Báo cáo khoa học: Nerve influence on myosin light chain phosphorylation in slow and fast skeletal muscles pdf

Nerve influence on myosin light chain phosphorylation
in slow and fast skeletal muscles
Cyril Bozzo
1,2
, Barbara Spolaore
3
, Luana Toniolo
1
, Laurence Stevens
2
, Bruno Bastide
2
,
Caroline Cieniewski-Bernard
2
, Angelo Fontana
3
, Yvonne Mounier
2
and Carlo Reggiani
1
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:
(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
)1
Æd
)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.
FEBS Journal 272 (2005) 5771–5785 ª 2005 FEBS 5771
of 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 phosphorylation
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 phosphorylation as it does with myosin
isoform expression [33]. To further confirm that low-
frequency neural discharge, typical of slow muscles, can
Myosin phosphorylation and denervation C. Bozzo et al.
5772 FEBS Journal 272 (2005) 5771–5785 ª 2005 FEBS
reduce 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
Table 1. Body mass (BM), muscle mass (MM), muscle mass ⁄ body mass ratio (MM ⁄ BM), and distribution of slow and fast myosin heavy
chain (MHC) and myosin light chain (MLC) isoforms determined by SDS ⁄ PAGE and densitometry in the soleus and EDL muscles of control
untreated rats (CONT, n ¼ 5, n ¼ 10 for muscles and myosin subunits), the controlateral leg of denervated rats (CODE, n ¼ 5), and in dener-
vated rats (DE, n ¼ 5). Each MHC isoform is expressed as a percentage of the total amount of MHC isoforms. Each alkali (MLC1 and ⁄ or
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.
Soleus EDL
CONT CODE DE CONT CODE DE
Body mass (BM, g) 307 ± 4 303 ± 13 307 ± 4 303 ± 13
Muscle mass (MM, mg) 123 ± 15 151 ± 21 82 ± 5* 144 ± 23 146 ± 11 82 ± 10*
MM ⁄ BM 0.40 ± 0.04 0.50 ± 0.05 0.27 ± 0.01* 0.47 ± 0.07 0.48 ± 0.02 0.27 ± 0.02*
MHC
MHCI 84.67 ± 3.01 84.54 ± 3.83 75.55 ± 3.29* 4.84 ± 4.70 2.22 ± 3.01 3.17 ± 2.79
MHCIIa 15.33 ± 3.01 15.46 ± 3.83 19.90 ± 3.00 1.20 ± 2.39 2.00 ± 0.39 7.78 ± 1.01*
MHCIId ⁄ x – – 4.56 ± 0.80* 38.25 ± 5.42 38.94 ± 4.34 35.66 ± 3.01
MHCIIb – – – 55.71 ± 5.30 56.84 ± 5.78 53.38 ± 3.11
Alkali light chains
MLC1slow 89.25 ± 2.86 91.20 ± 7.97 74.48 ± 2.53* 8.79 ± 6.97 5.57 ± 6.47 5.40 ± 6.33
MLC1fast + MLC3 10.75 ± 2.86 8.80 ± 7.97 25.52 ± 2.53* 91.21 ± 6.97 94.4 ± 6.47 94.6 ± 6.33
Regulatory light chains
MLC2slow 87.65 ± 2.96 88.57 ± 6.64 72.39 ± 2.76* 11.30 ± 4.97 8.30 ± 6.50 8.3 ± 8.02
MLC2fast 12.35 ± 2.96 11.43 ± 6.64 27.61 ± 2.76* 88.70 ± 4.97 91.70 ± 6.50 91.7 ± 8.02
*Significantly different (P<0.05) from CONT.
C. Bozzo et al. Myosin phosphorylation and denervation

