Spontaneous oscillation of tension and sarcomere length in skeletal myofibrils. Microscopic measurement and analysis.

We have devised a simple method for measuring tension development of single myofibrils by micromanipulation with a pair of glass micro-needles. The tension was estimated from the deflection of a flexible needle under an inverted phase-contrast microscope equipped with an image processor, so that the tension development is always accompanied by the shortening of the myofibril (auxotonic condition) in the present setup. The advantage of this method is that the measurement of tension (1/30 s for time resolution and about 0.05 micrograms for accuracy of tension measurement; 0.05 microns as a spatial resolution for displacement of the micro-needle) and the observation of sarcomere structure are possible at the same time, and the technique to hold myofibrils, even single myofibrils, is very simple. This method has been applied to study the tension development of glycerinated skeletal myofibrils under the condition where spontaneous oscillation of sarcomeres is induced, i.e., the coexistence of MgATP, MgADP and inorganic phosphate without free Ca2+. Under this condition, we found that the tension of myofibrils spontaneously oscillates accompanied by the oscillation of sarcomere length with a main period of a few seconds; the period was lengthened and shortened with stretch and release of myofibrils. A possible mechanism of the oscillation is discussed.     

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.     

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.     

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.     

Myofilament-generated tension oscillations during partial calcium activation and activation dependence of the sarcomere length-tension relation of skinned cardiac cells

During partial Ca2+ activation, skinned cardiac cells with sarcoplasmic reticulum destroyed by detergent developed spontaneous tension oscillations consisting of cycles (0.1-1 Hz) of rapid decrease of tension corresponding to the yield of some sarcomeres and slow redevelopment of tension corresponding to the reshortening of these sarcomeres. Such myofilament-generated tension oscillations were never observed during the full activation induced by a saturating [free Ca2+] or during the rigor tension induced by decreasing [MgATP] in the absence of free Ca2+ or when the mean sarcomere length (SL) of the preparation was greater than 3.10 microm during partial Ca2+ activation. A stiff parallel elastic element borne by a structure that could be digested by elastase hindered the study of the SL--active tension diagram in 8-13-microm-wide skinned cells from the rat ventricle, but this study was possible in 2-7-microm-wide myofibril bundles from the frog or dog ventricle. During rigor the tension decreased linearly when SL was increased from 2.35 to 3.80 microm. During full Ca2+ activation the tension decreased by less than 20% when SL was increased from 2.35 to approximately 3.10 microm. During partial Ca2+ activation the tension increased when SL was increased from 2.35 to 3.00 microm. From this observation of an apparent increase in the sensitivity of the myofilaments to Ca2+ induced by increasing SL during partial Ca2+ activation, a model was proposed that describes the tension oscillations and permits the derivation of the maximal velocity of shortening (Vmax). Vmax was increased by increasing [free Ca2+] or decreasing [free Mg2+] but not by increasing SL.     

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.     

Effects of phosphate and ADP on shortening velocity during maximal and submaximal calcium activation of the thin filament in skeletal muscle fibers.

The effects of added phosphate and MgADP on unloaded shortening velocity during maximal and submaximal Ca2+ activation of the thin filament were examined in skinned single skeletal fibers from rabbit psoas muscle. During maximal Ca2+ activation, added phosphate (10-30 mM) had no effect on unloaded shortening velocity as determined by the slack-test technique. In fibers activated at submaximal concentrations of Ca2+ in the absence of added phosphate, plots of slack length versus duration of unloaded shortening were biphasic, consisting of an initial high velocity phase of shortening and a subsequent low velocity phase of shortening. Interestingly, in the presence of added phosphate, biphasic slack-test plots were no longer apparent. This result was obtained in control fibers over a range of submaximal Ca2+ concentrations and in maximally Ca2+ activated fibers, which were first treated to partially extract troponin C. Thus, under conditions that favor the appearance of biphasic shortening (i.e., low [Ca2+], troponin C extraction), added phosphate eliminated the low velocity component. In contrast, in fibers activated in the presence of 5 mM added MgADP, biphasic slack-test plots were apparent even during maximal Ca2+ activation. The basis of biphasic shortening is not known but it may be due to the formation of axially compressed cross-bridges that become strained to bear a tension that opposes the relative sliding of the myofilaments. The present findings could be explained if added phosphate and MgADP bind to cross-bridges in a strain-dependent manner. In this case, the results suggest that phosphate inhibits the formation of cross-bridges that bear a compressive strain. Added MgADP, on the other hand, may be expected to detain cross-bridges in strong binding states, thus promoting an increase in the population of cross-bridges bearing a compressive strain. Alterations in the population of strained cross-bridges by added phosphate and MgADP would alter the internal load within the fiber and thus affect the speed of fiber shortening.     

