In particular, after some weeks, den-ervation [3,9–11] and CLFS [5–8] cause changes in the myosin heavy chain MHC distribution in slow and fast muscles, which validates the view that the
Trang 1in slow and fast skeletal muscles
Cyril Bozzo1,2, Barbara Spolaore3, Luana Toniolo1, Laurence Stevens2, Bruno Bastide2,
Caroline Cieniewski-Bernard2, Angelo Fontana3, Yvonne Mounier2and Carlo Reggiani1
1 Department of Anatomy and Physiology, University of Padova, Italy
2 Laboratory of Neuromuscular Plasticity, UPRES EA 1032, IFR118, USTL, Villeneuve d’Ascq, France
3 CRIBI Biotechnology Centre, University of Padova, Italy
Myosin isoforms are major determinants of the
con-tractile properties of skeletal muscle fibres [1], and the
neural discharge pattern has an important role in the
regulation of myosin isoform expression This has been
demonstrated by cross-innervation [2], denervation
[3,4] and chronic low-frequency stimulation (CLFS) [5–8] experiments In particular, after some weeks, den-ervation [3,9–11] and CLFS [5–8] cause changes in the myosin heavy chain (MHC) distribution in slow and fast muscles, which validates the view that the pattern
Keywords
CLFS; cyclosporin; denervation; myosin,
phosphorylation
Correspondence
C Reggiani, Department of Anatomy and
Physiology, University of Padova, Via
Marzolo 3, 35131 Padova, Italy
Fax: +39 049827 5301
Tel: +39 049827 5513
E-mail: carlo.reggiani@unipd.it
(Received 23 May 2005, revised 2 August
2005, accepted 12 September 2005)
doi:10.1111/j.1742-4658.2005.04965.x
Neural stimulation controls the contractile properties of skeletal muscle fibres through transcriptional regulation of a number of proteins, including myosin isoforms To study whether neural stimulation is also involved in the control of post-translational modifications of myosin, we analysed the phosphorylation of alkali myosin light chains (MLC1) and regulatory myo-sin light chains (MLC2) in rat slow (soleus) and fast (extensor digitorum longus EDL) muscles using 2D-gel electrophoresis and mass spectrometry
In control rats, soleus and EDL muscles differed in the proportion of the fast and slow isoforms of MLC1 and MLC2 that they contained, and also
in the distribution of the variants with distinct isoelectric points identified
on 2D gels Denervation induced a slow-to-fast transition in myosin iso-forms and increased MLC2 phosphorylation in soleus, whereas the oppos-ite changes in myosin isoform expression and MLC2 phosphorylation were observed in EDL Chronic low-frequency stimulation of EDL, with a pat-tern mimicking that of soleus, induced a fast-to-slow transition in myosin isoforms, accompanied by a decreased MLC2 phosphorylation Chronic administration (10 mgÆkg)1Æd)1 intraperitoneally) of cyclosporin A, a known inhibitor of calcineurin, did not change significantly the distribution
of fast and slow MLC2 isoforms or the phosphorylation of MLC2 All changes in MLC2 phosphorylation were paralleled by changes in MLC kinase expression without any variation of the phosphatase subunit, PP1
No variation in MLC1 phosphorylation was detectable after denervation
or cyclosporin A administration These results suggest that the low-fre-quency neural discharge, typical of soleus, determines low levels of MLC2 phosphorylation together with expression of slow myosin, and that MLC2 phosphorylation is regulated by controlling MLC kinase expression through calcineurin-independent pathways
Abbreviations
BAP, brightness-area product; CAM, calmodulin; CaN, calcineurin; CLFS, chronic low-frequency stimulation; COCsA, controls for CsA receiving cremophor A solution only; CODE, controlateral unoperated limb; CONT, control; CsA, cyclosporin A; ECL, enhanced
chemiluminescence; EDL, extensor digitorum longus; NFAT, nuclear factor of activated T cells; MS, mass spectrometry; IEF, isoelectric focusing; MHC, myosin heavy chain; MLC, myosin light chain.
