(Received for publication, October 25, 1995)
From the
We investigated the kinetics of Ca activation
of skeletal muscle contraction elicited by the photolysis of caged
Ca
. Previously we showed that partial extraction of
the 18-kDa regulatory light chains (RLCs) of myosin decreased the rate
of force development and was subsequently increased by
20%
following reconstitution with RLCs (Potter, J. D., Zhao, J. and Pan, B.
S.(1992) FASEB J. 6, A1240). We extend here the RLC-extraction
study to the complete removal of the RLCs. The complete removal of RLCs
was achieved by a combination of 5,5`-dithiobis-(2-nitrobenzoic acid)
and EDTA treatment followed by reduction of oxidized sulfydryl groups
by dithiothreitol. Under these conditions the complete extraction of
RLCs was accompanied by the extraction of endogenous troponin C,
resulting in the loss of isometric tension. Steady state force was
restored to 65-75% following troponin C reconstitution and
increased to 75-85% as a result of RLC reincorporation into the
fibers. The rates of force transients generated by UV-flash photolysis
of
1-(2-nitro-4,5-dimethoxyphenyl)-N,N,N`,N`-tetrakis[(oxycarbonyl)methyl]-1,2-ethanediamine)
or nitrophenyl-EGTA, photoliberating bound Ca
,
decreased 2-fold after RLC extraction and troponin C reconstitution and
then increased to the values of intact fibers after RLC reconstitution.
These results support our earlier findings that the regulatory light
chains of myosin play an important role in the kinetics of cross-bridge
cycling.
Vertebrate striated muscle contraction is triggered by the
binding of Ca to troponin C (TnC), (
)the
Ca
binding subunit of troponin, which together with
tropomyosin forms the regulatory system of the contractile apparatus
(for review see (2) and (3) ). Contraction of
molluscan and vertebrate smooth muscles is regulated via myosin, which
either binds Ca
directly (molluscan) (4, 5, 6) or undergoes a
Ca
/calmodulin activated phosphorylation at the myosin
regulatory light chains (RLCs) in smooth
muscle(7, 8, 9) . The recently determined
crystal structure of the ``regulatory domain'' of scallop
myosin reveals complexity in the structure of this myosin-linked
regulatory system. Although the activating binding site for
Ca
is on the essential light chain (ELC) of myosin,
the three protein complex (myosin, RLC, ELC) regulates
contraction(10) . It consists of intact ELC, part of myosin
heavy chain (6 amino acid residues), and almost entire (145 amino
acids) RLC, which contains a Ca
-Mg
binding site. Binding of Ca
to the
Ca
-specific site on the ELC of molluscan myosin is
accompanied by the cooperative interaction of RLC and the heavy chain.
Since RLCs appear to be a primary regulatory component of the thick
filament-linked systems it is of interest to understand their possible
role in thin filament regulated skeletal muscle. The crystal structure
of skeletal myosin subfragment 1 (11) reveals that RLCs are
located at the head-rod junction of the myosin molecule, implying a
possible role for RLCs in cross-bridge cycling in contracting
muscle(12) . Irving et al.(13) , demonstrated
that the RLC region of the myosin head tilts during active force in
skeletal muscle. RLCs exhibit a high level of primary sequence homology
with other Ca-binding proteins like calmodulin, TnC
and parvalbumin(14, 15, 16) . The high
affinity Ca
-Mg
binding site of RLCs
is occupied by either Mg
or Ca
and
is located in the first helix-loop-helix motif of the
NH
-terminal domain of the RLC. Under physiological
conditions in relaxed muscle this site is probably occupied by
Mg
(17) and may become saturated with
Ca
, depending on the time course of the
Ca
transient(18) . Since the process of
cation exchange occurs with a much slower rate than muscle activation,
the functional importance of this site is unknown. In a recently
published site-directed mutagenesis study, the high affinity
Ca
-Mg
site of RLC was replaced by
one of TnC's Ca
-specific sites, and this mutant
was unable to regulate contraction of scallop muscle whose endogenous
myosin RLC was replaced by this mutated one(19) .
In contrast to molluscan or smooth muscle myosin RLCs where their roles are quite well defined, their functional significance in skeletal muscle contraction is still not entirely clear. It has been shown that myosin RLCs may affect force development in skeletal muscle fibers (20, 21) or affect the conversion of chemical energy into movement in motility assays(22) . It has been also shown that the cation binding site of RLC may have a modulatory role in skeletal muscle contraction (23) .