FEBS Journal 272 (2005) 5771–5785 ª 2005 FEBS 5773
decreases 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), controlateral 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
the right. The isoelectric point and M
r
values are labelled on the
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
with SDS ⁄ PAGE in 8% gels, whereas separation of actin and MLC
isoforms was obtained with SDS ⁄ PAGE in 12% gels. Gels were
silver stained.
Myosin phosphorylation and denervation C. Bozzo et al.
5774 FEBS Journal 272 (2005) 5771–5785 ª 2005 FEBS

MLC3 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 control 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
was 1 : 4 in both MLC1 isoforms and did not change
after denervation.
Table 2. Identification of rat muscle proteins by mass spectrometry and database searching. Proteins were identified by ESI MS ⁄ MS and ⁄ or
MALDI-TOF. MLC, myosin light chain; PMF, peptide mass fingerprint; ESI-quadrupole-TOF analysis (MS ⁄ MS). Theor. pI, theoretical isoelec-
tric point.
Spots (Fig. 2) Protein name

Swiss-Prot
accession code
Matched peptides
by PMF
Peptides sequenced
by MS ⁄ MS
a
Sequence
coverage
Theor. pI;
M
r
b
Actin Actin, skeletal muscle P02568 15 2 46% 5.3 ⁄ 42.4
MLC1s MLC1s b, ventricular isoform P16409 9 2 50% 5.0 ⁄ 22.1
MLC1f MLC1f, skeletal muscle isoform P02600 5 2 30% 5.0 ⁄ 20.6
MLC1s1 MLC1s1 b, ventricular isoform P16409 9 1 50% 5.0 ⁄ 22.1
MLC1f1 MLC1f, skeletal muscle isoform P02600 5 2 30% 5.0 ⁄ 20.6
MLC2s MLC2 (MLC2v), myosin regulatory
light chain 2, ventricular ⁄ cardiac
muscle isoform
P08733 9 2 43% 4.9 ⁄ 18.7
MLC2f MLC2, myosin regulatory light chain 2,
skeletal muscle isoform
P04466 5 3 32% 4.8 ⁄ 18.9
MLC2s1 MLC2 (MLC2v), myosin regulatory
light chain 2, ventricular ⁄ cardiac
muscle isoform
P08733 5 — 26% 4.9 ⁄ 18.7
MLC2f1 Myosin regulatory light chain 2 MLC2 P04466 10 (12)

