Modulation of Contractile Activation in Skeletal Muscle by a Calcium-insensitive Troponin C Mutant*

Carl A. MorrisDagger §, Larry S. Tobacman, and Earl HomsherDagger

From the Dagger  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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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).

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 + Delta P (1 - e-kt), where P is the tension, Po is the initial force, Delta 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,


<AR><R><C><UP>W</UP><SUB>o</SUB>=(k<SUB>+2</SUB>*k<SUB>3</SUB>+k<SUB>−1</SUB>*k<SUB>3</SUB>+k<SUB>−1</SUB>*k<SUB>−2</SUB>)/D</C></R><R><C><UP>S</UP><SUB>o</SUB>=(k<SUB>+1</SUB>*k<SUB>3</SUB>+k<SUB>+1</SUB>*k<SUB>−2</SUB>)/D</C></R><R><C><UP>F</UP><SUB>o</SUB>=(k<SUB>+1</SUB>*k<SUB>+2</SUB>)/D</C></R></AR> (Eq. 1)
where
 D=k<SUB>+2</SUB>*k<SUB>3</SUB>+k<SUB>−1</SUB>*k<SUB>3</SUB>+k<SUB>−1</SUB>*k<SUB>−2</SUB>+k<SUB>+1</SUB>*k<SUB>3</SUB>+k<SUB>+1</SUB>*k<SUB>+2</SUB>+k<SUB>+1</SUB>*k<SUB>−2</SUB> (Eq. 2)
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,
<UP>F</UP>(t)=C+A*<UP>exp</UP>(&lgr;<SUB>1</SUB>*t)+B*<UP>exp</UP>(&lgr;<SUB>2</SUB>*t) (Eq. 3)
where
<AR><R><C>A=(&lgr;<SUB>1</SUB>*Z+&lgr;<SUB>1</SUB><SUP> 2</SUP>*<UP>F</UP><SUB>o</SUB>+(k<SUB>+1</SUB>+k<SUB>+2</SUB>))/(&lgr;<SUB>1</SUB>*(&lgr;<SUB>1</SUB>−&lgr;<SUB>2</SUB>))</C></R><R><C>B=(−&lgr;<SUB>2</SUB>*Z+&lgr;<SUB>2</SUB><SUP> 2</SUP>*<UP>F</UP><SUB>o</SUB>−(k<SUB>+1</SUB>+k<SUB>+2</SUB>))/(&lgr;<SUB>2</SUB>*(&lgr;<SUB>1</SUB>−&lgr;<SUB>2</SUB>))</C></R><R><C>C=(k<SUB>+1</SUB>+k<SUB>+2</SUB>)/(&lgr;<SUB>1</SUB>*&lgr;<SUB>2</SUB>)</C></R><R><C>Z=k<SUB>+2</SUB>*<UP>S</UP><SUB><UP>o</UP></SUB>+(k<SUB>+1</SUB>+k<SUB>−1</SUB>+k<SUB>+2</SUB>)*<UP>F</UP><SUB>o</SUB></C></R><R><C>&lgr;<SUB>1</SUB>=(<UP>−</UP>b+(b<SUP>2</SUP>−4c)<SUP>1/2</SUP>)/2</C></R><R><C>&lgr;<SUB>2</SUB>=(<UP>−</UP>b−(b<SUP>2</SUP>−4c)<SUP>1/2</SUP>)/2</C></R><R><C>b=k<SUB>+1</SUB>+k<SUB>−1</SUB>+k<SUB>+2</SUB>+k<SUB>−2</SUB>+k<SUB>3</SUB></C></R><R><C>c=k<SUB>+1</SUB>*(k<SUB>+2</SUB>+k<SUB>−2</SUB>)+k<SUB>3</SUB>*(k<SUB>+1</SUB>+k<SUB>−1</SUB>+k<SUB>+2</SUB>)+k<SUB>−1</SUB>*k<SUB>−2</SUB></C></R></AR> (Eq. 4)

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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

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 (open circle ) 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.

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.

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 (open circle ) 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."

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 (open circle ) 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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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
<UP>F</UP><SUB><UP>o</UP></SUB>=k<SUB>+1</SUB>/(3+1.25 k<SUB>+1</SUB>) (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(lambda 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 lambda 1 and lambda 2 produces a slowing of ktr to a value <15% different from that predicted by lambda 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.


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Scheme I.  

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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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