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1 Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Miami, Florida 33136; and 2 Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas 75390
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The role of phosphorylation of the
myosin regulatory light chains (RLC) is well established in smooth
muscle contraction, but in striated (skeletal and cardiac) muscle its
role is still controversial. We have studied the effects of RLC
phosphorylation in reconstituted myosin and in skinned skeletal muscle
fibers where Ca2+ sensitivity and the kinetics of
steady-state force development were measured. Skeletal muscle myosin
reconstituted with phosphorylated RLC produced a much higher
Ca2+ sensitivity of thin filament-regulated ATPase activity
than nonphosphorylated RLC (change in 
log of the Ca2+
concentration producing half-maximal activation = ~0.25). The same was true for the Ca2+ sensitivity of force in skinned
skeletal muscle fibers, which increased on reconstitution of the fibers
with the phosphorylated RLC. In addition, we have shown that the level
of endogenous RLC phosphorylation is a crucial determinant of the
Ca2+ sensitivity of force development. Studies of the
effects of RLC phosphorylation on the kinetics of force activation with
the caged Ca2+, DM-nitrophen, showed a slight
increase in the rates of force development with low statistical
significance. However, an increase from 69 to 84% of the initial
steady-state force was observed when nonphosphorylated
RLC-reconstituted fibers were subsequently phosphorylated with
exogenous myosin light chain kinase. In conclusion, our results suggest
that, although Ca2+ binding to the troponin-tropomyosin
complex is the primary regulator of skeletal muscle contraction, RLC
play an important modulatory role in this process.
steady-state force; calcium regulation; regulatory light chain depletion; myosin light chain kinase
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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THE REGULATION OF
CONTRACTION in molluscan or vertebrate smooth muscles occurs via
myosin, which either binds Ca2+ directly (molluscan)
(16, 49) or undergoes a Ca2+/calmodulin
(CaM)-activated phosphorylation at the myosin regulatory light chains
(RLC) in smooth muscle (1, 11, 39). Striated muscles are
activated by the binding of Ca2+ to troponin C (TnC), which
initiates a series of conformational changes within the proteins of the
thin filaments and leads to muscle contraction (29, 51).
Unlike molluscan or smooth muscles, the RLC of striated muscles do not
play a primary regulatory role, and therefore it is of interest to
understand their possible role in thin filament-regulated skeletal
muscles. The crystal structure of skeletal myosin subfragment 1 (35) reveals that the RLC are located at the head-rod
junction of the myosin molecule, implying their possible importance in
cross-bridge cycling in contracting muscle. The
NH2-terminal domain of RLC contains a divalent
cation-binding site that binds both Ca2+ and
Mg2+. Under physiological conditions, in relaxed muscle, it
is thought that this site is occupied by Mg2+
(14) and may become partially saturated with
Ca2+, depending on the length of the Ca2+
concentration ([Ca2+]) transient (37).
Analogous to smooth muscle myosin, the NH2-terminal domain
of RLC of skeletal myosin also contains two adjacent serine residues
located in the proximity of the cation-binding site. During muscle
contraction, the increase in [Ca2+] activates the
Ca2+/CaM-dependent myosin light chain kinase (MLCK) and
leads to phosphorylation of the RLC. In vivo phosphorylation of this
kind correlates with potentiation of the rate of force development and
maximal extent of isometric twitch tension (Ref. 43 and
references within). In vitro, the rate of isometric force redevelopment
of skinned muscle fibers was shown to increase with RLC phosphorylation
that also caused an increase in the Ca2+ sensitivity of
force (18-20, 24, 45). Although no effect on maximal
steady-state force (developed at maximal Ca2+ activation)
has been observed in skinned muscle fiber preparations under normal
conditions, a small effect of RLC phosphorylation has been observed
under fatigue conditions (8). The results presented in
this paper show a ~15% increase in maximal force developed by RLC
phosphorylated skinned fibers vs. those reconstituted with
nonphosphorylated RLC. Unlike the correlation of RLC phosphorylation and force potentiation in intact muscle or the increase in the Ca2+ sensitivity of force development in skinned muscle
fiber preparations, there is no clear understanding of the influence of
RLC phosphorylation on the actin-activated myosin ATPase activity
(28, 32, 41). The results presented here show that
phosphorylation of the RLC has a dramatic effect on the
Ca2+ sensitivity of the ATPase activity of reconstituted
thin filaments. Myosin depleted of endogenous RLC and reconstituted
with MLCK phosphorylated exogenous RLC increased the Ca2+
sensitivity of actin-tropomyosin (Tm)-troponin (Tn) activity by a
change of the log of the [Ca2+] producing half-maximal
activation (
pCa50) of ~0.25 vs. nonphosphorylated RLC.
This is the first report of such an effect of RLC phosphorylation in
the actomyosin ATPase system. It is worth mentioning that the magnitude
of the effect of RLC phosphorylation on the Ca2+
sensitivity of force development observed in our skinned skeletal muscle fibers was also more extensive than previously reported. Because
skinned fibers may contain different levels of RLC phosphorylation depending on how they are isolated, it will be critical in future experiments to measure the level of RLC phosphorylation to determine the contribution of this to any measured Ca2+ dependence.