b
— 54% 4.8 ⁄ 18.9
MLC 2s2 MLC2 (MLC-2v), myosin regulatory
light chain 2, ventricular ⁄ cardiac
muscle isoform
P08733 7 1 50% 4.9 ⁄ 18.7
a
Calculated for the corresponding Swiss-Prot entry.
b
Identified on the basis of mass determination with a score of 1.105 · 10
)6
on the
Swiss-Prot database.
C. Bozzo et al. Myosin phosphorylation and denervation
FEBS Journal 272 (2005) 5771–5785 ª 2005 FEBS 5775
Effects 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.
Table 3. Relative distribution of the variants of slow and fast isoforms of myosin light chain MLC1 and MLC2 in soleus and EDL muscles, separated with 2D-gel electrophoresis: control
untreated rats (CONT), the controlateral leg of denervated rats (CODE), and denervated rats (DE), rats served as controls for cyclosporin A (CsA) treatment (COCsA) and CsA treated rats
(CsA). The variants of MLC1 slow are indicated as ‘1s’ and ‘1s1’, whereas the variants of MLC1 fast are indicated as ‘1f’ and ‘1f1’. The variants of MLC2 slow are indicated as ‘2s’, ‘2s1’
and ‘2s2’, whereas the variants of MLC2 fast are indicated as ‘2f’ and ‘2f1’. Each variant of slow (or fast) MLC1 is expressed as a percentage of total slow (or fast) MLC1 isoforms. The
same expression is used for the variants of slow MLC2 and fast MLC2. Data are expressed as mean values ± SD.
Soleus EDL
CONT (n ¼ 4) CODE (n ¼ 4) DE (n ¼ 4) COCsA (n ¼ 4) CsA (n ¼ 4) CONT (n ¼ 4) CODE (n ¼ 4) DE (n ¼ 4) COCsA (n ¼ 4) CsA (n ¼ 4)
MLC1
1 s 79.63 ± 1.41 78.68 ± 3.32 79.89 ± 1.95 81.13 ± 4.56 80.04 ± 2.48 100 100 100 100 100
1s1 20.37 ± 1.41 21.32 ± 3.32 20.11 ± 1.95 18.87 ± 4.56 19.96 ± 2.48 – – – – –
1f 100 100 100 100 100 81.43 ± 0.76 79.77 ± 3.34 80.16 ± 4.91 78.56 ± 3.75 79.76 ± 4.11
1f1 – – – – – 18.57 ± 0.76 20.23 ± 3.34 19.84 ± 4.91 21.44 ± 3.75 20.24 ± 4.11
MLC2
2 s 74.09 ± 7.39 74.01 ± 3.16 66.09 ± 6.41 76.23 ± 8.19 77.27 ± 4.40 – – – – –
2s1 25.91 ± 7.39 25.99 ± 3.16 27.76 ± 4.34 23.77 ± 8.19 22.73 ± 4.40 66.84 ± 1.58 69.91 ± 3.89 77.73 ± 3.66* 67.83 ± 3.11 71.75 ± 3.49
2s2 – – 6.15 ± 2.93* – – 33.16 ± 1.58 30.09 ± 3.89 22.27 ± 3.66* 32.17 ± 3.11 28.25 ± 3.49
2f 100 100 79.85 ± 8.06* 100 100 74.25 ± 0.77 76.17 ± 4.95 74.72 ± 3.62 74.35 ± 7.77 74.00 ± 7.26
2f1 – – 20.15 ± 8.06* – – 25.65 ± 0.77 23.83 ± 4.95 25.28 ± 3.62 25.65 ± 7.77 26.00 ± 7.26
*Significantly different (P<0.05) from the respective control group.
Myosin phosphorylation and denervation C. Bozzo et al.
5776 FEBS Journal 272 (2005) 5771–5785 ª 2005 FEBS
Effects 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
after electrophoresis on 12% SDS ⁄ PAGE gels. (B) Mean values and SDs of the ratios of MLC kinase and PP1 signals to actin signal. n ¼ 5.
*Significantly different (P<0.05) from CONT.
Table 4. Body mass (BM), muscle mass (MM), muscle mass ⁄ body mass ratio (MM ⁄ BM), and distribution of slow and fast myosin heavy
chain (MHC) and myosin light chain (MLC) isoforms determined by SDS ⁄ PAGE and densitometry in soleus and EDL muscles of rats treated
with cyclosporin A (CsA, n ¼ 5) and rats serving as controls for cyclosporin A treatment (COCsA, n ¼ 5). Each MHC isoform is expressed as
a percentage of the total amount of MHC isoforms. Each alkali (MLC1 and ⁄ or MLC3) or regulatory (MLC2) isoform is expressed as a per-
centage of the total amount of alkali or regulatory MLC, respectively. Data are expressed as mean values ± SD.
Soleus EDL
COCsA CsA COCsA CsA
Body mass (BM, g) 309 ± 2 277 ± 22* 309 ± 2 277 ± 22*
Muscle mass (MM, mg) 127 ± 14 119 ± 24 146 ± 26 98 ± 5*
MM ⁄ BM 0.41 ± 0.05 0.43 ± 0.08 0.47 ± 0.09 0.43 ± 0.08
MHC
MHCI 85.11 ± 6.18 75.08 ± 5.25* 1.71 ± 3.41 2.69 ± 2.78
MHCIIa 14.89 ± 6.18 20.24 ± 4.81 3.01 ± 1.39 9.71 ± 2.02*
MHCIId ⁄ x – – 39.29 ± 2.11 45.75 ± 2.31*
MHCIIb – 4.68 ± 1.01* 55.99 ± 3.04 41.85 ± 2.87*
Alkali light chains