The question of the
physiological importance of the RLCs in skeletal muscle contraction is
addressed in the present study. To test if RLCs affect cross-bridge
cycling in force producing skeletal muscle fibers, we measured the
Ca-dependent activation elicited by the photolysis of
caged Ca
in the RLC-depleted fibers in comparison to
RLC-reconstituted fibers. Our preliminary study (1) in
agreement with the study of Hofmann et al.(20) , have
shown that partial extraction of RLCs from the fibers changes the rate
of force development. In this study we demonstrate that the complete
removal of RLCs from the fibres decreases the rate of force production
by about 2-fold and that reconstitution with rabbit skeletal myosin
RLCs restored the rate of force development to the value of intact
unextracted fibers.
Skeletal muscle fibers
isolated from rabbit psoas muscle were dissected into small bundles and
chemically skinned with 50% glycerol, 1% Triton X-100 in the relaxing
solution (the pCa 8 solution) containing 10M Ca
, 1 mM Mg
, 7 mM EGTA, 5 mM MgATP
, 20 mM imidazole, pH 7.0, 20
mM creatinine phosphate, and 15 units/ml of creatine
phosphokinase, ionic strength = 150 mM, for 24 h at 4
°C. Fibers were then stored at -20 °C in the same
solution without Triton X-100 for 2-4 weeks.
In previous experiments (1) the RLCs were extracted from the fibers by incubation in the solution of 2 mM EDTA, pH 7.8. This procedure resulted in only partial (20-40%) extraction of RLCs from the fibers.
Reconstitution of the TnC-replenished fibers with isolated rabbit skeletal RLCs was achieved by incubation of the fibers in a solution of 30 µM RLC (in pCa 8 buffer) for 1 h at room temperature. In the control experiments the TnC was present in the RLC's reconstitution solution to prevent the possible extraction of TnC from the fibers during the RLC incorporation.
In control experiments fibers were tested for the restoration of steady state force following the DTNB treatment. After the initial force measurements, the fibers were exposed to a solution containing 10 mM DTNB in the pCa 8 buffer, pH 7.5, for 5 min at 4 °C. This treatment completely lacked force development in the pCa 4 solution presumably due to oxidation of cystein SH- groups in the fiber proteins. The fibers were then immersed in the pCa 8 solution containing DTT as described earlier. After this treatment, the steady state force was restored to about 90%.
The
CaDependence of Force
Development-After measurement of the initial steady state
force of the unextracted fibers, they were relaxed in the pCa
8 buffer and then exposed to solutions of increasing
[Ca
] concentrations (from pCa 8 to pCa 4). The maximal force was measured in each
``pCa'' solution followed by a short relaxation of
the fibers in the pCa 8 solution. The same procedure was
applied to the RLC-depleted fibers following the TnC reconstitution and
again after the complete reconstitution of the fibers with RLCs. Data
were analyzed with the Hill equation: percent relative force = 100
[Ca
]
/([Ca
]
+ pCa
), where pCa
is the pCa of a solution which gives
50% of the force produced and n is the Hill coefficient.
Figure 1: The SDS-gel electrophoresis pattern of intact skinned muscle fibers (A), RLCs-extracted fibers (B), and the RLCs-reconstituted fibers (C). The migration of purified RLCs and TnC are presented in D and E, respectively. The electrophoresis was performed on the 12% acrylamide gels with a cross-linking ratio of acrylamide to N,N`-methylene bis-acrylamide = 61:1. The pH of the resolving gel was 9.3. The fibers presented in this figure were the same as those used for the force measurements described in Fig. 3. A, actin; Tm, tropomyosin; TnT and TnC, troponin T and C; LC1 and LC3, essential light chains of myosin; RLC, regulatory light chain of myosin.
Figure 3:
The protocol for steady state force
measurements following (i) extraction of RLCs and TnC from the fibers,
(ii) reconstitution of the fibers with TnC, and (iii) reconstitution
with RLCs and TnC. Incubation of the fibers with the RLC-extraction
solution (see ``Materials and Methods''), resulted in the
loss of the force development as a result of the parallel dissociation
of TnC from the fibers. Readdition of TnC back to the fibers restored
72% of the force as compared to the unextracted fibers. Further
reconstitution of the fibers with the RLCs (in the presence of TnC)
increased the force level to
82%. Between force measurements the
fibers were relaxed in the pCa 8
solution.
The reversible effect of the DTNB treatment
on the fibers was tested using steady state force measurements (Fig. 2). Incubation of the fibers with the pCa 8
solution containing 10 mM DTNB (pH 7.5) under nonextracting
conditions (no EDTA) resulted in the complete loss of isometric
tension. About 90% of force was recovered by the subsequent exposure of
the fibers to the solutions containing 2-30 mM DTT, as
shown in Fig. 2. This reversible effect of DTNB with DTT
treatment was also tested in the kinetic experiments where no
difference in the time course of force activation was seen after this
treatment, as determined by the photoliberation of Ca by DM-nitrophen (data not shown).
