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.     

Co-operative interactions between troponin-tropomyosin units extend the length of the thin filament in skeletal muscle

Ca2+ binding to troponin C (TnC), a subunit of the thin filament regulatory strand, activates vertebrate skeletal muscle contraction. Tension, however, increases with Ca2+ too abruptly to be the result of binding to sites on individual TnCs. Because extraction of one TnC on average per regulatory strand dramatically reduces the slope of the tension/Ca2+ relationship, we proposed that all 26 troponin-tropomyosin complexes of the regulatory strand form a co-operative system. This study of permeabilized (chemically skinned) rabbit psoas fibers analyzes the extraction time-course, the distribution of extraction sites on regulatory strands and the effects of extraction on the co-operativity of the tension/Ca2+ relationship. Two components of TnC are resolved in the time-course of extraction: a "rapidly extracting" component that can be selectively removed without affecting tension or co-operativity, and a "slow extracting" component whose loss reduces tension and co-operativity. Extraction of [14C]TnC shows that the slowly extracting component is lost randomly, so that, after removal of 5% of the TnC, most extracted strands have lost one TnC. Extraction interrupts the transmission of co-operativity by dividing the regulatory strand into smaller, independent co-operative systems; it reduces tension by preventing Ca2+ activation of TnC-depleted regulatory units. Co-operativity of the tension/Ca2+ relationship is modeled with the concerted-transition formalism for intact systems of 26 regulatory units, and for the smaller systems in extracted fibers.     

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.     

Role of creatine kinase in force development in chemically skinned rat cardiac muscle

The influence of phosphocreatine in the presence or absence of MgATP and MgADP was studied in Triton X-100-treated thin papillary muscles and ventricular strips of the rat heart. The pCa/tension relationships, the pMgATP/tension relationships, and the tension responses to quick length changes were analyzed. The results show three major consequences of the reduction of the phosphocreatine concentration in the presence of millimolar concentrations of the MgATP. (a) The resting tension and the maximal Ca2+-activated tension were increased, and the pCa/tension relationship was shifted toward higher pCa values and its steepness was decreased; these effects were enhanced by the inclusion of MgADP. (b) The time constant of tension recoveries after quick stretches applied during maximal activation was increased, while the extent of these recoveries was decreased. (c) The study of pMgATP/tension relationships in low Ca concentrations showed that the decrease in phosphocreatine induced a shift toward higher MgATP values with no changes in maximal rigor tension or the slope coefficient; these effects were increased by the increase in MgADP and were independent of the preparation diameter. Thus, modifications of the apparent Ca sensitivity and resting and maximal tension when phosphocreatine is decreased seem to be due to an increasing participation of rigor-like or slowly cycling cross-bridges spending more time in the attached state. These results suggest that endogenous creatine kinase is able to ensure maximal efficiency of myosin ATPase by producing a local high MgATP/MgADP ratio.     

Reciprocal coupling between troponin C and myosin crossbridge attachment

The attachment of cycling myosin crossbridges to actin and the resultant muscle contraction are regulated in skeletal muscle by the binding of Ca2+ to the amino-terminal, regulatory sites of the troponin C (TnC) subunit of the thin filament protein troponin. Conversely, the attachment of crossbridges to actin has been shown to alter the affinity of TnC for Ca2+. In this study, fluorescently labeled TnC incorporated into reconstituted thin filaments was used to investigate the relationship between crossbridge attachment to actin and structural changes in the amino-terminal region of TnC. Fluorescence intensity changes were measured under the following conditions: saturating [Ca2+] in the absence of crossbridges, rigor crossbridge attachment in the presence and absence of Ca2+, and cycling crossbridge attachment. The percent of heavy meromyosin crossbridges associated with the thin filaments under these conditions was also determined. The results show that, in addition to the binding of Ca2+ to TnC, the attachment of both rigor and cycling crossbridges to actin alters the structure of TnC near the regulatory, Ca2+-specific sites of the molecule. A differential coupling between weakly versus strongly bound crossbridge states and TnC structure was detected, suggesting a possible differential regulation of these states by conformational changes in TnC. These findings illustrate a reciprocal coupling, via thin filament protein interactions, between structural changes in TnC and the attachment of myosin crossbridges to actin, such that each can influence the other, and indicate that TnC is not simply an on-off switch but may exist in a number of different conformations.     

