Isometric force redevelopment of skinned muscle fibers from rabbit activated with and without Ca2+.

Fiber isometric tension redevelopment rate (kTR) was measured during submaximal and maximal activations in glycerinated fibers from rabbit psoas muscle. In fibers either containing endogenous skeletal troponin C (sTnC) or reconstituted with either purified cardiac troponin C (cTnC) or sTnC, graded activation was achieved by varying [Ca2+]. Some fibers were first partially, then fully, reconstituted with a modified form of cTnC (aTnC) that enables active force generation and shortening in the absence of Ca2+. kTR was derived from the half-time of tension redevelopment. In control fibers with endogenous sTnC, kTR increased nonlinearly with [Ca2+], and maximal kTR was 15.3 +/- 3.6 s-1 (mean +/- SD; n = 26 determinations on 25 fibers) at pCa 4.0. During submaximal activations by Ca2+, kTR in cTnC reconstituted fibers was approximately threefold faster than control, despite the lower (60%) maximum Ca(2+)-activated force after reconstitution. To obtain submaximal force with aTnC, eight fibers were treated to fully extract endogenous sTnC, then reconstituted with a mixture of a TnC and cTnC (aTnC:cTnC molar ratio 1:8.5). A second extraction selectively removed cTnC. In such fibers containing aTnC only, neither force nor kTR was affected by changes in [Ca2+]. Force was 22 +/- 7% of maximum control (mean +/- SD; n = 15) at pCa 9.2 vs. 24 +/- 8% (mean +/- SD; n = 8) at pCa 4.0, whereas kTR was 98 +/- 14% of maximum control (mean +/- SD; n = 15) at pCa 9.2 vs. 96 +/- 15% (mean +/- SD; n = 8) at pCa 4.0.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium-independent activation of skeletal muscle fibers by a modified form of cardiac troponin C.

A conformational change accompanying Ca2+ binding to troponin C (TnC) constitutes the initial event in contractile regulation of vertebrate striated muscle. We replaced endogenous TnC in single skinned fibers from rabbit psoas muscle with a modified form of cardiac TnC (cTnC) which, unlike native cTnC, probably contains an intramolecular disulfide bond. We found that such activating TnC (aTnC) enables force generation and shortening in the absence of calcium. With aTnC, both force and shortening velocity were the same at pCa 9.2 and pCa 4.0. aTnc could not be extracted under conditions which resulted in extraction of endogenous TnC. Thus, aTnC provides a stable model for structural studies of a calcium binding protein in the active conformation as well as a useful tool for physiological studies on the primary and secondary effects of Ca2+ on the molecular kinetics of muscle contraction.     

Influence of length on force and activation-dependent changes in troponin c structure in skinned cardiac and fast skeletal muscle

Linear dichroism of 5' tetramethyl-rhodamine (5'ATR) was measured to monitor the effect of sarcomere length (SL) on troponin C (TnC) structure during Ca2+ activation in single glycerinated rabbit psoas fibers and skinned right ventricular trabeculae from rats. Endogenous TnC was extracted, and the preparations were reconstituted with TnC fluorescently labeled with 5'ATR. In skinned psoas fibers reconstituted with sTnC labeled at Cys 98 with 5'ATR, dichroism was maximal during relaxation (pCa 9.2) and was minimal at pCa 4.0. In skinned cardiac trabeculae reconstituted with a mono-cysteine mutant cTnC (cTnC(C84)), dichroism of the 5'ATR probe attached to Cys 84 increased during Ca2+ activation of force. Force and dichroism-[Ca2+] relations were fit with the Hill equation to determine the pCa50 and slope (n). Increasing SL increased the Ca2+ sensitivity of force in both skinned psoas fibers and trabeculae. However, in skinned psoas fibers, neither SL changes or force inhibition had an effect on the Ca2+ sensitivity of dichroism. In contrast, increasing SL increased the Ca2+ sensitivity of both force and dichroism in skinned trabeculae. Furthermore, inhibition of force caused decreased Ca2+ sensitivity of dichroism, decreased dichroism at saturating [Ca2+], and loss of the influence of SL in cardiac muscle. The data indicate that in skeletal fibers SL-dependent shifts in the Ca2+ sensitivity of force are not caused by corresponding changes in Ca2+ binding to TnC and that strong cross-bridge binding has little effect on TnC structure at any SL or level of activation. On the other hand, in cardiac muscle, both force and activation-dependent changes in cTnC structure were influenced by SL. Additionally, the effect of SL on cardiac muscle activation was itself dependent on active, cycling cross-bridges.     

