1Department of Anatomy and Physiology, University of Padova, 35131 Padua, Italy; and 2Laboratory of Neuromuscular Plasticity, Institut Fédératif de Recherche 118, University of Sciences and Technologies, 59655 Villeneuve d'Ascq cedex, France
Submitted 25 September 2002 ; accepted in final form 10 May 2003
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ABSTRACT |
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muscle atrophy; muscle hypertrophy; clenbuterol; hindlimb suspension
MLC2 exists in several isoforms, of which two, coded by distinct genes, are expressed in mammalian skeletal muscles and described as slow MLC2 and fast MLC2 in relation to their predominant expression in slow or in fast muscle fibers (40). The slow MLC2 isoform is also expressed in ventricular myocardium, whereas a distinct isoform is expressed in atrial myocardium (40). In both slow and fast MLC2 phosphorylation occurs at the amino-terminal end, on serine residues 14 in slow MLC2 and 15 in fast MLC2 in rat muscles, and is catalyzed by the Ca2+/calmodulin-dependent MLC kinase (4). In smooth muscle MLC2, the primary phosphorylation site is located at serine 19, and evidence for a second site of phosphorylation at serine 12 or at threonine 9 has been given (16). Phosphorylation at serine 19 is catalyzed by Ca2+/calmodulin-dependent MLC kinase (16) and by Rho kinase (1) with an activating effect on actin-myosin interaction, whereas phosphorylation at serine 12 or threonine 9 is catalyzed by PKC with an inhibitory effect on myosin function (17). Whether a second site of phosphorylation is also present in sarcomeric MLC2 is still controversial (14, 29).
Changes in MLC2 phosphorylation pattern associated with variations of the functional state have been repeatedly demonstrated in cardiac and smooth muscles. In rat ventricular myocardium, MLC2 phosphorylation changes in connection with hypertrophy development (22,28), is positively correlated with blood pressure (12), and shows seasonal variations (26) in hibernating mammals. In human ventricle, a recent analysis of cardiac muscle strain pattern in vivo has shown that gradients of phosphorylation correlate with gradients of strain on cardiac myocytes (9). In tracheal and bronchial smooth muscle, phosphorylation level increases in the respiratory airways of dogs sensitized to ragweed pollen (18), whereas in arterial smooth muscles, phosphorylation levels increase in relation to high-mechanical stress conditions (2). All these changes can be interpreted in light of a double role of MLC2 phosphorylation, as considered above: a functional role of modulating calcium sensitivity and a structural role of contributing to assembly of cytoskeleton and myofibril filaments.
Less information on long-term changes in MLC2 phosphorylation is available for skeletal muscles. It is well known that phosphorylation of MLC2 increases during repeated electrical stimulation, but long-term effects of electrical stimulation (chronic low-frequency stimulation; CLFS) seem to include a reduction in the degree of phosphorylation (14, 20, 21). This would agree with the observation that CLFS induces a fast-to-slow transition of fiber types and that slow muscles have a lower level of phosphorylation than fast muscles (25).
The lack of information on the variation in MLC2 phosphorylation during
muscle adaptations involving changes in fiber size and fiber type, and the
relevance of MLC2 phosphorylation to the regulation of contractile
performance, prompted us to study MLC2 phosphorylation in two well-known
models of muscle plasticity, the atrophy induced by muscle disuse during
hindlimb suspension and the hypertrophy induced by clenbuterol administration.
Hindlimb suspension applied for 14 days causes atrophy, decrease in calcium
sensitivity, and slow-to-fast transition of the contractile protein isoforms
in the slow-twitch antigravitational soleus muscle
(44,
45). The
2-adrenergic agonist clenbuterol has a marked anabolic action
that produces muscle hypertrophy associated with an increase in the number of
fast fibers (34). Changes in
the myosin composition of soleus are consistent with slow-to-fast transitions
(8), and
Ca2+ sensitivity is increased. If the two treatments are
associated, clenbuterol reduces the soleus atrophy induced by hindlimb
suspension and increases the Ca2+ sensitivity in slow
and fast fibers, this effect being more marked in atrophied than in normal
muscles. In other words, clenbuterol has an anabolic action on muscle fibers
and appears to counteract to some extent the effects of unloading conditions
(37).