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 partial extraction of troponin complex upon the tension-pCa relation in rabbit skeletal muscle. Further evidence that tension development involves cooperative effects within the thin filament

Partial extraction of troponin C (TnC) decreases the Ca2+ sensitivity of tension development in mammalian skinned muscle fibers (Moss, R. L., G. G. Giulian, and M. L. Greaser. 1985. Journal of General Physiology. 86:585), which suggests that Ca2+-activated tension development involves molecular cooperativity within the thin filament. This idea has been investigated further in the present study, in which Ca2+-insensitive activation of skinned fibers from rabbit psoas muscles was achieved by removing a small proportion of total troponin (Tn) complexes. Ca2+-activated isometric tension was measured at pCa values (i.e., -log[Ca2+]) between 6.7 and 4.5: (a) in control fiber segments, (b) in the same fibers after partial removal of Tn, and (c) after recombination of Tn. Tn removal was accomplished using contaminant protease activity found in preparations of LC2 from rabbit soleus muscle, and was quantitated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and scanning densitometry. Partial Tn removal resulted in the development of a Ca2+-insensitive active tension, which varied in amount depending on the duration of the extraction, and concomitant decreases in maximal Ca2+-activated tensions. In addition, the tension-pCa relation was shifted to higher pCa values by as much as 0.3 pCa unit after Tn extraction. Readdition of Tn to the fiber segments resulted in the reduction of tension in the relaxing solution to control values and in the return of the tension-pCa relation to its original position. Thus, continuous Ca2+-insensitive activation of randomly spaced functional groups increased the Ca2+ sensitivity of tension development in the remaining functional groups along the thin filament. In addition, the variation in Ca2+-insensitive active tension as a function of Tn content after extraction suggests that only one-third to one-half of the functional groups within a thin filament need to be activated for complete disinhibition of that filament to be achieved.     

The thin filament of vertebrate skeletal muscle co-operatively activates as a unit

We find that extraction of as little as one troponin C molecule per troponin-tropomyosin strand on a thin filament reduces the slope of the pCa/tension relation. We interpret this to mean that the regulatory units along a thin filament of rabbit psoas fibers are linked co-operatively so that a thin filament activates as a unit. The presence of extended co-operativity explains why the pCa/tension relation in skinned fibers has a slope much higher than predicted by binding of Ca2+ to one regulatory unit. Replacement of the extracted troponin C with purified troponin C fully reverses the effect of extraction and shows it to be the essential Ca2+ binding protein responsible for the steep slope of the pCa/tension relation.     

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.     

Effect of different troponin T-tropomyosin combinations on thin filament activation

The response of permeabilized rabbit fast skeletal muscle fibers to calcium is determined by the troponin T (TnT) and tropomyosin (Tm) isoforms they express. Fibers expressing primarily TnT2f and alpha 2 Tm exhibit steeper pCa/tension relations than those in which either TnT1f or TnT3f and alpha beta Tm predominate. Troponin C extraction studies show that lower slopes do not result from a less concerted transition on the thin filament: the Tn-Tm regulatory strand activates as a unit in all fast fibers. Because the TnT variants differ in their N-terminal segments, and this region overlaps adjacent Tms on the regulatory strand, we propose that both the end-to-end overlap of Tm and the effect of TnT on that interaction are the basis of the concerted transition of the regulatory strand to the active state that occurs in the presence of calcium. Moreover, the effect of different Tn-Tm combinations on the ratio of the affinities of TnC for calcium in the relaxed and active states appears to be a significant determinant of the contractile properties of fast fibers in vivo.     