Troponin C modulates the activation of thin filaments by rigor cross-bridges.

Extraction of troponin C (TnC) from skinned muscle fibers reduces maximum Ca2+ and rigor cross-bridge (RXB)-activated tensions and reduces cooperativity between neighboring regulatory units (one troponin-tropomyosin complex and the seven associated actins) of thin filaments. This suggests that TnC has a determining role in RXB, as well as in Ca(2+)-dependent activation processes. To investigate this possibility further, we replaced fast TnC (fTnC) of rabbit psoas fibers with either CaM[3,4TnC] or cardiac TnC (cTnC) and compared the effects of these substitutions on Ca2+ and RXB activation of tension. CaM[3,4TnC] substitution has the same effect on Ca(2+)- and RXB-activated tensions; they are reduced 50%, and cooperativity between regulatory units is reduced 40%. cTnC substitution also reduces the maximum Ca(2+)-activated tension and cooperativity. But with RXB activation the effects on tension and cooperativity are opposite; cTnC substitution potentiates tension but reduces cooperativity. We considered whether tension potentiation could be explained by increased activation by cycling cross-bridges (CXBs), but the concerted transition formalism predicts fibers will fail to relax in high substrate and high pCa when CXBs are activator ligands. It predicts resting tension, which is not observed in either control or cTnC-substituted fibers. Rather, it appears that cTnC facilitates RXB activation of fast fibers more effectively than fTnC. The order of RXB-activated tension facilitation is cTnC > fTnC > CaM[3,4TnC] > empty TnC-binding sites. Comparison of the structures of fTnC, CaM[3,4TnC], and cTnC indicates that the critical region for this property lies in the central helix or N-terminal domain, including EF hand motifs 1 and 2.     

Function of the N-terminal calcium-binding sites in cardiac/slow troponin C assessed in fast skeletal muscle fibers

Fast skeletal troponin C (sTnC) has two low affinity Ca(2+)-binding sites (sites I and II), whereas in cardiac troponin C (cTnC) site I is inactive. By modifying the Ca2+ binding properties of sites I and II in cTnC it was demonstrated that binding of Ca2+ to an activated site I alone is not sufficient for triggering contraction in slow skeletal muscle fibers (Sweeney, H.L., Brito, R. M.M., Rosevear, P.R., and Putkey, J.A. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 9538-9542). However, a similar study using sTnC showed that Ca2+ binding to site I alone could partially activate force production in fast skeletal muscle fibers (Sheng, Z., Strauss, W.L., Francois, J.M., and Potter, J.D. (1990) J. Biol. Chem. 265, 21554-21560). The purpose of the current study was to examine the functional characteristics of modified cTnC derivatives in fast skeletal muscle fibers to assess whether or not either low affinity site can mediate force production when coupled to fast skeletal isoforms of troponin (Tn) I and TnT. Normal cTnC and sTnC were compared with engineered derivatives of cTnC having either both sites I and II active, or only site I active. In contrast to what is seen in slow muscle, binding of Ca2+ to site I alone recovered about 15-20% of the normal calcium-activated force and ATPase activity in skinned fast skeletal muscle fibers and myofibrils, respectively. This is most likely due to structural differences between TnI and/or TnT isoforms that allow for partial recognition and translation of the signal represented by binding Ca2+ to site I of TnC when associated with fast skeletal but not slow skeletal muscle.     