MLC1slow 90.85 ± 5.80 89.06 ± 5.11 6.81 ± 5.25 7.16 ± 6.18
MLC1fast + MLC3 9.15 ± 5.80 10.94 ± 5.11 93.19 ± 5.25 92.84 ± 6.18
Regulatory light chains
MLC2slow 86.35 ± 5.40 83.49 ± 9.95 9.16 ± 7.73 8.08 ± 4.14
MLC2fast 13.65 ± 5.40 16.51 ± 9.95 90.84 ± 7.73 91.92 ± 4.14
*Significantly different (P<0.05) from COCsA.
C. Bozzo et al. Myosin phosphorylation and denervation
FEBS Journal 272 (2005) 5771–5785 ª 2005 FEBS 5777
f1), 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
left to acidic on the right. M
r
and isoelectric point labels are placed on the upper and left edge of the MLC region. (B) Immunoblotting for
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.
Myosin phosphorylation and denervation C. Bozzo et al.
5778 FEBS Journal 272 (2005) 5771–5785 ª 2005 FEBS
acidic 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
C. Bozzo et al. Myosin phosphorylation and denervation
FEBS Journal 272 (2005) 5771–5785 ª 2005 FEBS 5779

obtained 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 muscles, 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 phosphorylated 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.
Myosin phosphorylation and denervation C. Bozzo et al.
5780 FEBS Journal 272 (2005) 5771–5785 ª 2005 FEBS
Experimental procedures
Animals and treatments
Experiments were performed on adult male Wistar rats
weighing  250 g. Animals were divided into five groups
for three comparisons, as described below. The experiments
and the treatment of the animals were approved by the
French Agricultural and Forest Ministry and the French
National Education Ministry (authorization 5900996).
Comparison 1: effects of denervation
Five animals with surgical section of the sciatic nerve (DE)
were compared with five control, untreated animals
(CONT). Rats of the DE group were anaesthetized with

Zoletil (10 mgÆkg
)1
; zolazepam ⁄ tiletamine 1 : 1, v ⁄ v) and
xilazine (2%, v ⁄ v) 0.06 mLÆkg
)1
, and  1 cm of the sciatic
nerve on the right hindlimb at the level of trochanter was
removed. The left hindlimb was used as a control of the
denervation (CODE). Two weeks after denervation, the ani-
mals were anaesthetized with an intraperitoneal injection of
ethylcarbamate and left in a completely relaxed state for
10 min, then the muscles (soleus and EDL) were dissected
and the animal killed by exsanguination. Once dissected,
soleus and EDL were quickly blotted, weighed and frozen
in liquid N
2
. The muscles were then pulverized in a small
steel mortar cooled with fluid nitrogen and stored at
)80 °C until analysed. The time from muscle dissection to
freezing never exceeded 10 min, and preliminary experi-
ments showed that no substantial differences in the propor-
tions of MLC variants were observed using a faster freezing
protocol.
Comparison 2: effects of CsA administration
A group of five animals received 10 mgÆday
)1
Ækg
)1
CsA,
diluted in a 10% cremophor A solution, by intraperitoneal

