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.     

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.     

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)     

Nitric oxide impairs Ca2+ activation and slows cross-bridge cycling kinetics in skeletal muscle.

The effects of the nitric oxide (NO) donor spermine NONOate (Sp-NO, 1.0 mM) on cross-bridge recruitment and cross-bridge cycling kinetics were studied in permeabilized rabbit psoas muscle fibers. Fibers were activated at various Ca2+ concentrations (pCa, negative logarithm of Ca2+ concentration), and the pCa at which force was maximal (pCa 4.0) and approximately 50% of maximal (pCa50 5.6) were determined. Fiber stiffness was determined using 1-kHz sinusoidal length perturbations, and the fraction of cross bridges in the force-generating state was estimated by the ratio of stiffness during maximal (pCa 4.0) and submaximal (pCa 5.6) Ca2+ activation to stiffness during rigor (at pCa 4.0). Cross-bridge cycling kinetics were evaluated by measuring the rate constant for force redevelopment after quick release (by 15% of optimal fiber length, L(o)) and restretch of the fiber to L(o). Exposing fibers to Sp-NO for 10 min reduced force and the fraction of cross bridges in the force-generating state at maximal and submaximal (pCa50) Ca2+ activation. However, the effects of Sp-NO were more pronounced during submaximal Ca2+ activation. Sp-NO also reduced the rate constant for force redevelopment but only during submaximal Ca2+ activation. We conclude that Sp-NO reduces Ca2+ sensitivity by decreasing the number of cross bridges in the strongly bound state and also impairs cross-bridge cycling kinetics during submaximal activation.     

Unloaded shortening of skinned muscle fibers from rabbit activated with and without Ca2+.

Unloaded shortening velocity (VUS) was determined by the slack method and measured at both maximal and submaximal levels of activation in glycerinated fibers from rabbit psoas muscle. Graded activation was achieved by two methods. First, [Ca2+] was varied in fibers with endogenous skeletal troponin C (sTnC) and after replacement of endogenous TnC with either purified cardiac troponin C (cTnC) or sTnC. Alternatively, fibers were either partially or fully reconstituted with a modified form of cTnC (aTnC) that enables force generation and shortening in the absence of Ca2+. Uniformity of the distribution of reconstituted TnC across the fiber radius was evaluated using fluorescently labeled sTnC and laser scanning fluorescence confocal microscopy. Fiber shortening was nonlinear under all conditions tested and was characterized by an early rapid phase (VE) followed by a slower late phase (VL). In fibers with endogenous sTnC, both VE and VL varied with [Ca2+], but VE was less affected than VL. Similar results were obtained after extraction of TnC and reconstitution with either sTnC or cTnC, except for a small increase in the apparent activation dependence of VE. Partial activation with aTnC was obtained by fully extracting endogenous sTnC followed by reconstitution with a mixture of aTnC and cTnC (aTnC:cTnC molar ratio 1:8.5). At pCa 9.2, VE and VL were similar to those obtained in fibers reconstituted with sTnC or cTnC at equivalent force levels. In these fibers, which contained aTnC and cTnC, VE and VL increased with isometric force when [Ca2+] was increased from pCa 9.2 to 4.0. Fibers that contained a mixture of a TnC and cTnC were then extracted a second time to selectively remove cTnC. In fibers containing aTnC only, VE and VL were proportional to the resulting submaximal isometric force compared with maximum Ca(2+)-activated control. With aTnC alone, force, VE, and VL were not affected by changes in [Ca2+]. The similarity of activation dependence of VUS whether fibers were activated in a Ca(2+)-sensitive or -insensitive manners implies that VUS is determined by the average level of thin filament activation and that, with sTnC or cTnC, VUS is affected by Ca2+ binding to TnC only.     