The three experimental treatments (clenbuterol, hindlimb suspension, and combined treatment) represent an optimal condition to assess 1) whether a slow-to-fast transition implies an increased phosphorylation of MLC2, as one might expect from the comparison between fast and slow muscles (25) and from the observation that CLFS causes a lower phosphorylation level associated with the appearance of a slow phenotype (see above), and 2) whether atrophy and hypertrophy are associated with different patterns of phosphorylation, as one might expect from studies on cardiac muscles (see above) and from the opposite effects on calcium sensitivity (decreased by hindlimb suspension and increased by clenbuterol treatment, see above). The results obtained showed that slow-to-fast transition is associated with increased levels of phosphorylation regardless of the development of atrophy or hypertrophy.
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MATERIALS AND METHODS |
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Protein extraction and sample preparation. Myofibrillar proteins were extracted from 710 mg of dry muscle powder as described previously (50), washing first with a solution containing 6.3 mM EDTA (pH 7), 0.1% pepstatin, and 1% phenylmethylsulfonyl fluoride (PMSF) and then with a second solution containing 50 mM KCl, 0.1% pepstatin, and 1% PMSF. The myofibrillar proteins were resuspended in 500 µl of milliQ water, and their concentration was determined with a protein assay kit (Dc Protein Assay, Bio-Rad) to prepare samples with a final quantity of 50 µg. The proteins were then precipitated for 2 h with acetone (8 vol (acetone)/vol (sample)), followed by centrifugation for 1 h at 13,000 rpm. The pellet was dissolved in Laemmli solution for SDS-PAGE or in rehydration buffer for two-dimensional gel electrophoresis.
Protein desphosphorylation. The samples (proteins suspended in water, see Protein extraction and sample preparation) were incubated for4hat37°C either with calf intestinal alkaline phosphatase (10 U/50 µg protein; Sigma P6772) or with serine/threonine phosphatase (PP1) extracted from rabbit skeletal muscle (1 U/50 µg protein; Upstate Cell Signaling Solutions). At the end of the incubation proteins were precipitated with acetone, centrifuged, and resuspended in rehydration buffer (see Protein extraction and sample preparation).
One-dimensional electrophoresis (SDS-polyacrylamide gel
electrophoresis). The isoform composition of myosin heavy chain (MHC) and
MLC was determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).
Separation of MHC isoforms was achieved by the method previously described
(46) with a 4.5% stacking gel
and a 7.5% separating gel. Electrophoresis was run for 18 h at low temperature
with 180 V (constant voltage; current 13 mA/gel). Separation of MLC
isoforms was achieved as described in Two-dimensional gel
electrophoresis for the second dimension of two-dimensional gel
electrophoresis. Gels were silver stained
(31) and digitized with an
Epson 1650 scanner with 200 dpi resolution. Identification of MHC and MLC
isoforms was based on the migration rate. The areas under the peaks
corresponding to the MHC or MLC isoforms were measured, and the area of each
peak was expressed as a fraction of the total area of the peaks corresponding
to MHC, to alkali MLC, or to regulatory MLC.
Two-dimensional gel electrophoresis. Proteins were separated by two-dimensional gel electrophoresis with a procedure similar to those previously used for MLC2 separation in sarcomeric muscles by Morano et al. (27) and Gonzalez et al. (14). For the first dimension, isoelectric focusing (IEF), proteins were solubilized in a buffer containing 8 M urea, 2% CHAPS, 0.01 M dithiothreitol (DTT), and 2% carrier ampholites (Amersham Biosciences) and then separated with the Ettan IPGphor Isoelectric Focusing System (Amersham Biosciences) on 3.5% acrylamide strips with immobilized pH gradients (47) (Amersham Biosciences). Strips were rehydrated at 50 V for 12 h, and proteins were focused under the following voltage conditions: 500 V for 1 h, 5001,000 V for 1 h, 5,000 V until reaching 100,000 V·h. Temperature was kept constant at 20°C.
