Modulation of Contractile Activation in Skeletal Muscle by a
Calcium-insensitive Troponin C Mutant*
Carl A.
Morris
§,
Larry S.
Tobacman¶, and
Earl
Homsher
From the
Department of Physiology, School of
Medicine, University of California, Los Angeles, CA 90095 and the
¶ Departments of Internal Medicine and Biochemistry, University of
Iowa, Iowa City, IA 52252
Received for publication, August 14, 2000, and in revised form, March 2, 2001
 |
ABSTRACT |
Calcium controls the level of muscle activation
via interactions with the troponin complex. Replacement of the native,
skeletal calcium-binding subunit of troponin, troponin C, with mixtures of functional cardiac and mutant cardiac troponin C insensitive to
calcium and permanently inactive provides a novel method to alter the
number of myosin cross-bridges capable of binding to the actin
filament. Extraction of skeletal troponin C and replacement with
functional and mutant cardiac troponin C were used to evaluate the
relationship between the extent of thin filament activation (fractional
calcium binding), isometric force, and the rate of force generation in
muscle fibers independent of the calcium concentration. The experiments
showed a direct, linear relationship between force and the number of
cross-bridges attaching to the thin filament. Further, above 35%
maximal isometric activation, following partial replacement with
mixtures of cardiac and mutant troponin C, the rate of force generation
was independent of the number of actin sites available for cross-bridge
interaction at saturating calcium concentrations. This contrasts with
the marked decrease in the rate of force generation when force was
reduced by decreasing the calcium concentration. The results are
consistent with hypotheses proposing that calcium controls the
transition between weakly and strongly bound cross-bridge states.
 |
INTRODUCTION |
The cyclic interaction of myosin and actin produces force and
shortening in contractile cells. In muscle fibers, actin and myosin
interaction is regulated by the intracellular calcium concentration acting through the thin filament regulatory proteins, the troponin complex, and tropomyosin. Until calcium binds to the troponin complex,
the muscle fiber remains relaxed with >95% of the myosin cross-bridges detached (1, 2). The influx of calcium into the filament
lattice of muscle fibers stimulates the association of actin and myosin
enabling the production of force or shortening and accelerating the
actomyosin ATPase rate by >100-fold during isometric contraction.
Several models have been postulated to account for this control. The
steric-blocking model of cross-bridge regulation asserts that
tropomyosin/troponin (Tm/Tn)1
prevents cross-bridge attachment in the absence of calcium by "blocking" cross-bridge access to binding sites on the thin
filament (3, 4). Alternatively, the kinetic regulation model assumes that the cross-bridges can, under all conditions, bind weakly to the
thin filament, and calcium controls the kinetics of cross-bridge turnover via changes in the weakly bound to strongly bound cross-bridge transition (5, 6). More recently, three-dimensional reconstructions of
electron micrographs have identified three distinct structural states
of the thin filament (7-9). In the absence of calcium, tropomyosin
blocks strong myosin binding sites on actin. Following Ca2+
binding to the troponin complex, the tropomyosin shifts away from the
myosin binding sites but does not completely expose all the putative
strong binding sites on actin. Further movement of the tropomyosin
requires strong cross-bridge binding to fully expose the myosin binding
sites. Full activation of the thin filament requires Ca2+
binding to the troponin complex and subsequent strong cross-bridge binding to the thin filament.
Brenner and Eisenberg (10) developed a method to measure the kinetics
of cross-bridge transitions from weakly bound to force-producing states
in activated muscle fibers and found the rate of tension development
(ktr) to be calcium-sensitive (6). This result is inconsistent with the steric-blocking mechanism, and Brenner (6)
suggested that Tm/Tn controlled the rate of Pi
release. However, subsequent work showed that the kinetics of
Pi release are independent of [Ca2+]
(11-14). Further, others have found the kinetics of cross-bridge cycling to be unaffected by compounds that affect thin filament dynamics (15). Taken together, these studies indicate that calcium is
regulating muscle activation by control of cross-bridge access. To
reconcile these observations with Brenner's data and hypothesis it was
proposed that [Ca2+] controls the transition from weak to
strong cross-bridge binding preceding the generation of
force (11, 16).
To this point, studies have investigated calcium regulation of muscle
contraction by adding various compounds, removing proteins, or
adjusting the free calcium concentration. These investigations have
left several issues unresolved. In particular, when the free calcium
concentration rises, it is unclear how this increases the rate of force
generation. Does the effect require a relatively high density of myosin
binding to actin, which tends to activate the thin filament, or does
calcium binding to troponin have a more direct effect on
ktr?
In the present study we describe a method to control the fraction of
troponin complexes to which calcium is bound, thereby also controlling
the fraction of the thin filament available for myosin binding while
maintaining the free calcium concentration constant at a high level.