The role of ATP in energy-deprivation contractures in unloaded rat ventricular myocytes

Energy-deprivation contractures were investigated in unloaded rat ventricular myocytes. Application of 2 mM cyanide in the presence of 10 mM 2-deoxyglucose (metabolic blockade) led to a rapid shortening "contracture" (maximum speed 1.5 +/- 0.2% control cell length/s). Cells shortened to a constant length of 69 +/- 1.6% of the control length. Removal of cyanide caused cells to shorten further ("recontracture"), before relaxing towards the control length. Cells shortened to 57 +/- 2.0% during the recontracture. Similar behaviour was observed in zero extracellular [Ca2+]. Cells permeabilized with saponin (0.1% w/v) responded to the removal of ATP from the bathing solution, and to readdition of ATP, as intact cells did to complete metabolic blockade and its removal. In these permeabilized cells, the extent and speed of contracture shortening were similar at pCa = 7 and pCa greater than 9. When the bath concentration of ATP ([ ATP]b) was lowered to zero, shortening stopped at about 70% of the control length. However, when [ATP]b was lowered to an intermediate level (4-20 microM), cells contracted to lengths as short as 30% of the control length. Similarly, when [ATP]b was restored from zero to an intermediate concentration (4-20 microM), recontracture shortening continued without relaxation. The peak speed of this Ca2(+)-independent shortening showed a sigmoidal dependence on pMgATP (pMgATP0.5 = 4.0). Phosphocreatine (10 mM) shifted the ATP dependence of Ca2(+)-independent shortening to lower [ATP]b (pMgATP0.5 = 5.0), suggesting that gradients of [ATP] could exist between the bath and the myofilaments. Ca2(+)-independent shortening was inhibited by the chemical phosphatase 2,3-butanedione monoxime (BDM), although BDM did not relax cells from the shortened state during energy deprivation. Using a simple model, we show that the results can be explained by cross-bridge cycling occurring independently of Ca2+ over a "window" range of [MgATP] (0.1-100 microM). Therefore, when [MgATP] falls, cross-bridge cycling occurs and the cell shortens. As [MgATP] falls to very low levels ([ MgATP] less than 1 microM), shortening ceases as the rate of cross-bridge cycling declines. Recontracture occurs on restoring ATP production, because stiffness falls and Ca2(+)-independent cross-bridge cycling initially increases. As [MgATP] rises above 100 microM, Ca2(+)-independent cross-bridge cycling ceases and the cell relaxes towards the control length. We conclude that energy-deprivation contractures, and recontractures, can result from changes in [MgATP] and do not necessarily require changes in [Ca2+]i.     

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.     

Activation kinetics of skinned cardiac muscle by laser photolysis of nitrophenyl-EGTA

The kinetics of Ca(2+)-induced contractions of chemically skinned guinea pig trabeculae was studied using laser photolysis of NP-EGTA. The amount of free Ca(2+) released was altered by varying the output from a frequency-doubled ruby laser focused on the trabeculae, while maintaining constant total [NP-EGTA] and [Ca(2+)]. The time courses of the rise in stiffness and tension were biexponential at 23 degrees C, pH 7.1, and 200 mM ionic strength. At full activation (pCa < 5.0), the rates of the rapid phase of the stiffness and tension rise were 56 +/- 7 s(-1) (n = 7) and 48 +/- 6 s(-1) (n = 11) while the amplitudes were 21 +/- 2 and 23 +/- 3%, respectively. These rates had similar dependencies on final [Ca(2+)] achieved by photolysis: 43 and 50 s(-1) per pCa unit, respectively, over a range of [Ca(2+)] producing from 15% to 90% of maximal isometric tension. At all [Ca(2+)], the rise in stiffness initially was faster than that of tension. The maximal rates for the slower components of the rise in stiffness and tension were 4.1 +/- 0.8 and 6.2 +/- 1.0 s(-1). The rate of this slower phase exhibited significantly less Ca(2+) sensitivity, 1 and 4 s(-1) per pCa unit for stiffness and tension, respectively. These data, along with previous studies indicating that the force-generating step in the cross-bridge cycle of cardiac muscle is marginally sensitive to [Ca(2+)], suggest a mechanism of regulation in which Ca(2+) controls the attachment step in the cross-bridge cycle via a rapid equilibrium with the thin filament activation state. Myosin kinetics sets the time course for the rise in stiffness and force generation with the biexponential nature of the mechanical responses to steps in [Ca(2+)] arising from a shift to slower cross-bridge kinetics as the number of strongly bound cross-bridges increases.     