Muscle force and stiffness during activation and relaxation. Implications for the actomyosin ATPase

Isolated skinned frog skeletal muscle fibers were activated (increasing [Ca2+]) and then relaxed (decreasing [Ca2+]) with solution changes, and muscle force and stiffness were recorded during the steady state. To investigate the actomyosin cycle, the biochemical species were changed (lowering [MgATP] and elevating [H2PO4-]) to populate different states in the actomyosin ATPase cycle. In solutions with 200 microM [MgATP], compared with physiological [MgATP], the slope of the plot of relative steady state muscle force vs. stiffness was decreased. At low [MgATP], cross-bridge dissociation from actin should be reduced, increasing the population of the last cross-bridge state before dissociation. These data imply that the last cross-bridge state before dissociation could be an attached low-force-producing or non-force-producing state. In solutions with 10 mM total Pi, compared to normal levels of MgATP, the maximally activated muscle force was reduced more than muscle stiffness, and the slope of the plot of relative steady state muscle force vs. stiffness was reduced. Assuming that in elevated Pi, Pi release from the cross-bridge is reversed, the state(s) before Pi release would be populated. These data are consistent with the conclusion that the cross-bridges are strongly bound to actin before Pi release. In addition, if Ca2+ activates the ATPase by allowing for the strong attachment of the myosin to actin in an A.M.ADP.Pi state, it could do so before Pi release. The calcium sensitivity of muscle force and stiffness in solutions with 4 mM [MgATP] was bracketed by that measured in solutions with 200 microM [MgATP], where muscle force and stiffness were more sensitive to calcium, and 10 mM total Pi, where muscle force and stiffness were less sensitive to calcium. The changes in calcium sensitivity were explained using a model in which force-producing and rigor cross-bridges can affect Ca2+ binding or promote the attachment of other cross-bridges to alter calcium sensitivity.     

MgADP promotes a catch-like state developed through force-calcium hysteresis in tonic smooth muscle.

Tonic rabbit femoral artery and phasic rabbit ileum smooth muscles permeabilized with Triton X-100 were activated either by increasing [Ca2+] from pCa > 8.0 to pCa 6.0 (calcium-ascending protocol) or contracted at pCa 6.0 before lowering [Ca2+] (calcium-descending protocol). The effects of, respectively, high [MgATP]/low [MgADP] [10 mM MgATP + creatine phosphate (CP) + creatine kinase (CK)] or low [MgATP]/[MgADP] (2 mM MgATP, 0 CP, 0 CK) on the "force-[Ca]" relationships were determined. In femoral artery at low, but not at high, [MgATP]/[MgADP] the force and the ratio of stiffness/force at pCa 7.2 were significantly higher under the calcium-descending than calcium-ascending protocols (54% vs. 3% of Po, the force at pCa 6.0) (force hysteresis); the levels of regulatory myosin light chain (MLC20) phosphorylation (9 +/- 2% vs. 10 +/- 2%) and the velocities of unloaded shortening V0 (0.02 +/- 0.004 l/s with both protocols) were not significantly different. No significant force hysteresis was detected in rabbit ileum under either of these experimental conditions. [MgADP], measured in extracts of permeabilized femoral artery strips by two methods, was 130-140 microM during maintained force under the calcium-descending protocol. Exogenous CP (10 mM) applied during the descending protocol reduced endogenous [MgADP] to 46 +/- 10 microM and abolished force hysteresis: residual force at low [Ca2+] was 17 +/- 5% of maximal force. We conclude that the proportion of force-generating nonphosphorylated (AMdp) relative to phosphorylated cross-bridges is higher on the Ca2+-descending than on the Ca2+-ascending force curve in tonic smooth muscle, that this population of positively strained dephosphorylated cross-bridges has a high affinity for MgADP, and that the dephosphorylated AMdp . MgADP state makes a significant contribution to force maintenance at low levels of MLC20 phosphorylation.     