The native skeletal TnC was extracted from thin filaments of skinned
muscle fibers and replaced with variable combinations of cardiac TnC
and an inactive cardiac mutant TnC, CBMIITnC (17, 18). Cardiac TnC has
a single regulatory calcium binding site (site II) as site I does not
contain the necessary charged residues to bind calcium (19, 20).
Mutation of two negatively charged residues (Asp-65 and Glu-66) to
neutral alanine residues prevents calcium binding to site II (18) and blocks activation of thin filament-myosin S1 MgATPase activity by Ca2+ (21). The CBMII TnC can be exchanged onto skinned
muscle fiber thin filaments following extraction of the native skeletal
TnC, effectively removing any calcium-dependent activation
of the muscle fiber. Complete replacement of endogenous TnC with a
similar CBMII TnC completely relaxed skeletal muscle fibers and
rendered them insensitive to [Ca2+] (17). In this way, we
have been able to investigate isometric force and the rate of force
generation as functions of the fraction of the thin filament that is
able to bind myosin under conditions where calcium binding to troponin
is fixed at a level determined by the CBMII TnC content.
Isometric tension and the rate of force redevelopment were measured in
muscle fibers following TnC replacement with ratios of cTnC and CBMII
TnC. The data show that isometric force is directly proportional to the
number of active thin filament units (A7TmTn) at saturating
calcium, and the rate of force redevelopment is unaffected by a
reduction in cross-bridge number. The results suggest that
ktr is primarily controlled by calcium binding
to troponin rather than the density of cross-bridges binding to the actin filament. Together, the results suggest that calcium controls cross-bridge access to the thin filament by regulating an equilibrium between weakly (non-productively) and strongly (productively) bound,
but non-force-bearing cross-bridges. The present work accounts for the
discrepancies between the opposing kinetic and steric-blocking models
of thin filament regulation. A preliminary report of this work was
published previously (22).
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EXPERIMENTAL PROCEDURES |
Solutions--
All fiber solutions contained 100 mM
N,N-bis[2hydroxyehtyl]-2-aminoethanesulfonic
acid (BES), pH 7.1, at 15 °C, 5 mM MgATP, 1 mM magnesium acetate, 20 mM potassium acetate,
15 mM creatine phosphate, 200 units/ml creatine
phosphokinase, and 1 mM dithiothreitol with an ionic
strength of 200 mM. Relaxing (REL) and activating solutions
also contained 20 mM EGTA. Calcium was added as
Ca2+-K+-EGTA, and the pCa was varied
by adjusting the proportions of K+-EGTA and
Ca2+-K+-EGTA. The preactivating solution
contained 2 mM K+-EGTA and 18 mM
1,6-diaminohexane-N,N,N',N'-tetraacetic
acid (HDTA). The composition of the solutions was calculated using the
QuickBasic program, SOLUTION (11).
Fiber Preparation and Mechanical Apparatus--
Psoas muscle
fibers were dissected from female New Zealand white rabbits,
glycerinated, and stored as described by Millar and Homsher (11).
Single fibers were dissected, and the ends were fixed by flowing 1%
glutaraldehyde in 50% glycerol over each end of the fiber.
Aluminum T-clips were then attached to the ends, and the fibers were
mounted between a force transducer (SensoNor AE801 strain gauge) and a
shaker motor (Ling 100A). The sarcomere length was set to 2.6 µm/sarcomere (measured by He:Ne laser diffraction), and the fiber
width and total length were measured. The rate of tension redevelopment
(ktr) was measured by activating the fiber, and
after reaching steady-state isometric tension, abruptly shortening the
fiber by ~20%, reducing force to zero. The fiber was held at this
length for 25 ms while the fiber shortened at maximal velocity, and
then the fiber was rapidly restretched to its original length (6). All
mechanical measurements were performed at 15 °C.
Data Acquisition and Curve Fitting--
Tension and displacement
signals were recorded and the digitized records were analyzed using
KFIT (11) and SigmaPlot 4.0 (SPSS Inc., Chicago, IL). The
ktr records were fit by a single exponential
equation of the form P = Po +
P (1
e
kt), where
P is the tension, Po is the initial
force,
P is the amplitude of the redeveloped tension, and
k is the ktr. Force-pCa curves were fit to the Hill Equation in the form
P = Po/(1 + 10nH(pK
pCa) where
Po is maximal force produced (pCa 4.5),
pK (pCa50) is the calcium
concentration yielding 0.5 Po and
nH is the Hill coefficient. Significance was
determined using Student's t test and the confidence level
was set at p < 0.05. The data were reported as
mean ± S.E. with (n) the number of fibers analyzed.