injection. This group was compared with a group of five
rats receiving the vehicle alone (i.e. 10% cremophor A solu-
tion) (COCsA). The volume of the cremophor solution
injected was calculated according to the animal weight, as
for the CsA group. At the end of 2 weeks of treatment, ani-
mals were killed and muscles prepared exactly as described
for denervation experiments (see comparison 1, above).
Comparison 3: CLFS
Finally, a group of five rats were treated at the Institute of
Neurophysiology, University of Oslo, according to the pro-
tocol indicated as CLFS. After sciatic nerve section, EDL
was stimulated with trains at 20 Hz for 10 s, every 20 s for
three weeks [29,36], a period necessary to obtain an optimal
fast-to-slow phenotype transition. EDL was prepared at the
end of the treatment, precisely as indicated for denervation
(see comparison 1, above). The CLFS group was compared
with the CONT group described in comparison 1.
Protein extraction for 1D- and 2D-electrophoreses
Muscle powder was used to extract myofibrillar proteins
for MHC and MLC analysis by electrophoresis. Myofibril-
lar proteins were extracted from 7 to 10 mg of dry muscle
powder, as described previously [45], washed first with a
solution containing 6.3 mm EDTA (pH 7), 0.1% (v ⁄ v)
pepstatine and 1% (v ⁄ v) phenylmethanesulfonyl fluoride,
and then with a second solution containing 50 mm KCl,
0.1% (v ⁄ v) pepstatine and 1% (v ⁄ v) phenylmethanesulfo-
nyl fluoride. The proteins were resuspended in 1mL of
sterile MilliQ water and their concentration was deter-
mined by a protein assay kit (Dc Protein Assay; BioRad,
Ivry s ⁄ Seine, France) to prepare samples with a final pro-

tein content of 10 lg for 1D-electrophoresis, 70 lg for
2D-electrophoresis, and 150 lg for mass spectrometry.
This last protein quantity was chosen to optimize the mass
spectrometry analysis by intensifying the less abundant
spots, and avoiding contamination between spots migra-
ting in very near positions. Then, the proteins were preci-
pitated for 2 h with acetone (8 : 1 v ⁄ v), followed by
centrifugation for 1 h at 13 000 g. The pellet was dissolved
in Laemmli solution for SDS ⁄ PAGE or in rehydration
buffer for 2D-electrophoresis.
1D-electrophoresis for MHC and MLC analyses
MHC isoform composition was determined by SDS ⁄ PAGE
on a 4.5% stacking gel and a 8% separating gel. Electro-
phoresis was run for 20 h at low temperature (180 V con-
stant, 13 mA per gel). For the analysis of MLC isoform
composition, 12% separating gels with 4% stacking gels
were used. The electrophoresis was run for 8 h at low tem-
perature (150 V, 13 mA per gel). After the run, gel slabs
were silver-stained, as described previously [24]. The relative
proportions of MHC or MLC isoforms in each sample
were determined by densitometry (GS-700 Imaging Densi-
tometer; BioRad). At least two independent measurements
were performed on each sample. The mean value was used
as an individual measurement.
2D-electrophoresis for MLC analysis
Proteins were separated by 2D-gel electrophoresis, using a
procedure similar to that previously described [24]. For the
first dimension, or IEF, proteins were solubilized in 8 m
urea, 2% (v ⁄ v) Chaps, 0.01 m dithiothreitol and 2% carrier
ampholites (buffer, and then separated using the Ettan

IPGphor Isoelectric Focusing System on 3.5% acrylamide
strips with immobilized pH gradients (pH 4–7) (all
C. Bozzo et al. Myosin phosphorylation and denervation
FEBS Journal 272 (2005) 5771–5785 ª 2005 FEBS 5781
Amersham Biosciences, Bucks, UK). Strips were rehydrated
at 50 V for 12 h and proteins were focused under the fol-
lowing voltage conditions: 500 V for 1 h, 500–1000 V for
1 h, and 8000 V until reaching 100 000 VÆh. The tempera-
ture was kept constant at 20 °C. After reduction with a
buffer comprising 6 m urea, 30% (v ⁄ v) glycerol, 2% (v ⁄ v)
dithiothreitol and 0.375 m Tris ⁄ HCl, pH 8.8, and alkylation
with the same buffer containing 2.5% (v ⁄ v) iodoacetamide,
the strips were embedded in a 4% polyacrylamide stacking
gel and the proteins separated by SDS ⁄ PAGE (12% gel)
for 8 h at 150 V at low temperature (4 °C). Following elec-
trophoresis, the gels were silver-stained, as described previ-
ously [24] or stained with Coomassie Brilliant Blue, as
specified. The positions of slow and fast isoforms of MLC
on 2D gels were identified according to their isoelectric
point in the first dimension, and to appropriate markers of
molecular weight in the second dimension, and confirmed
by immunostaining [24] and by mass spectrometry (see
below).
Protein transfer and immunoblotting
Proteins separated on 12% gels by SDS ⁄ PAGE were trans-
ferred to nitrocellulose membrane to analyse the expression
of MLC kinase, PP1 and actin by immunostaining. Proteins
separated on 2D gels were transferred to nitrocellulose mem-
brane to characterize the spots corresponding to the MLC.
In both cases, transfer was obtained by a semidry transfer