Mechanical transients initiated by photolysis of caged ATP within fibers of insect fibrillar flight muscle

Kinetics of the cross-bridge cycle in insect fibrillar flight muscle have been measured using laser pulse photolysis of caged ATP and caged inorganic phosphate (Pi) to produce rapid step increases in the concentration of ATP and Pi within single glycerol-extracted fibers. Rapid photochemical liberation of 100 microM-1 mM ATP from caged ATP within a fiber caused relaxation in the absence of Ca2+ and initiated an active contraction in the presence of approximately 30 microM Ca2+. The apparent second order rate constant for detachment of rigor cross-bridges by ATP was between 5 x 10(4) and 2 x 10(5) M-1s-1. This rate is not appreciably sensitive to the Ca2+ or Pi concentrations or to rigor tension level. The value is within an order of magnitude of the analogous reaction rate constant measured with isolated actin and insect myosin subfragment-1 (1986. J. Muscle Res. Cell Motil. 7:179-192). In both the absence and presence of Ca2+ insect fibers showed evidence of transient cross-bridge reattachment after ATP-induced detachment from rigor, as found in corresponding experiments on rabbit psoas fibers. However, in contrast to results with rabbit fibers, tension traces of insect fibers starting at different rigor tensions did not converge to a common time course until late in the transients. This result suggests that the proportion of myosin cross-bridges that can reattach into force-generating states depends on stress or strain in the filament lattice. A steady 10-mM concentration of Pi markedly decreased the transient reattachment phase after caged ATP photolysis. Pi also decreased the amplitude of stretch activation after step stretches applied in the presence of Ca2+ and ATP. Photolysis of caged Pi during stretch activation abruptly terminated the development of tension. These results are consistent with a linkage between Pi release and the steps leading to force production in the cross-bridge cycle.     

Role of MgATP and MgADP in the cross-bridge kinetics in chemically skinned rabbit psoas fibers. Study of a fast exponential process (C)

The role of the substrate (MgATP) and product (MgADP) molecules in cross-bridge kinetics is investigated by small amplitude length oscillations (peak to peak: 3 nm/cross-bridge) and by following amplitude change and phase shift in tension time courses. The range of discrete frequencies used for this investigation is 0.25-250 Hz, which corresponds to 0.6-600 ms in time domain. This report investigates the identity of the high frequency exponential advance (process C), which is equivalent to "phase 2" of step analysis. The experiments are performed in maximally activated (pCa 4.5-5.0) single fibers from chemically skinned rabbit psoas fibers at 20 degrees C and at the ionic strength 195 mM. The rate constant 2 pi c deduced from process (C) increases and saturates hyperbolically with an increase in MgATP concentration, whereas the same rate constant decreases monotonically with an increase in MgADP concentration. The effects of MgATP and MgADP are opposite in all respects we have studied. These observations are consistent with a cross-bridge scheme in which MgATP and MgADP are in rapid equilibria with rigorlike cross-bridges, and they compete for the substrate site on myosin heads. From our measurements, the association constants are found to be 1.4 mM-1 for MgATP and 2.8 mM-1 for MgADP. We further deduced that the composite second order rate constant of MgATP binding to cross-bridges and subsequent isomerization/dissociation reaction to be 0.57 x 10(6)M-1s-1.     