After reduction with a buffer containing 6 M urea, 30% glycerol, 0.375 M Tris · HCl (pH 8.8), and 2% DTT and alkylation with the same buffer with the addition of 2.5% iodoacetamide, the strips were embedded in 4% polyacrylamide stacking gels and the proteins were separated in 12% polyacrylamide gels (SDS-PAGE) for 8 h at 150 V and low temperature (4°C). After electrophoresis gels were silver stained according to Oakley et al. (31).
Image analysis and quantification. Two-dimensional gels were digitized with an Epson 1650 scanner at a resolution of 1,200 dpi. The spots were analyzed densitometrically, determining brightness-area product (BAP) with a constant threshold after black/white inversion with Adobe Photoshop software. In each gel, the BAP values of the spots identified as slow and fast MLC2 were summed to give a total for slow MLC2 and a total for fast MLC2: the value of each spot identified as slow MLC2 was expressed as a percentage of total slow MLC2, and the same was done for the spots identified as fast MLC2. From percentage values obtained in different gels means ± SE were calculated. The quantification procedure was validated by running mixtures of a constant amount of purified actin and increasing amounts of purified slow MLC2 (kindly donated by Dr. Monica Canepari and Dr. Daniela Romano, University of Pavia, Pavia, Italy) on separate gels and determining the ratio between the BAP values of MLC2 and actin. Examples of the linear relation between the ratio of MLC2 and actin BAP values and the actual amounts of MLC2 loaded are shown in Fig. 1B.
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Statistical analysis. Data are expressed as means ± SE and were compared by one-way ANOVA followed by Bonferroni test. The level of significance was set at P < 0.05.
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RESULTS |
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MLC2 isoform identification. The positions of slow and fast isoforms of MLC2 on two-dimensional gels were determined according to their molecular weight, using appropriate markers of molecular weight in the second dimension, and to their isoelectric point in the first dimension (IEF) and were confirmed by immunoblotting (see Fig. 1A). The spots of MLC2 on the two-dimensional gel detectable in soleus and EDL of control rats are shown in Fig. 3 and indicated as s and s1 and f and f1. In the soleus of control rats, slow MLC2 was the predominant regulatory light chain and appeared separated into two spots: s and s1 (see Fig. 3, left); a third almost undetectable more acidic spot was occasionally present. Only a minor amount of fast MLC2 was present in the soleus and it appeared as a single spot, located close to the s1 spot of slow MLC2 in a position corresponding to the spot indicated as f in the EDL. In the EDL, the fast isoform of MLC2 was pre-dominant and appeared composed of two spots (f and f1) of similar size with lower molecular weights and more acidic isoelectric points than the spots of the slow isoform (see Fig. 3, right).
Effect of clenbuterol administration on MLC2 isoform distribution.
As reported in Table 2 and
shown in Fig. 4
(left), clenbuterol administration stimulated the expression of fast
MLC2 in the soleus. In control soleus muscles the slow MLC2 isoform showed the
following distribution: s represented 74%, s1 represented 26%, and the third
more acidic spot, s2, was almost undetectable (see
Table 3 and
Fig. 4). Fast MLC2 was scarcely
expressed and was represented by the less acidic form f. In CB soleus muscles,
the most acidic form (s2) was consistently present and reached a relative
proportion of 6%. The proportion of s1 was not significantly modified.
Furthermore, clenbuterol treatment induced not only the expression of fast
MLC2 but also the concomitant appearance of the acidic form f1, which
represented 30% of the total vs. 70% of the less acidic form f (see
Table 3 and
Fig. 4). The incubation with
alkaline phosphatase removed the most acidic forms of both slow and fast MLC2
isoforms (see Fig. 4,
right), making the pattern of CB soleus in two-dimensional
electrophoresis similar to that of the control soleus, except for the greater
presence of the fast MLC2 isoform.
