Effect of P(i) on unloaded shortening velocity of slow and fast mammalian muscle fibers

Chemically skinned muscle fibers,prepared from the rat medial gastrocnemius and soleus, were subjectedto four sequential slack tests in Ca²⁺-activating solutionscontaining 0, 15, 30, and 0 mM added P_i. P_i (15 and 30 mM) had no effect on the unloaded shortening velocity (V_o) of fibers expressing type IIb myosin heavychain (MHC). For fibers expressing type I MHC, 15 mM P_i didnot alter V_o, whereas 30 mM P_ireduced V_o to 81 ± 1% of the original 0 mM P_i value. This effect was readily reversible whenP_i was lowered back to 0 mM. These results are notcompatible with current cross-bridge models, developed exclusively fromdata obtained from fast fibers, in which V_o isindependent of P_i. The response of the type I fibers at 30 mM P_i is most likely the result of increased internal drag opposing fiber shortening resulting from fiber type-specific effects ofP_i on cross bridges, the thin filament, or therate-limiting step of the cross-bridge cycle.

     

Effect of an ADP analog on isometric force and ATPase activity of active muscle fibers

The role played by ADP in modulatingcross-bridge function has been difficult to study, because it is hardto buffer ADP concentration in skinned muscle preparations. To solvethis, we used an analog of ADP, spin-labeled ADP (SL-ADP). SL-ADP bindstightly to myosin but is a very poor substrate for creatine kinase orpyruvate kinase. Thus ATP can be regenerated, allowing well-definedconcentrations of both ATP and SL-ADP. We measured isometric ATPaserate and isometric tension as a function of both [SL-ADP], 0.1-2mM, and [ATP], 0.05-0.5 mM, in skinned rabbit psoas muscle,simulating fresh or fatigued states. Saturating levels of SL-ADPincreased isometric tension (by P'), the absolute value of P' beingnearly constant, ~0.04 N/mm², in variable ATP levels, pH7. Tension decreased (50-60%) at pH 6, but upon addition ofSL-ADP, P' was still ~0.04 N/mm². The ATPase wasinhibited competitively by SL-ADP with an inhibition constant,K_i, of ~240 and 280 µM at pH 7 and 6, respectively. Isometric force and ATPase activity could both be fit bya simple model of cross-bridge kinetics.

     

Cross-bridge blocker BTS permits direct measurement of SR Ca2+ pump ATP utilization in toadfish swimbladder muscle fibers

Because the major processes involved in muscle contraction require rapid utilization of ATP, measurement of ATP utilization can provide important insights into the mechanisms of contraction. It is necessary, however, to differentiate between the contribution made by cross-bridges and that of the sarcoplasmic reticulum (SR) Ca²⁺ pumps. Specific and potent SR Ca²⁺ pump blockers have been used in skinned fibers to permit direct measurement of cross-bridge ATP utilization. Up to now, there was no analogous cross-bridge blocker. Recently, N-benzyl-p-toluene sulfonamide (BTS) was found to suppress force generation at micromolar concentrations. We tested whether BTS could be used to block cross-bridge ATP utilization, thereby permitting direct measurement of SR Ca²⁺ pump ATP utilization in saponin-skinned fibers. At 25 µM, BTS virtually eliminates force and cross-bridge ATP utilization (both <4% of control value). By taking advantage of the toadfish swimbladder muscle's unique right shift in its force-Ca²⁺ concentration ([Ca²⁺]) relationship, we measured SR Ca²⁺ pump ATP utilization in the presence and absence of BTS. At 25 µM, BTS had no effect on SR pump ATP utilization. Hence, we used BTS to make some of the first direct measurements of ATP utilization of intact SR over a physiological range of [Ca²⁺]at 15°C. Curve fits to SR Ca²⁺ pump ATP utilization vs. pCa indicate that they have much lower Hill coefficients (1.49) than that describing cross-bridge force generation vs. pCa (5). Furthermore, we found that BTS also effectively eliminates force generation in bundles of intact swimbladder muscle, suggesting that it will be an important tool for studying integrated SR function during normal motor behavior. muscle energetics; skinned muscle fibers; sarcoplasmic reticulum calcium ion pumps; cross bridges     

An expanded latch-bridge model of protein kinase C-mediated smooth muscle contraction.

