95, as RyR1 is probably not a substrate of endogenously expressed CaMKII in skeletal muscle (Sacchetto et al. 2005a). Furthermore, these data suggest that the activation of CaMKII may play a protective role in the development of fatigue during strenuous contractile activity, a phenomenon that is not entirely inconceivable given that failure of Ca2+ release from the SR is a likely cause of fatigue (Dahlstedt & Westerblad, 2001; Dutka et al. 2005). However, as CaMKII is a mulitifunctional kinase, and probably affects other substrates that are involved in excitation–contraction coupling (Sacchetto et al. 2005a) as well as catabolism (Wright et al. 2004, 2005; Singh et al. 2004; Sacchetto et al. 2005b), it is possible that CaMKII affects skeletal muscle fatigue rate by multiple mechanisms, perhaps not involving trisk95. Clearly, further work is required to examine the precise role and mechanism of CaMKII-mediated phosphorylation on skeletal muscle Ca2+ release and fatigue.
When comparing the in situ and ex vivo models it is apparent that there are stark differences in the tension–time profiles. In particular, even though the stimulation protocols were the same, there was a small decline (i.e. 10–15%) in tension development during the 30 min in situ hindlimb stimulation whereas there was up to a 70% decline in tension development after 1 min of EDL stimulation ex vivo. This is likely to be largely explained by the dependence of diffusion for O2 transport when muscles are incubated ex vivo in that the large fall in tension during stimulation can be attributed to the development of hypoxia due to inadequate O2 diffusion (Barclay, 2005). Indeed, when single muscle fibres are contracted ex vivo, O2 diffusion distances are relatively negligible, and substantial falls in tension are not observed until after several minutes of stimulation (Westerblad & Allen, 1991).
It is questionable as to whether trisk95 phosphorylation has a physiological role during all types of exercise. For instance, it was observed that there was a large increase in phosphorylation of trisk95 with no recorded increase in tension when muscles were contracted in situ. Furthermore, stimulus-induced intracellular Ca2+ levels increase in the early phase of repetitive tetanic contractions, and this phenomenon may be explained by trisk95 phosphorylation; however, the putative increase in Ca2+ release during this situation may be physiologically irrelevant as this increase may be supramaximal relative to the sensitivity of the myofibrils to Ca2+ (Westerblad & Allen, 1991). Thus, it may be that only when the stimulation of muscle is severe so that fatigue develops, that trisk95 phosphorylation becomes physiologically relevant to excitation–contraction coupling.
In summary, the activation of skeletal muscle CaMKII is rapid and sustained during contractions. Trisk95 is a substrate of CaMKII in predominantly fast-twitch skeletal muscle, and CaMKII may function to modulate Ca2+ release and fatigue via phosphorylation of Trisk95.
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References |
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different from KN62 or myr-AIP2, P < 0.001.