The effect of the lattice spacing change on cross-bridge kinetics in chemically skinned rabbit psoas muscle fibers. II. Elementary steps affected by the spacing change.

The actin-myosin lattice spacing of rabbit psoas fibers was osmotically compressed with a dextran T-500, and its effect on the elementary steps of the cross-bridge cycle was investigated. Experiments were performed at the saturating Ca (pCa 4.5-4.9), 200 mM ionic strength, pH 7.0, and at 20 degrees C, and the results were analyzed by the following cross-bridge scheme: [formula: see text] where A = actin, M = myosin head, S = MgATP, D = MgADP, and P = Pi = phosphate. From MgATP and MgADP studies on exponential process (C) and (D), the association constants of cross-bridges to MgADP (K0), MgATP (K1a), the rate constants of the isomerization of the AM S state (k1b and k-1b), and the rate constants of the cross-bridge detachment step (k2 and k-2) were deduced. From Pi study on process (B), the rate constants of the cross-bridge attachment (power stroke) step (k4- and k-4) and the association constant of Pi ions to cross-bridges (K5) were deduced. From ATP hydrolysis measurement, the rate constant of ADP-isomerization (rate-limiting) step (k6) was deduced. These kinetic constants were studied as functions of dextran concentrations. Our results show that nucleotide binding, the ATP-isomerization, and the cross-bridge detachment steps are minimally affected by the compression. The rate constant of the reverse power stroke step (k-4) decreases with mild compression (0-6.3% dextran), presumably because of the stabilization of the attached cross-bridges in the AM*DP state. The rate constant of the power stroke step (k4) does not change with mild compression, but it decreases with higher compression (> 6.3% dextran), presumably because of an increased difficulty in performing the power stroke. These results are consistent with the observation that isometric tension increases with a low level of compression and decreases with a high level of compression. Our results also show that the association constant K5 of Pi with cross-bridge state AM*D is not changed with compression. Our result further show that the ATP hydrolysis rate decreased with compression, and that the rate constants of the ADP-isomerization step (k6) becomes progressively less with compression. The effect of compression on the power stroke step and rate-limiting step implies that a large-scale molecular rearrangement in the myosin head takes place in these two slow reaction steps.     

Effects of Ca2+ on the kinetics of phosphate release in skeletal muscle.

The process of phosphate dissociation during the muscle cross-bridge cycle has been investigated by photoliberation of inorganic phosphate (Pi) within skinned fibers of rabbit psoas muscle. This permitted a test of the idea that Ca2+ controls muscle contraction by regulating the Pi release step of the cycle. Photoliberation of Pi from structurally distinct "caged" Pi precursors initiated a rapid tension decline of up to 12% of active tension, and this was followed by a slower tension decline. The apparent rate constant of the fast phase, kPi, depended on both [Pi] and [Ca2+], whereas the slow phase generally occurred at 2-4 s-1. At maximal Ca2+, kPi increased in a nonlinear manner from 43 +/- 2 s-1 to 118 +/- 7 s-1, as Pi was raised from 0.9 to 12 mM. This was analyzed in terms of a three-state kinetic model in which a force-generating transition is coupled to Pi dissociation from the cross-bridge. As Ca(2+)-activated tension was reduced from maximal (Pmax) to 0.1 Pmax, (i) kPi decreased by up to 2.5-fold, (ii) the relative amplitude of the rapid phase increased 2-fold, and (iii) the relative amplitude of the slow phase increased about 6-fold. Changes in the rapid phase are compatible with Ca2+ influencing an apparent equilibrium constant for the force-generating transition. By comparison, kPi was faster than the rate constant of tension redevelopment, ktr, and was influenced less by Ca2+. Ca2+ effects on the caged Pi transient cannot account for the large effects of Ca2+ on actomyosin ATPase rates or cross-bridge cycling kinetics but may be a manifestation of reciprocal interactions between the thin filament and force-generating cross-bridges, and may represent Ca2+ regulation of the distribution of cross-bridges between non-force-and force-generating states.     