The effect of phosphate and calcium on force generation in glycerinated rabbit skeletal muscle fibers. A steady-state and transient kinetic study

The effect of varying concentrations of Pi and Ca2+ on isometric force and on the rate of force development in skinned rabbit psoas muscle fibers has been investigated. Steady-state results show that the three parameters that define the force-pCa relation (Po, pK, and n) all vary linearly with log [Pi]. As [Pi] increases, Po and pK decrease while n increases. The kinetics of force generation in isometrically contracting fibers were studied by laser flash photolysis of caged phosphate. The observed rate of the resulting tension transient, kPi, is 23.5 +/- 1.7 s-1 at 10 degrees C, 0.7 mM Pi, and is independent of [Ca2+] over the range pCa 4.5-7.2. By contrast, kTR, the rate of tension redevelopment following a period of isotonic shortening, is sensitive to [Ca2+] and is slower than kPi (kTR = 13.6 +/- 0.2 s-1 at pCa 4.5, 0.7 mM Pi). The results show that [Ca2+] does not directly affect the Pi release or force-generating steps of the cross-bridge cycle and show that the observed rate of force development depends on how the measurement is made. The data can be interpreted in terms of a model in which strong cross-bridges activate the thin filament, this activation being modulated by Ca2+ binding to troponin.     

Cross-bridge scheme and force per cross-bridge state in skinned rabbit psoas muscle fibers.

The rate and association constants (kinetic constants) which comprise a seven state cross-bridge scheme were deduced by sinusoidal analysis in chemically skinned rabbit psoas muscle fibers at 20 degrees C, 200 mM ionic strength, and during maximal Ca2+ activation (pCa 4.54-4.82). The kinetic constants were then used to calculate the steady state probability of cross-bridges in each state as the function of MgATP, MgADP, and phosphate (Pi) concentrations. This calculation showed that 72% of available cross-bridges were (strongly) attached during our control activation (5 mM MgATP, 8 mM Pi), which agreed approximately with the stiffness ratio (active:rigor, 69 +/- 3%); active stiffness was measured during the control activation, and rigor stiffness after an induction of the rigor state. By assuming that isometric tension is a linear combination of probabilities of cross-bridges in each state, and by measuring tension as the function of MgATP, MgADP, and Pi concentrations, we deduced the force associated with each cross-bridge state. Data from the osmotic compression of muscle fibers by dextran T500 were used to deduce the force associated with one of the cross-bridge states. Our results show that force is highest in the AM*ADP.Pi state (A = actin, M = myosin). Since the state which leads into the AM*ADP.Pi state is the weakly attached AM.ADP.Pi state, we confirm that the force development occurs on Pi isomerization (AM.ADP.Pi --> AM*ADP.Pi). Our results also show that a minimal force change occurs with the release of Pi or MgADP, and that force declines gradually with ADP isomerization (AM*ADP -->AM.ADP), ATP isomerization (AM+ATP-->AM*ATP), and with cross-bridge detachment. Force of the AM state agreed well with force measured after induction of the rigor state, indicating that the AM state is a close approximation of the rigor state. The stiffness results obtained as functions of MgATP, MgADP, and Pi concentrations were generally consistent with the cross-bridge scheme.     

Activation of skinned trabeculae of the guinea pig induced by laser photolysis of caged ATP.

The kinetics of force production in chemically skinned trabeculae from the guinea pig were studied by laser photolysis of caged ATP in the presence of Ca2+. Preincubation of the tissue during rigor with the enzyme apyrase was used to reduce the population of MgADP-bound cross-bridges (Martin and Barsotti, 1994). In untreated tissue, tension remained constant or dipped slightly below the rigor level immediately after ATP release, before increasing to the maximum measured in pCa 4.5 and 5 mM MgATP. The in-phase component stiffness, which is a measure of cross-bridge attachment, exhibited a large decrease before increasing to 55% of that measured in rigor. Neither the rate of the decline nor of the rise in tension was sensitive to the concentration of photolytically released ATP. The rate of the decline in stiffness was found to be dependent on [ATP]: 1.8 x 10(4) M-1/s-1, a value more than four times higher than that previously measured in similar experiments in the absence of Ca2+. The rate of tension development averaged 14.9 +/- 2.5 s-1. Preincubation with apyrase altered the mechanical characteristics of the early phase of the contraction. The rate and amplitude of the initial drop in both tension and stiffness after caged ATP photolysis increased and became dependent on [ATP]. The second-order rate constants measured for the initial drop in tension and stiffness were 8.4 x 10(4) M-1 s-1 and 1.5 x 10(5) M-1 s-1. These rates are more than two times faster than those previously measured in the absence of Ca2+. The effects of apyrase incubation on the time course of tension and stiffness were consistent with the hypothesis that during rigor, skinned trabeculae retain a significant population of MgADP-bound cross-bridges. These in turn act to attenuate the initial drop in tension after caged ATP photolysis and slow the apparent rate of rigor cross-bridge detachment. The results also show that Ca2+ increases the rate of cross-bridge detachment in both untreated and apyrase-treated tissue, but the effect is larger in untreated tissue. This suggests that in cardiac muscle Ca2+ modulates the rate of cross-bridge detachment.     