A thin-filament-regulated latch-bridge model of smooth muscle contraction is proposed to integrate thin-filament-based inhibition of actomyosin ATPase activity with myosin phosphorylation in the regulation of smooth muscle mechanics. The model included two latch-bridge cycles, one of which was identical to the four-state model as proposed by Hai and Murphy (Am J Physiol Cell Physiol 255: C86-C94, 1988), whereas the ultraslow cross-bridge cycle has lower cross-bridge cycling rates. The model-fitted phorbol ester induced slow contractions at constant myosin phosphorylation and predicted steeper dependence of force on myosin phosphorylation in phorbol ester-stimulated smooth muscle. By shifting cross bridges between the two latch-bridge cycles, the model predicts that a smooth muscle cell can either maintain force at extremely low-energy cost or change its contractile state rapidly, if necessary. Depending on the fraction of cross bridges engaged in the ultraslow latch-bridge cycle, the model predicted biphasic kinetics of smooth muscle mechanics and variable steady-state dependencies of force and shortening velocity on myosin phosphorylation. These results suggest that thin-filament-based regulatory proteins may function as tuners of actomyosin ATPase activity, thus allowing a smooth muscle cell to have two discrete cross-bridge cycles with different cross-bridge cycling rates.     

Inorganic phosphate speeds loaded shortening in rat skinned cardiac myocytes

Force generation in striated muscle is coupled with inorganic phosphate (P_i) release from myosin, because force falls with increasing P_i concentration ([P_i]). However, it is unclear which steps in the cross-bridge cycle limit loaded shortening and power output. We examined the role of P_i in determining force, unloaded and loaded shortening, power output, and rate of force development in rat skinned cardiac myocytes to discern which step in the cross-bridge cycle limits loaded shortening. Myocytes (n = 6) were attached between a force transducer and position motor, and contractile properties were measured over a range of loads during maximal Ca²⁺ activation. Addition of 5 mM P_i had no effect on maximal unloaded shortening velocity (V_o) (control 1.83 ± 0.75, 5 mM added P_i 1.75 ± 0.58 muscle lengths/s; n = 6). Conversely, addition of 2.5, 5, and 10 mM P_i progressively decreased force but resulted in faster loaded shortening and greater power output (when normalized for the decrease in force) at all loads greater than 10% isometric force. Peak normalized power output increased 16% with 2.5 mM added P_i and further increased to a plateau of 35% with 5 and 10 mM added P_i. Interestingly, the rate constant of force redevelopment (k_tr) progressively increased from 0 to 10 mM added P_i, with k_tr 360% greater at 10 mM than at 0 mM added P_i. Overall, these results suggest that the P_i release step in the cross-bridge cycle is rate limiting for determining shortening velocity and power output at intermediate and high relative loads in cardiac myocytes. muscle mechanics; force-velocity relationship; cross-bridge cycle     

Non-cross-bridge calcium-dependent stiffness in frog muscle fibers

At the end of the force transient elicited by a fast stretch applied to an activated frog muscle fiber, the force settles to a steady level exceeding the isometric level preceding the stretch. We showed previously that this excess of tension, referred to as "static tension," is due to the elongation of some elastic sarcomere structure, outside the cross bridges. The stiffness of this structure, "static stiffness," increased upon stimulation following a time course well distinct from tension and roughly similar to intracellular Ca²⁺ concentration. In the experiments reported here, we investigated the possible role of Ca²⁺ in static stiffness by comparing static stiffness measurements in the presence of Ca²⁺ release inhibitors (D600, Dantrolene, ²H₂O) and cross-bridge formation inhibitors [2,3-butanedione monoxime (BDM), hypertonicity]. Both series of agents inhibited tension; however, only D600, Dantrolene, and ²H₂O decreased at the same time static stiffness, whereas BDM and hypertonicity left static stiffness unaltered. These results indicate that Ca²⁺, in addition to promoting cross-bridge formation, increases the stiffness of an (unidentified) elastic structure of the sarcomere. This stiffness increase may help in maintaining the sarcomere length uniformity under conditions of instability. intact muscle fiber; static stiffness; tension inhibitors; titin     