Characteristics of Ca2+- and Mg2+-induced tension development in chemically skinned smooth muscle fibers

Chemically skinned fibers from guinea pig taenia caecum were prepared by saponin treatment to study the smooth muscle contractile system in a state as close to the living state as posible. The skinned fibers showed tension development with an increase of Ca2+ in the solution, the threshold tension occurring as 5 X 10(-7) M Ca2+. The maximal tension induced with 10(-4) M Ca2+ was as large and rapid as the potassium-induced contracture in the intact fibers. The slope of the pCa tension curve was less steep than that of skeletal muscle fibers and shifted in the direction of lower pCa with an increase of MgATP. The presence of greater than 1 mM Mg2+ was required for Ca2+-induced contraction in the skinned fibers as well as for the activation of ATPase and superprecipitation in smooth muscle myosin B. Mg2+ above 2 mM caused a slow tension development by itself in the absence of Ca2+. Such a Mg2+-induced tension showed a linear relation to concentrations up to 8 mM in the presence of MgATP. Increase of MgATP concentration revealed a monophasic response without inhibition of Ca2+-induced tension development, unlike the biphasic response in striated muscle. When MgATP was removed from the relaxing solution, the tension developed slowly and slightly, even though the Mg2+ concentrations was fixed at 2 mM. These results suggest a substantial difference in the mode of actin-myosin interaction between smooth and skeletal muscle.     

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.     

The effects of partial extraction of TnC upon the tension-pCa relationship in rabbit skinned skeletal muscle fibers

The activation of contraction in vertebrate skeletal muscle involves the binding of Ca2+ to low-affinity binding sites on the troponin C (TnC) subunit of the regulatory protein troponin. The present study is an investigation of possible cooperative interactions between adjacent functional groups, composed of seven actin monomers, one tropomyosin, and one troponin, along the same thin filament. Single skinned fibers were obtained from rabbit psoas muscles and were then placed in an experimental chamber containing relaxing solution maintained at 15 degrees C. Isometric tension was measured in solutions containing maximally and submaximally activating levels of free Ca2+ (a) in control fiber segments, (b) in the same segments after partial extraction of TnC, and finally (c) after recombination of TnC into the segments. The extraction was done at 11-13 degrees C in 20 mM Tris, 5 mM EDTA, pH 7.85 or 8.3, a procedure derived from that of Cox et al. (1981. Biochem. J. 195:205). Extraction of TnC was quantitated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the control and experimental samples. Partial extraction of TnC resulted in reductions in tension during maximal Ca activation and in a shift of the relative tension-pCa (i.e., -log[Ca2+]) relationship to lower pCa's. The readdition of TnC to the extracted fiber segments resulted in a recovery of tension to near-control levels and in the return of the tension-pCa relation to its original position. On the basis of these findings, we conclude that the sensitivity to Ca2+ of a functional group within the thin filament may vary depending upon the state of activation of immediately adjacent groups.     

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.     

Alterations in Ca2+ sensitive tension due to partial extraction of C- protein from rat skinned cardiac myocytes and rabbit skeletal muscle fibers