Modeling Equations--
The steady-state solutions for the
fraction of the cross-bridges in the weakly bound (Wo),
strongly bound (So), and force-exerting (Fo) states
shown in Scheme 1 are given by the equation,
|
(Eq. 1)
|
where
|
(Eq. 2)
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The solution for the fraction of cross-bridges in the
force-exerting state F(t) at anytime, t, after
changing any of the rate constants from a steady-state value is given
by the equation,
|
(Eq. 3)
|
where
|
(Eq. 4)
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TnC Extraction/Reconstitution--
To extract the endogenous
skeletal TnC from the fibers, the sarcomere length was increased to
>3.0 µm in REL (23), and the fibers were transferred to a solution
containing 5 mM EDTA, 20 mM Tris·HCl (pH
7.2), and 0.5 mM trifluoperazine dihydrochloride at
15-17 °C (24). The fibers were incubated until the
Ca2+-activated isometric tension fell below 10%
Po, generally within 10 min. The fibers were
reconstituted by incubation in REL containing 0.5 mg/ml TnC for 1 min
followed by a wash in REL for 30 s. This was repeated until
Ca2+-dependent tension reached a maximal,
constant value. Skeletal TnC was kindly provided by Marion Greaser
(University of Wisconsin) and purified as described by Greaser and
Gergely (25). Cardiac TnC (cTnC and CBMIITnC) were isolated as
described (18).
SDS-PAGE--
The extraction and reconstitution of the troponin
C was quantified using SDS-PAGE. Muscle fibers containing sTnC, cTnC,
or ratios of CBMII TnC:cTnC were placed in sample buffer, heated to
95 °C for 3 min, and sonicated to denature and solubilize the muscle
fiber. The samples were loaded onto a 12% Tris·HCl separating gel.
Following electrophoresis, the gels were stained using the silver stain
technique of Guilian et al. (26) with minor modifications. After staining, the gels were dried and scanned. The
apparent molecular mass of CBMIITnC is 1 kDa < purified cTnC, allowing quantitative separation of the two proteins by
their mobilities. Gels were analyzed using a GS-700 scanning
densitometer (Bio-Rad) normalizing bands to the area under the myosin
light chain 1 peak. The relative content of cTnC and CBMII TnC was
determined from the ratio of cTnC:(cTnC + CBMII TnC) after accounting
for differences in staining intensity and a background peak at the same
position as CBMII TnC.
 |
RESULTS |
Extraction/Replacement of TnC--
To investigate the effects of
cardiac and CBMII TnC replacement on muscle function, it was necessary
to effectively remove the native sTnC from the muscle fibers.
Extraction reduced the Ca2+-dependent force to
8.1% (±1.1) of the maximal force obtained at pCa 4.5 (Po = 146.7 (±3.8) kN/m2,
n = 45). Extraction of the sTnC and subsequent
reconstitution with purified sTnC returned maximal
Ca2+-dependent force to 125.5 ± 10.3 kN/m2 or 86 ± 3% Po
(n = 5). Reconstitution with cTnC returned maximal
isometric force to 96.9 (±4.7) kN/m2 (n = 17) or 65.4% (± 5.0) of that observed prior to extraction.
It was important to determine whether the cardiac TnC or CBMII TnC
preferentially bound to the fiber thin filaments under the
extraction/replacement procedure. The difference in the apparent molecular weights of cTnC and CBMII TnC made quantification possible. Fig. 1 shows two lanes (A) and
the associated profiles (B) of a gel containing fiber
segments reconstituted with 100% cTnC and 50% cTnC:50% CBMII TnC.
Determination of the relative proportion of cTnC in fibers
reconstituted with various ratios of cTnC and CBMII TnC as a function
of the relative amount of cTnC added to the reconstitution solution is
shown in Fig. 2. The results demonstrate that the binding of cTnC and CBMII TnC to the thin filaments of fibers
is similar under the reconstitution conditions used (pCa 9.0), which is consistent with the binding studies performed using cTn
and CBMII Tn complexes (18).

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Fig. 1.
A, SDS-polyacrylamide gel obtained from
fiber segments after extraction and replacement with either 100% cTnC
(lane 1) or 50% cTnC:50% CBMII TnC (lane 2).
Both lanes contain ~1 cm of fiber. B, densitometric
profiles of gel lanes shown in A. Calculation of the area
under the TnC peaks yields a value of 0.42 for the cTnC in
lane 1 and values of 0.192 for cTnC and 0.213 for CBMII TnC
in lane 2. The relative cTnC content (cTnC-cTnC+ CBMII TnC))
of lane 2 is 0.47.
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Fig. 2.
Relative cTnC content of fibers reconstituted
with various ratios of cTnC:CBMII TnC. Each point
represents a single fiber segment analyzed as shown in Fig. 1 and
described under "Experimental Procedures." Analysis was performed
on three separate gels. The solid line indicates the linear
regression through the data and the dashed lines represent
the 95% confidence levels. Linear regression of the data yields a
line (R2 = 0.93) with a slope of
0.887 ± 0.1 and a y intercept of 0.076 ± 0.069.