In summary, the role of RLC phosphorylation, as demonstrated here and
earlier by others (Ref. 43 and references within) is more
important in modulating skeletal muscle contraction than originally
suspected or appreciated.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Rabbit Skeletal Phosphorylated and Nonphosphorylated RLC
RLC of myosin were isolated and purified as described by Wagner et al. (50). Briefly, myosin at a concentration of 12-15 mg/ml in 0.5 M KCl, 10 mM EDTA, and 10 mM phosphate buffer, pH 8.5, was incubated with 10 mM DTNB for 15 min on ice. Myosin was then precipitated by addition of 13 vol of 10 mM EDTA, pH 7.0, and centrifuged. The RLC-depleted myosin was then resuspended in 0.5 M KCl, 10 mM dithiothreitol (DTT), and 10 mM phosphate buffer, pH 7.0. This myosin was further redialyzed and used in the actin-activated ATPase assays as RLC-depleted myosin and used in the reconstitution experiments with nonphosphorylated and/or phosphorylated RLC. The supernatant containing the dissociated RLC was dialyzed against 2 M urea, 25 mM Tris·HCl (pH 7.5), 0.1 mM phenylmethylsulfonyl fluoride, 0.02% NaN3, and 1 mM DTT, and then it was applied to a Q-Sepharose column equilibrated with the same buffer. The RLC was eluted by using a salt gradient of 0-0.45 M KCl in the above buffer. Fractions containing the purified protein (96-98% purity, as determined by SDS-PAGE) were pooled and stored at80°C. The
concentrations of the proteins were determined by using the Coomassie
Plus Assay (Pierce).
Phosphorylation of the Nonphosphorylated RLC with Ca2+/CaM-Activated MLCK
RLC (100-160 µM) was dialyzed against 20 mM phosphate buffer (pH 8.0), and 30 mM KCl. Phosphorylation of the protein was generated by the addition of 0.1 mM CaCl2, 12 mM MgCl2, 5 mM ATP, 5 µM bovine testicular CaM, and 0.5 µM MLCK. A catalytically active truncated fragment of the rabbit skeletal muscle MLCK was used in this study (12). The MLCK, missing the first 256 amino acids, was expressed in Sf9 cells infected with a recombinant virus (7). After 2 h of room temperature incubation, the level of RLC-phosphorylation was checked by 12.5% urea-SDS-PAGE. These conditions were shown to achieve 100% RLC phosphorylation. Phosphorylated RLC (P-RLC) was further purified on a Q-Sepharose column, pooled, and stored at80°C (as previously described).
Reconstitution of RLC-depleted Myosin With Nonphosphorylated and/or Phosphorylated RLC
RLC-depleted myosin was dialyzed into a buffer containing 0.4 M KCl, 50 mM MOPS, pH 7.0, 2 mM MgCl2, and 10 mM DTT, whereas RLC and P-RLC were dialyzed against the same buffer with the exception of the KCl being 0.1 M. Protein concentrations after dialysis were determined as stated earlier. RLC-depleted myosin and the P-RLC or RLC were then mixed in a 1:2 molar ratio and incubated on ice for 2 h to allow for reconstitution to take place. The P-RLC and/or RLC reconstituted myosins were then dialyzed against 0.4 M KCl, 50 mM MOPS (pH 7.0), and 10 mM DTT. To remove any excess RLC after dialysis, the reconstituted myosins were precipitated by the addition of 13 vol of cold H2O and collected by centrifugation. Pellets were resuspended into minimal volumes of myosin dialysis buffer and dialyzed for an additional 2 h. Protein concentrations were determined, and 0.1 mg/ml samples were used in the K+ EDTA and Ca2+ ATPases assays (22) to check the enzymatic activity of the native (undepleted), RLC-depleted, and P-RLC/RLC reconstituted myosins. Dialysis was continued until K+ EDTA and Ca2+ ATPase activity levels approached the published values (22).Ca2+ Binding Studies
Fluorescence measurements. P-RLC and RLC were dialyzed against a solution of 90 mM KCl, 120 mM MOPS (pH 7.0), and 2 mM EGTA. For measurements in the presence of Mg2+, the dialysis buffer also contained 2 mM MgCl2. Measurements were performed by using SLM Spectrofluorometer model 8100 (SLM Instruments). The proteins (5 µM) were placed in a 2-ml quartz cuvette, and the tryptophan fluorescence of RLC was excited at 290 nm. Emission spectra were acquired in the range of 310 to 410 nm. All measurements were performed at room temperature (22°C). For Ca2+ titrations, peak fluorescence between 341 and 344 nm was acquired and averaged for each [Ca2+]. Data were fitted to the Hill equation, analyzed, and plotted by using SigmaPlot 2000. The amount of Ca2+ added to achieve a desired free [Ca2+] was calculated according to Robertson and Potter (36).
Flow dialysis.
