Search

The effects of 2,3-butanedione monoxime (BDM) on intracellular Ca2+ transient and cross-bridge function were studied in frog single fibres from the anterior tibialis muscle of Rana temporaria (sarcomere length, 2.2 m; temperature, 24 C). The fluorescent dye fluo-3 was used to monitor the intracellular free calcium concentration ([Ca2+i) during isometric contractions. BDM (15 m M) reduced the amplitude of the Ca2+ transient during twitches, but this effect was too small to explain the marked inhibition of BDM on twitch force. [Ca2+i reached at the end of 1-s tetanic stimulation was not significantly affected by BDM (1.0 and 1.8 m M) while the maximum tetanic tension was substantially reduced. The rate of relaxation during isometric tetanus was increased by BDM whereas the rate of decay of the Ca2+ transient was reduced in the presence of BDM. The results strongly suggest that BDM, under the experimental conditions used, mainly affects the contractile machinery resulting in altered performance of the cross-bridges. These effects of BDM were evaluated in terms of the cross-bridge model of Huxley (1957) which was fitted to the experimental forcevelocity data in the presence and absence of BDM. (Less)

Background Isoflurane depresses the intracellular Ca2+ transient and force development during a twitch, but its effects on crossbridge cycling rates are difficult to predict because of the transient nature of the twitch. Measurements of the effects of isoflurane on crossbridge cycling kinetics during tetanic contractions, which provide a steady state level of activation in intact cardiac muscle, have not been previously reported. Methods Ferret right ventricular papillary muscles were isolated, and superficial cells were microinjected with the bioluminescent photoprotein aequorin to monitor the intracellular Ca2+ concentration. The rate of tension redevelopment (kTR) was measured during steady state isometric activation (tetanic stimulation, frequency 20 Hz, 1 microM ryanodine, temperature = 30 degrees C) in the absence of isoflurane (2, 6, and 12 mM extracellular [Ca2+]) and in the presence of 0.5, 1.0, and 1.5 minimum alveolar concentration isoflurane (12 mM extracellular [Ca2+]). Results Intracellular [Ca2+], isometric force, and kTR all increased when the extracellular [Ca2+] increased. Isoflurane (0.5, 1.0, and 1.5 minimum alveolar concentration) caused intracellular [Ca2+], isometric force, and kTR to decrease in a dose-dependent manner in the presence of 12 mM extracellular [Ca2+]. In the presence of increasing concentrations of isoflurane, the relation between intracellular [Ca2+] and force remained unchanged, whereas the relation between intracellular [Ca2+] and kTR was shifted toward higher [Ca2+]. Conclusions These results indicate that isoflurane depresses myocardial crossbridge cycling rates. It appears that this effect is partially mediated by a decrease in the intracellular [Ca2+]. However, additional mechanisms must be considered to explain the shift of the relation between intracellular [Ca2+] and kTR toward higher [Ca2+].

Numerous studies have explored the energetic properties of skeletal and cardiac muscle fibers. In this mini-review, we specifically explore the interactions between actin and myosin during cross-bridge cycling and provide a conceptual framework for the chemomechanical transduction that drives muscle fiber energetic demands. Because the myosin heavy chain (MHC) is the site of ATP hydrolysis and actin binding, we focus on the mechanical and energetic properties of different MHC isoforms. Based on the conceptual framework that is provided, we discuss possible sites where muscle remodeling may impact the energetic demands of contraction in skeletal and cardiac muscle.

We examined whether the three states model can explain the systolic and relaxation properties of cardiac muscle to clarify what factors affect these properties. Changing the values of the parameters describing the calcium transient and calcium sensitivity, we estimated the effects of these parameters on the systolic and relaxation properties of twitch contraction. The simulations showed the following four features: 1) An increase in the maximum calcium concentration and calcium sensitivity, and a prolongation of the calcium transient led to an increase in peak tension associated with an increase in the time to peak tension. 2) An increase in myosin ATPase activity led to an increase in peak tension associated with a decrease in the time to peak tension. 3) An increase of peak tension was accompanied by a prolongation of the late systolic period. 4) The constant of the late tension relaxation from 25% to 10% of the peak tension was altered when the crossbridge cycling rate, the resting calcium concentration or the late decline of the calcium transient was changed. The simulation were not contradictory to the experimental results and showed that three state muscle model can provide qualitative descriptions on the systolic and relaxation characteristics of cardiac muscle.

Ca2+ sensitizers act on the central mechanism (Ca2+ binding affinity of troponin C) and/or downstream mechanisms (thin filament regulation of actin and direct action on crossbridge cycling) of cardiac E–C coupling. Ca2+ sensitizers have mechanistic and energetic advantages over the agents that act through the upstream mechanism (intracellular Ca2+ mobilization). Ca2+ sensitizers and the agents that act through cyclic AMP-mediated signaling process have been postulated to belong to different classes, however, recent experimental findings revealed that certain Ca2+ sensitizers, such as levosimendan, OR 1896 and UD-CG 212 Cl, require cyclic AMP-mediated signaling for induction of the Ca2+ sensitizing effect. No clinically available agents act primarily via Ca2+ sensitization, but the positive inotropic effect of pimobendan and levosimendan is partly due to an increase in myofilament Ca2+ sensitivity. These agents are the hybrid of Ca2+ sensitizer and PDE III inhibitor. The extent of contribution of Ca2+ sensitizing effect of these agents to the clinical effectiveness to improve the hemodynamics in patients with heart failure is uncertain. Nevertheless pieces of evidence have been accumulating that these agents with Ca2+ sensitizing effect are clinically more effective than the agents that act purely via the upstream mechanism.

It has been found that maximum specific force (Fmax; force per cross-sectional area) of rat diaphragm muscle doubles from birth to 84 days (adult). We hypothesize that this developmental change in Fmax reflects an increase in myosin heavy chain (MHC) content per half-sarcomere (an estimate of the number of cross bridges in parallel) and/or a greater force per cross bridge in fibers expressing fast MHC isoforms compared with slow and neonatal MHC isoforms (MHCslow and MHCneo, respectively). Single Triton 100-X-permeabilized fibers were activated at a pCa of 4.0. MHC isoform expression was determined by SDS-PAGE. MHC content per half-sarcomere was determined by densitometric analysis and comparison to a standard curve of known MHC concentrations. MHC content per half-sarcomere progressively increased during early postnatal development. When normalized for MHC content per half-sarcomere, fibers expressing MHCslow and coexpressing MHCneoproduced less force than fibers expressing fast MHC isoforms. We conclude that lower force per cross bridge in fibers expressing MHCslow and MHCneo contributes to the lower Fmax seen in early postnatal development.