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Steady-state Isometric Tension--
As CBMII TnC is a cardiac TnC
mutant, it was necessary to determine the effects on the isometric
tension and the calcium sensitivity in fibers after extraction of
native TnC and replacement with cTnC. The difference in calcium
sensitivity between the control fibers containing native sTnC and those
after cTnC replacement is shown in Fig.
3. Following replacement of sTnC with
cTnC the Hill coefficient was reduced from 2.83 (±0.11) to 1.95 (±0.13) and the pCa50 shifted from 6.72 (±0.01) to 6.64 (±0.02); both changes are significant
(p < 0.05). The reduction in the Hill coefficient and
shift in pCa50 after replacement with cTnC have been reported previously (27, 28). Extraction of endogenous sTnC and
replacement with purified sTnC produced no significant differences
(p < 0.01) in the Ca2+ sensitivity
(nH = 2.87 (±0.43);
pCa50 = 6.74 (±0.03); n = 5).

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Fig. 3.
Relative force-pCa
relationship from muscle fibers before extraction of skeletal TnC and
after reconstitution with cardiac TnC. Values in the plots are
shown as mean ± S.E. The values obtained were normalized against
the maximum force produced by the individual fiber at pCa
4.5 prior to extraction. The data were fit to the Hill equation (see
"Experimental Procedures") with R2 > 0.99. The sTnC ( ) yielded a maximum relative force of 1.00 ± 0.01, a
Hill coefficient of 2.83 ± 0.11, and a
pCa50 of 6.72 ± 0.01. The cTnC ( )
produced a maximum relative force of 0.98 ± 0.03, a Hill
coefficient of 1.95 ± 0.13, and a
pCa50 of 6.64 ± 0.02.
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Isometric force is believed to be dependent on the number of
cross-bridges attached to the thin filament (29-31). To determine whether reduction in the level of thin filament activation directly correlates with a decrease in the number of thin filament sites, fibers
were reconstituted with various ratios of CBMII:cTnC, and the isometric
tension was measured. Fig. 4 demonstrates
that tension fell in direct proportion to the reduction in cTnC content
of the fiber. Because the cTnC content added correlated well with the
amount of cTnC bound to the thin filaments (Fig. 2), the cTnC content
is given as the ratio of cTnC added. The tension measured after
replacement with CBMII:cTnC was normalized against the average maximal
force produced after replacement with 100% cTnC (65.4% of sTnC;
n = 17). Regression analysis indicated that the slope was not different from 1 (p > 0.3) and the
y intercept was not different from 0 (p > 0.2). The direct proportionality of the force reduction to the
fractional content of cTnC suggests that there is little cooperativity
between the steady-state isometric force and the number of attached
cross-bridges at saturating [Ca2+].

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Fig. 4.
Relative tension as a function of the cardiac
TnC content. The data are normalized to the value obtained for
100% cTnC (96.9 kN/m2; n = 17). Each
point represents 5-10 fibers and is shown as mean ± S.E. The solid line is a linear fit to the data, yielding a
slope of 0.966 ± 0.049 and a y intercept of 0.023 ± 0.026 (R2 = 0.999). The dashed
line indicates the 95% confidence levels.
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The Rate of Tension Redevelopment (ktr)--
The rate
of tension redevelopment (6) is controlled by [Ca2+],
which implies regulation of a cross-bridge transition involving force
generation (11, 12, 32). Representative traces of ktr as a function of pCa are shown in
Fig. 5A. It is evident that the Ca2+ sensitivity of force redevelopment is affected by
the troponin C isoform. These differences are seen more clearly in Fig.
5B, which shows ktr as a function of
the relative tension. Skeletal TnC produced a greater rate of force
redevelopment at high calcium concentrations (18.1 ± 0.46 s
1, n = 45 at pCa 4.5) but was
reduced almost 10-fold at low [Ca2+]. At pCa
6.0, the force was still ~98% of that at pCa 4.5 but ktr fell to only 14.8 (±1.2) s
1
(n = 10). The ktr fell to near
minimal values (2.89 ± 0.4 s
1; n = 10) at pCa 6.7 even though isometric force was still ~50% Po.

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Fig. 5.
A, representative traces of
ktr measurements in a single skinned fiber
before extraction of skeletal TnC and after replacement with cardiac
TnC. The level of activation was varied by adjusting the pCa
in the activating solution. The cardiac TnC traces were normalized to
the maximal force obtained in the fiber at pCa 4.5 prior to
extraction. The fiber was 55 µm in diameter and 2.6 mm in length and
produced a maximal tension of 138.7 kN/m2 at pCa
4.5. B, the effects of [Ca2+] on
ktr as a function of relative tension. The
tension was reduced by decreasing the [Ca2+]. The sTnC
data ( ) are normalized to the tension obtained in the same fiber at
pCa 4.5 prior to extraction. The cTnC data ( ) are
normalized against the tension measured at pCa 4.5 in the
same fiber after replacement. The data are shown as mean ± S.E.
with n > 5 for each point. The solid lines indicate
the relationship predicted based on the model presented in Scheme 1 and
detailed in equations under "Experimental Procedures."