Effects of MgATP, MgADP, and Pi on actin movement by smooth muscle myosin.

To test the idea that the in vitro motility assay is a simplified model system for muscle contraction, the MgATP-dependent movement of actin filaments by thiophosphorylated smooth muscle myosin was characterized in the presence of the products MgADP and inorganic phosphate. The dependence of actin filament velocity on MgATP concentration was hyperbolic with a maximum velocity of 0.6 micron/s and an apparent Km = 40 microM (30 degrees C). MgADP competitively inhibited actin movement by MgATP with a Ki = 0.25 mM. Inorganic phosphate did not affect actin filament velocity in the presence of 1 mM MgATP, but competitively inhibited movement in the presence of 50 microM MgATP with a Ki = 9.5 mM. The effects of ADP and Pi on velocity agree with fiber mechanical studies, confirming that the motility assay is an excellent system to investigate the molecular mechanisms of force generation and shortening in smooth muscle. The rate at which rigor cross-bridges can be recruited to move actin filaments was observed by initiating cross-bridge cycling from rigor by flash photolysis of caged MgATP. Following the flash, which results in a rapid increase in MgATP concentration, actin filaments experienced a MgATP-dependent delay prior to achieving steady state velocity. The delay at low MgATP concentrations was interpreted as evidence that motion generating cross-bridges are slowed by a load due to a transiently high percentage of rigor cross-bridges immediately following MgATP release.     

Spontaneous oscillatory contraction without regulatory proteins in actin filament-reconstituted fibers.

Skinned skeletal and cardiac muscle fibers exhibits spontaneous oscillatory contraction (SPOC) in the presence of MgATP, MgADP, and inorganic phosphate (Pi)1 but the molecular mechanism underlying this phenomenon is not yet clear. We have investigated the role of regulatory proteins in SPOC using cardiac muscle fibers of which the actin filaments had been reconstituted without tropomyosin and troponin, according to a previously reported method (Fujita et al., 1996. Biophys. J. 71:2307-2318). That is, thin filaments in glycerinated cardiac muscle fibers were selectively removed by treatment with gelsolin. Then, by adding exogenous actin to these thin filament-free cardiac muscle fibers under polymerizing conditions, actin filaments were reconstituted. The actin filament-reconstituted cardiac muscle fibers generated active tension in a Ca(2+)-insensitive manner because of the lack of regulatory proteins. Herein we have developed a new solvent condition under which SPOC occurs, even in actin filament-reconstituted fibers: the coexistence of 2,3-butanedione 2-monoxime (BDM), a reversible inhibitor of actomyosin interactions, with MgATP, MgADP and Pi. The role of BDM in the mechanism of SPOC in the actin filament-reconstituted fibers was analogous to that of the inhibitory function of the tropomyosin-troponin complex (-Ca2+) in the control fibers. The present results suggest that SPOC is a phenomenon that is intrinsic to the actomyosin motor itself.     

Creatine kinase in regulation of heart function and metabolism. II. The effect of phosphocreatine on the rigor tension of EGTA-treated rat myocardial fibers

Bundles of rat cardiac fibers were treated with EGTA to increase the permeability of the sarcolemma to ions and small molecules. In the medium without calcium, the EGTA-treated fibers developed rigor tension dependent on the concentration of MgATP in the bathing solution: half-maximal tension was recorded at 2.5 mM MgATP and maximal tension at 0.1 mM MgATP in the medium. However, in the presence of 15 mM phosphocreatine without added creatine kinase a decrease of MgATP concentration to 0.1 mM did not result in any development of rigor tension. Phosphocreatine prevented rigor tension development in the absence of added MgATP when MgADP was added. In the presence of MgADP, phosphocreatine decreased rigor tension more rapidly and to a higher extent than added MgATP. At 5 mM MgADP, half-maximal rigor tension was observed in the presence of 2 mM phosphocreatine which is close to the Km value for phosphocreatine in the creatine-kinase reaction. These results demonstrate that the intact creatine kinase in the EGTA-treated fibers with increased sarcolemmal permeability is able to ensure rapid replenishment of MgATP in the myofibrillar compartment at the expense of phosphocreatine. The data obtained conform completely to the concept of adenine-nucleotide compartmentation in cardiac cells and of energy channelling by the phosphocreatine-creatine shuttle mechanism.     