Figure 2: Restoration of steady state force after DTNB treatment. The skinned muscle fibers were tested for steady state force development in the pCa 4 solution and then transferred to the solution (see ``Materials and Methods'') containing 10 mM DTNB, pH 7.5, for 5 min at 4 °C. The force measurements were then repeated. No isometric tension was developed by the fibers when tested in the pCa 4 solution after blocking the SH- groups. Reduction of the sulfydryl groups with the 2-30 mM DTT solutions for 1 h resulted in restoration of 89% force of the original intact skinned fiber.
As shown, extraction of the RLCs from the fibers was paralleled by the dissociation of TnC what subsequently resulted in the loss of force (data of 15-20 experiments). After readdition of TnC back to the fibers force was restored to 65-75% and increased to 80 ± 5% following full reconstitution of the fibers with TnC and RLCs.
Figure 4:
The Ca dependence of
force development of unextracted fibers (
); RLCs-depleted (TnC
reconstituted) fibers (
); and RLCs reconstituted fibers
(
). The pCa
of unextracted fibers was 5.39
± 0.03 and the Hill coefficient n = 1.87.
RLC-depleted fibers demonstrated a decreased Ca
sensitivity of the force development. The pCa-force
relationship shifted toward higher Ca
concentrations
with pCa
= 4.80 ± 0.06 and the Hill
coefficient n = 1.6. Full reconstitution of the fibers
with RLCs resulted in pCa
= 4.93 ±
0.04 and n = 1.82. Data points are the average of four
independent experiments.
Figure 5:
Ca activated force
transients measured with DM-nitrophen (left panels) or NP-EGTA (right panels) of untreated (A), RLC-depleted (TnC
reconstituted) (B), and RLC- and TnC-reconstituted (C) fibers.
We have shown here that the regulatory light chains of myosin
significantly affect the rate of activation of skeletal muscle fibers.
The regulation of muscle contraction in vertebrate striated muscles
occurs via Ca binding to the thin filament regulatory
protein TnC(2) . The results presented here show additionally
that RLCs play an important modulatory role in skeletal muscle
contraction. The obvious question that arises from this is what is the
mechanism of this RLC-induced modulation of skeletal muscle
contraction? In the present study we investigated the structural role
of the RLCs in skeletal muscle activation, utilizing skinned skeletal
muscle fibers where steady state force development and the kinetics of
muscle activation were readily determined. The method we have developed
allows for the efficient extraction of RLCs from these skinned fibers
and their subsequent reconstitution with isolated RLCs. This method
resulted in the complete removal of RLCs (Fig. 1) at room
temperature, conditions which did not require heating of the fibers to
30-37 °C as in previous studies(21, 31) .
Extraction of RLCs did not influence the maximal steady state force (Fig. 3) but significantly affected the rate constants of the
force activation transient (Table 1). The kinetics of muscle
activation monitored with two different Ca
chelators
(NP-EGTA and DM-nitrophen) showed the same decrease in the rate of
force development in RLC-depleted fibers compared to nonextracted
fibers. Further reconstitution of the fibers with RLCs restored the
rate of force development to about 80% of the values of intact fibers.
The decreased rates in the RLC-extracted (TnC-reconstituted) fibers
were accompanied by a decrease in the Ca sensitivity
of steady state force development where the force-pCa
relationship shifted toward higher Ca
concentrations,
(
pCa
= -0.59). Although the
RLC-reconstituted fibers had essentially the same rates of activation
as the intact fibers, the Ca
dependence of steady
state force development did not return to that of the nonextracted
fibers. This observed decrease in the Ca
sensitivity
of force development affected the magnitude of the Ca
activated force transient (Fig. 5). Due to this rightward
shift in the force-pCa relationship toward higher
Ca
concentrations, the amplitude of the force
transients of the treated fibers dropped to
10% of the value of
untreated fibers exposed to the same [Ca
]
transient. To exclude the possible effect of irreversible oxidation of
sulfydryl groups on the force-pCa relationship, the
Ca
dependence of force development was measured after
a combined DTNB/DTT treatment under nonextracting conditions (absence
of EDTA). No difference between untreated and DTNB/DTT-treated fibers
was observed (data not shown). Thus the rightward shift toward higher
[Ca
] observed for RLC-depleted (TnC
reconstituted) fibers is due to extraction of the RLCs from the fibers
and is not due to the reversible modification of sulfydryl groups. This
result of decreased Ca
sensitivity of force
development is not consistent with the findings of Metzger and
Moss(21) , who utilized a different extraction procedure that
removed 60% of the RLCs. In those experiments, they observed a leftward
shift of the tension-pCa relationship to lower Ca
concentrations. Further studies will be necessary to explain
these differences. Also, further experiments will be performed to
determine why the force-pCa relationship does not return to
the initial values upon full reconstitution of the fibers with RLCs and
TnC.