Trang 2of neural discharge is the main determinant of nerve
influence on myosin expression
Whereas the transcriptional control of myosin
iso-form expression in muscle plasticity is generally
accep-ted [1], it has still not been established whether
myofilament functions can be the target of long-term
regulation based on post-translational protein
modifi-cations The recent observation that, during aging,
cross-bridge kinetics in slow fibres change as a result
of myosin nonenzymatic glycosylation [12,13],
demon-strates that post-translational modifications can be
relevant to regulate contractile properties over long
time periods
Phosphorylation of the light chain subunits is the
most studied post-translational modification of myosin
Phosphorylation of the regulatory myosin light chain
(MLC) is catalysed by a calmodulin-dependent kinase
(MLC kinase), which is activated by the increase in
cytosolic calcium [14] Thus, a repetitive or tetanic
sti-mulation causes a transient increase of phosphorylation
of regulatory MLC Phosphorylation is then removed
by a phosphatase composed of PP1 associated with
MYPT2, a targeting subunit specific to skeletal muscle
MLC [15–17] Myosin phosphorylation enhances force
development at submaximal calcium concentrations (i.e
induces a shift of the force–pCa curve towards lower
calcium concentrations) [18,19] and, through this
mech-anism, offers a plausible explanation for the
phenom-enon of post-tetanic potentiation [20]
There is evidence in favour of the existence of a
long-term regulation of MLC2 phosphorylation, besides
the short-term regulation that is dependent on calcium
released during contraction Long-term regulation
means that the phosphorylation levels at rest and
dur-ing contraction change over periods of days or weeks,
and this might be considered as a special case of
skel-etal muscle differentiation and plasticity In substantial
agreement with early observations that the
phosphory-lation level is higher in fast than in slow muscles [20],
recent studies have shown that phosphorylation
decrea-ses with CLFS, which induces a fast-to-slow
transfor-mation [21–23] An increase in myosin phosphorylation
during adaptive responses, such as hindlimb unloading,
which implies a slow-to-fast transformation, has been
demonstrated in a recent study [24] A decrease in the
MLC phosphorylation after 7 days of denervation in
the fast extensor digitorum longus (EDL) has also been
described [25] Taken together, these findings suggest
that slow-to-fast transformations are associated with
increased phosphorylation and that fast-to-slow
trans-formations are associated with reduced
phosphoryla-tion The finding that MLC2 phosphorylation decreases
with CLFS suggests that contractile activity may cause
contrasting variations of the degree of myosin phos-phorylation during short and long time intervals In fact, during short-term regulation, repetitive or tetanic stimulation (duration: seconds or fractions of seconds) leads to a transient increased phosphorylation, whereas
in long-term regulation, CLFS (duration: days or weeks) causes a reduced phosphorylation
We designed this study to further investigate the relevance of neural stimulation on long-term changes
in myosin phosphorylation using, as a model, the den-ervation of fast and slow muscles Only a few studies have analysed MLC phosphorylation in skeletal muscle after denervation and, to our best knowledge, those studies were only focused on fast muscles, such as EDL [25,26] or gastrocnemius [27], perhaps because
of the high level of phosphorylation in fast muscles [18,19] In fast muscles, the basal level of phosphoryla-tion [25], and the transient increase in phosphorylaphosphoryla-tion after electrostimulation [26,27], are reduced after
7 days [25,26] or 2 weeks [27], respectively, of denerva-tion The decrease in phosphorylation in denervated fast muscles, where genes coding for fast myosins are down-regulated [9], supports the hypothesis of a strong link between fast isoform expression and high phos-phorylation level Although slow muscle fibres are believed to be more dependent on nerve influence than fast fibres [28,29] no study has investigated the changes