Flow dialysis was performed in a solution of 100 mM KCl and 20 mM
imidazole, pH 7.0 (22°C). The proteins were equilibrated in this
buffer before measurements. The flow-dialysis experiments were
performed according to Colowick and Womack (4) with
modifications. Briefly, the upper chamber of the apparatus containing
the protein and the labeled substrate (45Ca2+)
was separated by a membrane from the lower chamber. The buffer was
pumped through the lower chamber at a constant rate of 1.5 ml/30 s. The
upper chamber was first equilibrated with 0.4 ml buffer for 15 min
followed by the protein (0.4 ml) for 5 min. After
45Ca2+ was added, equilibrium was attained by
flowing buffer through the lower chamber for 5 min. After steady state
was reached, unlabeled substrate (Ca2+) was added at
regular intervals and in varying concentrations. Fractions were
collected every 30 s, and the effluent was sampled for measurement
of radioactivity. The specific radioactivity of 45Ca2+ used in the experiment was 12-16
mCi/mg (from NEN Life Science Products), and 2 µCi of
45Ca2+ per experiment gave sufficient
radioactivity in the dialysate for accurate measurements. Data were
analyzed by using Scatchard analysis (34, 38)
![[Ca-bound]/[Ca-free]/[protein]](/content/vol92/issue4/fulltext/1661/img001.gif)
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![= −<IT>K</IT><SUB>Ca</SUB> × [Ca-bound]/[protein] + &eegr;<IT>K</IT><SUB>Ca</SUB>](/content/vol92/issue4/fulltext/1661/img002.gif)
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is the total number of
Ca2+ binding sites, and KCa is the
Ca2+ binding constant.
Actin-Activated ATPase Assays
Rabbit skeletal myosin was obtained as described earlier (26, 41). F-actin, Tm, and Tn were isolated and purified from rabbit skeletal muscle according to Strzelecka-Golaszewska et al. (42), Potter (33), and Smillie (40), respectively. Myosin was dialyzed to 0.4 M KCl, 50 mM MOPS (pH 7.0), and 1 mM DTT, whereas F-actin, Tm, and Tn were homogenized together in a ratio of 7:1:1, respectively, and dialyzed in the same buffer as the myosin but with 0.1 M KCl. Actin-activated ATPase assays were performed by using 1 µM myosin-4 µM F-actin-0.6 µM Tm-0.6 µM Tn in a solution containing 20 mM MOPS (pH 7.0), 35 mM KCl, 2 mM EGTA, 2.5 mM MgCl2, and increasing [Ca2+] from pCa 8 to 4 (36). The reaction was initiated with 2.5 mM ATP, and after 5 min incubation at 30°C terminated with 5% trichloroacetic acid. Inorganic phosphate was measured according to Fiske and SubbaRow (6).Skinned Fiber Preparation and Force Measurements
Experiments were performed with glycerinated rabbit psoas muscle fibers dissected from rabbits and chemically skinned (as described in Refs. 17, 48). Fiber bundles of three to five single fibers were mounted on a force transducer [assembled according to Guth and Potter (10)]. The fibers were then treated with the pCa 8 relaxing solution, containing 1% Triton X-100, for 15 min. The composition of the pCa 8 solution was as follows: 10
8 M [Ca2+], 1 mM Mg2+, 7 mM
EGTA, 5 mM MgATP2+, 20 mM imidazole (pH 7.0), 20 mM
creatinine phosphate, and 15 units/ml of creatine phosphokinase (ionic
strength = 150 mM) (5). To judge the quality of the
fibers, fibers were contracted (in the pCa 4 solution containing the
same composition as the pCa 8 solution except [Ca2+] = 104 M) and relaxed several times to obtain stable force
values. Sarcomere length was adjusted to 2.4 µm. Sarcomere length
control was not available. The maximal force per cross-sectional area,
calculated for all newly mounted fibers before the reconstitution
experiments, was 301 ± 29 kN/m2. To further evaluate
the fibers suitability for further testing, the Ca2+
dependence of force development was measured twice to make certain that
it was consistent with our established Ca2+ dependence for
these solutions and stable. All measurements were performed at room
temperature (22°C). Once the fibers passed these initial tests, they
were then used for RLC treatment (see below). Fibers not passing these
initial tests were rejected and new ones were tested.
Preparation of Skinned Fibers Containing Fully Dephosphorylated RLC
The protocol described earlier (17, 48) for skeletal muscle fiber preparation resulted in various levels of RLC phosphorylation. To obtain fully dephosphorylated fibers, small strips of psoas muscle (~2 mm in diameter) were dissected from rabbit psoas muscle and incubated in (in mM) 60 KPr, 2 MgCl2, 5 EGTA, 25 MOPS (pH 7.0), 1 NaN3, and 25% glycerol for 1 h at 4°C. The fibers were then transferred to a fresh solution for an additional 24 h, followed by incubation in the same solution containing 50% glycerol. The latter solution was changed every 12 h for 48 h, after which the fibers were transferred to this solution and stored at20°C for no longer than 6 wk.
Phosphorylation of Endogenous RLC in Skinned Fibers With Ca2+/CaM-Activated MLCK
Phosphorylation of RLC in the skeletal muscle fibers was performed in the pCa 6 solution (the composition of this solution was the same as the pCa 8 buffer except [Ca2+] = 106 M), plus 5 µM bovine CaM and 0.5 µM MLCK. The
same catalytically active truncated fragment of the rabbit skeletal
muscle MLCK (12) was used to phosphorylate the RLC in the
fibers or in the isolated state (7). After 30 min
of phosphorylation, the fibers were washed with the pCa 8 solution and
subjected to force measurements.