Ca2+ - and cross-bridge-dependent changes in N- and C-terminal structure of troponin C in rat cardiac muscle

Linear dichroism of 5'-tetramethylrhodamine (5'ATR)-labeled cardiac troponin C (cTnC) was measured to monitor cTnC structure during Ca2+-activation of force in rat skinned myocardium. Mono-cysteine mutants allowed labeling at Cys-84 (cTnC(C84), near the D/E helix linker); Cys-35 (cTnC(C35), at nonfunctional site I); or near the C-terminus with a cysteine inserted at site 98 (cTnC-C35S,C84S,S98C, cTnC(C98)). With 5'ATR-labeled cTnC(C84) and cTnC(C98) dichroism increased with increasing [Ca2+], while rigor cross-bridges caused dichroism to increase more with 5'ATR-labeled cTnC(C84) than cTnC(C98). The pCa50 values and n(H) from Hill analysis of the Ca2+-dependence of force and dichroism were 6.4 (+/-0.02) and 1.08 (+/-0.04) for force and 6.3 (+/-0.04) and 1.02 (+/-0.09) (n = 5) for dichroism in cTnC(C84) reconstituted trabeculae. Corresponding data from cTnC(C98) reconstituted trabeculae were 5.53 (+/-0.03) and 3.1 (+/-0.17) for force, and 5.39 (+/-0.03) and 1.87 (+/-0.17) (n = 5) for dichroism. The contribution of active cycling cross-bridges to changes in cTnC structure was determined by inhibition of force to 6% of pCa 4.0 controls with 1.0 mM sodium vanadate (Vi). With 5'ATR-labeled cTnC(C84) Vi caused both the pCa50)of dichroism and the maximum value at pCa 4.0 to decrease, while with 5'ATR-labeled cTnC(C98) the pCa50 of dichroism decreased with no change of dichroism at pCa 4.0. The dichroism of 5'ATR-labeled cTnC(C35) was insensitive to either Ca2+ or strong cross-bridges. These data suggest that both Ca2+ and cycling cross-bridges perturb the N-terminal structure of cTnC at Cys-84, while C-terminal structure is altered by site II Ca2+-binding, but not cross-bridges.     

Ca2+ and cross-bridge-induced changes in troponin C in skinned skeletal muscle fibers: effects of force inhibition

Changes in skeletal troponin C (sTnC) structure during thin filament activation by Ca2+ and strongly bound cross-bridge states were monitored by measuring the linear dichroism of the 5' isomer of iodoacetamidotetramethylrhodamine (5'IATR), attached to Cys98 (sTnC-5'ATR), in sTnC-5'ATR reconstituted single skinned fibers from rabbit psoas muscle. To isolate the effects of Ca2+ and cross-bridge binding on sTnC structure, maximum Ca2+-activated force was inhibited with 0.5 mM AlF4- or with 30 mM 2,3 butanedione-monoxime (BDM) during measurements of the Ca2+ dependence of force and dichroism. Dichroism was 0.08 +/- 0.01 (+/- SEM, n = 9) in relaxing solution (pCa 9.2) and decreased to 0.004 +/- 0.002 (+/- SEM, n = 9) at pCa 4.0. Force and dichroism had similar Ca2+ sensitivities. Force inhibition with BDM caused no change in the amplitude and Ca2+ sensitivity of dichroism. Similarly, inhibition of force at pCa 4.0 with 0.5 mM AlF4- decreased force to 0.04 +/- 0.01 of maximum (+/- SEM, n = 3), and dichroism was 0.04 +/- 0.03 (+/- SEM, n = 3) of the value at pCa 9.2 and unchanged relative to the corresponding normalized value at pCa 4.0 (0.11 +/- 0.05, +/- SEM; n = 3). Inhibition of force with AlF4- also had no effect when sTnC structure was monitored by labeling with either 5-dimethylamino-1-napthalenylsulfonylaziridine (DANZ) or 4-(N-(iodoacetoxy)ethyl-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole (NBD). Increasing sarcomere length from 2.5 to 3.6 microm caused force (pCa 4.0) to decrease, but had no effect on dichroism. In contrast, rigor cross-bridge attachment caused dichroism at pCa 9.2 to decrease to 0.56 +/- 0.03 (+/- SEM, n = 5) of the value at pCa 9. 2, and force was 0.51 +/- 0.04 (+/- SEM, n = 6) of pCa 4.0 control. At pCa 4.0 in rigor, dichroism decreased further to 0.19 +/- 0.03 (+/- SEM, n = 6), slightly above the pCa 4.0 control level; force was 0.66 +/- 0.04 of pCa 4.0 control. These results indicate that cross-bridge binding in the rigor state alters sTnC structure, whereas cycling cross-bridges have little influence at either submaximum or maximum activating [Ca2+].     