1 The role of h1-calponin in regulating the contractile properties of smooth muscle was investigated in bladder and vas deferens of mice carrying a targeted mutation in both alleles designed to inactivate the basic calponin gene. These calponin knockout (KO) mice displayed no detectable h1-calponin in their smooth muscles. 2 The amplitudes of Ca2+ sensitization, force and Ca2+ sensitivity were not significantly different in permeabilized smooth muscle of KO compared with wild-type (WT) mice, nor were the delays in onset and half-times of Ca2+ sensitization, initiated by flash photolysis of caged GTPγS, different. 3 The unloaded shortening velocity (Vus) of thiophosphorylated fibres was significantly (P < 0.05) faster in the smooth muscle of KO than WT animals, but could be slowed by exogenous calponin to approximate WT levels; the concentration dependence of exogenous calponin slowing of Vus was proportional to its actomyosin binding in situ. 4 Actin expression was reduced by 25-50%, relative to that of myosin heavy chain, in smooth muscle of KO mice, without any change in the relative distribution of the actin isoforms. 5 We conclude that the faster Vus of smooth muscle of the KO mouse is consistent with, but does not prove without further study, physiological regulation of the crossbridge cycle by calponin. Our results show no detectable role of calponin in the signal transduction of the Ca2+-sensitization pathways in smooth muscle.

Recent findings indicate that mechanical strain (deformation) exerted by the extracellular matrix modulates activation of airway smooth muscle cells. Furthermore, cytoskeletal organization in airway smooth muscle appears to be dynamic, and subject to modulation by receptor activation and mechanical strain. Mechanosensitive modulation of crossbridge activation and cytoskeletal organization may represent intracellular feedback mechanisms that limit the shortening of airway smooth muscle during bronchoconstriction. Recent findings suggest that receptor-mediated signal transduction is the primary target of mechanosensitive modulation. Mechanical strain appears to regulate the number of functional G-proteins and/or phospholipase C enzymes in the cell membrane possibly by membrane trafficking and/or protein translocation. Dense plaques, membrane structures analogous to focal adhesions, appear to be the primary target of cytoskeletal regulation. Mechanical strain and receptor-binding appear to regulate the assembly and phosphorylation of dense plaque proteins in airway smooth muscle cells. Understanding these mechanisms may reveal new pharmacological targets for controlling airway resistance in airway diseases.

To elucidate the function of calponin in intact contracting smooth muscle cells in vivo, we generated mice with a mutated basic calponin (h1) locus (Yoshikawa et al., Genes Cells 3, 685–695, 1998). Crossbridge cycling rates were estimated in aortic smooth muscle by the force redevelopment following an isometric step shortening as a function of time after K+ depolarization. Evidence is presented that calponin is involved in the inhibition of shortening velocity in the tonic phase of contraction. The phosphorylation levels of myosin regulatory light chain and cytosolic calcium concentrations were not significantly different in paired comparisons between calponin-deficient (−/−) and wild-type (+/+) muscles at any time point after stimulation. The force-velocity relationships in vas deferens smooth muscle showed that the maximum shortening velocity of −/− muscle was significantly faster than that of +/+ muscle. There was no change in the length-force relationships in both −/− and +/+ muscles of aorta and vas deferens. The results suggest that calponin plays a role in regulation of the crossbridge cycling and that it may be responsible for reduced shortening velocity during a maintained contraction of mammalian smooth muscle.

Abstract —This study investigated the possible roles of strongly binding myosin crossbridges in determining loaded shortening and power output in cardiac myocytes. Single skinned cardiac myocytes were attached between a force transducer and position motor, and shortening velocities were measured over a range of loads during varying levels of Ca 2+ activation. Lowering the [Ca 2+ ] slowed shortening velocities, decreased relative power output, and increased the curvature of length traces. We tested the hypothesis that Ca 2+ activation dependence of loaded shortening is determined primarily by strongly binding crossbridges or by [Ca 2+ ] per se, which was done by measuring loaded shortening before and after addition of N -ethylmaleimide–conjugated myosin subfragment-1 (NEM-S1), a strongly binding myosin analogue that cooperatively enhances thin filament activation. At fixed [Ca 2+ ], NEM-S1 reduced the curvature of length traces and sped loaded shortening velocities. Even when [Ca 2+ ] was adjusted so that force was equal with and without NEM-S1, myocyte shortening was faster and exhibited less curvature with NEM-S1. In the presence of NEM-S1, peak relative power output was also significantly greater during activations either at the same [Ca 2+ ] or when [Ca 2+ ] was adjusted to achieve the same force. Consequently, NEM-S1 eliminated any Ca 2+ dependence of relative power output that is normally observed in cardiac myocytes. These results indicate that strongly binding crossbridges play a significant role in determining loaded shortening and power output and suggest that previously observed Ca 2+ dependence of power output is mediated by alterations in numbers of crossbridges bound to the thin filament.

Myocardial relaxation is governed by the interplay of two macromolecular systems: (1) myofilaments and (2) calcium extruding pumps/exchangers. In myocardium from failing hearts, both systems act more slowly than normal, and cause relaxation to decelerate, which may impede early rapid filling and can often limit cardiac pumping ability--especially during exercise. Gene-based therapy to augment sluggish SERCA pumps is a possibility being currently investigated in research laboratories. In normal myocardium, the rate of dissociation of myosin crossbridges sets the rate of relaxation. In this case, relaxation is characterized by two features: (1) load-dependence and (2) displacement-dependence. Load-dependence derives from cooperative mechanisms acting among ensembles of crossbridges and myofilament regulatory proteins (troponin, tropomyosin); it allows contraction to be prolonged when more crossbridges are attached and mutually support each other. The rate of relaxation can still be rapid, however, as this cooperative system begins to collapse. Displacement-dependence is more important later in contraction, because tenuous crossbridge attachments cannot easily re-form after being disrupted when myofilaments slide along each other. Myofilaments control normal relaxation because the calcium extruding systems reduce calcium to near diastolic levels relatively early; however, when the relative timing of crossbridge dissociation versus calcium sequestration is altered, and calcium uptake is slowed (relative to crossbridges), then removal of calcium can become rate limiting instead. In this case, load- and displacement-dependence are less marked. Both the timing of calcium removal and the sensitivity of the myofilaments to calcium affect relaxation timing.