Reconstitution of RLC-Depleted Skinned Fibers With Exogenous RLC
After initial force measurements on the control, untreated skinned skeletal muscle fibers, the endogenous RLC were extracted according to the protocol described in Szczesna et al. (48). Because of the partial extraction of TnC, the RLC-depleted fibers were first incubated with 20 µM rabbit skeletal TnC in the pCa 8 solution for 30 min at room temperature (22°C), rinsed in the pCa solution, and tested for Ca2+-dependent force development. RLC-depleted (TnC-reconstituted) fibers were then incubated with 30 µM RLC dissolved in the pCa 8 buffer, rinsed with the same buffer without protein added, and tested for force development. P-RLC or nonphosphorylated RLC were used in the reconstitution experiments; when the nonphosphorylated RLC were utilized, the fibers were further treated with Ca2+/CaM-activated MLCK to phosphorylate the reconstituted RLC.Steady-State Force Measurements
As described above, bundles of three to five single fibers were mounted on a force transducer with stainless steel clips and incubated for 15 min at room temperature (22°C) in the pCa 8 solution, containing 1% Triton X-100. Maximal force was measured in the pCa 4 solution, and the fibers were then relaxed in the pCa 8 solution. The effect of RLC phosphorylation was tested in a series of parallel experiments performed on fibers reconstituted with various RLC. These measurements were performed on the control, RLC-, and TnC-depleted fibers, RLC-depleted and TnC-reconstituted fibers, and finally on the RLC- and TnC-reconstituted fibers. Fibers were reconstituted with either P-RLC or nonphosphorylated RLC that were further phosphorylated in the fibers with Ca2+/CaM-activated MLCK (for 30 min at room temperature). Ca2+ dependence of force development was measured in solutions of increasing [Ca2+] (from pCa 8 to 4). The data were fitted to the following equation
![Y<SUB>H</SUB> = 100 × [Ca<SUP>2+</SUP>]<SUP><IT>n</IT><SUB>H</SUB></SUP>/([Ca<SUP>2+</SUP>]<SUP><IT>n</IT><SUB>H</SUB></SUP>+[Ca<SUP>2+</SUP>]<SUB>50</SUB><SUP><IT>n</IT><SUB>H</SUB></SUP> )](/content/vol92/issue4/fulltext/1661/img003.gif)
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Measurements of the Rate of Force Development
Before the kinetics measurements, the fibers were contracted (pCa 4) and relaxed (pCa 8) several times to reach a stable force level, and their sarcomere length was reset to 2.4 µm. Then they were treated with the pCa 8 solution containing 1% of Triton X-100 for 15 min at room temperature (22°C), contracted in the pCa 4 solution, and incubated with a "low EGTA" pCa 8 solution (same as pCa 8 solution except containing 0.5 mM instead of 7 mM EGTA). Fibers were then exposed to (in mM) 2.5 DM-nitrophen, 1.002 CaCl2, 100 TES, 1.2 MgCl2, 1.4 ATP, 10 glutathione, 29.4 1,6-hexamethylenediamine-2-aminoethane sulfonic acid, and 20 creatine phosphate (pH 7.1, ionic strength = 150 mM adjusted with potassium propionate) (9). After that, the entire fibers were illuminated in air with an ultraviolet (UV) light pulse xenon flash lamp (model XFL-355-3017, Advanced Radiation). Total air exposure was ~3 s. The duration of the UV pulse from the xenon lamp was ~2 ms with the total UV energy (<400 nm) equal to ~43 mJ. Average energy density exposure of the fiber was ~3.4 mJ/mm2 (light spot diameter = 4mm). To minimize the period of high-tension development, fibers were transferred to relaxing solution (pCa 8) within 1.5 s of the initial flash. As a result of the rapid Ca2+ release, fibers developed isometric tension, characterized by a double-exponential time course (48). The rate constants of activation were calculated according to the equation: y = A(1 ek1t) + B(1 ek2t) + C, where k1 and
k2 are the rate constants, A and
B are the amplitudes of the force transient, C is
a constant, and t is time of force transient. The
rates of force activation were measured before and after
phosphorylation of the fibers with Ca2+/CaM-activated MLCK.