Calcium-dependent protein-protein interactions induce changes in proximity relationships of Cys48 and Cys64 in chicken skeletal troponin I.

The goal of this study was to relate conformational changes in the N-terminal domain of chicken troponin I (TnI) to Ca2+ activation of the actin-myosin interaction. The two cysteine residues in this region (Cys48 and Cys64) were labeled with two sulfhydryl-reactive pyrene-containing fluorophores [N-(1-pyrene)maleimide, and N-(1-pyrene)iodoacetamide]. The labeled TnI showed a typical fluorescence spectrum: two sharp peaks of monomer fluorescence and a broad peak of excimer fluorescence arising from the formation of an excited dimer (excimer). Results obtained show that forming a binary complex of labeled TnI with skeletal TnC (sTnC) in the absence of Ca2+ decreases the excimer fluorescence, indicating a separation of the two residues. This reduction in excimer fluorescence does not occur when labeled TnI is complexed with cardiac TnC (cTnC). The latter causes only partial activation of the Ca2+-dependent myofibrillar ATPase. The binding of Ca2+ to the two N-terminal sites of sTnC causes a significant decrease in excimer fluorescence and an increase in monomer fluorescence in complexes of labeled TnI with skeletal TnC or TnC/TnT, while Ca2+ binding to site II of cTnC only causes an increase in monomer fluorescence but no change in excimer fluorescence. Thus a conformational change in the N-terminal region of TnI may be necessary for full activation of muscle contraction.     

The role of the NH(2)- and COOH-terminal domains of the inhibitory region of troponin I in the regulation of skeletal muscle contraction.

The role of the inhibitory region of troponin (Tn) I in the regulation of skeletal muscle contraction was studied with three deletion mutants of its inhibitory region: 1) complete (TnI-(Delta96-116)), 2) the COOH-terminal domain (TnI-(Delta105-115)), and 3) the NH(2)-terminal domain (TnI-(Delta95-106)). Measurements of Ca(2+)-regulated force and relaxation were performed in skinned skeletal muscle fibers whose endogenous TnI (along with TnT and TnC) was displaced with high concentrations of added troponin T. Reconstitution of the Tn-displaced fibers with a TnI.TnC complex restored the Ca(2+) sensitivity of force; however, the levels of relaxation and force development varied. Relaxation of the fibers (pCa 8) was drastically impaired with two of the inhibitory region deletion mutants, TnI-(Delta96-116).TnC and TnI-(Delta105-115).TnC. The TnI-(Delta95-106).TnC mutant retained approximately 55% relaxation when reconstituted in the Tn-displaced fibers. Activation in skinned skeletal muscle fibers was enhanced with all TnI mutants compared with wild-type TnI. Interestingly, all three mutants of TnI increased the Ca(2+) sensitivity of contraction. None of the TnI deletion mutants, when reconstituted into Tn, could inhibit actin-tropomyosin-activated myosin ATPase in the absence of Ca(2+), and two of them (TnI-(Delta96-116) and TnI-(Delta105-115)) gave significant activation in the absence of Ca(2+). These results suggest that the COOH terminus of the inhibitory region of TnI (residues 105-115) is much more critical for the biological activity of TnI than the NH(2)-terminal region, consisting of residues 95-106. Presumably, the COOH-terminal domain of the inhibitory region of TnI is a part of the Ca(2+)-sensitive molecular switch during muscle contraction.     

Calcium-binding properties of troponin C in detergent-skinned heart muscle fibers