Variations in cross-bridge attachment rate and tension with phosphorylation of myosin in mammalian skinned skeletal muscle fibers. Implications for twitch potentiation in intact muscle

The Ca2+ sensitivities of the rate constant of tension redevelopment (ktr; Brenner, B., and E. Eisenberg. 1986. Proceedings of the National Academy of Sciences. 83:3542-3546) and isometric force during steady-state activation were examined as functions of myosin light chain 2 (LC2) phosphorylation in skinned single fibers from rabbit and rat fast-twitch skeletal muscles. To measure ktr the fiber was activated with Ca2+ and steady isometric tension was allowed to develop; subsequently, the fiber was rapidly (less than 1 ms) released to a shorter length and then reextended by approximately 200 nm per half sarcomere. This maneuver resulted in the complete dissociation of cross-bridges from actin, so that the subsequent redevelopment of tension was related to the rate of cross-bridge reattachment. The time course of tension redevelopment, which was recorded under sarcomere length control, was best fit by a first-order exponential equation (i.e., tension = C(1 - e-kt) to obtain the value of ktr. In control fibers, ktr increased sigmoidally with increases in [Ca2+]; maximum values of ktr were obtained at pCa 4.5 and were significantly greater in rat superficial vastus lateralis fibers (26.1 +/- 1.2 s-1 at 15 degrees C) than in rabbit psoas fibers (18.7 +/- 1.0 s-1). Phosphorylation of LC2 was accomplished by repeated Ca2+ activations (pCa 4.5) of the fibers in solutions containing 6 microM calmodulin and 0.5 microM myosin light chain kinase, a protocol that resulted in an increase in LC2 phosphorylation from approximately 10% in the control fibers to greater than 80% after treatment. After phosphorylation, ktr was unchanged at maximum or very low levels of Ca2+ activation. However, at intermediate levels of Ca2+ activation, between pCa 5.5 and 6.2, there was a significant increase in ktr such that this portion of the ktr-pCa relationship was shifted to the left. The steady-state isometric tension-pCa relationship, which in control fibers was left shifted with respect to the ktr-pCa relationship, was further left-shifted after LC2 phosphorylation. Phosphorylation of LC2 had no effect upon steady-state tension during maximum Ca2+ activation. In fibers from which troponin C was partially extracted to disrupt molecular cooperativity within the thin filament (Moss et al. 1985. Journal of General Physiology. 86:585-600), the effect of LC2 phosphorylation to increase the Ca2+ sensitivity of steady-state isometric force was no longer evident, although the effect of phosphorylation to increase ktr was unaffected by this maneuver.(ABSTRACT TRUNCATED AT 400 WORDS)     

Backward movements of cross-bridges by application of stretch and by binding of MgADP to skeletal muscle fibers in the rigor state as studied by x-ray diffraction