Transverse tubular system depolarization reduces tetanic force in rat skeletal muscle fibers by impairing action potential repriming

When muscle fibers are repeatedly stimulated, they may become depolarized and force output decline. Excitation of the transverse tubular system (T-system) is critical for activation, but its role in muscle fatigue is poorly understood. Here, mechanically skinned fibers from rat fast-twitch muscle were used, because the sarcolemma is absent but the T-system retains normal excitability and its properties can be studied in isolation. The T-system membrane was fully polarized by bathing the skinned fiber in an internal solution with 126 mM K⁺ (control solution) or set at partially depolarized levels (approximately –63 and –58 mV) in solutions with 66 or 55 mM K⁺, respectively, and action potentials (APs) were triggered in the sealed T-system by field stimulation. Prolonged depolarization of the T-system reduced tetanic force proportionately more than twitch force, with greater effect at higher stimulation frequency (responses at 20 and 100 Hz reduced to 71 and 62% in 66 mM K⁺ and to 54 and 35% in 55 mM K⁺, respectively). Double-pulse stimulation showed that depolarization increased the repriming period (estimated minimum time before a second AP can be produced) from 4 ms to 7.5 and 15 ms in the 66 and 55 mM K⁺ solutions, respectively. These results demonstrate that T-system depolarization reduces tetanic force by impairing AP repriming, rather than by preventing AP generation per se or by inactivating the T-system voltage sensors. The findings also explain why it is advantageous to reduce the rate of motoneuron stimulation to muscles during repeated or prolonged periods of activity. T-system; muscle fatigue; excitation-contraction coupling     

Ionic Strength and the Contraction Kinetics of Skinned Muscle Fibers

The influence of KCl concentration on the contraction kinetics of skinned frog muscle fibers at 5–7°C was studied at various calcium levels. The magnitude of the calcium-activated force decreased continuously as the KCl concentration of the bathing solution was increased from 0 to 280 mM. The shortening velocity at a given relative load was unaffected by the level of calcium activation at 140 mM KCl, as has been previously reported by Podolsky and Teichholz (1970. J. Physiol. [Lond.]. 211: 19), and was independent of ionic strength when the KCl concentration was increased from 140 to 280 mM. In contrast, the shortening velocity decreased as the KCl concentration was reduced below 140 mM; the decrease in velocity was enhanced when the fibers were only partially activated. In the low KCl range, the resting tension of the fibers increased after the first contraction cycle. The results suggest that in fibers activated at low ionic strength some of the cross bridges that are formed are abnormal in the sense that they retard shortening and persist in relaxing solution.     

Parallel inhibition of active force and relaxed fiber stiffness by caldesmon fragments at physiological ionic strength and temperature conditions: additional evidence that weak cross-bridge binding to actin is an essential intermediate for force generation.

Previously we showed that stiffness of relaxed fibers and active force generated in single skinned fibers of rabbit psoas muscle are inhibited in parallel by actin-binding fragments of caldesmon, an actin-associated protein of smooth muscle, under conditions in which a large fraction of cross-bridges is weakly attached to actin (ionic strength of 50 mM and temperature of 5 degrees C). These results suggested that weak cross-bridge attachment to actin is essential for force generation. The present study provides evidence that this is also true for physiological ionic strength (170 mM) at temperatures up to 30 degrees C, suggesting that weak cross-bridge binding to actin is generally required for force generation. In addition, we show that the inhibition of active force is not a result of changes in cross-bridge cycling kinetics but apparently results from selective inhibition of weak cross-bridge binding to actin. Together with our previous biochemical, mechanical, and structural studies, these findings support the proposal that weak cross-bridge attachment to actin is an essential intermediate on the path to force generation and are consistent with the concept that isometric force mainly results from an increase in strain of the attached cross-bridge as a result of a structural change associated with the transition from a weakly bound to a strongly bound actomyosin complex. This mechanism is different from the processes responsible for quick tension recovery that were proposed by Huxley and Simmons (Proposed mechanism of force generation in striated muscle. Nature. 233:533-538.) to represent the elementary mechanism of force generation.     