C-protein, a substantial component of muscle thick filaments, has been postulated to have various functions, based mainly on results from biochemical studies. In the present study, effects on Ca(2+)-activated tension due to partial removal of C-protein were investigated in skinned single myocytes from rat ventricle and rabbit psoas muscle. Isometric tension was measured at pCa values of 7.0 to 4.5: (a) in untreated myocytes, (b) in the same myocytes after partial extraction of C-protein, and (c) in some myocytes, after readdition of C-protein. The solution for extracting C-protein contained 10 mM EDTA, 31 mM Na2HPO2, 124 mM NaH2PO4, pH 5.9 (Offer et al., 1973; Hartzell and Glass, 1984). In addition, the extracting solution contained 0.2 mg/ml troponin and, for skeletal muscle, 0.2 mg/ml myosin light chain-2 in order to minimize loss of these proteins during the extraction procedure. Between 60 and 70% of endogenous C-protein was extracted from cardiac myocytes by a 1-h soak in extracting solution at 20-23 degrees C; a similar amount was extracted from psoas fibers during a 3-h soak at 25 degrees C. For both cardiac myocytes and skeletal muscle fibers, partial extraction of C-protein resulted in increased active tension at submaximal concentrations of Ca2+, but had little effect upon maximum tension. C-protein extraction also reduced the slope of the tension-pCa relationships, suggesting that the cooperativity of Ca2+ activation of tension was decreased. Readdition of C-protein to previously extracted myocytes resulted in recovery of both tension and slope to near their control values. The effects on tension did not appear to be due to disruption of cooperative activation of the thin filament, since C-protein extraction from cardiac myocytes that were 40-60% troponin-C (TnC) deficient produced effects similar to those observed in cells that were TnC replete. Measurements of the tension-pCa relationship in skeletal muscle fibers were also made at a sarcomere length of 3.5 microns which, because of the distribution of C-protein on the thick filament, should eliminate any interaction between C-protein and actin. The effects of C-protein extraction were similar at long and short sarcomere lengths. These data are consistent with a model in which C-protein modulates the range of movement of myosin, such that the probability of myosin binding to actin is increased after its extraction.     

Conformational change of skeletal muscle troponin

The fluorescence titration curve of skeletal muscle troponin containing TnI with 2-[4'-iodoacetamido)anilino)naphthalene-6-sulfonic acid-labeled Cys-48 and/or Cys-64 was composed of two transition curves. One transition occurred at the pCa region higher than 8.0, and the other between pCa 8.0 and 6.0. The transition at the lower pCa region had a midpoint of pCa 6.85, and the midpoint did not depend on Mg2+. The time course of the fluorescence change subsequent to the rapid pCa-jump of the solution was biphasic. The fast phase was due to the transition at the lower pCa region, and the rate constant of the process was characteristic of the conformational change of the protein induced by Ca2+ binding to the low affinity Ca2+-binding sites of TnC. The slow phase was from the transition at the higher pCa region, and its rate constant was characteristic of the conformational change of the protein induced by Ca2+ binding to the high affinity Ca2+-binding sites of TnC. Therefore we can conclude that the fluorescence probe bound to Cys-48 and/or Cys-64 of TnI detects the conformational change of the Tn complex induced by Ca2+ binding to both the low and high affinity Ca2+-binding sites of TnC. The fluorescence probe bound to Cys-133 of TnI or Met residues of TnT detected the conformational change of the Tn complex induced by Ca2+ binding to the low affinity Ca2+-binding sites of TnC.     

Biological function and site II Ca2+-induced opening of the regulatory domain of skeletal troponin C are impaired by invariant site I or II Glu mutations

To investigate the roles of site I and II invariant Glu residues 41 and 77 in the functional properties and calcium-induced structural opening of skeletal muscle troponin C (TnC) regulatory domain, we have replaced them by Ala in intact F29W TnC and in wild-type and F29W N domains (TnC residues 1-90). Reconstitution of intact E41A/F29W and E77A/F29W mutants into TnC-depleted muscle skinned fibers showed that Ca(2+)-induced tension is greatly reduced compared with the F29W control. Circular dichroism measurements of wild-type N domain as a function of pCa (= -log[Ca(2+)]) demonstrated that approximately 90% of the total change in molar ellipticity at 222 nm ([theta](222 nm)) could be assigned to site II Ca(2+) binding. With E41A, E77A, and cardiac TnC N domains this [theta](222 nm) change attributable to site II was reduced to < or =40% of that seen with wild type, consistent with their structures remaining closed in +Ca(2+). Furthermore, the Ca(2+)-induced changes in fluorescence, near UV CD, and UV difference spectra observed with intact F29W are largely abolished with E41A/F29W and E77A/F29W TnCs. Taken together, the data indicate that the major structural change in N domain, including the closed to open transition, is triggered by site II Ca(2+) binding, an interpretation relevant to the energetics of the skeletal muscle TnC and cardiac TnC systems.     