In order to obtain information with regard to behavior of the Ca2+ receptor, troponin C (TnC), in intact myofilament lattice of cardiac muscle, we investigated Ca2+-binding properties of canine ventricular muscle fibers skinned with Triton X-100. Analysis of equilibrium Ca2+-binding data of the skinned fibers in ATP-free solutions suggested that there were two distinct classes of binding sites which were saturated over the physiological range of negative logarithm of free calcium concentration (pCa): class I (KCa = 7.4 X 10(7) M-1, KMg = 0.9 X 10(3) M-1) and class II (KCa = 1.2 X 10(6) M-1, KMg = 1.1 X 10(2) M-1). The class I and II were considered equivalent, respectively, to the Ca2+-Mg2+ and Ca2+-specific sites of TnC. The assignments were supported by TnC content of the skinned fibers determined by electrophoresis and 45Ca autoradiograph of electroblotted fiber proteins. Dissociation of rigor complexes by ATP caused a downward shift of the binding curve between pCa 7 and 5, an effect which could be largely accounted for by lowering of KCa of the class II sites. When Ca2+ binding and isometric force were measured simultaneously, it was found that the threshold pCa for activation corresponds to the range of pCa where class II sites started to bind Ca2+ significantly. We concluded that the low affinity site of cardiac TnC plays a key role in Ca2+ regulation of contraction under physiological conditions, just as it does in the regulation of actomyosin ATPase. Study of kinetics of 45Ca washout from skinned fibers and myofibrils revealed that cardiac TnC in myofibrils contains Ca2+-binding sites whose off-rate constant for Ca2+ is significantly lower than the Ca2+ off-rate constant hitherto documented for the divalent ion-binding sites of either cardiac/slow muscle TnC or fast skeletal TnC.     

Binding of cardiac troponin-I147-163 induces a structural opening in human cardiac troponin-C.

The interaction of troponin-C (TnC) with troponin-I (TnI) plays a central role in skeletal and cardiac muscle contraction. We have recently shown that the binding of Ca2+ to cardiac TnC (cTnC) does not induce an "opening" of the regulatory domain in order to interact with cTnI [Sia, S. K., et al. (1997) J. Biol. Chem. 272, 18216-18221; Spyracopoulos et al. (1997) Biochemistry 36, 12138-12146], which is in contrast to the regulatory N-domain of skeletal TnC (sTnC). This implies that the mode of interaction between cTnC and cTnI may be different than that between sTnC and sTnI. In sTnI, a region downstream from the inhibitory region (residues 115-131) has been shown to bind the exposed hydrophobic pocket of Ca2+-saturated sNTnC [McKay, R. T., et al. (1997) J. Biol. Chem. 272, 28494-28500]. The present study demonstrates that the corresponding region in cTnI (residues 147-163) binds to the regulatory domain of cTnC only in the Ca2+-saturated state to form a 1:1 complex, with an affinity approximately six times weaker than that between the skeletal counterparts. Thus, while Ca2+ does not cause opening, it is required for muscle regulation. The solution structure of the cNTnC.Ca2+.cTnI147-163 complex has been determined by multinuclear multidimensional NMR spectroscopy. The structure reveals an open conformation for cNTnC, similar to that of Ca2+-saturated sNTnC. The bound peptide adopts a alpha-helical conformation spanning residues 150-157. The C-terminus of the peptide is unstructured. The open conformation for Ca2+-saturated cNTnC in the presence of cTnI (residues 147-163) accommodates hydrophobic interactions between side chains of the peptide and side chains at the interface of A and B helices of cNTnC. Thus the mechanistic differences between the regulation of cardiac and skeletal muscle contraction can be understood in terms of different thermodynamics and kinetics equilibria between essentially the same structure states.     

Cooperative effects of rigor and cycling cross-bridges on calcium binding to troponin C

The effects of rigor and cycling cross-bridges on distributions of calcium (Ca) bound within sarcomeres of rabbit psoas muscle fibers were compared using electron probe x-ray microanalysis. Calcium in the overlap region of rigor fibers, after correction for that bound to thick filaments, was significantly higher than in the I-band at all pCa levels tested between 6.9 and 4.8, but the difference was greatest at pCa 6.9. With addition of MgATP, differences were significant at high levels of activation (pCa 5.6 and 4.9); near and below the threshold for activation, Ca was the same in I-band and overlap regions. Comparison of Ca and mass profiles at the A-I junction showed elevation of Ca extending 55-110 nm (up to three regulatory units) into the I-band. Extraction of TnC-reduced I-band and overlap Ca in rigor fibers at pCa 5.6 to the same levels found in unextracted fibers at pCa 8.9, suggesting that variations reported here reflect changes in Ca bound to troponin C (TnC). Taken together, these observations provide evidence for near-neighbor cooperative effects of both rigor and cycling cross-bridges on Ca(2+) binding to TnC.     