Effect of temperature on cross-bridge properties in intact frog muscle fibers

It is well known that the force developed by skeletal muscles increases with temperature. Despite the work done on this subject, the mechanism of force potentiation is still debated. Most of the published papers suggest that force enhancement is due to the increase of the individual cross-bridge force. However, reports on skinned fibers and single-molecule experiments suggest that cross-bridge force is temperature independent. The effects of temperature on cross-bridge properties in intact frog fibers were investigated in this study by applying fast stretches at various tension levels (P) on the tetanus rise at 5 degrees C and 14 degrees C to induce cross-bridge detachment. Cross-bridge number was measured from the force (critical force, P(c)) needed to detach the cross-bridge ensemble, and the average cross-bridge strain was calculated from the sarcomere elongation needed to reach P(c) (critical length, L(c)). Our results show that P(c) increased linearly with the force developed at both temperatures, but the P(c)/P ratio was considerably smaller at 14 degrees C. This means that the average force per cross bridge is greater at high temperature. This mechanism accounts for all the tetanic force enhancement. The critical length L(c) was independent of the tension developed at both temperatures but was significantly lower at high temperature suggesting that cross bridges at 14 degrees C are more strained. The increased cross-bridge strain accounts for the greater average force developed.     

Effect of lactate on depolarization-induced Ca(2+) release in mechanically skinned skeletal muscle fibers

Itis unclear whether accumulation of lactate in skeletal muscle fibersduring intense activity contributes to muscle fatigue. Usingmechanically skinned fibers from rat and toad muscle, we were able toexamine the effect of L(+)-lactate onexcitation-contraction coupling independently of other metabolicchanges. We investigated the effects of lactate on the contractileapparatus, caffeine-induced Ca²⁺ release from thesarcoplasmic reticulum, and depolarization-induced Ca²⁺release. Lactate (15 or 30 mM) had only a small inhibitory effect directly on the contractile apparatus and caused appreciable(20-35%) inhibition of caffeine-induced Ca²⁺ release,seemingly by a direct effect on the Ca²⁺ release channels.However, 15 mM lactate had no detectable effect on Ca²⁺release when it was triggered by the normal voltage sensor mechanism, and 30 mM lactate reduced such release by only <10%. These results indicate that lactate has only a relatively small inhibitory effect onnormal excitation-contraction coupling, indicating that lactate accumulation per se is not a major factor in muscle fatigue.

     

Secophalloidin as a novel activator of skinned cardiac muscle

Secophalloidin (SPH) is known to activate skinned cardiac muscle in the absence of Ca(2+). We hypothesized that SPH-induced changes in cross-bridge properties underlie muscle activation. We found that force responsiveness to orthovanadate was drastically reduced in SPH activated muscles compared to Ca(2+)-activated contraction. Moreover, SPH caused approximately 30% increase in Ca(2+)-independent force in muscles where Ca(2+) sensitivity was totally destroyed by troponin I extraction with 10mM vanadate. Thus, SPH and Ca(2+) activation differ in both properties of the cross-bridge cycle and protein requirements for thin filament regulation. In addition, we tested the relationship between the activating effects SPH and EMD 57033, a Ca(2+) sensitizer that increases resting force in cardiac muscle. After maximal activation by either SPH or EMD 57033, the other compound was found to further increase force, indicating that SPH activates muscle via a novel mechanism.     