procedure by applying a current of 0.8 mAÆcm
)2
for 6 h.
Nitrocellulose sheets were reacted first with the primary anti-
bodies for 1 h at 37 °C. The following primary antibodies
were used: monoclonal anti-actin clone AC-40 (A3853;
Sigma Aldrich, St Louis, MO, USA), polyclonal anti-(MLC
kinase) (sc9456), polyclonal anti-(PP1) (sc6106), polyclonal
anti-(MLC2) (FL-172sc15370) (Santa Cruz Biotechnology,
Santa Cruz, CA, USA), monoclonal anti-(phospho-serine)
(PSR-45) and monoclonal anti-(phospho-threonine) (PTR-8)
(both Sigma Aldrich). Then, a rabbit anti-mouse immuno-
globulin (P260; Dako, Glostrup, Denmark) for anti-actin, a
goat anti-mouse immunoglobulin (Chemicon International,
Hants, UK) for anti-phosphoserine and anti-phosphothreo-
nine, and a rabbit anti-goat immunoglobulin (A5420; Sigma
Aldrich) for anti-(MLC kinase), anti-PP1 and anti-MLC2
were employed as secondary antibodies. The bands or the
spots were visualized by an enhanced chemiluminescence
(ECL) method. Preliminary tests were carried out for each
antibody to check any cross-reactivity and to verify the exact
position of each protein on the gel, particularly actin and
PP1 that had a very similar molecular mass.
Image analysis and quantification
2D gels were digitized with a scanner (EPSON 1650;
Epson, Meerbusch, Germany) at a resolution of 1200 dots
per inch. The spots were analysed densitometrically and
each spot was characterized by a value of brightness-area
product (BAP) with a constant threshold after black ⁄ white
inversion using Adobe Photoshop Software (Adobe, San

Jose, CA, USA). In each gel, the BAP values of the spots
identified as slow and, respectively, fast isoforms of MLC1
and MLC2 were summed to give a total for each isoform:
slow MLC1, fast MLC1, slow MLC2 and fast MLC2. The
value of each spot was expressed as a percentage of the
total value for that particular isoform. From percent-
age values obtained in different gels, the mean values ±
standard deviation were calculated. The quantification pro-
cedure had been validated previously [24] by running, on
separate gels, known mixtures of a constant amount of
purified actin and increasing amounts of purified slow
MLC2, and determining the ratio between the BAP values
of MLC2 and actin (Fig. 1B in Bozzo et al. [24]). The reli-
ability of the silver staining was further validated by com-
paring the spot quantification on silver-stained and on
Coomassie blue-stained gels. Both staining protocols lead
to similar values of the percentage distribution of the
MLC variants.
Mass spectrometry for identification of the 2D
spots
Tryptic in-gel digestion
Selected spots were excised from 2D gels stained with Coo-
massie Brilliant Blue, and proteins were in-gel digested, as
previously described [46]. Briefly, gel pieces were destained
and the proteins were reduced with dithiothreitol, alkylated
with iodoacetamide and digested with porcine trypsin (modi-
fied sequencing grade; Promega, Madison, WI, USA) over-
night at 37 °C. The supernatant was then transferred to
another tube and residual tryptic peptides were extracted
upon incubation of gel spots, first with 25 mm NH