Effects of mutations in the central helix of troponin C on its biological activity

To investigate the role of the central helix of skeletal muscle troponin C (TnC), five deletion mutants (Dobrowolski, Z., Xu, G.Q., and Hitchcock-DeGregori, S.E. (1991) J. Biol. Chem. 266, 5703-5710) of chicken TnC in the D/E linker region (K87EDAKGKSEEE97), dEDA, dKG, dKGK, dSEEE, and dKED-AKGK, were assayed for their ability to regulate muscle contraction by testing their effectiveness in restoring force and Ca2+ regulation to TnC-depleted rabbit skinned skeletal muscle fibers. By comparison with rabbit skeletal TnC, wild-type TnC, and chicken TnC, all mutants except dKG equally restored force development and Ca2+ regulation to TnC-depleted skinned muscle fibers. In contrast, approximately 4 times more dKG than rabbit skeletal TnC was required to reach 50% force restoration. Also, the pCa50 for dKG activation of force was significantly decreased. Thus, most of the TnC mutants that we studied did not have significantly altered biological activity in the skinned fiber assay. However, the 2-residue deletion in the central helix (dKG) significantly affected TnC activity. This deletion would be expected to produce a 160 degree rotation in the alpha-helix versus 60 degrees for dKGK and dEDA, 40 degrees in dSEEE, and 20 degrees in dKEDAKGK. Therefore, the change in orientation of the two Ca2(+)-binding domains appears to be a major parameter affecting TnC activity. The shift in the Ca2+ dependence in force activation may result from the inability of the Ca2(+)-specific domain to properly interact with its binding site on troponin I, an interaction which is known to increase the affinity of TnC for Ca2+ (Potter, J.D., and Gergely, J. (1975) J. Biol. Chem. 250, 4628-4633). In addition, the length of the central helix of TnC, Gly92, and the negatively charged cluster, EEE, appear not to be crucial for TnC activity.     

Effect of troponin I phosphorylation by protein kinase A on length-dependence of tension activation in skinned cardiac muscle fibers

We examined the effect of troponin I (TnI) phosphorylation by cAMP-dependent protein kinase (PKA) on the length-dependent tension activation in skinned rat cardiac trabeculae. Increasing sarcomere length shifted the pCa (-log[Ca2+])-tension relation to the left. Treatment with PKA decreased the Ca2+ sensitivity of the myofilament and also decreased the length-dependent shift of the pCa-tension relation. Replacement of endogenous TnI with phosphorylated TnI directly demonstrated that TnI phosphorylation is responsible for the decreased length-dependence. When MgATP concentration was lowered in the absence of Ca2+, tension was elicited through rigorous cross-bridge-induced thin filament activation. Increasing sarcomere length shifted the pMgATP (-log[MgATP])-tension relation to the right, and either TnI phosphorylation or partial extraction of troponin C (TnC) abolished this length-dependent shift. We conclude that TnI phosphorylation by PKA attenuates the length-dependence of tension activation in cardiac muscle by decreasing the cross-bridge-dependent thin filament activation through a reduction of the interaction between TnI and TnC.     

Force-calcium relationship depends on myosin heavy chain and troponin isoforms in rat diaphragm muscle fibers.

The present study examined Ca(2+) sensitivity of diaphragm muscle (Dia(m)) fibers expressing different myosin heavy chain (MHC) isoforms. We hypothesized that Dia(m) fibers expressing the MHC(slow) isoform have greater Ca(2+) sensitivity than fibers expressing fast MHC isoforms and that this fiber-type difference in Ca(2+) sensitivity reflects the isoform composition of the troponin (Tn) complex (TnC, TnT, and TnI). Studies were performed in single Triton-X-permeabilized Dia(m) fibers. The Ca(2+) concentration at which 50% maximal force was generated (pCa(50)) was determined for each fiber. SDS-PAGE and Western analyses were used to determine the MHC and Tn isoform composition of single fibers. The pCa(50) for Dia(m) fibers expressing MHC(slow) was significantly greater than that of fibers expressing fast MHC isoforms, and this greater Ca(2+) sensitivity was associated with expression of slow isoforms of the Tn complex. However, some Dia(m) fibers expressing MHC(slow) contained the fast TnC isoform. These results suggest that the combination of TnT, TnI, and TnC isoforms may determine Ca(2+) sensitivity in Dia(m) fibers.     

Calmidazolium alters Ca2+ regulation of tension redevelopment rate in skinned skeletal muscle.