The myosin cross–bridge exists in two conformations, which differ in the orientation of a long lever arm. Since the lever arm undergoes a 60° rotation between the two conformations, which would lead to a displacement of the myosin filament of about 11nm, the transition between these two states has been associated with the elementary ‘power stroke’ of muscle. Moreover, this rotation is coupled with changes in the active site (CLOSED to OPEN), which probably enable phosphate release. The transition CLOSED to OPEN appears to be brought about by actin binding. However, kinetics shows that the binding of myosin to actin is a two–step process which affects both ATP and ADP affinity and vice versa. The structural basis of these effects is only partially explained by the presently known conformers of myosin. Therefore, additional states of the myosin cross–bridge should exist. Indeed, cryoelectron microscopy has revealed other angles of the lever arm induced by ADP binding to a smooth muscle actin–myosin complex.

Work performing heart preparations from hypercontractile, phospholamban deficient mouse hearts showed no change in parameters of contraction or relaxation in response to isoproterenol stimulation. Thus, the aim of the present study was to investigate whether or not changes at the level of the contractile apparatus occur in addition to the altered expression of Ca2+-regulating proteins observed in these mouse models, e.g., phospholamban, ryanodine receptors. Triton-X skinned fiber preparations from phospholamban deficient (n = 9) and wild-type (n = 10) mice were used and the Ca2+-activated force as well as the myosin ATPase-activity were simultaneously measured. The tension dependent ATPase-activity was unchanged in phospholamban deficient animals when compared to controls. The SERCA 2a-inhibitor cyclopiazonic acid did not affect myosin ATPase-activity in this system. The Ca2+-sensitivity of Ca2+-activated force and myosin ATPase were unchanged as well. Comparison of the concentrations needed to achieve half maximal activation of the myosin ATPase-activity and force demonstrated that the Ca2+-sensitivity of the myosin ATPase was higher compared to the Ca2+-sensitivity of tension development. This holds true for phospholamban deficient mice (EC50 ATPase: 0.9 +/- 0.2 micromol/l; tension: 1.7 +/- 0.3 micromol/l; p0.001) and wild-type controls (1.1 +/- 0.01 micromol/l; 2.2 +/- 0.4 micromol/l; p0.01). The myosin ATPase-activity and force were correlated to each other in both, phospholamban deficient mice and controls and did not change at submaximal Ca2+ concentrations. The ATPase/ force-ratio, as a parameter of tension cost, was similar in either phospholamban deficient mice or controls. Thus, the present study provides evidence that at the level of the contractile proteins regulation of Ca2+-activated force and energy demand of force development are not altered in phospholamban deficient mice with enhanced myocardial performance. At the level of the regulation of crossbridge interaction, no adaptive or compensatory mechanisms have been initiated by ablation of phospholamban.

We examined the effects of redox modulation on single membrane-permeabilized fibre segments from the fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus muscles of adult rats to determine whether the contractile apparatus was the redox target responsible for the increased contractility of muscles exposed to low concentrations of H2O2. The effects of H2O2 on maximum Ca2+-activated force were dose-dependent with 30 min exposure to 5 mM H2O2 causing a progressive decrease by 22 ± 3 and 13 ± 2% in soleus and EDL permeabilized muscle fibres, respectively. Lower concentrations of exogenous H2O2 (100 μM and 1 mM) had no effect on maximum Ca2+-activated force. Subsequent exposure to the reductant dithiothreitol (DTT, 10 mM, 10 min) fully reversed the H2O2-induced depression of force in EDL, but not in soleus muscle fibres. Incubation with DTT alone for 10 min did not alter Ca2+-activated force in either soleus or EDL muscle fibres. The sensitivity of the contractile filaments to Ca2+ (pCa50) was not altered by exposure to any concentration of exogenous H2O2. However, all concentrations of H2O2 diminished the Hill coefficient in permeabilized fibres from the EDL muscle, indicating that the cooperativity of Ca2+ binding to troponin is altered. H2O2 (5 mM) did not affect rigor force, which indicates that the number of crossbridges participating in contraction was not reduced. In conclusion, H2O2 may reduce the maximum Ca2+ activated force production in skinned muscle fibres by decreasing the force per crossbridge.

Direct measurements of a relationship between force and actin–myosin biochemistry in muscle suggest that molecular forces in active muscle rapidly equilibrate among, not within, individual myosin crossbridges [Baker et al. (1999) Biophys J 77: 2657–2664]. This observation suggests a thermodynamic model of muscle contraction in which muscle, not an individual myosin crossbridge, is treated as a near-equilibrium system. The general approach can be applied to any ensemble of molecular motors that undergo a physicochemical step against a constant external potential. In this paper we apply the model to a simple two-state crossbridge scheme like that proposed by A.F. Huxley (1957) [Prog Biophys 7: 255–317], and we immediately obtain A.V. Hill's muscle equation. We show that this equation accurately describes steady-state muscle mechanics, biochemistry and energetics. This thermodynamic model provides a novel description of force-dependent actin–myosin kinetics in muscle and provides precise chemical expressions for myosin cooperativity, myosin duty ratios, the number of working strokes per ATP hydrolyzed, muscle efficiency, and energy transfer.

It is unclear whether preceding repetitive brief ischemia causes any improvement in the energy efficiency of intracellular calcium cycling or crossbridge cycling that may lead to cardioprotection after subsequent sustained ischemia/reperfusion, a phenomenon called ischemic preconditioning. To address this issue, left ventricular (LV) contractility (E(max)) and the relation between myocardial oxygen consumption (VO(2)) and pressure-volume area (PVA, a measure of LV total mechanical energy) were assessed before (Control) and 20 min (Rep-20) and 60 min (Rep-60) after repetitive brief ischemia in 11 isolated, blood-perfused dog hearts. At Rep-20, E(max) and PVA-independent VO(2) (nonmechanical energy expenditure) decreased by 23.0 +/- 19.5 and 13.9 +/- 18.0%, respectively (both p < 0.05). However, at Rep-60, both E(max) and PVA-independent VO(2) recovered to their respective control levels. The oxygen cost of contractility (the slope of the PVA-independent VO(2)-E(max) relation during CaCl(2) loading) remained constant (Control 0.0019 +/- 0.0009 vs. Rep-60 0.0018 +/- 0.0013 ml O(2) x ml x mmHg(-1) x beat(-1) x 100 g(-2), ns), suggesting unchanged efficiency in Ca(2+) cycling. Also, the contractile efficiency (the reciprocal of the slope of the VO(2)-PVA relation, reflecting the efficiency of crossbridge cycling) was the same between the Control and Rep-60 (53.7 +/- 16.7 vs. 55.4 +/- 14.4%, ns). Basal metabolism VO(2) during KCl arrest was also similar to that in the normal heart. Nonmechanical energy expenditure was reduced in proportion to the decrease in LV contractility after repetitive brief ischemia, while both the contractile efficiency and oxygen cost of contractility remained constant. These results indicate that the heart, after repetitive brief ischemia but before sustained ischemia, has normal efficiencies of crossbridge cycling and Ca(2+) cycling despite the transiently reduced contractility.