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Replacement with cTnC reduced the maximal ktr at
pCa 4.5 to 10.8 (±0.5) s
1 (n = 17). However, reducing the calcium concentration from pCa 4.5 to pCa 7.0 caused the rate of tension redevelopment to
fall to 3.32 (±0.36) s
1 (n = 7), only a
3-fold reduction. These results indicate that the rate of tension
redevelopment is markedly affected by [Ca2+] regardless
of TnC isoform. The TnC isoform bound to the thin filament, however,
modulates the rate of tension redevelopment in active muscle fibers
(33).
Although ktr is highly sensitive to variations
in the [Ca2+], it is unclear whether the effect is caused
by [Ca2+]-dependent limits on cross-bridge
cycling, cross-bridge number, or both. To evaluate the effects of
reducing the cross-bridge number independent of [Ca2+],
ktr was measured after extraction and
replacement with different ratios of CBMII:cTnC at constant, saturating
[Ca2+]. Representative traces of
ktr with different ratios of cTnC and CBMII TnC
are shown in Fig. 6A. It is
apparent that ktr was largely unaffected by the
reduction in the number of actin monomers available for myosin
cross-bridge interaction. Isometric tension fell in direct proportion
to the addition of CBMII TnC (Fig. 4) whereas
ktr remained unchanged and near maximal for all
conditions except for 75% CBMII TnC (7.24 ± 0.21 s
1; n = 7). Fig. 6B plots
ktr as a function of relative tension in fibers
containing 100% cTnC with tension varied by altering the
pCa of the activating solution and fibers containing various ratios of CBMII:cTnC. The results indicate that
ktr is greatly affected by changes in
[Ca2+] but is not sensitive to reductions in the fraction
of the thin filament that can be activated and bind cross-bridges until
tension is reduced to <35% Po.

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Fig. 6.
A, representative traces of
ktr measurements after replacement with cardiac
TnC or ratios of CBMII and cardiac TnC. For the 25% CBMII and 75%
CBMII records, the force has been normalized to 65% of the maximal
force obtained at pCa 4.5 in the fiber prior to
extraction/replacement with CBMII. The traces are taken from three
separate fibers. B, ktr as a function
of relative tension. Tension was reduced by decreasing the
[Ca2+] for cTnC fibers ( ) and by varying the
cTnC:CBMII TnC ratio for the CBMII TnC fibers ( ). All data are shown
as mean ± S.E. with n > 5 for each
point. The solid line through the cTnC
corresponds to the prediction from the equations below for cTnC. The
CBMII TnC data are fit to a hyperbolic equation.
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 |
DISCUSSION |
Huxley's (29) two-state model of muscle contraction assumes that
isometric force is produced by S1 interactions with the thin
filament and therefore is dependent on the number of attached cross-bridges. Gordon et al. (30) and Edman (31) found that isometric force is proportional to the degree of thick-thin filament overlap and hence the number of cross-bridges. In this study, we have
shown that isometric tension under saturating calcium conditions
declines in proportion to calcium binding to troponin.
Prior studies have reduced the fraction of the thin filament that can
be activated by partial extraction of the endogenous TnC (23, 27, 34,
35). The removal of TnC from the troponin complex prevents calcium
binding and inactivates the thin filament in the regions containing the
partial troponin complexes (TnI-TnT-Tm units). Because TnC and TnI
interact with each other in undefined ways (36), TnC extraction may
alter several of these interactions and affect other aspects of the
thin filament regulatory mechanism (37). Replacement of sTnC by either
cTnC or CBMII TnC avoids this complication by maintaining a full
complement of TnC on the thin filament while reducing the number of
potentially active regulatory units.
The force-pCa plot of fibers containing sTnC and cTnC (Fig.
3) is similar to those obtained previously (11, 27). The force is
reduced cooperatively as the calcium concentration is lowered in fibers
containing either sTnC or cTnC. The reduction in cooperativity (nH) upon reconstitution with cTnC is
qualitatively consistent with the presence of only one Ca2+
binding site in cTnC. However, the force-pCa relationship in skinned fibers is more cooperative than can be explained by
Ca2+ binding to individual troponin subunits. If this were
the only cooperative mechanism involved, the maximal
nH would be 1.0 for cardiac and 2.0 for skeletal
TnC (19). Adjacent troponin subunits are connected by tropomyosin, and
enhancement of Ca2+ binding via cooperative strong
cross-bridge binding may influence the degree of thin filament
activation and therefore the mechanism of regulation (21, 38, 39).