in phosphorylation after denervation in slow muscles Following the above reasoning, an increase in myosin phosphorylation in the slow soleus muscle after dener-vation might be anticipated
Therefore, the first aim of this study was to assess whether denervation modifies the level of MLC2 phos-phorylation in soleus and in EDL, used as representative slow and fast muscles, respectively (i.e two muscles where specific patterns of neural stimulation determine and maintain opposite structural and functional charac-teristics) The second aim of this study was to under-stand the molecular mechanisms that determine the changes in phosphorylation level To achieve this, we tested the hypothesis that changes in phosphorylation were caused by variations in the amount of MLC kinase and phosphatase The available evidence points to a role
of the calcineurin–nuclear activated factor of T-cells (NFAT) pathway in mediating the effect of the low-fre-quency pattern of neural discharge on the transcription
of genes typical of a slow muscle phenotype, such as slow myosin subunits [30–32] In the frame of this model
we explored whether the inhibition of calcineurin with cyclosporin A (CsA) could mimic the effects of denerva-tion on myosin phosphoryladenerva-tion as it does with myosin isoform expression [33] To further confirm that low-frequency neural discharge, typical of slow muscles, can
Trang 3reduce myosin phosphorylation in fast muscle, we
exam-ined the effects of CLFS on the fast EDL Finally,
taking into account the recent evidence of MLC1
phos-phorylation in cardiac muscle [34], we extended our
investigation, based on 2D-gel electrophoresis and mass
spectrometry, to MLC1 isoforms
The results obtained after denervation and CLFS
confirmed the hypothesis of a close connection
between fast myosin expression and high
phosphoryla-tion level of MLC2, both in soleus and in EDL The
expression of MLC kinase was found to vary in direct
association with the degree of phosphorylation,
provi-ding a possible explanation of the regulatory
mechan-ism Treatment with CsA was sufficient to modify
myosin isoform expression, but did not change MLC2
phosphorylation or MLC kinase expression, suggesting
that the regulatory mechanism was not
calcineurin-dependent In addition, although 2D gels gave
evi-dence in favour of MLC1 phosphorylation in skeletal
muscles, no variation in its degree of phosphorylation
was found in slow and fast muscles after denervation
or CsA administration
Results
Effects of denervation on rat soleus and EDL
The effects of denervation were studied by comparing
five rats with surgical interruption of the sciatic nerve
and five control rats of similar age and body mass (see the Experimental procedures) As seen in Table 1, two weeks after sciatectomy, denervated soleus and EDL (DE) showed atrophy (i.e decrease of mass) when compared with the corresponding muscles of the control animals (CONT) or with the muscles of the controlateral unoperated limb (CODE)
The distribution of MHC and MLC isoforms in the soleus and EDL of control and treated rats were analysed by SDS⁄ PAGE Four bands, correspond-ing to MHCI (slow isoforms) and to MHCIIa, MHCIId⁄ x and MHCIIb (fast isoform), were separ-ated on 8% gels (Fig 1) The results of densitometry are reported in Table 1 As can be seen for both soleus and EDL muscles, no difference in MHC iso-form distribution was present between control, untreated rats (CONT) and the contro-lateral leg of denervated rats (CODE) Soleus showed a predomin-ant MHCI band and a minor MHCIIa band, whereas
in EDL, the bands corresponding to MHCIId⁄ x and MHCIIb were predominant, in accordance with previ-ous observations [35]
In soleus, 14 days after denervation, a significant change in MHC isoform distribution was detectable (Fig 1 and Table 1) The slow MHC isoform was less abundant in DE than in CONT and CODE, and this was accompanied by expression of the fast MHCIId⁄ x isoform In EDL, denervation was followed by an increase in MHCIIa expression, with corresponding
MLC3) or regulatory (MLC2) isoform is expressed as a percentage of the total amount of alkali or regulatory MLC, respectively Data are expressed as mean value ± SD.
MHC
Alkali light chains
Regulatory light chains
*Significantly different (P < 0.05) from CONT.