Single fibres isolated from frog muscle were tetanically stimulated at 14°C to produce isometric tetani at a sarcomere length of about 2.16 μm, using a striation follower device to measure the sarcomere length of a selected segment of fibre. Force–velocity data were obtained by applying ramp releases at pre-set velocity at the tetanus plateau. Sarcomere stiffness was measured at isometric plateau and during isotonic shortening by using sinusoidal length changes at 2 kHz frequency and about 1 nm per half sarcomere (hs) peak to peak amplitude. A correction method was used to compensate for the force truncation due to the quick recovery. After data collection, the bathing solution was substituted with Ringer plus ethylene glycol (EG) at 2 M (11.2% v/v). When the fibre was fully equilibrated with the new solution, the measurements were repeated. Ethylene glycol reduced the speed of the tetanus rise and tetanus relaxation without altering the isometric tension, and reduced the maximum shortening velocity by about 20%. During isotonic contraction tension and stiffness at each given shortening velocity were reduced by about the same amount, so that the stiffness/tension ratio remained almost unaltered. Force–velocity and stiffness data in both standard and EG Ringer were analysed in terms of a two state model (Huxley, 1957). The analysis showed that our results can be accounted for by assuming that EG at 2 M concentration reduces all the rate constants for crossbridges interaction by about the same amount.

Despite extensive knowledge of many muscle A-band proteins (myosin molecules, titin, C-protein (MyBP-C)), details of the organization of these molecules to form myosin filaments remain unclear. Recently the myosin head (crossbridge) configuration in a relaxed vertebrate muscle was determined from low-angle X-ray diffraction (Hudson et al. (1997), J Mol Biol273: 440–455). This showed that, even without C-protein, the myosin head array displays a characteristic polar pattern with every third 143 A-spaced crossbridge level particularly prominent. However, X-ray diffraction cannot determine the polarity of the crossbridge array relative to the neighbouring actin filaments; information crucial to a proper understanding of the contractile event. Here, electron micrographs of negatively-stained goldfish A-segments and of fast-frozen, freeze-fractured plaice A-bands have been used to determine the resting myosin head polarity relative to the M-band. In agreement with the X-ray data, the prominent 429 A-spaced striations are seen outside the C-zone, where no non-myosin proteins apart from titin are thought to be located. The head orientation is with the concave side of the curved myosin heads (containing the entrance to the ATP-binding site) facing towards the M-band and the convex surface (containing the actin-binding region at one end) facing away from the M-band.

Steady-state conditions of contraction, at maximal and submaximal forces, were produced in intact single muscle fibres, from Rana esculenta, using full tetani and graded K+-contractures. The uniformity in radial direction, of spreading of activation produced in K+-contractures, was checked in relation to the fibre diameters. The absolute isometric force was similar in tetani and maximal contractures, for fibres with diameters between 40 and 60 μm, but not for fibres with diameters greater than about 70 μm in which contracture force never reached tetanic force. The force–[K+]o relation was similar for fibres with diameters between 40 and 60 μm, but it was right shifted and it had a minor slope for fibres with diameters greater than 65–70 μm. This suggests that only in the small diameter fibres (40–60 μm) the activation does not fail to penetrate uniformly from the surface towards the fibre core. For fibres selected in the diameter range between 40 and 60 μm, force–velocity relations and unloaded shortening velocities were determined in tetani and maximal and submaximal contractures. Data were obtained across a force range of 0.3 to 1 P 0 (tetanic plateau force). Controlled velocity method was used to obtain force–velocity relations, and slack test to determine the unloaded shortening velocity (V U). The values of the parameters characterising the force–velocity relation (V 0 and a/P 0) and V U as determined by the slack test did not differ significantly in tetani and contractures, independent of the activation level or absolute force developed by the fibre. These results show that, at least within the range of forces tested, crossbridge kinetics is independent of the number of cycling crossbridges, in agreement with the prediction of the ‘recruitment’ model of myofilament activation.

The gaseous free radical nitric oxide (NO*) has been implicated in a wide range of physiological functions and also has a role in the pathogenesis of cellular injury. It has been suggested that NO* and its congeners may exert effects on actin-myosin crossbridge cycling by modulating critical thiols on the myosin head. To understand the mode and site of actin of NO* in myofibrils, the effects of the NO* donor 3-(2-hydroxy-1-methyl-2-nitrosohydrazine)-N-methyl-1-propanamine (NOC-7) have been studied in Triton X-100-treated rabbit cardiac fibers, in which isometric force was measured at controlled degrees of activation. Experiments were undertaken after previous exposure of the preparations to NOC-7 (for 30 min). We found that NO* induced several alterations of myofibrillar function, i.e., decrease in Ca2+ sensitivity and Hill coefficient and potentiation of rigor contracture. We attributed the effect on rigor contracture to strong inhibition of myofibrillar creatine kinase (CK) activity, because it could be prevented by exogeneously added CK; such CK inactivation afforded by NO* may result in the myofibrillar ATP-to-ADP ratio. In further experiments, concentration of NO* released from NOC-7 was determined by the electron spin resonance spin-trapping technique; N-(dithiocarboxy)sarcosine-Fe2+ complex was used as the spin-trap. NO* at cumulative concentration of 0.69 microM was effective in producing both enhancement of rigor contracture and decrease of myofibrillar-bound CK activity; however, Ca2+-sensitivity (pCa50) was significantly decreased at5.6 microM of NO*, suggesting a result from different mechanisms. Thus, the observed decrease in Ca2+ sensitivity seems to be associated with direct modification of the regulatory proteins by a relatively higher concentration of NO*, and possibly not via inhibition of myofibrillar CK activity. The data reported here indicate that CK may be a pathophysiologically main target for increased NO* formation at low molecular range in the disease state in cardiac muscle.

Superfast muscles power high-frequency motions such as sound production and visual tracking. As a class, these muscles also generate low forces. Using the toadfish swimbladder muscle, the fastest known vertebrate muscle, we examined the crossbridge kinetic rates responsible for high contraction rates and how these might affect force generation. Swimbladder fibers have evolved a 10-fold faster crossbridge detachment rate than fast-twitch locomotory fibers, but surprisingly the crossbridge attachment rate has remained unchanged. These kinetics result in very few crossbridges being attached during contraction of superfast fibers (only ≈1/6 of that in locomotory fibers) and thus low force. This imbalance between attachment and detachment rates is likely to be a general mechanism that imposes a tradeoff of force for speed in all superfast fibers.