SDS-Urea Gel Electrophoresis
The level of RLC phosphorylation in skinned skeletal muscle fibers was tested by using 11% polyacrylamide-SDS gels containing 6 M urea and 0.08 M Tris-glycine buffer, pH 8.6. Control (not treated) fibers as well as the MLCK-treated fibers were sonicated in a solution of 8 M urea for 25 min at room temperature and loaded onto mini-slab gels. The electrode buffer contained 0.08 M Tris-glycine, pH 8.6. The gels were silver stained (23) to increase the visibility of the protein bands, and the level of RLC-phosphorylation was quantified by densitometry of the stained gels.Statistical Analysis
Differences between the measurements of the Ca2+ sensitivity of the thin filament ATPase activity, force development, and kinetics of force activation of nonphosphorylated RLC vs. P-RLC were determined by using an unpaired Student's t-test (Sigma Plot 2000), with significance defined as P < 0.05.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Ca2+ Binding to Isolated RLC
The binding of Ca2+ to P-RLC or nonphosphorylated RLC and the effect of Mg2+ on this binding were studied with the fluorescence method, in which the Trp fluorescence of the single Trp residue of the RLC was monitored. The Ca2+ affinity to the single Ca2+-Mg2+ binding site of the RLC was confirmed with the flow-dialysis method. In agreement with Alexis and Gratzer (2), the KCa for nonphosphorylated rabbit skeletal RLC was ~2.26 × 105 M1 (reported 2.5 × 105 M1), as determined by the fluorescence
method. Phosphorylation of the RLC with Ca2+/CaM-activated
MLCK only slightly decreased its Ca2+ affinity
(KCa ~1.83 × 105
M1). In the presence of 2 mM Mg2+, the
apparent equilibrium constant of Ca (K'Ca)
decreased to 1.28 × 105 M1 and
3.89 × 104 M1 for nonphosphorylated RLC
and P-RLC, respectively. Similar KCa and
K'Ca values were obtained with the flow-dialysis
method. KCa was ~1.50 × 105
M1 for nonphosphorylated RLC and ~1.04 × 105 M1 for P-RLC in the absence of
Mg2+. K'Ca was ~6.02 × 104 M1 for nonphosphorylated RLC and
~5.19 × 104 M1 for P-RLC in the
presence of 2 mM Mg2+. Low KCa
values monitored either by the flow-dialysis or fluorescence method and
the low sensitivity to Mg2+ suggest that the binding of
Ca2+ to the isolated RLC may not reflect the physiological
situation observed in muscle. The Ca2+ affinity to the RLC
bound to myosin has been reported to be 100-fold higher than to
isolated RLC (14).
Regulation of Actin-Tm-Tn-Activated Myosin ATPase Activity by RLC Phosphorylation
DTNB treatment of skeletal muscle myosin resulted in ~70% RLC-deficient myosin (data not shown). This RLC-depleted myosin bound nonphosphorylated RLC and/or P-RLC with the same stoichiometry of intact untreated myosin. Figure 1 demonstrates the effect of the RLC phosphorylation on actin-Tm-Tn-activated myosin ATPase activity. Ca2+ regulation of the ATPase activity was determined for the control rabbit skeletal myosin (nonphosphorylated), RLC-depleted myosin, and myosin reconstituted with either P-RLC or nonphosphorylated RLC. Ca2+ sensitivity of actin-activated ATPase activity of myosin depleted of the decreased RLC by apCa50 of
approximately 0.15 ± 0.02 compared with untreated,
nonphosphorylated myosin. Reconstitution of the RLC-depleted myosin
with nonphosphorylated RLC did not significantly change the
pCa50 value (pCa50 = ~6.55 ± 0.02, n = 3, P > 0.1); however,
P-RLC-reconstituted myosin dramatically increased Ca2+
sensitivity of the actin-Tm-Tn ATPase activity. The difference expressed in the pCa50 units between P-RLC-reconstituted
and RLC-depleted myosin was a pCa50 of ~0.29
(n = 3, P < 0.01), and between P-RLC and the myosin reconstituted with nonphosphorylated RLC was a pCa50 of ~0.25 (n = 3, P < 0.01; Fig. 1).
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Skinned Fiber Studies
Steady-state maximal force.
To study the effect of phosphorylation of RLC, we have performed a
series of experiments by measuring the steady-state force development
in skinned skeletal muscle fibers with the use of the following
conditions (Table 1). 1)
Nonextracted fibers were phosphorylated with
Ca2+/CaM-activated MLCK; 2) RLC and TnC were
extracted from the fibers according to our previously published
procedure (48), and the fibers were reconstituted with
TnC; 3) after TnC reconstitution, the fibers were
reconstituted with nonhosphorylated RLC; 4) fibers were
phosphorylated with Ca2+/CaM-activated MLCK; or
5) alternatively the fibers were reconstituted with
prephosphorylated P-RLC. The experiments described here, including the
RLC extraction, reconstitution with exogenous TnC and RLC, and then
phosphorylation with Ca2+/CaM-activated MLCK, were time
consuming (3-4 h), and fiber rundown was observed. Therefore, the
level of maximal force after protein reconstitution was ~20-30%
lower compared with control fibers. All measurements were performed at
room temperature. As shown in Table 1, treatment of the control,
nonextracted skinned fibers with Ca2+/CaM-activated MLCK
slightly increased maximal steady-state force to 105 ± 4%
(n = 10). Reconstitution of the RLC-depleted fibers with nonphosphorylated RLC (and TnC) resulted in 68.9 ± 8.9%
force recovery compared with control fibers. Subsequent incubation of these reconstituted fibers with Ca2+/CaM-activated MLCK
resulted in an additional 15.4% increase in force (n = 7; Table 1). The observed increase in maximal force (to 84.3 ± 10.1%, P < 0.02) was clearly a result of the RLC
phosphorylation. Alternatively, when RLC-depleted fibers were
reconstituted with prephosphorylated RLC, the force recovery was higher
than for nonphosphorylated RLC (80 ± 6.9 vs. 68.9 ± 8.9%,
0.02 < P < 0.05; Table 1).