Functional aspects of skeletal muscle contractile apparatus and sarcoplasmic reticulum after fatigue

This study examined the effects of fatigue on the functionalaspects of the contractile apparatus and sarcoplasmic reticulum (SR).Frog semitendinosus muscles were stimulated to fatigue, and skinnedfibers or a homogenate fraction was prepared from both fatigued andrested contralateral muscles. In fatigued fibers, maximalCa²⁺-activated force of thecontractile apparatus was unaltered, whereas maximal actomyosin-ATPaseactivity was depressed by 20%. TheCa²⁺ sensitivity of force wasincreased, whereas that of actomyosin-ATPase was not altered. Also, therate constant for tension redevelopment was decreased at submaximalCa²⁺ concentration. These latterfindings suggest that fatigue slows the dissociation offorce-generating myosin cross bridges.Ca²⁺ uptake andCa²⁺-ATPase activity of the SRwere depressed by 46 and 21%, respectively, in the fatigued muscles.Fatigue also reduced the rates of SR Ca²⁺ release evoked byAgNO₃ and4-chloro-m-cresol by 38 and 45%, respectively. During fatigue, the contractile apparatus and SR undergointrinsic functional alterations. These changes likely result inaltered force production and energy consumption by the intact muscle.

     

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.     

Peak power output is maintained in rabbit psoas and rat soleus single muscle fibers when CTP replaces ATP

The chemomechanicalcoupling mechanism in striated muscle contraction was examined bychanging the nucleotide substrate from ATP to CTP. Maximum shorteningvelocity [extrapolation to zero force from force-velocity relation(V_max) andslope of slack test plots (V₀)], maximumisometric force (P_o), power, andthe curvature of the force-velocity curve[a/P_o(dimensionless parameter inversely related to the curvature)] weredetermined during maximumCa²⁺-activated isotoniccontractions of fibers from fast rabbit psoas and slow rat soleusmuscles by using 0.2 mM MgATP, 4 mM MgATP, 4 mM MgCTP, or 10 mM MgCTPas the nucleotide substrate. In addition to a decrease in the maximumCa²⁺-activated force in both fibertypes, a change from 4 mM ATP to 10 mM CTP resulted in a decrease inV_max in psoasfibers from 3.26 to 1.87 muscle length/s. In soleus fibers,V_max was reduced from 1.94 to 0.90 muscle length/s by this change in nucleotide. Surprisingly, peak power was unaffected in either fiber type by thechange in nucleotide as the result of a three- to fourfold decrease inthe curvature of the force-velocity relationship. The results areinterpreted in terms of the Huxley model of muscle contraction as anincrease in f₁and g₁ coupled toa decrease in g₂(where f₁ is therate of cross-bridge attachment and g₁ andg₂ are rates ofdetachment) when CTP replaces ATP. This adequately accounts for theobserved changes in P_o,a/P_o,and V_max.However, the two-state Huxley model does not explicitly reveal thecross-bridge transitions that determine curvature of the force-velocityrelationship. We hypothesize that a nucleotide-sensitive transitionamong strong-binding cross-bridge states followingP_i release, but before the release of the nucleotide diphosphate, underlies the alterations ina/P_o reported here.

     

Myosin molecular motor dysfunction in dystrophic mouse diaphragm

Cross-bridge properties and myosin heavy chain (MHC) compositionwere investigated in isolated diaphragm from 6-mo-old control (n = 12) andmdx(n = 12) mice. Compared with control,peak tetanic tension fell by 50% inmdx mice(P < 0.001). The total number ofcross bridges per square millimeter(×10⁹), the elementaryforce per cross bridge, and the peak mechanical efficiency were lowerin mdx than in control mice (eachP < 0.001). The duration of thecycle and the rate constant for cross-bridge detachment weresignificantly lower in mdx than incontrol mice. In the overall population, there was a linearrelationship between peak tetanic tension and either total number ofcross bridges per square millimeter or elementary force per crossbridge (r = 0.996 andr = 0.667, respectively, eachP < 0.001). Themdx mice presented a higher proportionof type IIA MHC (P < 0.001) thancontrol mice and a reduction in type IIX MHC(P < 0.001) and slowmyosin isoforms (P < 0.01) comparedwith control mice. We concluded that, inmdx mice, impaired diaphragm strengthwas associated with qualitative and quantitative changes in myosin molecular motors. It is proposed that reduced force generated per crossbridge contributed to diaphragm weakness inmdx mice.