4
HCO
3
at
37 °C for 15 min followed by shrinking of gel pieces with
acetonitrile, and then upon incubation with 5% (v ⁄ v) formic
acid at 37 °C for 15 min and shrinking with acetonitrile.
The extracts were combined with the primary supernatant
and dried in a SpeedVac centrifuge (Savant Instrument
Inc., NY, USA). Protein digests were then resuspended in
20 lLof1%(v⁄ v) trifluoroacetic acid and purified on Zip
Tip C
18
microcolumns (Millipore, Bedford, MA, USA)
according to the instructions provided by the manufacturer.
Peptides were eluted in 50% (v ⁄ v) methanol containing
0.1% (v ⁄ v) formic acid, and analysed directly by mass
spectrometry.
Mass spectrometry
Mass spectra were acquired on a tandem mass spectrometer
Q-TOF Micro (Micromass, Manchester, UK) equipped
with a Z-spray nanoflow electrospray interface. NanoESI
capillaries were prepared in-house from borosilicate glass
Myosin phosphorylation and denervation C. Bozzo et al.
5782 FEBS Journal 272 (2005) 5771–5785 ª 2005 FEBS
tubes of 1 mm outer diameter (OD) and 0.78 mm inner dia-
meter (ID) (Harvard Apparatus, Holliston, MA, USA)
using a Flaming ⁄ Brown P-80 PC micropipette puller (Sutter
Instruments, Hercules, CA, USA), and gold coated using
an Edwards S150B sputter coater (Edwards High Vacuum,

Crawley, West Sussex, UK). The capillary tips were cut
under a stereomicroscope to give inner diameters of
1–5 lm. Typically, 2 lL of solution eluted from the Zip
Tip C
18
microcolumn was loaded directly onto the capillary
tips and analysed using the following experimental parame-
ters (positive ion mode): capillary voltage, 1.5 kV; sample
cone, 30.0 V; extractor cone, 1.0 V. In the collision cell,
argon was at an indicated inlet pressure of 10 p.s.i. and the
collision energy setting was 4.0 V. The electrospray source
was heated at 40 °C and the desolvatation gas (nitrogen)
was set at a flow of 50 LÆh
)1
. When MS ⁄ MS experiments
were performed, the collision gas was typically used at an
indicated inlet pressure of 30 p.s.i. and the collision energy
setting was 30 V. External calibration was performed using
a solution of 0.1% (v ⁄ v) phosphoric acid in 50% (v ⁄ v)
aqueous acetonitrile. Instrument control, data acquisition
and processing were achieved with masslynx software
(Micromass). Deionized water from the MilliQ water sys-
tem (Millipore) was always used. HPLC-grade methanol
and acetonitrile, trifluoroacetic acid, dithiothreitol and
iodoacetamide were purchased from Fluka (Buchu, Switzer-
land). Formic acid was obtained from Sigma.
Database searching
Molecular mass values of individual tryptic peptides, and
MS ⁄ MS spectra used for protein identification, were
searched using the MASCOT search engine (http://

www.matrixscience.com) against the SWISS-PROT data-
base, with trypsin plus potentially one missed cleavage.
Peptide mass fingerprint and MS ⁄ MS spectra analysis used
the assumption that peptides were monoisotopic, carbami-
domethylated at cysteine residues and, as a variable modifi-
cation, oxidized at methionine residues.
Statistical analysis
All data were expressed as mean values ± standard devi-
ation. The statistical significance of the difference between
means was determined using the t-test or ANOVA followed
by the Bonferroni test. Differences at or above the 95%
confidence level were considered significant (P<0.05).
Acknowledgements
This work was partially supported by EU grant
HPRN-CT-2000-0091 to CB, Italian Ministry of Uni-
versity, through PRIN (Research Project of National
Interest) grant 2004, CNES (Centre National d’Etudes
Spatiales, grant 3194), Conseil Regional du Nord
Pas-de-Calais. The authors wish to express their gratit-
ude to Professor T. Lomo, Institute of Neurophysiol-
ogy, University of Oslo, for valuable help with CLFS
experiments, and to Novartis Pharma AG, Basel for
the gift of CsA.
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