Force regulation by Ca2+ in skinned single cardiac myocytes of frog.

Atrial and ventricular myocytes 200 to 300 microm long containing one to five myofibrils are isolated from frog hearts. After a cell is caught and held between two suction micropipettes the surface membrane is destroyed by briefly jetting relaxing solution containing 0.05% Triton X-100 on it from a third micropipette. Jetting buffered Ca2+ from other pipettes produces sustained contractions that relax completely on cessation. The pCa/force relationship is determined at 20 degrees C by perfusing a closely spaced sequence of pCa concentrations (pCa = -log[Ca2+]) past the skinned myocyte. At each step in the pCa series quick release of the myocyte length defines the tension baseline and quick restretch allows the kinetics of the return to steady tension to be observed. The pCa/force data fit to the Hill equation for atrial and ventricular myocytes yield, respectively, a pK (curve midpoint) of 5.86 +/- 0.03 (mean +/- SE.; n = 7) and 5.87 +/- 0.02 (n = 18) and an nH (slope) of 4.3 +/- 0.34 and 5.1 +/- 0.35. These slopes are about double those reported previously, suggesting that the cooperativity of Ca2+ activation in frog cardiac myofibrils is as strong as in fast skeletal muscle. The shape of the pCa/force relationship differs from that usually reported for skeletal muscle in that it closely follows the ideal fitted Hill plot with a single slope while that of skeletal muscle appears steeper in the lower than in the upper half. The rate of tension redevelopment following release restretch protocol increases with Ca2+ >10-fold and continues to rise after Ca2+ activated tension saturates. This finding provides support for a strong kinetic mechanism of force regulation by Ca2+ in frog cardiac muscle, at variance with previous reports on mammalian heart muscle. The maximum rate of tension redevelopment following restretch is approximately twofold faster for atrial than for ventricular myocytes, in accord with the idea that the intrinsic speed of the contractile proteins is faster in atrial than in ventricular myocardium.     

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.     

Tension transients initiated by photogeneration of MgADP in skinned skeletal muscle fibers

Addition of MgADP to skinned skeletal muscle fibers causes a rise in Ca(2+)-activated isometric tension. Mechanisms underlying this tension increase have been investigated by rapid photogeneration of ADP within skinned single fibers of rabbit psoas muscle. Photolysis of caged ADP (P2-1(2-nitrophenyl)ethyladenosine 5'-diphosphate) resulted in an exponential increase in isometric tension with an apparent rate constant, kADP, of 9.6 +/- 0.3 s-1 (mean +/- SE, n = 28) and an amplitude, PADP, of 4.9 +/- 0.3% Po under standard conditions (0.5 mM photoreleased MgADP, 4 mM MgATP, pH 7.0, pCa 4.5, 0.18 M ionic strength, 15 degrees C). PADP depended upon the concentration of photoreleased MgADP as well as the concentration of MgATP. A plot of 1/PADP vs. 1/[MgADP] at three MgATP concentrations was consistent with competition between MgADP and MgATP for the same site on the crossbridge. The rate of the transient, kADP, also depended upon the concentration of MgADP and MgATP. At both 4 and 1 mM MgATP, kADP was not significantly different after photorelease of 0.1-0.5 mM MgADP, but was reduced by 28-40% when 3.5 mM MgADP was added before photorelease of 0.5 mM MgADP. kADP was accelerated by about twofold when MgATP was varied from 0.5 to 8 mM MgATP. These effects of MgATP and MgADP were not readily accounted for by population of high force-producing states resulting from reversal of the ADP dissociation process. Rather, the results suggest that competition between MgADP and MgATP for crossbridges at the end of the cycle slows detachment leading to accumulation of force-generating crossbridges. Elevation of steady- state Pi concentration from 0.5 to 30 mM caused acceleration of kADP from 10.2 +/- 0.5 to 27.8 +/- 1.8 s-1, indicating that the tension rise involved crossbridge flux through the Pi dissociation step of the cycle.