     

Impact of beta-myosin heavy chain isoform expression on cross-bridge cycling kinetics

Myosin heavy chain (MHC) isoforms alpha and beta have intrinsically different ATP hydrolysis activities (ATPase) and therefore cross-bridge cycling rates in solution. There is considerable evidence of altered MHC expression in rodent cardiac disease models; however, the effect of incremental beta-MHC expression over a wide range on the rate of high-strain, isometric cross-bridge cycling is yet to be ascertained. We treated male rats with 6-propyl-2-thiouracil (PTU; 0.8 g/l in drinking water) for short intervals (6, 11, 16, and 21 days) to generate cardiac MHC patterns in transition from predominantly alpha-MHC to predominantly beta-MHC. Steady-state calcium-dependent tension development and tension-dependent ATP consumption (tension cost; proportional to cross-bridge cycling) were measured in chemically permeabilized (skinned) right ventricular muscles at 20 degrees C. To assess dynamic cross-bridge cycling kinetics, the rate of force redevelopment (ktr) was determined after rapid release-restretch of fully activated muscles. MHC isoform content in each experimental muscle was measured by SDS-PAGE and densitometry. alpha-MHC content decreased significantly and progressively with length of PTU treatment [68 +/- 5%, 58 +/- 4%, 37 +/- 4%, and 27 +/- 6% for 6, 11, 16, and 21 days, respectively; P < 0.001 (ANOVA)]. Tension cost decreased, linearly, with decreased alpha-MHC content [6.7 +/- 0.4, 5.6 +/- 0.5, 4.0 +/- 0.4, and 3.9 +/- 0.3 ATPase/tension for 6, 11, 16, and 21 days, respectively; P < 0.001 (ANOVA)]. Likewise, ktr was significantly and progressively depressed with length of PTU treatment [11.1 +/- 0.6, 9.1 +/- 0.5, 8.2 +/- 0.7, and 6.2 +/- 0.3 s(-1) for 6, 11, 16, and 21 days, respectively; P < 0.05 (ANOVA)] Thus cross-bridge cycling, under high strain, for alpha-MHC is three times higher than for beta-MHC. Furthermore, under isometric conditions, alpha-MHC and beta-MHC cross bridges hydrolyze ATP independently of one another.     

Strain sensitivity and turnover rate of low force cross-bridges in contracting skeletal muscle fibers in the presence of phosphate.

Inorganic phosphate (Pi) decreases the isometric tension of skinned skeletal muscle fibers, presumably by increasing the relative fraction of a low force quaternary complex of actin, myosin, ADP, and Pi (A.M.ADP.Pi). At the same time, Pi gives rise to a fast relaxing mechanical component as detected by oscillations at 500 Hz. To characterize the dynamic properties of this A.M.ADP.Pi complex, the effect of Pi on the tension response to stretch was investigated with rabbit psoas fibers. A ramp stretch applied in the presence of 20 mM Pi increased tension more than in the control solution (0 mM Pi) but reduced the fast relaxing component to the control level. Thus, a stretch seems to convert the low force, fast relaxing A.M.ADP.Pi complex to a high force, slow relaxing form. However, the Pi-induced enhancement of the tension response was not observed until the fibers were stretched more than 0.4% of their length, suggesting that a critical cross-bridge extension of approximately 4 nm is required for this conversion. The rate constant of the attachment/detachment of this low force complex was estimated from the velocity dependence of the enhancement. It was approximately 10 s-1, in marked contrast to the A.M.ADP.Pi complex under low salt, relaxed conditions (approximately 10,000 s-1). The enhancement of the tension response was not observed when isometric tension was reduced by lowering free calcium, implying that calcium and Pi affect different steps in the actomyosin ATPase cycle during contraction.     

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.     

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.