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Effect of hindlimb suspension on MLC2 isoform distribution. The inactivity caused by hindlimb suspension induced the appearance of s2 as observed in CB muscles (see Table 3 and Fig. 4). The proportions of s and s1, however, were not significantly reduced in HS soleus compared with control soleus. Hindlimb suspension also induced the slow-to-fast transition, with a moderate increase of the proportion of the fast MLC2 isoform (24%, see Table 2), which appeared separated into two spots (see Table 3 and Fig. 4). In agreement with the results obtained for CB soleus, alkaline phosphatase incubation of HS soleus removed the more acidic spots, leaving a pattern similar to that observed in control soleus: both s2 and f1 disappeared.
Effect of clenbuterol administration combined with hindlimb suspension on MLC2 isoform distribution. The combined treatment of clenbuterol and hindlimb suspension caused muscle atrophy slightly but significantly lower than that induced by hindlimb suspension alone (Table 1) and a shift toward fast isoforms comparable to that induced by clenbuterol alone (Table 2). Two-dimensional electrophoresis (see Fig. 4 and Table 3) revealed that the shift toward acidic forms of the slow MLC2 isoform was more pronounced than with hindlimb suspension or clenbuterol alone: s decreased significantly, and both s1 and s2 increased. Two spots corresponding to the fast MLC2 isoform, f and f1, were present (see Fig. 4 and Table 3). As in the two treatments described above, the phosphatase action removed the more acidic spots, thus determining a distribution reminiscent of that of control soleus (see Fig. 4).
Effects of PP1 dephosphorylation. Samples of soleus muscles from
rats exposed to the experimental treatments were incubated with PP1. The
results obtained in HS soleus are shown in
Fig. 5: after PP1 incubation
two acidic spots, s2 and f1, were completely removed, whereas the proportion
of the intermediate acidic form s1 was reduced (to 10% of total slow
MLC2) but not eliminated. Similar results were obtained with CB and CB-HS
soleus samples (data not shown). The specificity of the phosphatase action was
tested by incubating the samples with heat-inactivated PP1: no change in spot
distribution occurred after incubation with heat-inactivated PP1 (see
Fig. 5).
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DISCUSSION |
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Most of the classic studies on MLC2 phosphorylation in skeletal muscles
have been based on isoelectrofocusing (IEF) on polyacrylamide gels after
nondenaturing pyrophosphate electrophoresis of myosin
(41). Two-dimensional
electrophoresis has been applied first to smooth muscle
(11), then to cardiac muscle
(27), and only more recently
to skeletal muscle (10,
14). The use of
two-dimensional electrophoresis has definitely improved the resolution of the
protein separation, particularly with the use of narrow and immobilized pH
gradients (39). The two
isoforms of MLC2 were unambiguously identified on the basis of their molecular
weight and their reactivity with specific antibodies; they appeared divided
into different spots, among which the more acidic forms could be considered as
phosphorylated forms. In rat skeletal muscles fast MLC2 appeared to be
composed of two spots and slow MLC2 of three spots
(14). Interestingly, if we
assume that the more acidic spots represent the phosphorylated forms, the
picture emerging from two-dimensional electrophoresis is slightly different
from that obtained with more classic methods
(41). Separation on
two-dimensional gels indicates that in the rat 30% of the slow MLC2
isoform in soleus and 3040% of the fast MLC2 isoform in EDL are in
phosphorylated forms (see also Refs.
10 and
14). By contrast, no
significant amount of phosphorylated MLC2 in the rat soleus and only 0.1
mol/mol in rat white gastrocnemius were found by Moore and Stull
(25). Similar ratios (0.10 and
0.19 mol/mol, respectively) have been reported for rat gastrocnemius
(21,
23).