The results obtained here present a clearer picture of what occurs
during force generation in isometrically contracting skinned muscle
fibers. The most straightforward, although not the only, interpretation
of Fig. 4 is that under conditions of saturating calcium
(pCa 4.5), CBMII TnC replacement of cTnC limits the number of cross-bridges capable of binding to the thin filament, and the
isometric force decreases in direct proportion to the reduction in
active thin filament regulatory units. Isometric force, a steady-state measurement, is linearly dependent on the number of thin filament actin
monomers available for cross-bridge interaction. This suggests that
reduction in the number of active thin filament units, by either
lowering [Ca2+] or increasing the proportion of CBMIITnC,
directly limits the number of cross-bridges capable of binding to the
thin filament.
The relationship between force and Ca2+ binding is linear
(Fig. 4) and therefore seems unaffected by cooperative interactions between adjacent regulatory units along the length of the thin filament. Either a concave or a convex relationship between the fractional occupancy and the isometric force would be evidence for such
cooperativity but was not observed. The absence of this behavior does
not prove, however, that force generation is unaffected by longitudinal
cooperativity along the thin filament. One reason for this is that the
linear behavior in Fig. 4 may reflect a balance of compensating
cooperative effects in which regulatory units with calcium induce
myosin binding on adjacent units and units without calcium restrict
myosin binding on adjacent units. Each phenomenon has been reported in
other types of experiments with partial extraction of either whole
troponin (40) or TnC (35). Further, any non-linearity between the
fractional Ca2+ binding and isometric force would be
difficult to detect for thin filament occupancies less than 25% and
therefore cannot be excluded with the present data. Finally, the
use of CBMII TnC precludes a possible source of cooperativity that can
occur in normal thin filaments; cross-bridge binding to a regulatory
unit where Ca2+ is bound may induce Ca2+
binding on adjacent regulatory units (21). Despite these caveats about
an underlying complexity in the system, the linear results in Fig. 4
imply that Ca2+ is controlling the steady-state isometric
force by limiting cross-bridge access to the thin filament.
Is a similar mechanism at work during transient events (e.g.
ktr) in the muscle fiber? In this study, we
tested whether ktr is dependent on
[Ca2+] or the number of cross-bridges attached to the
thin filament. Brenner (6) showed that the rate of tension
redevelopment is highly dependent on the [Ca2+] with a
non-linear decline in ktr as calcium levels were
reduced and suggested that regulation occurred during a weak to strong transition. If Ca2+ specifically controls the transition
from a weakly bound to a strongly bound, force-generating state, then
ktr and
kPi, the rate of the
tension decline following photogeneration of Pi from caged-Pi, would be the same. However, measurements of
kPi revealed little or no
Ca2+ dependence (11, 12, 14, 16) even though
ktr measured in the same preparation exhibited a
strong dependence on [Ca2+]. In the present study,
decreasing the number of force-generating cross-bridges reduced
steady-state tension but did not greatly affect the rate of force
generation until the level of thin filament activation was less than
~35%. These results are consistent with the hypothesis that
[Ca2+] controls a cross-bridge transition
preceding force generation, proposed to be a transition from
a weakly bound to a strongly bound, non-force-bearing cross-bridge
state (11, 14, 16, 41).
If ktr actually measures a two-step process as
suggested, (i.e. a weakly to strongly bound transition
followed by the force-generating isomerization or Pi
release), the differing effects of CBMII or decreasing the calcium
concentration on cross-bridge function can be explained by the model
described below.
In this model, cross-bridges are detached or weakly attached (W),
strongly attached but not generating force (S), or strongly attached
and generating forcing (F). The weakly attached states (M·ATP,
M·ADP·Pi, or A-M·ADP·Pi) attach and
detach from the actin filament rapidly and do not sustain significant
force (the hyphen indicates a weakly attached state). The strongly
attached state (AM·ADP·Pi) does not generate force.
Entry into the strongly bound state (S) involves a thin filament
isomerization controlled by troponin and tropomyosin with the forward
rate k+1 (increased by elevations in
[Ca2+]) and the reverse rate k
1.
The strongly attached and force-exerting state (F), AM·ADP (and its
isomers), are generated by an isomerization and/or the release of
Pi from the strongly bound AM·ADP·Pi state controlled by k+2 and
k
2. The rigor cross-bridge, AM, is also a
strongly bound, force-exerting cross-bridge. The rate of
force-generating cross-bridge detachment to the detached/weakly attached cross-bridges (W) is defined by k3 and
is slow (2-4 s
1) under isometric conditions as estimated
from the steady-state isometric ATPase rate (6). At low
[Pi] (~1 mM), k+2
and k
2 are ~20 s
1 and 3 s
1, respectively (11, 12, 16). We assume that the TnC
isoform and pCa have no direct effect on
k+2, k
2, or
k3 as these rates should only depend on the
myosin and actin present, neither of which changed during our
experiments. We also assume that addition of calcium and/or replacement
of regulatory proteins changes only k+1 and/or
k
1. Assuming that k
1, k+2, k
2, and
k3 are constant in skeletal muscle and that
k
1 is 4 s
1, then steady-state
isometric force, Fo, is a hyperbolic function of
k+1 defined by
|
(Eq. 5)
|
The analytical expressions used to determine the steady-state
isometric force, Fo (assumed to be proportional to the fraction
of cross-bridges attached in the force-generating states) and the time
course of force production, F(t), either from rest or after
a period of rapid shortening and re-stretch are given under
"Experimental Procedures." The time course of force generation is
dominated by the A exp(
1t) term as
B is insignificant compared with A at relative
forces of <90%. At larger values of k+1 (>15
s
1 when Fo > 90%), the difference in magnitude
of
1 and
2 produces a slowing of
ktr to a value <15% different from that predicted by
1 alone. The consequence of this behavior
is that the rise of force subsequent to rapid shortening (during which k3 is >100 s
1) is well fit by a
single exponential term (R2 > 0.95).