Abstract —We compared the dynamics of the contraction and relaxation of single myocytes isolated from nontransgenic (NTG) mouse hearts and from transgenic (TG-β-Tm) mouse hearts that overexpress the skeletal isoform of tropomyosin (Tm). Compared with NTG controls, TG-β-Tm myocytes showed significantly reduced maximal rates of contraction and relaxation with no change in the extent of shortening. This result indicated that the depression in contraction dynamics determined in TG-β-Tm isolated hearts is intrinsic to the cells. To further investigate the effect of Tm isoform switching on myofilament activity and regulation, we measured myofilament force and ATPase rate as functions of pCa (−log of [Ca 2+ ]). Compared with controls, force generated by myofilaments from TG-β-Tm hearts and myofibrillar ATPase activity were both more sensitive to Ca 2+ . However, the shift in pCa 50 (half-maximally activating pCa) caused by changing sarcomere length from 1.8 to 2.4 μm was not significantly different between NTG and TG-β-Tm fiber preparations. To test directly whether isoform switching affected the economy of contraction, force versus ATPase rate relationships were measured in detergent-extracted fiber bundles. In both NTG and TG-β-Tm preparations, force and ATPase rate were linear and identically correlated, which indicated that crossbridge turnover was unaffected by Tm isoform switching. However, detergent extracted fibers from TG-β-Tm demonstrated significantly less maximum tension and ATPase activity than NTG controls. Our results provide the first evidence that the Tm isoform population modulates the dynamics of contraction and relaxation of single myocytes by a mechanism that does not alter the rate-limiting step of crossbridge detachment. Our results also indicate that differences in sarcomere-length dependence of activation between cardiac and skeletal muscle are not likely due to differences in the isoform population of Tm.

We discuss the force-velocity relations obtained in a two-state crossbridge model for molecular motors. They can be calculated analytically in two limiting cases: for a large number and for one pair of motors. The effect of the strain-dependent detachment rate on the motor characteristics is studied. It can lead to linear, myosin-like, kinesin-like and anomalous curves. In particular, we specify the conditions under which oscillatory behavior may be found., 5 pages, 4 figures, REVTeX; thoroughly revised version; also available at http://www.physik.tu-muenchen.de/~frey/

The proposed model is characterized by the constant r (Eq. 2-1), the induced potential (Fig. 1), two attached states of a myosin head (Fig. 1), the nonlinear elastic property of the crossbridge (Eq. 2-7), and the expression of U* (Eqs. 3-8 and 3-9), which led us to the following conclusions. 1. The following various magnitudes of myosin head motion are compatible with each other: about 2 nm of the quantity called power stroke by Irving (27), which is the mean moving distance of myosin head in the isometric tension in our model, 4-5 nm of the displacement of a single myosin head during one ATP hydrolysis cycle (Molloy et al. (20)) or a few tens of nm when the actin and myosin filaments are set parallel (Tanaka et al. (21) and Kitamura et al. (42)), and more than 200 nm of the myosin head displacement in a multi-myosin head system below 22 degrees C (Harada et al. (19)). 2. There is one-to-one coupling between the ATP hydrolysis cycle and the attachment-detachment cycle of a myosin head in accordance with the generally accepted concept of chemical reactions, since the head is trapped in the spatially shifting wide potential well (Fig. 1) until epsilon ATP is exhausted. Here, an actin filament interacts with a myosin head like a single molecule. 3. The calculated tension dependence of muscle stiffness agrees well with the observations by Ford et al. (12), as shown in Fig. 9. 4. The calculated shortening velocity V of muscle as a function of P/P0 agreed very well with experimental results as shown in Fig. 13. The deviation from the Hill equation (34) observed by Edman (32) is related with U* being effectively infinite for f1 < kappa b yc0 (Fig. 10). 5. Calculated energy liberation rate W + H as a function of P/P0 has characteristics almost the same as the Hill equation (33), and agrees well with the experimental results as shown in Fig. 14. 6. The time course of tension recovery after a quick length change is determined by four parameters: kappa f, kappa b, a, and Z0. Among them, kappa f, kappa b (Eq. 2-22) and a (Eq. 4-21) are readily determined by analysis of the steady filament sliding and p0. Calculations of T1/T0 and T2/T0 with these three parameters are in very good agreement with experimental data (Fig. 21). Calculated tension variations by assigning the value in Eq. 4-23 to Z0 agree with the observation (Fig. 17). 7. The model suggests that large fluctuations exist in relative positions between the actin and myosin filaments even when the load on a muscle is kept constant (Fig. 23). Taking this fluctuation into account, the time course of the isotonic velocity transient shown in Fig. 22 becomes understandable referring to Fig. 24. 8. The experimental data of the delta yhs vs. delta P/P0 relationship (Fig. 25) is explained. The delta yhs value at delta P/P0 = 0 (about 5 nm) supports the two-attached-state model and thus indicates that the incremental unit step of a myosin head motion along an actin filament is close to L (5.46 nm).

The structure of truncated, recombinant Dictyostelium myosin motor domain complexed with Mg.ADP and slowly dissociating analogues of Pi has previously been characterized as two main states (S1-MgADP plus BeFx vs. A1F4- or Vi). The BeFx bound state is thought to mimic the weak actin-binding M.ATP complex, while the states with A1F4- or Vi bound mimic the M.ADP.Pi state. While the effects of A1F4- and Vi on fibre mechanics have been previously described (Chase et al., 1994, 1993), the effects of BeFx have not been characterized in detail. At pCa 4.5 (12 degrees C), we measured (i) steady-state isometric tension, (ii) stiffness (KS; 1 kHz sinusoids), and (iii) unloaded shortening velocity (Vu; slack test) in single skinned muscle fibres from rabbit psoas. Results were compared when tension was inhibited with either BeFx or 2,3-butanedione-monoxime (BDM) or modulated by altering myoplasmic [Ca2+]. With 3 mM total fluoride, 1 mM BeFx inhibited both tension and KS by approximately 50% (compared to 7-10 mM BDM and 50-100 microM A1F4-). Increasing [BeFx] to 10 mM further reduced tension to approximately 15% P0, but had little further effect on KS; with BDM and altered [Ca2+], KS scaled more proportionately with tension. Inhibition of tension and KS by BeFx was more rapidly reversible, compared with slow recovery from tension inhibition with A1F4- or Vi. Vu exhibited a complex dependence on [BeFx], being relatively unaffected by concentrations < or = 1 mM, and becoming inhibited steeply for [BeFx] above this level. With BDM, Vu co-varied more directly with force. Our results suggest that BeFx may induce a different cross-bridge state in fibres than do A1F4- or Vi, but all three analogues of Pi form complexes that mimic crossbridge states that follow ATP hydrolysis.