Recording of intracellular Ca2+ from smooth muscle cells by sub-micron tip, double-barrelled CA2+-selective microelectrodes

Novel, double-barrelled Ca2+-selective microelectrodes with tip diameters of approximately 0.1 micron were constructed by using Simon's neutral Ca2+ ligand (ETH 1001). Concentric micropipettes were utilized for the first time for Ca2+-selective microelectrodes in which the Ca2+ ligand was incorporated into a protruding inner pipette, surrounded by an outer reference electrode. In addition, they were made from high resistance aluminosilicate glass tubing (Corning Code 1724). These Ca2+-selective electrodes had linear responses from pCa 3 to pCa 7 in the presence of constant [K+]. They provided on-line observation of changes in intracellular [Ca2+] and in the resting membrane potential in single smooth muscle cells isolated from toad stomach. The mean concentration of intracellular Ca2+ in resting cells was 163.6 +/- 20 nM (+/- SEM, n = 16). Doubling the intracellular Ca2+ level by exposure of cells to elevated [K+] was sufficient to cause shortening.     

Rate of phosphate release after photoliberation of adenosine 5'-triphosphate in slow and fast skeletal muscle fibers.

Inorganic phosphate (Pi) release was determined by means of a fluorescent Pi-probe in single permeabilized rabbit soleus and psoas muscle fibers. Measurements of Pi release followed photoliberation of approximately 1.5 mM ATP by flash photolysis of NPE-caged ATP in the absence and presence of Ca2+ at 15 degrees C. In the absence of Ca2+, Pi release occurred with a slow rate of 11 +/- 3 microM . s-1 (n = 3) in soleus fibers and 23 +/- 1 microM . s-1 (n = 10) in psoas fibers. At saturating Ca2+ concentrations (pCa 4.5), photoliberation of ATP was followed by rapid force development. The initial rate of Pi release was 0.57 +/- 0.05 mM . s-1 in soleus (n = 13) and 4.7 +/- 0.2 mM . s-1 in psoas (n = 23), corresponding to a rate of Pi release per myosin head of 3.8 s-1 in soleus and 31.5 s-1 in psoas. Pi release declined at a rate of 0.48 s-1 in soleus and of 5.2 s-1 in psoas. Pi release in soleus was slightly faster in the presence of an ATP regenerating system but slower when 0.5 mM ADP was added. The reduction in the rate of Pi release results from an initial redistribution of cross-bridges over different states and a subsequent ADP-sensitive slowing of cross-bridge detachment.     

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.     

Ca2+ regulation of rabbit skeletal muscle thin filament sliding: role of cross-bridge number

We investigated how strong cross-bridge number affects sliding speed of regulated Ca(2+)-activated, thin filaments. First, using in vitro motility assays, sliding speed decreased nonlinearly with reduced density of heavy meromyosin (HMM) for regulated (and unregulated) F-actin at maximal Ca(2+). Second, we varied the number of Ca(2+)-activatable troponin complexes at maximal Ca(2+) using mixtures of recombinant rabbit skeletal troponin (WT sTn) and sTn containing sTnC(D27A,D63A), a mutant deficient in Ca(2+) binding at both N-terminal, low affinity Ca(2+)-binding sites (xxsTnC-sTn). Sliding speed decreased nonlinearly as the proportion of WT sTn decreased. Speed of regulated thin filaments varied with pCa when filaments contained WT sTn but filaments containing only xxsTnC-sTn did not move. pCa(50) decreased by 0.12-0.18 when either heavy meromyosin density was reduced to approximately 60% or the fraction of Ca(2+)-activatable regulatory units was reduced to approximately 33%. Third, we exchanged mixtures of sTnC and xxsTnC into single, permeabilized fibers from rabbit psoas. As the proportion of xxsTnC increased, unloaded shortening velocity decreased nonlinearly at maximal Ca(2+). These data are consistent with unloaded filament sliding speed being limited by the number of cycling cross-bridges so that maximal speed is attained with a critical, low level of actomyosin interactions.     

Faster force transient kinetics at submaximal Ca2+ activation of skinned psoas fibers from rabbit.