The interpretation of the three spots observed in rat slow MLC2 is still controversial. A convincing demonstration that all three spots correspond to MLC2 has been given by Gonzalez et al. (14), using specific antibodies in Western blots. The three spots (indicated as s, s1, and s2 in this study) might correspond to two variants of slow MLC2, each of them under unphosphorylated and phosphorylated states, with the assumption that the intermediate spot (s1) contains one unphosphorylated and one phosphorylated form (14). Evidence in favor of the existence of two distinct variants of slow MLC2 was first given for the ventricular myocardium (27, 35). Two variants of slow MLC2 have been also observed in human vastus lateralis muscle (15). It is worthwhile to recall that only one gene coding for the slow isoform of MLC2 is expressed in ventricular myocardium and in slow skeletal muscle fibers (40). The presence of two variants must therefore be attributed to posttranscriptional or posttranslational modifications. In the latter alternative the hypothesis of double phosphorylation can be considered, especially if we refer to the demonstration of several phosphorylation sites in smooth muscle MLC2 (16). In this case, the increase of phosphorylation observed in this study might be explained by phosphorylation of the fast MLC2 and a second phosphorylation of the phosphorylated slow MLC2. Against the hypothesis of the double phosphorylation of slow MLC2 is the observation that only the most acidic spot (s2) of the slow MLC2 is completely removed by phosphatase action, whereas the intermediate acidic spot (s1) is greatly reduced but does not disappear. The spot of fast MLC2 considered as phosphorylated (f1) is completely removed (see also Ref. 14). Both alkaline phosphatase and PP1 were tested and were found insufficient to completely dephosphorylate slow MLC2. The specificity of PP1 is determined by the regulatory subunit (7), and the commercially available muscle PP1 used in this study contains a mixture of regulatory subunits, as stated by the manufacturer. Thus the partial effect of phosphatase might be due to a limitation to the access of the enzyme, for instance, by a structural constraint: only a very specific phosphatase, for example, PP1-M containing the myosin-specific regulatory subunit (MYPT2; Ref. 7) might produce a pattern with only one spot for the slow MLC2 and one spot for the fast MLC2.
The increase in MLC2 phosphorylation was associated with the slow-to-fast
transition induced by all three treatments considered in this study; this is
in agreement with the fact that MLC2 phosphorylation is higher in fast than in
slow muscles, the difference being in turn dependent on different levels of
calmodulin-dependent kinase activity
(25). An inhibition of kinase
gene expression (20) has been
reported in relation to the decrease of the phosphorylation level observed
after CLFS (14,
20,
21). One might speculate that,
if the fast-to-slow transition induced by CLFS implies the inhibition of the
calmodulin-dependent MLC kinase, the slow-to-fast transition determined by
clenbuterol or by hindlimb suspension could include the activation of the same
gene, as a part of a general "fast muscle fiber" program. This is,
however, a simplified view: there are possibly also different isoforms of MLC
kinase and regulatory mechanisms based on phosphorylation and
autophosphorylation. The autophosphorylation reaction depends on
calcium/calmodulin activation
(13) with a rate of
phosphorylation different from that of MLC2 phosphorylation but does not seem
to modulate the catalytic function of the kinase. It is yet not known whether
Rho kinase (1) can
phosphorylate MLC2 in skeletal muscle. A recent study, however, showed
variations of Rho kinase in relation to muscle atrophy
(6). Finally, an increase of
MLC2 phosphorylation might be caused by a decreased activity of specific
phosphatase: the possible regulation of the MLC phosphatase (or PP1-M) in
relation with -adrenergic stimulation was recently discussed by Decostre
et al. (10).
There is convincing evidence that phosphorylation of MLC2 increases the calcium sensitivity of the myofibrils and therefore force development at submaximal activation (33, 47, 49). As mentioned in the introduction, previous work by our group (44, 45) showed that calcium sensitivity decreased in atrophy induced by hindlimb suspension, whereas it increased after clenbuterol administration and after the combined treatment (37). Surprisingly, in both cases the phosphorylation level was found to be increased. This suggests that the decrease in calcium affinity in muscle fibers atrophied by hindlimb suspension in the presence of a high degree of MLC2 phosphorylation is due to other factors.
In conclusion, this study represents one of the first demonstrations that the long-term adaptation mechanisms of the contractile performance of skeletal muscles, although mainly based on regulation of myosin isoform expression and therefore operated at the transcriptional level, can be based partly on posttranslational modifications, phosphorylation in this case or glycosylation in other cases (36), as discussed in a recently published review (5). This finding is of special interest considering that phosphorylation represents the way of regulation used by a large number of intracellular signaling pathways.
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DISCLOSURES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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