The present work extends our previous findings that even
partial extraction of RLCs from the fibers changes the kinetics of
force activation(1) . Previously, 35 ± 5% extraction of
RLCs from the fibers was achieved with a solution containing 2 mM EDTA, pH 7.8, as compared to the 90 ± 5% extraction
obtained in the present study. Moreover, in this work we employed the
specific Ca chelator, NP-EGTA which binds
Mg
with a much lower affinity (K
= 9
10
M) than
DM-nitrophen (K
= 2.5
10
M) (30) used in the earlier
study. The reason for this was to test whether the observed differences
in the rates of force development were exclusively due to the
extraction of RLCs from the fibers and not to the changes in
[Mg
] that, although minimized, occur with
DM-nitrophen solution. The results demonstrate that both caged
compounds gave similar force transients. Thus, the different rates of
activation seen for the native fibers, RLC-depleted (TnC-reconstituted)
and RLC-reconstituted fibers are clearly related to the presence or
absence of the myosin regulatory light chains.
In accord with our results, Lowey et al.(22) , demonstrated that removal of the RLCs from skeletal myosin reduced the velocity of single actin filaments migrating on a myosin coated surface in an in vitro motility assay. The velocity of the actin movement was restored when myosin was reconstituted with RLCs. The effect was even larger when myosin was extracted and then reconstituted with both RLC and ELCs. Interestingly, the actin-activated ATPase activity of the light chain-depleted myosin did not follow the changes in the sliding velocity of single actin filaments. Based on these interesting results the authors concluded that even though the light chains were not important for the enzymatic activity of skeletal myosin, their interactions with the heavy chain of myosin played an important role in the conversion of chemical energy into movement. Moreover, as was demonstrated by VanBuren et al.(32) , the extraction of the regulatory light chains of myosin had no effect (about 3%) on isometric force as measured in an in vitro motility assay utilizing the force measurement method developed and described by Kishino and Yanagida(33) . Our data fully support these findings in that although RLCs do not effect the maximal steady state isometric force development, they are important determinants of the kinetics of cross-bridge cycling in skeletal muscle fibers.
The
location of the RLCs within the myosin head implies a possible
significance of RLCs for the interaction of cross-bridges with actin
during contraction(11, 12, 13) . The crystal
structure of skeletal myosin S1 (11) shows that RLCs are
wrapped around a 10-nm long single -helix of the heavy chain near
the COOH-terminal region of the myosin head. It has been hypothesized
that this part of the heavy chain acts like a lever arm of the working
cross-bridge(34) . Regarding the structural organization of the
COOH-terminal region of the myosin head, it is possible that the RLCs
located in this region may affect force-generating cross-bridges.
However, the slower rate of the Ca
-dependent
activation of skeletal muscle fibers after RLCs extraction (Table 1) with little effect on maximal steady state force (Fig. 3) suggests that rather than the strength and stability of
the myosin lever arms, RLCs affect their kinetics.
Further
experiments will be necessary to investigate the role of the
Ca-Mg
binding site located in the
NH
-terminal region of RLC in force generation and the
Ca
-dependent activation of muscle contraction.
Interestingly, as was recently shown by Diffee et
al.(23) , an avian mutant of RLC having reduced affinity
for Ca
, did not restore maximal tension when
exchanged with endogenous RLC in skeletal muscle fibers. We do not
believe that Ca
binding to the RLCs influences the
rate of force development in the photolysis, since the time course for
the exchange of Mg
for Ca
would be
very slow, primarily due to the slow dissociation of
Mg
(17, 18, 35) , compared
to the time course of muscle activation that we measured. We can not
make the same argument in the steady state force-pCa
measurements in this case since there would be sufficient time for the
exchange of Mg
for Ca
and it is
possible that Ca
binding to the RLCs may affect this
process. Further experiments with mutant light chains will hopefully
answer this question.
It would also be interesting to know if
phosphorylation of RLCs at the serine residues (Ser-14, Ser-15) affects
the Ca-dependence of force development and the rate
of activation in skeletal muscle(36) . Phosphorylation of RLCs
plays a primary role in the Ca
regulation of smooth
muscle contraction (7, 8, 9) and may somehow
further modulate the activation of skeletal muscle contraction (37, 38, 39) possibly by influencing the
interaction of skeletal myosin with
actin(24, 40, 41) . In summary, although the
Ca
control of skeletal muscle contraction is
regulated by the thin filament proteins, troponin and tropomyosin, the
regulatory light chains appear to modulate this process once activated.