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Ca2+ Sensitivity of Force Development
Figure 2A demonstrates the effect of Ca2+/CaM-activated MLCK in skinned (not RLC extracted) skeletal muscle fibers on force-pCa dependence. In this example, a leftward shift, toward a lower [Ca2+], ofpCa50 ~0.14, was observed. No
shift (pCa50 of approximately 0.06 ± 0.05, data
not shown) was observed when the fibers were treated with MLCK buffer
alone (minus MLCK). Because control fibers used in the experiments
presented in Fig. 2A were partially phosphorylated, this
yielded a significant variation (standard deviation) in the pCa50 values (5.58 ± 0.08). Figure 2B
summarizes the effect of phosphorylation on the force-pCa relationship
for fibers 1) depleted of RLC, 2) reconstituted
with nonphosphorylated rabbit skeletal RLC (and TnC), and 3)
were then phosphorylated with the Ca2+/CaM MLCK. Extraction
of the RLC led to a rightward shift in the force-pCa relationship, as
shown previously (48). When nonphosphorylated RLC
was reincorporated into the fibers, there was no further change in the
force-pCa dependence; however, when the reconstituted fibers were
exposed to Ca2+/CaM-activated MLCK, the force-pCa
relationship matched that of the unextracted skinned fibers. Again,
this suggests that the control fibers used in this experiment were
partially phosphorylated. This result accounts for our original results
(48), where we were unable to fully restore the
Ca2+ dependence of the extracted fibers to the control
values with rabbit nonphosphorylated RLC. Similarly, when the
RLC-extracted fibers were reconstituted with rabbit P-RLC, the original
Ca2+ dependence of force was restored (Fig.
2C). Interestingly, the fibers chosen for the experiments
shown in Fig. 2C, where P-RLC was reconstituted into
RLC-depleted fibers, were phosphorylated during skinning procedure with
endogenous MLCK. The pCa50 = 5.67 of these control
fibers was then different from these from Fig. 2, A and
B (pCa50 = 5.58).
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Effect of Phosphorylation of Endogenous RLC on the Ca2+ Sensitivity of Force Development in Untreated Skinned Fibers
A very strong correlation exists between the level of endogenous phosphorylation of the RLC in muscle fibers and the force-pCa relationship. SDS-urea gel electrophoresis was used to measure the level of RLC phosphorylation in the control skinned skeletal muscle fibers. As shown in Fig. 3, the fibers used in steady-state measurements had different levels of RLC phosphorylation. Fresh fibers usually demonstrated a high level of RLC phosphorylation, which decreased gradually as the fibers were stored over time in 50% glycerol at20°C. Figure 3A
demonstrates an example of the fully phosphorylated (lane 5)
or fully dephosphorylated (lane 4) fibers. Figure
3B is an example of 60% phosphorylated fibers (lane
2). The force-pCa measurements clearly indicated that as the level
of phosphorylation of the RLC was increased, so was the
Ca2+ sensitivity of force development and vice versa. This
was confirmed in experiments with fully phosphorylated vs. fully
dephosphorylated fibers (Fig. 4). These
fibers were not treated with exogenous MLCK but phosphorylated during
their preparation (skinning) with endogenous MLCK. As expected,
RLC-phosphorylated fibers (pCa50 = 5.68 ± 0.02, n = 4) demonstrated higher sensitivity to
Ca2+ than RLC-dephosphorylated fibers
(pCa50 = 5.53 ± 0.03, n = 4), with pCa50 ~0.15 (P < 0.01).
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Effect of Phosphorylation of the Endogenous RLC on the Kinetics of Force Development in Skinned Fibers
Interestingly, studies of the effect of RLC phosphorylation on the kinetics of force activation with the caged Ca2+, DM-nitrophen, showed no significant change in the rates of force development (Table 2). The protocol for these experiments (Ca2+ dependence of steady-state force and the kinetics of force activation on phosphorylation of the RLC) is presented in Fig. 5. It demonstrates a typical flash photolysis experiment that was reproduced several times (n = 7; Table 2), with slight variations between experiments. As shown, the transient force was lower after the second UV exposure. This is probably due to either fiber rundown or to the incomplete equilibration of the caged chelator before the first flash. After the first flash, the subsequent flashes yielded the same force transient amplitude. Table 2 summarizes the activation constants (k1 and k2) calculated by using a two-exponential fit analysis of the experimental data. The k1 constant expresses a rapid activation rate of contraction, whereas the k2 constant is a slow component, possibly due to diffusion processes related to reequilibration of the fiber with the bulk solution after the flash. Even though there was a significant change in the force-pCa relationship with RLC phosphorylation (P < 0.01), only a slight phosphorylation-dependent increase of low statistical significance (0.05 < P < 0.1) in the rates of force activation was observed (Table 2).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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RLC of myosin are a member of the Ca2+-binding
"EF-hand" protein family like TnC, CaM, parvalbumin, or the alkali
light chains of myosin (3, 21). The divalent cation
binding site of RLC is located in the first 
-helix-loop--helix
motif in the NH2-terminal domain of RLC. In agreement with
previous studies (2), we have shown that this site in
isolated RLC binds either Ca2+ or Mg2+. The low
affinity for Ca2+ (in the range of 105
M1) slightly decreased on the phosphorylation of the RLC.