Trang 4decreases in MHCIId⁄ x and MHCIIb, these latter
being below statistical significance
As seen in Fig 1, five bands corresponding to MLC
isoforms were identified on 12% gels and
densito-metrically quantified: three alkali MLC isoforms
(MLC1slow, MLC1fast and MLC3) and two regulatory
MLC isoforms (MLC2slow and MLC2fast) As
expec-ted [35], fast isoforms were predominant in EDL,
whereas slow isoforms were predominant in soleus As
described above for MHC isoforms, no difference in
MLC distribution was present between CONT and
CODE In soleus, denervation caused a change in the
distribution of both alkali and regulatory MLC
iso-forms, with a decrease in slow isoforms and an increase
in fast isoforms No significant changes were observed
in denervated EDL (Table 1)
Separation of MLCs by 2D-gel electrophoresis
MLCs were analysed by 2D-gel electrophoresis to detect
possible variants of the five isoforms separated by
1D gels (Fig 1): MLC1slow, MLC1fast, MLC2slow,
MLC2fast and MLC3 In 2D gels (Fig 2), MLC1slow
was divided by isoelectric focusing (IEF) into two spots, named 1s and 1s1 (1s1 being more acidic than 1s), and MLC1fast was similarly divided into 1f and 1f1 MLC2slow was separated into three spots, indicated as 2s, 2s1 and 2s2, in order from basic to acidic isoelectric point, whereas MLC2fast was divided into two spots, namely 2f and 2f1, 2f1 being a more acidic variant
Fig 2 Myosin light chain (MLC) region in silver-stained 2D gels from soleus (left column) and EDL (right column) muscles of con-trol rats (CONT), concon-trolateral leg (CODE) and denervated leg (DE)
of denervated rats, in rats receiving daily cremophor injections (COCsA) and in rats receiving daily injections of cyclosporin A (CsA) The pH gradient extends from basic on the left to acidic on
uppermost panels Two variants (1s and 1s1) of MLC1slow, two variants (1f and 1f1) of MLC1fast, three variants (2s, 2s1, 2s2) of MLC2 slow, and two variants (2f and 2f1) of MLC2 fast were sep-arated on 2D gels Note the different pattern of MLC1 and MLC2 spots in control soleus and EDL muscles The changes in pattern
of MLC2 variants in DE muscles are detectable both in soleus (from a three-spot pattern to a five-spot pattern) and in EDL (decrease in the acidic variants) 2s2 and 2f1 spots in denervated soleus are encircled An arrow indicates the position of 2s2 in den-ervated EDL.
Fig 1 Myosin heavy chain (MHC) and myosin light chain (MLC)
expression in soleus (left side of the figure) and EDL (right side of
the figure) muscles in control, untreated rats (CONT), in the
contro-lateral leg (CODE) and in the denervated leg (DE) of rats with
surgi-cal section of sciatic nerve, in rats receiving cremophor A solution
only (COCsA) and in rats receiving cyclosporin A diluted in
cremo-phor A solution (CsA) Separation of MHC isoforms was obtained
silver stained.
Trang 5MLC3 appeared as a single spot (not shown in Fig 2).
The spots corresponding to MLC isoforms and their
variants were identified and classified, as previously
des-cribed [24], on the basis of their molecular weight
(sec-ond dimension), isoelectric point (first dimension) and
immunoblotting The reactivity with the antibody
FL-172sc15370, specific for MLC2, showed that the
spots 2s, 2s1, 2s2, 2f and 2f1 were variants of either
MLC2slow or MLC2fast [24], whereas the reactivity
with the antibody PSR-45, specific to P-serine, showed
that the spots 1s1, 1f1, 2s1, 2s2 and 2f1 contained
phos-phorylated serine residues No spots were reactive to
the antibody PTR-8, specific to P-threonine Finally,
the identity of the spots was confirmed with good scores
by mass spectrometry, as shown in Table 2
The relative proportions of the spots corresponding
to MLC isoforms and their variants were
densito-metrically quantified, as described in the
Experi-mental procedures, and the results are shown in
Table 3 In control soleus (CONT, CODE), the
pre-dominant isoform, MLC2slow (Table 1), was composed
of two spots, the less acidic (2s) being more abundant
than the more acidic (2s1) MLC2fast was also present
and appeared to be composed of only one spot (2f)
Denervation of soleus caused not only a shift from
MLC2slow to MLC2fast, as described above (Table 1),
but also a significant increase of the more acidic forms
for both slow and fast MLC2 A third, more acidic, spot
(2s2) appeared in MLC2slow, and a second, acidic spot (2f1) appeared in MLC2fast (Fig 2 where 2s2 and 2f1 are circled, and Table 3)
In control EDL, MLC2fast was predominant (see also Table 1) and was divided by IEF into two vari-ants (2f and 2f1), the less acidic variant being more abundant (Table 3) MLC2slow was also present and composed of two spots Importantly, careful analysis
of the relative positions of the spots in 2D gels of con-trol EDL compared with concon-trol soleus showed that the two variants of MLC2slow present in EDL corres-ponded to 2s1 and 2s2, whereas the less acidic variant, 2s, was not detectable Denervation of EDL modified the relative proportion of the variants of MLC2slow,
as the more acidic spot (2s2) significantly decreased (Fig 2, arrow, and Table 3)
The proportions of the spots corresponding to MLC1slow and MLC1fast were determined in soleus and EDL of control and treated animals (Fig 2 and Table 3) Interestingly, only the predominant isoform appeared divided into two spots, both in soleus (1s and 1s1), where MLC1slow was more abundant, and
in EDL (1f and 1f1), where MLC1fast was predomin-ant The less abundant isoform appeared as a single spot, both in soleus (1f) and in EDL (1s) The ratio between the more acidic spot and the less acidic spot was1 : 4 in both MLC1 isoforms and did not change after denervation
isoelec-tric point.