The effects of the applied stretch and MgADP binding on the structure of the actomyosin cross-bridges in rabbit and/or frog skeletal muscle fibers in the rigor state have been investigated with improved resolution by x-ray diffraction using synchrotron radiation. The results showed a remarkable structural similarity between cross-bridge states induced by stretch and MgADP binding. The intensities of the 14.4- and 7.2-nm meridional reflections increased by approximately 23 and 47%, respectively, when 1 mM MgADP was added to the rigor rabbit muscle fibers in the presence of ATP-depletion backup system and an inhibitor for muscle adenylate kinase or by approximately 33 and 17%, respectively, when rigor frog muscle was stretched by approximately 4.5% of the initial muscle length. In addition, both MgADP binding and stretch induced a small but genuine intensity decrease in the region close to the meridian of the 5.9-nm layer line while retaining the intensity profile of its outer portion. No appreciable influence was observed in the intensities of the higher order meridional reflections of the 14.4-nm repeat and the other actin-based reflections as well as the equatorial reflections, indicating a lack of detachment of cross-bridges in both cases. The changes in the axial spacings of the actin-based and the 14.4-nm-based reflections were observed and associated with the tension change. These results indicate that stretch and ADP binding mediate similar structural changes, being in the correct direction to those expected for that the conformational changes are induced in the outer portion distant from the catalytic domain of attached cross-bridges. Modeling of conformational changes of the attached myosin head suggested a small but significant movement (about 10-20 degrees) in the light chain-binding domain of the head toward the M-line of the sarcomere. Both chemical (ADP binding) and mechanical (stretch) intervensions can reverse the contractile cycle by causing a backward movement of this domain of attached myosin heads in the rigor state.     

Regulation of force development studied by photolysis of caged ADP in rabbit skinned psoas fibers.

The present study examined the effects of Ca(2+) and strongly bound cross-bridges on tension development induced by changes in the concentration of MgADP. Addition of MgADP to the bath increased isometric tension over a wide range of [Ca(2+)] in skinned fibers from rabbit psoas muscle. Tension-pCa (pCa is -log [Ca(2+)]) relationships and stiffness measurements indicated that MgADP increased mean force per cross-bridge at maximal Ca(2+) and increased recruitment of cross-bridges at submaximal Ca(2+). Photolysis of caged ADP to cause a 0.5 mM MgADP jump initiated an increase in isometric tension under all conditions examined, even at pCa 6.4 where there was no active tension before ADP release. Tension increased monophasically with an observed rate constant, k(ADP), which was similar in rate and Ca(2+) sensitivity to the rate constant of tension re-development, k(tr), measured in the same fibers by a release-re-stretch protocol. The amplitude of the caged ADP tension transient had a bell-shaped dependence on Ca(2+), reaching a maximum at intermediate Ca(2+) (pCa 6). The role of strong binding cross-bridges in the ADP response was tested by treatment of fibers with a strong binding derivative of myosin subfragment 1 (NEM-S1). In the presence of NEM-S1, the rate and amplitude of the caged ADP response were no longer sensitive to variations in the level of activator Ca(2+). The results are consistent with a model in which ADP-bound cross-bridges cooperatively activate the thin filament regulatory system at submaximal Ca(2+). This cooperative interaction influences both the magnitude and kinetics of force generation in skeletal muscle.     

Ionic interventions that alter the association of troponin C C-domain with the thin filaments of vertebrate striated muscle

The regulatory complex of vertebrate skeletal muscle integrates information about cross-bridge binding, divalent cations and other intracellular ionic conditions to control activation of muscle contraction. Relatively little is known about the role of the troponin C (TnC) C-domain in the absence of Ca2+. Here, we use a standardized condition for measuring isometric tension in rabbit psoas skinned fibers to track TnC attachment and detachment in the absence of Ca2+ under different conditions of ionic strength, pH and MgATP. In the presence of MgATP and Mg2+, TnC detaches more readily and has a 1.5- to 2-fold lower affinity for the intact thin filament at pH 8 and 250 mM K+ than at pH 6 or in 30 mM K+; changes in affinity are fully reversible. The response to ionic strength is lost when Mg2+ and MgATP are absent, whereas the response to pH persists, suggesting that weaker electrostatic TnC-TnI-TnT interactions can be overridden by strongly bound cross-bridges. In solution, titration of a fluorescent C-domain mutant (F154W TnC) with Mg2+ reveals no significant changes in Mg2+ affinity with pH or ionic strength, suggesting that these parameters influence TnC binding by acting directly on electrostatic forces between TnC and TnI rather than by changing Mg2+ binding to C-domain sites III and IV.