To examine if the Ca2(+)-binding kinetics of troponin C (TnC) can influence the rate of cross-bridge force production, we studied the effects of calmidazolium (CDZ) on steady-state force and the rate of force redevelopment (ktr) in skinned rabbit psoas muscle fibers. CDZ increased the Ca2(+)-sensitivity of steady-state force and ktr at submaximal levels of activation, but increased ktr to a greater extent than can be explained by increased force alone. This occurred in the absence of any significant effects of CDZ on solution ATPase or in vitro motility of fluorescently labeled F-actin, suggesting that CDZ did not directly influence cross-bridge cycling. CDZ was strongly bound to TnC in aqueous solutions, and its effects on force production could be reversed by extraction of CDZ-exposed native TnC and replacement with purified (unexposed) rabbit skeletal TnC. These experiments suggest that the method of CDZ action in fibers is to bind to TnC and increase its Ca2(+)-binding affinity, which results in an increased rate of force production at submaximal [Ca2+]. The results also demonstrate that the Ca2(+)-binding kinetics of TnC influence the kinetics of ktr.     

Cooperative mechanisms in the activation dependence of the rate of force development in rabbit skinned skeletal muscle fibers

Regulation of contraction in skeletal muscle is a highly cooperative process involving Ca(2+) binding to troponin C (TnC) and strong binding of myosin cross-bridges to actin. To further investigate the role(s) of cooperation in activating the kinetics of cross-bridge cycling, we measured the Ca(2+) dependence of the rate constant of force redevelopment (k(tr)) in skinned single fibers in which cross-bridge and Ca(2+) binding were also perturbed. Ca(2+) sensitivity of tension, the steepness of the force-pCa relationship, and Ca(2+) dependence of k(tr) were measured in skinned fibers that were (1) treated with NEM-S1, a strong-binding, non-force-generating derivative of myosin subfragment 1, to promote cooperative strong binding of endogenous cross-bridges to actin; (2) subjected to partial extraction of TnC to disrupt the spread of activation along the thin filament; or (3) both, partial extraction of TnC and treatment with NEM-S1. The steepness of the force-pCa relationship was consistently reduced by treatment with NEM-S1, by partial extraction of TnC, or by a combination of TnC extraction and NEM-S1, indicating a decrease in the apparent cooperativity of activation. Partial extraction of TnC or NEM-S1 treatment accelerated the rate of force redevelopment at each submaximal force, but had no effect on kinetics of force development in maximally activated preparations. At low levels of Ca(2+), 3 microM NEM-S1 increased k(tr) to maximal values, and higher concentrations of NEM-S1 (6 or 10 microM) increased k(tr) to greater than maximal values. NEM-S1 also accelerated k(tr) at intermediate levels of activation, but to values that were submaximal. However, the combination of partial TnC extraction and 6 microM NEM-S1 increased k(tr) to virtually identical supramaximal values at all levels of activation, thus, completely eliminating the activation dependence of k(tr). These results show that k(tr) is not maximal in control fibers, even at saturating [Ca(2+)], and suggest that activation dependence of k(tr) is due to the combined activating effects of Ca(2+) binding to TnC and cross-bridge binding to actin.     

Effect of rigor and cycling cross-bridges on the structure of troponin C and on the Ca2+ affinity of the Ca2+-specific regulatory sites in skinned rabbit psoas fibers

Intrinsic troponin C (TnC) was extracted from small bundles of rabbit psoas fibers and replaced with TnC labeled with dansylaziridine (5-dimethylaminonaphthalene-1-sulfonyl). The flourescence of incorporated dansylaziridine-labeled TnC was enhanced by the binding of Ca2+ to the Ca2+-specific (regulatory) sites of TnC and was measured simultaneously with force (Zot, H.G., Güth, K., and Potter, J.D. (1986) J. Biol. Chem. 261, 15883-15890). Various myosin cross-bridge states also altered the fluorescence of dansylaziridine-labeled TnC in the filament, with cycling cross-bridges having a greater effect than rigor cross-bridges; and in both cases, there was an additional effect of Ca2+. The paired fluorescence and tension data were used to calculate the apparent Ca2+ affinity of the regulatory sites in the thin filament and were shown to increase at least 10-fold during muscle activation presumably due to the interaction of cycling cross-bridges with the thin filament. The cross-bridge state responsible for this enhanced Ca2+ affinity was shown to be the myosin-ADP state present only when cross-bridges are cycling. The steepness of the pCa force curves (where pCa represents the -log of the free Ca2+ concentration) obtained in the presence of ATP at short and long sarcomere lengths was the same, suggesting that cooperative interactions between adjacent troponin-tropomyosin units may spread along much of the actin filament when cross-bridges are attached to it. In contrast to the cycling cross-bridges, rigor bridges only increased the Ca2+ affinity of the regulatory sites 2-fold. Taken together, the results presented here indicate a strong coupling between the Ca2+ regulatory sites and cross-bridge interactions with the thin filament.     