The overall cross-bridge cycling rate is slow and limited by an
irreversible isomerization step preceding ADP release defined by
k3 (42). The measurement of the rate of tension
redevelopment isolates the force-generating step
(k+2 and k
2) and the
preceding equilibrium
(k+1/k
1) from the
overall cross-bridge cycle. Thus when k+1 and
k
1 are both small, ktr
approaches k3 as a limit. Modeling of this
mechanism suggests that Ca2+ activates the muscle by
increasing k+1 while not affecting k
1. By varying k+1 from
0 to 20 s
1 to simulate changes in the free calcium
concentration, the model correctly defines the observed non-linear
behavior of ktr as a function of relative
isometric force in fibers containing sTnC (Fig. 5B,
solid line labeled sTnC). The changes in
ktr produced by replacing sTnC with cTnC (Figs.
5B and 6B, solid lines labeled cTnC)
can be produced by raising k
1 from 4 s
1 to 10 s
1 and allowing
k+1 to vary from 0 to 13 s
1 as the
calcium concentration is raised. Therefore, the model suggests that the
rate detached or weakly bound cross-bridges productively bind to the
thin filament determines the rate of force generation.
In the present experiments, as [Ca2+] was reduced in
fibers containing sTnC, ktr fell from ~18 to 2 s
1. After extraction of endogenous sTnC and replacement
with cTnC, the Ca2+-induced reduction in
ktr was smaller, from ~11 to 3 s
1. Cardiac muscle exhibits a smaller, 3-6-fold increase
in ktr as [Ca2+] is raised from
submaximal to maximal levels (43, 44). Although ktr depends on [Ca2+] and the
myosin isoform (6, 32), it is significant that substitution of TnC
alone causes large changes in the sensitivity and rates associated with
force generation. Because incorporation of different TnC isoforms
should not alter the cross-bridge structure or the intrinsic
cross-bridge cycling rate of myosin, the changes must be caused by
TnC-dependent effects. The proposed model correctly accounts for the observed differences of fibers containing sTnC or cTnC
in ktr as a function of relative force. As shown
in Fig. 5B, the cTnC data are well fit by simply increasing
k
1 from 4 s
1 to 10 s
1 and reducing the maximal rate of
k+1 to 13 s
1 leaving the other
rate constants unchanged.
Why is ktr reduced in the presence of cTnC
compared with sTnC? The most likely reason is that the TnC interaction
with Ca2+ and signaling to the other Tn subunits, Tm and
actin, play a role in controlling the state of the thin filament
activation, which is a complex and incompletely understood process
(45-47). Although differences in the Ca2+ affinity between
the two TnC isoforms may contribute to this behavior, it is more likely
that the changes are due to the markedly different structure of the
cardiac TnC stalk and regulatory domain from that of skeletal TnC. The
structural differences may alter the ability of the TnC to interact
with TnI and effect sequential changes in tropomyosin position that
influence the rate of cross-bridge attachment. Also, NMR studies reveal
that the cTnC structure shows a more closed conformation than sTnC and
that the Ca2+ binding and dissociation produces slower
conformational changes in cTnC (48, 49). Thus, the rate at which TnC
can undergo the required conformational changes to affect the inherent
properties of the thin filament are TnC isoform-dependent
and therefore alter fiber function. Such changes have physiological
implications because cardiac muscle does not require the rapid and
complete activation necessary for normal physiological function in
skeletal muscle.
The presence of CBMII TnC affects only the regulatory units containing
the mutant TnC while the other regulatory units are all potentially
fully active. If the [Ca2+] is saturating, most of the
native TnC molecules will be in their calcium-bound state and the
tropomyosin will be oscillating primarily over the
Ca2+-induced position providing cross-bridge access to the
actin binding sites. Thus, ktr will not be
limited by calcium binding and the weak to strong cross-bridge
transition. The mechanism described in Scheme 1 successfully
predicts the behavior shown in Fig. 5B in which
ktr changes little until the steady-state
isometric force rises to values greater than ~50% maximal. This way
of thinking about the regulatory mechanism indicates that regulation
involves kinetic regulation of the transition from a weakly bound to a strongly bound state as first suggested by Brenner (6). It also
suggests that kinetic and steric mechanisms are not truly separate
because steric effects from tropomyosin positioning on the thin
filament affect the weak to strong cross-bridge transition.