The dynamics of the interaction of fast skeletal muscle myosin subfragment-1 with pyrene-labelled actin were examined using both stopped-flow and pressure relaxation methods. The data suggest a four-step model i.e.: $$A + M.\left( N \right)\mathop \Leftrightarrow \limits^{K_0 } $$ $$\mathop {A + M.\left( N \right)}\limits_{collisioncomplex} \mathop \Leftrightarrow \limits^{K_1 } $$ $$\mathop {A - M.\left( N \right)}\limits_{A - state} \mathop \Leftrightarrow \limits^{K_2 } $$ $$A - M.\left( N \right)\mathop {\mathop \Leftrightarrow \limits^{K_3 } }\limits_{R - states} A.M\left( N \right)\# $$ ADP weakens the acto-S1 affinity via a reduction in K0, with no apparent effect on K1 and no effect on K2, whilst k+2 and k−2 are both markedly reduced. Increased ionic strength reduces both K0 and k+2 with no major effect on k+1. Step 3 represents an extension to previous models and is ADP-dependent. The present work is discussed in relation to earlier studies which led to somewhat different conclusions (Taylor EW (1991) J Biol Chem266: 294–302; Geeves MA (1989) Biochemistry28: 5864–5871). It is likely that the interaction proceeds via formation of a disordered complex stabilised by ionic interactions (corresponding to step 0), followed by a disordered-to-ordered transition involving additional hydrophobic contacts (step 1) after which further contacts of both types are made coupled to internal conformational changes (steps 2 and 3). Step 3 could have a role in extending the lifetime of force-generating crossbridges and limiting ATP turnover during contraction against a load, and may be equivalent to a structural change observed in recent cryo-EM studies on the smooth muscle system (Whittaker M, Wilsonkubalek EM, Smith JE, Faust L, Milligan RA and Sweeney HL (1995) Nature378: 748–751). Cooperative interactions between the two myosin heads also appear to have a role in this putative latch mechanism.

This study aimed to establish whether quantitatively accurate predictions of the rate of crossbridge-dependent energy output from shortening muscle could be made on the basis of a 2-state model of crossbridge kinetics incorporating weak coupling between mechanical cycles and ATP hydrolysis. The model was based on Huxley's (1957) model but included rapid detachment, without ATP hydrolysis, of crossbridges when their strain energy increased sufficiently that crossbridge free energy exceeded that of the unbound state (Cooke et al., 1994). An expression was derived relating force to steady-state velocity in terms of the model's rate constants. The values of the rate constants that both provided the best fit through force–velocity data and correctly predicted crossbridge-dependent rate of energy output during an isometric contraction were found and used to predict the variation in rate of energy liberation with shortening velocity. The model predictions closely matched the estimated crossbridge energetics of frog sartorius muscle, including the decline in rate of enthalpy output at high shortening velocities. Data from fast- and slow-twitch muscles of the mouse were also simulated. The velocity-dependence of rate of energy liberation from fast-twitch EDL muscle was well described by the model. The model overestimated crossbridge-dependent energy output from slow-twitch soleus at low shortening velocities but provided accurate predictions of energy output at high velocities. In terms of this model, the distinctive energetics of fast and slow muscles cannot be explained exclusively by differences in cross-bridge detachment rate; differences in the relative rates of crossbridge attachment must also be considered to explain the different relations between energy output and shortening velocity.

In both cardiac and skeletal muscle, the force-generating molecular motors (crossbridges) are turned on by increasing the intracellular free calcium level that regulates the troponin-tropomyosin system. However, calcium activation is a two-way process in the sense that activated crossbridges also affect the troponin-tropomyosin system. Here we review the mechanism of calcium action on myofilament proteins, particularly tropomyosin, that affects both the extent and the rate of force development and hence the contractility of the myocardium. At low myoplasmic Ca2+ concentrations tropomyosin is located at the edge of the thin filament, thereby interfering with the formation of strong actin-myosin linkages (blocked state). An increase in Ca2+ activity causes an azimuthal shift of tropomyosin around the filament (by about 30 degrees), thereby increasing the probability of low-force crossbridge interaction, a process which by cooperative effects induces further tropomyosin movement (by an additional 10 degrees) which results in the open state of the filament characterized by forceful crossbridge interaction. (This mechanism may be analogous to that in ligand-gated ion channels, where ligand binding increases the open probability of the pore.) The extent of activation then depends on the free Ca2+ concentration and on the calcium sensitivity of the thin filament that may be affected by protein phosphorylation, crossbridge attachment, the troponin isoform composition of the filament, and novel calcium-sensitizing drugs that act on the contractile or regulatory proteins and thus increase the force of the heart.

Abstract —Reductions in sarcomere length (SL) and concomitant increases in interfilament lattice spacing have been shown to decrease the Ca 2+ sensitivity of tension in myocardium. We tested the idea that increased lattice spacing influences the SL dependence of isometric tension by reducing the probability of strong interactions of myosin crossbridges with actin, thereby decreasing cooperative activation of the thin filament. Single ventricular myocytes were isolated by enzymatic digestion of rat hearts and were subsequently rapidly skinned. Maximal tension and Ca 2+ sensitivity of tension (ie, pCa 50 ) were measured in the absence and presence of N -ethylmaleimide–modified myosin subfragment 1 (NEM-S1) at both short and long SLs. NEM-S1, a strong-binding non–tension-generating derivative of the myosin head, was applied to single skinned myocytes to cooperatively promote strong binding of endogenous myosin crossbridges. Compared with control myocytes at SL of ≈1.90 μm, application of NEM-S1 markedly increased submaximal Ca 2+ -activated tensions and thereby increased Ca 2+ sensitivity; ie, pCa 50 increased from 5.40±0.02 to 5.52±0.02 pCa units in the presence of NEM-S1. Furthermore, NEM-S1 treatment reversibly eliminated the SL dependence of the Ca 2+ sensitivity of tension, in that the ΔpCa 50 between short and long lengths was 0.02±0.01 pCa units in the presence of NEM-S1 compared with a ΔpCa 50 of 0.10±0.01 pCa units in control myocytes. From these results we conclude that the decrease in the Ca 2+ sensitivity of tension at short SL results predominantly from decreased cooperative activation of the thin filament due to reductions in the number of strong-binding crossbridges.