Swiss-Prot accession code
Matched peptides
by PMF
Peptides sequenced
Sequence coverage
Theor pI;
muscle isoform
skeletal muscle isoform
muscle isoform
muscle isoform
Swiss-Prot database.
Trang 6Effects of CsA treatment
To study whether CsA administration could reproduce the effects of denervation on the expression and phos-phorylation of myosin subunits, a group of five rats was treated for 2 weeks with CsA, as described in the Experimental procedures As shown in Table 4, at the end of two weeks of treatment, body mass was signifi-cantly lower in rats that received CsA than in rats receiving vehicle alone (COCsA) As initial body mass did not differ among the groups of animals, the differ-ence observed at the end of the treatment pointed to
a significant impairment in body mass growth (10%) caused by CsA After CsA treatment, EDL mass was significantly decreased ()32%), whereas soleus mass was similar to that of the control (Table 4) The higher atrophy induced by CsA in fast compared with slow muscles has been reported in previous studies [28] MHC expression was modified by CsA treatment in both soleus and EDL (Fig 1 and Table 4) In soleus, CsA induced a reduction of MHCI, associated with a surprising increase in MHCIIb expression In EDL, CsA caused a significant increase of MHCIIa and MHCIId⁄ x expression, accompanied by a significant decrease in the expression of MHCIIb Interestingly, CsA administration did not cause any change in the distribution of MLC isoforms in the two muscles ana-lysed Furthermore, no changes in the distribution of the variants separated with 2D-electrophoresis were detected (Fig 2 and Table 3)
MLC kinase and PP1 expression The expression of the skeletal MLC kinase and the phosphatase subunit, PP1, were determined by SDS⁄ PAGE, western blot and densitometry, using actin band as a reference signal The results are shown in Fig 3 In soleus, denervation significantly increased MLC kinase expression by 2.5-fold, but did not influ-ence PP1 expression, which remained similar to the values obtained in CONT and in CODE CsA adminis-tration did not affect either MLC kinase or PP1 expres-sion In control EDL (CONT, CODE and COCsA), the level of MLC kinase expression was 1.5-fold higher than in control soleus, but lower than the MLC kin-ase level reached in denervated soleus Denervation reduced MLC kinase expression in EDL by 30%, so that the MLC kinase level in denervated EDL was sim-ilar to that measured in control soleus The addition of CsA did not change the expression of MLC kinase The level of PP1 expression was similar in soleus and EDL, and no variation in PP1 expression was observed after EDL denervation and CsA treatment
MLC1 1
MLC2 2
Trang 7Effects of CLFS on MLC2 phosphorylation and
MLC kinase and PP1 expression in EDL
CLFS of EDL for three weeks induced a fast-to-slow
transition in MHC isoforms, with a decrease in
MHCIId⁄ x and MHCIIb and an increase in MHCIIa
(data not shown) (29,36) 2D-electrophoresis (Fig 4A) showed that CLFS caused pronounced changes in the distribution of the variants of both MLC2slow and MLC2fast Whereas four MLC2 variants were detect-able in CONT EDL (left panel of Fig 4A, see also Fig 2) (i.e the slow 2s1 and 2s2 and the fast 2f and
A
B
Fig 3 Myosin light chain kinase (MLC kinase) and phosphatase regulatory subunit (PP1) in soleus and EDL muscles of control untreated rats (CONT), controlateral leg (CODE) and denervated leg (DE) of rats with surgical section of the sciatic nerve, rats receiving a daily injection of cremophor A solution (COCsA) and rats receiving cyclosporin A diluted in cremophor A solution (CsA) (A) Representative immunoblots
*Significantly different (P < 0.05) from CONT.
per-centage of the total amount of alkali or regulatory MLC, respectively Data are expressed as mean values ± SD.