The KCa of phosphorylated RLC was ~1.4-fold
lower than that of nonphosphorylated RLC. It has been shown that the
affinity of RLC for both cations increases by a factor of 100 in
skeletal myosin (14). Because RLC binds Ca2+
and Mg2+ in a competitive way, it was expected that the
K'Ca determined in the presence of Mg2+ was
smaller for both P-RLC and nonphosphorylated RLC. However, the extent
of the Mg2+-induced change in the Ca2+ affinity
of isolated RLC was not large (2- to 4.7-fold). This suggests that the
RLC specificity for Ca2+ may change depending on the
complexity of the system (isolated state, bound to myosin, bound to
myosin in muscle). Likewise, the effect of RLC phosophorylation could
be different in the isolated state and when bound to myosin in the
muscle cell.
The structural significance of RLC in skeletal muscle contraction has been addressed in previous studies (48) and has been studied intensively by others using various extraction/reconstitution methods applied to skinned muscle fibers. The studies of Moss et al. (30), Hofmann et al. (13), Metzger and Moss (25), and Patel et al. (31) have shown that partial extraction of RLC from skeletal muscle fibers increased the rate of tension redevelopment at submaximal [Ca2+]. Our laboratory's studies (48) have revealed that removal of RLC decreased the rate of force development by a factor of two and that this could be restored by reincorporation of RLC in the fibers. In the present work, we have investigated the effect of RLC phosphorylation and Ca2+ binding to RLC on the regulation of skeletal muscle contraction (46). Although regulation in vertebrate striated muscles occurs via Ca2+ binding to the thin filament proteins, the binding of Ca2+ to RLC and phosphorylation of RLC with Ca2+/CaM-activated MLCK seem to play a role in these regulatory processes (Ref. 43 and references within). Numerous in vivo studies, which have utilized intact skeletal muscles, have demonstrated that the level of myosin phosphorylation significantly increases after tetanic stimulation (15, 19, 27) or a low-frequency repetitive stimulus train (staircase potentiation) (43). Studies on skinned skeletal muscle fibers have shown that phosphorylation of RLC slightly increased the Ca2+ sensitivity of isometric tension and the rate of force development (24, 43, 44). On the basis of this information, it was proposed that RLC phosphorylation causes potentiation of isometric twitch tension by increasing the sensitivity of the contractile proteins to Ca2+.
Consistent with Metzger et al. (24) and Sweeney
et al. (43), we have shown that the force-pCa relationship
was shifted toward lower concentrations of Ca2+ as a result
of RLC phosphorylation, although the effect seen by these authors was
much smaller than the one observed in this study. Likewise, our
laboratory's preliminary work (46, 47) and this study
demonstrate that phosphorylation of the RLC not only increases the
Ca2+ sensitivity of force development (pCa50
~0.15) but also raises the maximal steady-state force (Table 1). Godt
and Nosek (8) also reported phosphorylation-dependent
increases in maximal force in frog muscle fibers under fatigue
conditions. It is worth mentioning that the RLC
phosphorylation-dependent changes in the contractility of skinned
skeletal muscle fibers assessed in this study were much larger than had
been reported by others. The effect of RLC phosphorylation was even
more pronounced in the ATPase activity assays performed on
reconstituted thin filaments with the use of the RLC (with or without
phosphorylation)-reconstituted myosin (Fig. 1). P-RLC induced a large
shift toward lower [Ca2+] in the actin-Tm-Tn-activated
myosin ATPase activity. Compared with the myosin reconstituted with
nonphosphorylated RLC, pCa50 was ~0.25. The initial
maximal level of the ATPase activity determined for untreated
nonphosphorylated myosin was decreased after RLC depletion and fully
recovered after RLC reconstitution. To our knowledge, this is the first
report of such a significant effect of RLC phosphorylation on the
Ca2+ sensitivity of the Tm/Tn-regulated actomyosin ATPase
activity. An important factor that may influence the effect of RLC
phosphorylation on force/ATPase measurements is the initial level of
RLC phosphorylation without MLCK added. Our skinned fiber results
suggest that the endogenous level of RLC phosphorylation is crucial for
determining the level of the Ca2+ sensitivity of force and
important for the proper evaluation of the effects of the RLC
extraction/reconstitution on the Ca2+-sensitivity of the
force. As demonstrated in our laboratory's previous study
(48), depletion of the RLC from skinned fibers resulted in
a decrease in the Ca2+ sensitivity of force development.
Our laboratory also demonstrated that reconstitution of RLC-depleted
fibers with nonphosphorylated RLC did not restore the Ca2+
sensitivity of force development. The results presented in this paper
have shed light on this initial observation. As shown in Fig. 2,
B and C, only P-RLC was able to restore the
force-pCa dependence to the level of not extracted fibers when
reconstituted in RLC-depleted fibers. We also found that the protocol
for the preparation of skinned fibers utilized previously
(48) resulted in various levels of RLC phosphorylation
(usually >50%), whereas the protein used for reconstitution was
always nonphosphorylated. The protocol for the preparation of fully
dephosphorylated skinned fibers used in this study (see MATERIALS
AND METHODS) resulted in dephosphorylated RLC and allowed us to
investigate the effect of RLC phosphorylation in nonextracted skinned
fibers. Figure 4 demonstrates the force-pCa relationship for two types
of skinned skeletal fibers (obtained with two different methods)
containing either fully phosphorylated or fully dephosphorylated RLC.