Skinned muscle fibres from the gracilis muscle of the rabbit were used to record small angle X-ray diffraction spectra under various contractile conditions. The intracellular calcium concentration, expressed as pCa, was varied between 8.0 and 5.74. Equatorial diffraction spectra were fitted by a function consisting of five Gaussian curves and a hyperbola to separate the (1.0), (1.1), (2.0), (2.1) and Z-line diffraction peaks. The hyperbola was used to correct for residual scattering in the preparation. The ratio between the intensities of the (1.1) and (1.0) peaks was defined as the relative transfer of mass between myosin and actin, due to crossbridge formation after activation by calcium. The relation between the ratio and the relative force of the fibre (normalized to the force at pCa 5.74 and sarcomere length 2.0 microns) was linear. At high pCa (from pCa 6.34 to 8.0) no active force was observed, while the ratio still decreased. Sarcomere length was recorded by laser diffraction. The laser diffraction patterns did not show changes in sarcomere length due to activation in the high pCa range (between 8.0 and 6.34). From these results the conclusion is drawn that crossbridge movement occurs even at subthreshold calcium concentrations in the cell, when no active force is exerted. Since no force is generated this movement may be related to crossbridges in the weakly bound state.

This study examined the effects of fatigue on the functional aspects of the contractile apparatus and sarcoplasmic reticulum (SR). Frog semitendinosus muscles were stimulated to fatigue, and skinned fibers or a homogenate fraction was prepared from both fatigued and rested contralateral muscles. In fatigued fibers, maximal Ca2+-activated force of the contractile apparatus was unaltered, whereas maximal actomyosin-ATPase activity was depressed by 20%. The Ca2+sensitivity of force was increased, whereas that of actomyosin-ATPase was not altered. Also, the rate constant for tension redevelopment was decreased at submaximal Ca2+concentration. These latter findings suggest that fatigue slows the dissociation of force-generating myosin cross bridges. Ca2+uptake and Ca2+-ATPase activity of the SR were depressed by 46 and 21%, respectively, in the fatigued muscles. Fatigue also reduced the rates of SR Ca2+release evoked by AgNO3and 4-chloro- m-cresol by 38 and 45%, respectively. During fatigue, the contractile apparatus and SR undergo intrinsic functional alterations. These changes likely result in altered force production and energy consumption by the intact muscle.

Crossbridge (CB) properties were investigated in isolated diaphragm of rabbits during congestive heart failure (CHF, n=9) induced by chronic volume and pressure overload. This model induced cardiac hypertrophy and heart failure. Controls (C) were prepared (n=14). Compared to C, peak tension in CHF fell by 57% in twitch and by 40% in tetanus; Vmax declined by 47% in twitch and by 48% in tetanus. Our study provided an analytical means of calculating from A. F. Huxley's equations the rate constants for CB attachment and detachment, CB single force (II), CB number per mm3 (m'), peak mechanical efficiency (Effmax), and turnover rate of myosin ATPase (kcat); m', II, and Effmax were lower in CHF than in C in both twitch and tetanus. The marked decline in m' and II accounted for the fall in diaphragm strength. In the overall population of C and CHF, Effmax was linearly related to II. Conversely, there was no relationship between Vmax and kcat. Dissociation between Vmax and kcat might be explained by the crucial role attributed to two apparently nonconserved surface 'loops' on the motor domain of myosin head.

To clarify the structure of the ATP hydrolysis intermediates (ADP.Pi bound state) formed by actomyosin crossbridges, the effects of various phosphate analogs in the presence of MgADP on the structures of the thin and thick filaments in glycerinated rabbit psoas muscle fibers in the rigor state have been investigated by X-ray diffraction with a short exposure time using synchrotron radiation. When MgADP and phosphate analogs such as metallofluorides (BeFx = 3,4 and AlF4) and vanadate (VO4(Vi)) were added to rigor fibers in the presence of the ATP-depletion backup system, the intensities of the actin-based layer lines were markedly weakened. The greatest effect (approximately 50% decrease in intensity) was observed in the presence of BeFx among the analogs examined. The intensity distribution of the 5.9 nm actin-based layer line shifted towards that observed in the Ca(2+)-activated fibers, while the first actin layer line at approximately 1/36.7 nm-1 retained a rigor-like profile with an intensity weakened by approximately 50%. The intensity of the equatorial 10 reflection increased while that of the 11 reflection changed little, resulting in only a small increase (approximately 1.7 fold) in the intensity ratio of the 10 to the 11 reflection. No resting-like pattern appeared upon the addition of MgADP and BeFx. These results indicate that a substantial fraction (approximately 40%) of the myosin heads dissociate from actin but the detached heads remain in the vicinity of the actin filaments when MgADP and BeFx bind. The states produced by binding phosphate analogs to a rigor muscle differ from the resting-like state produced by adding them to a contracting muscle (Takemori et al., J. Biochem. (Tokyo) 117 (1995) 603-608). Our conclusion put forward to explain the data is that one of the two heads of a crossbridge is detached and the other retains a rigor-like attachment.

Abstract —Cardiac myofilaments contain proteins that regulate the interaction between actin and myosin. In the thick filament, there are several proteins that may contribute to the regulation of the contraction. The myosin binding protein C, or C protein, has 4 sites that can be phosphorylated by a Ca 2+ -calmodulin–controlled kinase, protein kinase A or protein kinase C. Using electron microscopy and optical diffraction, we examined the structure of thick filaments isolated from rat ventricles with either the α or β isoform of myosin heavy chain (MHC) and the effect of specific phosphorylation of C protein on the structure. In thick filaments with α-MHC, crossbridges were clearly visible. Phosphorylation of C protein by protein kinase A extended the crossbridges from the backbone of the filament, changed their orientation, increased the degree of order of the crossbridges, and decreased the flexibility of the crossbridges. Crossbridges in filaments with β-MHC were less ordered and apparently more flexible. Phosphorylation of C protein in β-MHC–containing filaments did not extend the crossbridges and did not alter degree of order or flexibility. The relative flexibility of the crossbridges inferred from the optical diffraction pattern correlated well with the rate of ATP hydrolysis by actomyosin. These results suggest that (1) crossbridge flexibility is an important parameter in setting the rate of crossbridge cycling, and (2) C protein–mediated control of the position and flexibility of crossbridges may regulate actomyosin ATPase activity by modifying the kinetics of crossbridge cycling.