MHC
Alkali light chains
Regulatory light chains
*Significantly different (P < 0.05) from COCsA.
Trang 8f1), only two major spots were detected after CLFS:
the slow 2s and the fast 2f (middle panel of Fig 4A)
This pattern resembled that present in CONT soleus
(right panel of Fig 4A, see also Fig 2) Interestingly,
the variant 2s, typical of soleus, appeared in CLFS
EDL, and the acidic variants 2s1 and 2s2 disappeared
Among the variants of MLC2 fast, only 2f was present
in CLFS EDL The expression of MLC kinase in
CLFS EDL was reduced to approximately two-thirds
of the level measured in CONT EDL, becoming
sim-ilar to the level measured in CONT soleus (Fig 4B)
No change in PP1 expression was observed
Discussion
The major goal of this study was to examine the nerve
influence on MLC phosphorylation in slow and fast
muscles In both cases we found that, in addition to
the known effects on myosin isoform expression,
den-ervation was able to modify the degree of MLC2
phos-phorylation Whereas in EDL, denervation caused a
shift towards less acidic variants of MLC2, in soleus,
denervation caused a shift towards more acidic
vari-ants of MLC2 No variations in the distribution of
MLC1 variants were detected
2D-gel electrophoresis with IEF based on strips
with immobilized pH gradients was used to separate
the phosphorylated and unphosphorylated forms A detailed analysis using mass spectrometry was per-formed to reinforce the identification of the individ-ual spots based on isoelectric point (first dimension), molecular mass (second dimension) and immunostain-ing The spots corresponding to actin and the vari-ants of MLC isoforms, were identified The reactivity with anti-(P-serine) immunoglobulin provided evi-dence to identify the more acidic variants as phos-phorylated forms No attempt was made to determine which residues undergo phosphorylation,
as the focus of the study was the long-term changes
in the ratios between more acidic and less acidic vari-ants Both the slow and the fast isoforms of alkali MLC (MLC1slow and MLC1fast) were present in two discrete variants (1s and 1s1 and, respectively, 1f and 1f1) with slightly different isoelectric points and similar molecular weights Interestingly, both in so-leus and in EDL, only the more abundant MLC1 isoform appeared divided in two spots and therefore the three spot pattern detectable in soleus was the mirror image of the three spot pattern present in EDL (Fig 2) Three variants of MLC2 slow (2s, 2s1, 2s2) were separated on 2D gels The identification had been achieved in our previous study by immuno-blotting with antibody specific to MLC2 [24] and was confirmed, in this study, by MS The two more
A
B
Fig 4 Effects of chronic low frequency stimulation (CLFS) on myosin light chain MLC2 phosphorylation in EDL muscle (A) Separation of the MLC2 variants in silver-stained 2D gels in CONT EDL, in EDL after CLFS and in CONT soleus Three variants (2s, 2s1, and 2s2) of MLC2 slow, and two variants (2f and 2f1) of MLC2 fast, were separated on 2D gels; also see Fig 2 The pH gradient extends from basic on the
the detection of MLC kinase, PP1 and actin expression (left), and ratios of MLC kinase and PP1 signals to actin signal (right) from CONT EDL, CLFS EDL and CONT soleus *Significant difference (P < 0.05) between CONT EDL and CLFS EDL.