The early, rapid phase of tension recovery (phase 2) after a step change in sarcomere length is thought to reflect the force-generating transition of myosin bound to actin. We have measured the relation between the rate of tension redevelopment during phase 2 (r), estimated from the half-time of tension recovery during phase 2 (r = t0.5(-1)), and steady-state force at varying [Ca2+] in single fibers from rabbit psoas. Sarcomere length was monitored continuously by laser diffraction of fiber segments (length approximately 1.6 mm), and sarcomere homogeneity was maintained using periodic length release/restretch cycles at 13-15 degrees C. At lower [Ca2+] and forces, r was elevated relative to that at pCa 4.0 for both releases and stretches (between +/- 8 nm). For releases of -3.4 +/- 0.7 nm.hs-1 at pCa 6.6 (where force was 10-20% of maximum force at pCa 4.0), r was 3.3 +/- 1.0 ms-1 (mean +/- SD; N = 5), whereas the corresponding value of r at pCa 4.0 was 1.0 +/- 0.2 ms-1 for releases of -3.5 +/- 0.5 nm.hs-1 (mean +/- SD; N = 5). For stretches of 1.9 +/- 0.7 nm.hs-1, r was 1.0 +/- 0.3 ms-1 (mean +/- SD; N = 9) at pCa 6.6, whereas r was 0.4 +/- 0.1 ms-1 at pCa 4.0 for stretches of 1.9 +/- 0.5 (mean +/- SD; N = 14). Faster phase 2 transients at submaximal Ca(2+)-activation were not caused by changes in myofilament lattice spacing because 4% Dextran T-500, which minimizes lattice spacing changes, was present in all solutions.(ABSTRACT TRUNCATED AT 250 WORDS)     

The relationship between contractile force and intracellular [Ca2+] in intact rat cardiac trabeculae

The control of force by [Ca2+] was investigated in rat cardiac trabeculae loaded with fura-2 salt. At sarcomere lengths of 2.1-2.3 microns, the steady state force-[Ca2+]i relationship during tetanization in the presence of ryanodine was half maximally activated at a [Ca2+]i of 0.65 +/- 0.19 microM with a Hill coefficient of 5.2 +/- 1.2 (mean +/- SD, n = 9), and the maximal stress produced at saturating [Ca2+]i equalled 121 +/- 35 mN/mm2 (n = 9). The dependence of steady state force on [Ca2+]i was identical in muscles tetanized in the presence of the Ca(2+)-ATPase inhibitor cyclopiazonic acid (CPA). The force-[Ca2+]i relationship during the relaxation of twitches in the presence of CPA coincided exactly to that measured at steady state during tetani, suggesting that CPA slows the decay rate of [Ca2+]i sufficiently to allow the force to come into a steady state with the [Ca2+]i. In contrast, the relationship of force to [Ca2+]i during the relaxation phase of control twitches was shifted leftward relative to the steady state relationship, establishing that relaxation is limited by the contractile system itself, not by Ca2+ removal from the cytosol. Under control conditions the force-[Ca2+]i relationship, quantified at the time of peak twitch force (i.e., dF/dt = 0), coincided fairly well with steady state measurements in some trabeculae (i.e., three of seven). However, the force-[Ca2+]i relationship at peak force did not correspond to the steady state measurements after the application of 5 mM 2,3-butanedione monoxime (BDM) (to accelerate cross-bridge kinetics) or 100 microM CPA (to slow the relaxation of the [Ca2+]i transient). Therefore, we conclude that the relationship of force to [Ca2+]i during physiological twitch contractions cannot be used to predict the steady state relationship.     

Depression of force production and ATPase activity in different types of human skeletal muscle fibers from patients with chronic heart failure.