Cardiac troponin T isoforms affect the Ca(2+) sensitivity of force development in the presence of slow skeletal troponin I: insights into the role of troponin T isoforms in the fetal heart

In this study we investigated the physiological role of the cardiac troponin T (cTnT) isoforms in the presence of human slow skeletal troponin I (ssTnI). ssTnI is the main troponin I isoform in the fetal human heart. In reconstituted fibers containing the cTnT isoforms in the presence of ssTnI, cTnT1-containing fibers showed increased Ca(2+) sensitivity of force development compared with cTnT3- and cTnT4-containing fibers. The maximal force in reconstituted skinned fibers was significantly greater for the cTnT1 (predominant fetal cTnT isoform) when compared with cTnT3 (adult TnT isoform) in the presence of ssTnI. Troponin (Tn) complexes containing ssTnI and reconstituted with cTnT isoforms all yielded different maximal actomyosin ATPase activities. Tn complexes containing cTnT1 and cTnT4 (both fetal isoforms) had a reduced ability to inhibit actomyosin ATPase activity when compared with cTnT3 (adult isoform) in the presence of ssTnI. The rate at which Ca(2+) was released from site II of cTnC in the cTnI.cTnC complex (122/s) was 12.5-fold faster than for the ssTnI.cTnC complex (9.8/s). Addition of cTnT3 to the cTnI.cTnC complex resulted in a 3.6-fold decrease in the Ca(2+) dissociation rate from site II of cTnC. Addition of cTnT3 to the ssTnI.cTnC complex resulted in a 1.9-fold increase in the Ca(2+) dissociation rate from site II of cTnC. The rate at which Ca(2+) dissociated from site II of cTnC in Tn complexes also depended on the cTnT isoform present. However, the TnI isoforms had greater effects on the Ca(2+) dissociation rate of site II than the cTnT isoforms. These results suggest that the different N-terminal TnT isoforms would produce distinct functional properties in the presence of ssTnI when compared with cTnI and that each isoform would have a specific physiological role in cardiac muscle.     

Evidence that both Ca(2+)-specific sites of skeletal muscle TnC are required for full activity

To investigate the role of the Ca2(+)-specific (I and II) sites of fast skeletal muscle troponin C (TnC) in the regulation of contraction, we have produced two TnC mutants which have lost the ability to bind Ca2+ at either site I (VG1) or at site II (VG2). Both mutants were able to partially restore force to TnC-depleted skinned muscle fibers (approximately 25% for VG1 and approximately 50% for VG2). In contrast, bovine cardiac TnC (BCTnC), which like VG1 binds Ca2+ only at site II, could fully reactivate the contraction of TnC-depleted fibers. Higher concentrations of both mutants were required to restore force to the TnC-depleted fibers than with wild type TnC (WTnC) or BCTnC. VG1 and VG2 substituted fibers could not bind additional WTnC, indicating that all of the TnC-binding sites were saturated with the mutant TnC's. The Ca2+ concentration required for force activation was much higher for VG1 and VG2 substituted fibers than for WTnC or BCTnC substituted fibers. Also, the steepness of force activation was much less in VG1 and VG2 versus WTnC and BCTnC substituted fibers. These results suggest cooperative interactions between sites I and II in WTnC. In contrast, BCTnC has essentially the same apparent Ca2+ affinity and steepness of force activation as does WTnC. Thus, cardiac TnC must have structural differences from WTnC which compensate for the lack of site I, while in WTnC, both Ca2(+)-specific sites are probably crucial for full functional activity.