Trang 9acidic spots (2s1 and 2s2) were stained by
anti-(P-ser-ine) immunoglobulin The comparison between soleus
and EDL of control animals revealed a new and
unexpected difference as the two less acidic spots (2s
and 2s1) were present in soleus, whereas the two
more acidic spots (2s1 and 2s2) were present in EDL
(Figs 2 and 4) After denervation, 2s2 became
detect-able in soleus and became smaller in EDL The
ori-gin of the three spots remains controversial [24],
regarding whether they represent unphosphorylated
(2s), monophosphorylated (2s1) or di-phosphorylated
(2s2) variants, as observed in smooth muscle [37], or
the combination of two distinct post-translational
modifications In favour of the second explanation is
the complete removal of 2s2 and the incomplete
removal of 2s1 by phosphatase [24], and the recent
observation of a de-amidation site [38] in MLC2slow,
which gives origin to a more acidic form Finally,
two distinct variants (2f and 2f1) of the fast MLC2
isoform were separated by IEF and their
identifica-tion confirmed by MS The more acidic spot (2f1)
was reactive with anti-(P-serine) immunoglobulin
If the more acidic variants, reactive with
anti-(P-serine) immunoglobulin, can be considered as
phosphor-ylated forms of MLC2 slow or MLC2 fast, the extent of
MLC2 phosphorylation in the soleus and EDL of
con-trol animals found in this study was generally higher
than the values for resting muscles reported in other
studies The difference might be a result of the
proce-dure used for muscle sampling or the methods employed
for separation of the phosphorylated variants In fact,
the values found in this study are very similar to those
reported by Gonzalez and coworkers [23], who used a
sampling protocol and a separation based on
2D-elec-trophoresis that were very similar to those used in the
present study 2D-electrophoresis has often been used to
separate phosphorylated variants in cardiac and smooth
muscles [39] but seldom in skeletal muscle [23] IEF after
electrophoresis in pyrophosphate gels [20] and
urea-gly-cerol-acrylamide gel electrophoresis [26] have been more
often used in skeletal muscle In many studies, muscles
have been allowed to rest for a given time interval,
either in vivo or in vitro, before freezing In this study,
deep anaesthesia was expected to induce prolonged
muscle relaxation and give time sufficient to reach low
and steady levels of phosphorylation We believe that
the most important condition for reliable comparison is
to follow carefully the protocols chosen for muscle
sampling and phosphorylated myosin determination In
our view, the high reproducibility of the data and the
similarity with the data obtained with comparable
pro-tocols [23] supports the reliability of our determination
of the basal level of myosin phosphorylation
The changes of the phosphorylation level after den-ervation are examples of long-term post-translational modification, clearly different from the increase in phosphorylation that occurs after repetitive stimulation and which is responsible for post-tetanic potentiation [20] It has been known for many years that the phos-phorylation level is higher in fast than in slow muscles [20] A decrease in phosphorylation level has been pre-viously described in muscles that are subjected to CLFS [21–23], a condition which is known to induce a fast-to-slow transformation Our previous work [24] has shown that slow-to-fast transformation, induced
by either disuse (hindlimb unloading) or clenbuterol administration, is associated with an increased phos-phorylation level Denervation is known to affect myosin isoforms, the transcriptional changes being detectable at the mRNA level after a few days and at the protein level within a few weeks [3,9,10] The chan-ges in myosin subunit expression observed in this study were in complete agreement with previous observa-tions, as, in denervated soleus, slow myosin was expressed to a lower extent (both MHCI and slow MLC) than fast myosin (MHCIId⁄ x and fast MLC), which increased In denervated EDL, only an increase
in the expression of MHCIIa, indicative of a moderate transition towards a slow phenotype, was observed In accordance with previous observations on long-term changes in myosin phosphorylation, the slow-to-fast transition in soleus was associated with an increased level of phosphorylation, and the fast-to-slow (or fast-to-less fast) transition in EDL with a decrease in phosphorylation level
CLFS experiments confirmed the link between fast-to-slow transformation and a decrease in the phos-phorylation level, by showing that in a fast muscle the stimulation, according to a pattern typical of a slow muscle, induced, at the same time, changes in myosin isoform expression and decreased myosin phosphoryla-tion This latter was related to a decreased expression
of MLC kinase Taken together, the changes following denervation of soleus, and the changes following CLFS
of EDL, demonstrate that the stimulation pattern is essential for the long-term regulation of myosin phos-phorylation Interestingly, the spot 2s, which is the most abundant in soleus, also appears in EDL after CLFS
The results obtained provide clear evidence that the long-term changes in phosphorylation level are caused by changes in MLC kinase, without signifi-cant variations of the phosphatase, or at least of the phosphatase subunit, PP1 Western blot analysis showed that upon denervation, MLC kinase increases