Isometric force production and ATPase activity were determined simultaneously in single human skeletal muscle fibers (n = 97) from five healthy volunteers and nine patients with chronic heart failure (CHF) at 20 degrees C. The fibers were permeabilized by means of Triton X-100 (1% vol/vol). ATPase activity was determined by enzymatic coupling of ATP resynthesis to the oxidation of NADH. Calcium-activated actomyosin (AM) ATPase activity was obtained by subtracting the activity measured in relaxing (pCa = 9) solutions from that obtained in maximally activating (pCa = 4.4) solutions. Fiber type was determined on the basis of myosin heavy chain isoform composition by polyacrylamide SDS gel electrophoresis. AM ATPase activity per liter cell volume (+/-SE) in the control and patient group, respectively, amounted to 134 +/- 24 and 77 +/- 9 microM/s in type I fibers (n = 11 and 16), 248 +/- 17 and 188 +/- 13 microM/s in type IIA fibers (n = 14 and 32), 291 +/- 29 and 126 +/- 21 microM/s in type IIA/X fibers (n = 3 and 5), and 325 +/- 32 and 205 +/- 21 microM/s in type IIX fibers (n = 7 and 9). The maximal isometric force per cross-sectional area amounted to 64 +/- 7 and 43 +/- 5 kN/m(2) in type I fibers, 86 +/- 11 and 58 +/- 4 kN/m(2) in type IIA fibers, 85 +/- 6 and 42 +/- 9 kN/m(2) in type IIA/X fibers, and 90 +/- 5 and 59 +/- 5 kN/m(2) in type IIX fibers in the control and patient group, respectively. These results indicate that, in CHF patients, significant reductions occur in isometric force and AM ATPase activity but that tension cost for each fiber type remains the same. This suggests that, in skeletal muscle from CHF patients, a decline in density of contractile proteins takes place and/or a reduction in the rate of cross-bridge attachment of approximately 30%, which exacerbates skeletal muscle weakness due to muscle atrophy.     

Role of Ca2+ and cross-bridges in skeletal muscle thin filament activation probed with Ca2+ sensitizers

Thin filament regulation of contraction is thought to involve the binding of two activating ligands: Ca2+ and strongly bound cross-bridges. The specific cross-bridge states required to promote thin filament activation have not been identified. This study examines the relationship between cross-bridge cycling and thin filament activation by comparing the results of kinetic experiments using the Ca2+ sensitizers caffeine and bepridil. In single skinned rat soleus fibers, 30 mM caffeine produced a leftward shift in the tension-pCa relation from 6.03 +/- 0.03 to 6.51 +/- 0.03 pCa units and lowered the maximum tension to 0.60 +/- 0.01 of the control tension. In addition, the rate of tension redevelopment (ktr) was decreased from 3.51 +/- 0.12 s-1 to 2.70 +/- 0.19 s-1, and Vmax decreased from 1.24 +/- 0.07 to 0.64 +/- 0.02 M.L./s. Bepridil produced a similar shift in the tension-pCa curves but had no effect on the kinetics. Thus bepridil increases the Ca2+ sensitivity through direct effects on TnC, whereas caffeine has significant effects on the cross-bridge interaction. Interestingly, caffeine also produced a significant increase in stiffness under relaxing conditions (pCa 9.0), indicating that caffeine induces some strongly bound cross-bridges, even in the absence of Ca2+. The results are interpreted in terms of a model integrating cross-bridge cycling with a three-state thin-filament activation model. Significantly, strongly bound, non-tension-producing cross-bridges were essential to modeling of complete activation of the thin filament.     

Cardiac length dependence of force and force redevelopment kinetics with altered cross-bridge cycling

We examined the influence of cross-bridge cycling kinetics on the length dependence of steady-state force and the rate of force redevelopment (k(tr)) during Ca(2+)-activation at sarcomere lengths (SL) of 2.0 and 2.3 microm in skinned rat cardiac trabeculae. Cross-bridge kinetics were altered by either replacing ATP with 2-deoxy-ATP (dATP) or by reducing [ATP]. At each SL dATP increased maximal force (F(max)) and Ca(2+)-sensitivity of force (pCa(50)) and reduced the cooperativity (n(H)) of force-pCa relations, whereas reducing [ATP] to 0.5 mM (low ATP) increased pCa(50) and n(H) without changing F(max). The difference in pCa(50) between SL 2.0 and 2.3 microm (Delta pCa(50)) was comparable between ATP and dATP, but reduced with low ATP. Maximal k(tr) was elevated by dATP and reduced by low ATP. Ca(2+)-sensitivity of k(tr) increased with both dATP and low ATP and was unaffected by altered SL under all conditions. Significantly, at equivalent levels of submaximal force k(tr) was faster at short SL or increased lattice spacing. These data demonstrate that the SL dependence of force depends on cross-bridge kinetics and that the increase of force upon SL extension occurs without increasing the rate of transitions between nonforce and force-generating cross-bridge states, suggesting SL or lattice spacing may modulate preforce cross-bridge transitions.