Both types of fibers were prepared under conditions that either
activated or deactivated endogenous MLCK with no exogenous enzymes
added. As expected, the fibers containing dephosphorylated RLC were
less sensitive to Ca2+ than the phosphorylated ones, by
pCa50 ~0.15 ± 0.02. In summary, as the level of
phosphorylation of the RLC was increased, so was the Ca2+
sensitivity of force development and vice versa. Thus, depending on the
level of endogenous RLC phosphorylation, the rightward shift in
Ca2+ dependence after RLC extraction could vary. Perhaps
various phosphorylation-dependent changes in the Ca2+
sensitivity of force development presented by other laboratories resulted from different levels of initial phosphorylation of the RLC.
This seems to be a crucial issue in the proper determination of the
maximal effect of RLC phosphorylation on the Ca2+
sensitivity of force development in skinned skeletal muscle fibers. As
we have shown, endogenous RLC phosphorylation (in the control fibers)
can vary, and this effect may attenuate the difference in
pCa50 between different fibers containing either P-RLC or
nonphosphorylated RLC. Therefore, the initial level of the RLC
phosphorylation should be controlled or at least known in all studies
involving measurements of the Ca2+ sensitivity of force
development in skinned muscle fibers. Interestingly, consistent with
previous reports (24, 45), phosphorylation of the RLC in
skinned skeletal muscle fibers only slightly increased the kinetics of
force activation; however, this change was of low significance
(0.05 < P < 0.1; Table 2). We are planning more detailed future experiments to assess the effect of RLC phosphorylation on the kinetics of force development in this skeletal muscle system. The question remaining is: How does phosphorylation of the RLC modulate
skeletal muscle contraction? Does the phosphorylation of the RLC
directly affect the interaction of myosin and actin or is it an
indirect allosteric effect of the RLC on the Ca2+ binding
to TnC in the thin filaments of skeletal muscle? Additionally, the
relationship between phosphorylation of RLC and metal binding to the
single Ca2+ and Mg2+ binding site on the RLC
needs to be established. It is possible that these two important
regions of RLC, the Ca2+-binding and the phosphorylation
sites, are communicating with each other and that the binding of
Ca2+ to this site is required to see the
phosphorylation-dependent effects in muscle contraction. Although the
RLC phosphorylation-dephosphorylation process is too slow to be an
obligatory mechanism for skeletal muscle contraction, it plays a role
in maintaining a specific level of force at a lower
[Ca2+] and could be important for working muscle (e.g.,
improving performance, etc.). The phosphorylation-dependent enhancement
of muscle function through increases in actomyosin ATPase activity and
tension as the free [Ca2+] progressively rises are
especially important under kinetic conditions in which the
[Ca2+] in the muscle cell does not saturate the thin
filament regulatory system. At the molecular level, the
phosphorylation-dependent force potentiation may simply result from the
recruitment of more strongly bound cross bridges as the phosphorylation
of the RLC causes cross bridges to move away from the thick filament
backbone and become more accessible to actin (43).
In summary, phosphorylation of the RLC had a dramatic effect on the
Ca2+ sensitivity of the ATPase activity of reconstituted
thin filaments (pCa50 ~0.25). Likewise, the
Ca2+ sensitivity of force development also increased on RLC
phosphorylation (pCa50 ~0.15). In addition, maximal
steady-state force of the P-RLC fibers was ~15% higher than fibers
reconstituted with nonphosphorylated RLC. These results suggest that
although the thin filament proteins Tn and Tm mediate the regulation of
skeletal muscle contraction, the role of RLC in these processes cannot
be ignored and need to be further explored.
| |
ACKNOWLEDGEMENTS |
|---|
This work was supported by National Institutes of Health Grants AR-45183 (to J. D. Potter), HL-06296 and HL-26043 (to J. T. Stull), and AHA Grant 9808237V (to D. Szczesna).
| |
FOOTNOTES |
|---|
Address for reprint requests and other correspondence: J. D. Potter, Professor and Chairman, Dept. of Molecular and Cellular Pharmacology, Univ. of Miami School of Medicine, 1600 N.W. 10th Ave., Miami, FL 33136 (E-mail: jdpotter{at}miami.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published December 21, 2001;10.1152/japplphysiol.00858.2001
Received 15 August 2001; accepted in final form 17 December 2001.
| |
REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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): pCa50 = 6.66 ± 0.05, n = 2; RLC-depleted myosin
(
): pCa50 = 6.51 ± 0.03, n = 3; myosin reconstituted with nonphosphorylated RLC
(
): pCa50 = 6.55 ± 0.02, n = 3; myosin reconstituted with P-RLC
(
): pCa50 = 6.80 ± 0.04, n = 2. The difference between P-RLC and
RLC-reconstituted myosins was ~0.25 (n = 3, P < 0.01). pCa, pH of Ca2+. Data are
means ± SD of n = 2-3 experiments.
; pCa50 = 5.39 ± 0.04, n = 6); (2),
TnC-reconstituted fibers were subsequently reconstituted with
nonphosphorylated RLC (
; pCa50 = 5.41 ± 0.08, n = 6); (3), fibers were
then treated with MLCK (