in soleus and decreases in EDL Preliminary results
Trang 10obtained in our laboratory with quantitative PCR
confirm that both denervation and hindlimb
unload-ing cause an increase in the mRNA of MLC kinase
(data not shown) In agreement with these
observa-tions, a moderate increase in transcription of the
MLC kinase gene is reported in the supplementary
data of a microarray study of the transcriptional
changes occurring in soleus during hindlimb
unload-ing [40]
To shed some light on the intracellular signaling
pathway controlling MLC2 phosphorylation and
MLC kinase expression, we explored whether the
degree of phosphorylation and MLC kinase
concen-tration were affected by 2 weeks of CsA
administra-tion, a condition that is expected to reproduce the
transcriptional changes caused by denervation
According to a widely accepted model, the
transcrip-tional effects of neural discharge pattern are
medi-ated by an intracellular signalling pathway that links
cytosolic calcium increase to calmodulin (CAM),
cal-cineurin (CaN) and NFAT [30,33] Dephosphorylated
by CaN, NFAT translocates into the nucleus and
contributes to activate the transcription of genes
spe-cific for the slow phenotype [30,32,41,42] CsA is
expected to inhibit the phosphatase action of CaN
and therefore to block the signalling pathway
con-necting neural stimulation and transcription The
results obtained in this study confirmed that CsA
administration induces a slow-to-fast transition in
soleus, as previously observed by Bigard et al [33],
and also a fast-to-less fast transition in EDL;
how-ever, no significant changes in MLC2
phosphoryla-tion and MLC kinase expression were detected
Whereas the observed changes in MHC isoform
expression suggest that CsA administration was
effective at the transcriptional level, the lack of effect
on MLC2 phosphorylation and MLC kinase
expres-sion supports the view that these two parameters
were regulated by a pathway different from CAM–
CaN–NFAT This conclusion needs to be considered
with caution as CsA treatment and denervation
might be not completely overlapping, in view of the
following reasons (a) whereas CsA should only
inter-fere with the signalling pathway mediating neural
discharge inside muscle fibres, denervation achieved
by severing the sciatic nerve not only interrupts
neural discharge on muscles, but also causes
unload-ing as activity of both agonist and antagonist
mus-cles is removed, (b) fast musmus-cles, such as EDL, are
more responsive to CsA than slow muscles with
regard to atrophy and to myosin isoform transition,
as previously observed by Bigard et al [33], in
agree-ment with the higher concentration of CaN in fast
than in slow muscles [43], and (c) recent studies on the promoter region of MHCI [44] cast some doubts
as to whether transcriptional CsA effects are only mediated by interruption of the CaN–NFAT path-way
In this study, not only MLC2 phosphorylation, but also MLC1 phosphorylation, was taken into considera-tion In cardiac muscle [34], three variants of MLC1 slow exist and the more acidic forms are
phosphorylat-ed either in Ser200 or in Thr69 Our observations are,
to the best of our knowledge, the first demonstration that two variants with different isoelectric points exist also in skeletal muscle The reactivity with anti-(P-ser-ine) immunoglobulin suggests that a serine residue is phosphorylated The ratio between the unphosphoryl-ated and phosphorylunphosphoryl-ated variants is similar in cardiac and in skeletal muscle as for both fast and slow MLC1 the more acidic form represents 25% of the total In cardiac muscle, ischemic preconditioning has been shown to modify the ratio from 1 : 4 to 1 : 3 [34] Our results show that in skeletal muscles neither denerva-tion nor hindlimb unloading (our unpublished data) were able to modify the ratio between the variants of MLC1
In conclusion, this study provides evidence which strongly suggests that changes in fibre type are asso-ciated with changes in myosin phosphorylation level, with an increase associated with slow-to-fast trans-ition and a decrease associated with fast-to-slow transition In particular, the comparison between den-ervation of soleus and CLFS of EDL shows that the pattern of low frequency neural stimulation, typical
of slow muscles, determines low levels of phosphory-lation together with the expression of the typical slow fibre genes The mechanism and the time course
of this regulation needs to be further clarified, although the parallel variations of phosphorylation and MLC kinase amount point to the transcriptional regulation of MLC kinase as a possible mechanism, and the lack of effect of CsA administration suggests
a calcineurin-independent intracellular signalling The physiological relevance of the association between fast fibre phenotype and higher phosphorylation lev-els can be understood, taking into account that repetitive stimulation induces, at the same time, a decrease in force development through the fatigue mechanism and an increase in force development through phosphorylation Thus, the presence of a more effective phosphorylation mechanism in fast fibres, which are more prone to fatigue, might repre-sent a useful mechanism to counteract the quick reduction of force that, in fast fibres, follows con-tractile activity