Evidence for a potential role of strongly bound cross-bridges
contributing to thin filament activation at lower thin filament occupancies is given by the data in Fig. 6. ktr
is plotted as a function of the isometric force at pCa 4.5 in fibers containing various fractions of CBMII TnC. At forces >25%
of maximal, ktr is independent of isometric
force. However, at 25% isometric force ktr is
markedly reduced even though [Ca2+] is saturating. This
could occur if reduction in the level of thin filament activation is
accelerated by the decline in productively attached cross-bridges. The
results imply that Ca2+ controls the steady-state isometric
force over the range of 25-100% force by limiting cross-bridge access
to the thin filament.
How do these results relate to biochemical and structural mechanisms
thought to underlie the regulation of muscle contraction? Biochemical
investigations have revealed the presence of three thin filament
states: blocked, closed, and open, (45) while more recent cryo-electron
microscopy studies have identified three structural states of the thin
filament: off, Ca2+-induced, and myosin-induced (7-9) that
may correspond to the biochemical states. The initiation of contraction
involves calcium binding to the low affinity site(s) of the troponin C
subunit (site I in cTnC and both sites I and II in sTnC) on the
troponin complex. The binding of calcium initiates a structural change in the interaction between TnC and TnI resulting in a relaxation of the
TnI-based inhibition. The tropomyosin is now able to shift from the
blocked or "off" position to the closed or
"Ca2+-induced", intermediate position closer to the
groove between the actin strands. This shift in the average
position of the Tm molecule opens myosin binding sites on the actin
filaments important for strong, stereo-specific cross-bridge
attachment. Cross-bridges will be able to bind productively,
i.e. proceed to a strong binding conformation and continue
through the actomyosin ATPase cycle unimpeded by the presence or
absence of calcium. The attachment of strongly bound cross-bridges is
associated with a further shift in the average position of tropomyosin
to the open or "myosin-induced" state, increasing the probability
of other cross-bridges binding to the thin filament. For each state,
tropomyosin position is probably a dynamic oscillation in which Tm does
not occupy a single static position on the actin surface but
continually shifts back and forth over the actin surface. As
[Ca2+] falls, the probability that Tm is in the open
position falls, reducing the rate of strong, stereo-specific
cross-bridge binding. Strong cross-bridge binding likely stabilizes the
Tm position that makes available further myosin binding sites on nearby
actin monomers (8, 47).
Scheme 1 quantifies these relationships and together with the
structural interpretation of regulation described above leads to
explanations for the curvilinear relationship between force and
ktr and for the decline of
ktr at high concentrations of CBMII. At thin
filament occupancy >25% of maximal there is sufficient cooperativity
along the thin filament that Ca2+ simply controls the rate
of attachment (k+1) of myosin to the thin
filament and ktr is varied with
Ca2+. At low thin filament occupancy by cross-bridges, the
proportion of time Tm spends in the closed or blocked positions will be
greater and the thin filament will be partially inactivated. This will reduce the cooperativity of calcium binding to the thin filament and
the spread of activation along the thin filament. It will therefore
produce a decline in ktr (even at saturating
Ca2+) (Fig. 6B, open circles).
 |
ACKNOWLEDGEMENTS |
We acknowledge the assistance of Dr. Richard
Moss (Univ. of Wisconsin) with single muscle fiber SDS-PAGE. We are
grateful to Dr. Marion Greaser (Univ. of Wisconsin) for the kind gift
of skeletal troponin C and to Will Silverman (UCLA) for his assistance with gel analysis.
 |
FOOTNOTES |
*
This work was supported in part by National Institutes of
Health Grants AR-30988 (to E. H.) and HL38834 (to L. S. T.).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.
§
Partially supported by National Institutes of Health Predoctoral
Training Grant GM08496. To whom correspondence should be addressed:
Dept. of Physiology, Pennsylvania Muscle Institute, University of
Pennsylvania, Philadelphia, PA 19104. Tel: 215-898-0046; Fax:
215-573-8871; E-mail: camorris@mail.med.upenn.edu.
Published, JBC Papers in Press, March 21, 2001, DOI 10.1074/jbc.M007371200
 |
ABBREVIATIONS |
The abbreviations used are:
Tm, tropomyosin;
Tn, troponin;
TnC, troponin C;
CBMII, cardiac binding
mutant (site II);
REL, relaxing solution;
ktr,
rate of tension redevelopment;
PAGE, polyacrylamide gel
electrophoresis;
kN, kilonewton;
cTnC, cardiac TnC;
sTnC, skeletal TnC;
a, actin;
m, myosin.
 |
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