Force generation during muscle contraction can be understood in terms of cyclical length changes in segments of actin thin filaments moving through the three-dimensional lattice of myosin thick filaments. Recent anomalies discovered in connection with analysis of myosin step sizes in in vitro motility assays and with skinned fibres can be rationalized by assuming that ATP hydrolysis on actin accompanies these length changes. The paradoxically rapid regeneration of tension in quick release experiments, as well as classical energetic relationships, such as Hill's force-velocity curve, the Fenn effect, and the unexplained enthalpy of shortening, can be given mutually self-consistent explanations with this model. When muscle is viewed as a Markov process, the vectorial process of chemomechanical transduction can be understood in terms of lattice dependent transitions, wherein the phosphate release steps of the myosin and actin ATPases depend only on occurrence of allosteric changes in neighbouring molecules. Tropomyosin has a central role in coordinating the steady progression of these cooperative transitions along actin filaments and in gearing up the system in response to higher imposed loads.

The role of dityrosine as a fluorescent crossbridge between adjacent calmodulin molecules within the high molecular mass polymers that are generated by Arthromyces peroxidase-catalyzed cross-linking [Malencik, D. A., and Anderson, S. R. (1996) Biochemistry 35, 4375] has been examined in frequency domain fluorescence anisotropy studies. Measurements on a polymer fraction possessing a range of molecular masses > 96 000 in NaDodSO4 polyacrylamide gel electrophoresis demonstrate predominating fast local rotations involving the dityrosyl moieties. Normal distribution analyses of the results show peak rotational correlation times of 0.6 ns (zero Ca2+) and 1.2 ns (+Ca2+), values that are smaller than the principal correlation times determined for the global rotation of the free calmodulin monomer in either the presence or absence of Ca2+. The intermolecularly cross-linked segments of the polymers retain a degree of the mobility that is characteristic of the tyrosine-containing sequences of native calmodulin. The half-widths of the normal distribution curves range from 13 ns (zero Ca2+) to approximately 90 ns (5 mM Ca2+), thus encompassing varying rates of segmental motion within the polymers. When Ca2+ is present, possible contributions from the global rotations of polymer molecules are detected near the operating limits of the method. Experiments with the intramolecularly cross-linked calmodulin monomer give global rotational correlation times of 7.9 ns (zero Ca2+) and 11.4 ns (+Ca2+), which compare to values of 7.2 ns and 9.9 ns found previously in time domain measurements [Small, E. W., and Anderson, S. R. (1988) Biochemistry 27, 419]. Rotations of apparent phi2 = 0.2 to 0.3 ns also are detected, accounting for 31% (-Ca2+) to 23% (+Ca2+) of the anisotropy.

We previously established a new measuring method of the myocardial O2 consumption of mechanically unloaded rat left-ventricular slices. O 2 consumption of unstimulated myocardium corresponds to basal metabolism. We have found O2 consumption of stimulated myocardium to include basal metabolism and O 2 consumption for Ca2+ handling in the excitation-contraction coupling, but not for crossbridge cycling. Thus, O2 consumption for the excitation-contraction coupling is obtained by subtracting basal metabolism from O2 consumption of the stimulated myocardium. We have shown that O2 consumption for the excitation-contraction coupling corresponds to 40% of basal metabolism. The purpose of the present study was to analyse the component of myocardial O2 consumption for the excitation-contraction coupling by this method. Blockade of the sarcoplasmic reticulum Ca2+ pump by thapsigargin (0.1-1 micro mol/l), or by cycloplazonic acid (10 micro mol/l), significantly reduced O2 consumption for the excitation-contraction coupling by 40 or 70% of the respective controls. Neither thapsigargin nor cyclopliazonic acid reduced basal metabolism O2 consumption. The magnitude of free shortening of the unloaded myocardial slices, quantified by slice surface area reduction, was small (about 1.5%) because of the lack of external preload. Thapsigargin (1 micro mol/l) and cycloplazonic acid (10 micro mol/l) markedly attenuated the already reduced free shortening. 2,3-butanedione monoxime (5 mmol/l) also largely suppressed the free shortening, although this agent did not alter the O2 consumption of either unstimulated or stimulated myocardium. Some residual cross-bridge cycling may occur without detectable O2 consumption. Our present energetic results revealed that the O2 consumption of myocardial slices for the Ca2+ handling in the excitation-contraction coupling was mainly used for the sarcoplasmic reticulum Ca2+ pump.

A model of passive and active cardiac muscle mechanics is presented, suitable for use in continuum mechanics models of the whole heart. The model is based on an extensive review of experimental data from a variety of preparations (intact trabeculae, skinned fibres and myofibrils) and species (mainly rat and ferret) at temperatures from 20 to 27 degrees C. Experimental tests include isometric tension development, isotonic loading, quick-release/restretch, length step and sinusoidal perturbations. We show that all of these experiments can be interpreted with a four state variable model which includes (i) the passive elasticity of myocardial tissue, (ii) the rapid binding of Ca2+ to troponin C and its slower tension-dependent release, (iii) the kinetics of tropomyosin movement and availability of crossbridge binding sites and the length dependence of this process and (iv) the kinetics of crossbridge tension development under perturbations of myofilament length.

Force responses to fast ramp stretches at various velocities were recorded in single muscle fibres isolated from tibialis anterior muscle of the frog (Rana esculenta) at a sarcomere length between 2.15 and 3.25 microns at 15 degrees C. Stretches were applied at the tetanus plateau and during tetanus rise. Length changes were recorded at the sarcomere level using either a laser diffractometer or a striation follower apparatus. The immediate force response to the stretch is not simply elastic, as is usually assumed, but is composed of the sum of at least two components: (i) elastic (force proportional to the amount of stretch); and (ii) viscous (force proportional to the rate of stretch). The viscous response is associated with a short (about 10 microseconds) relaxation time. The amplitude of the viscous component increases progressively with tension during the tetanus rise and scales down with sarcomere length approximately in the same way as the tetanic tension. These results suggest that the viscosity of activated fibres may arise from crossbridge kinetics.

In smooth muscle, the state of prolonged contraction (latch state) is associated with very slow energy turnover and cycling of crossbridges that are dephosphorylated. A similar state may be reproduced in skinned fibres when the calcium-induced contraction is terminated by calcium removal with ethylenebis(oxonitrilo)tetraacetate (EGTA) and, during the slow relaxation that follows, force is maintained by dephosphorylated crossbridges that cycle slowly or not at all and may cooperatively reattach after detachment (Khromov et al. 1995, Biophys J 69:2611-2622). In guinea-pig skinned taenia coli that has been pretreated by prolonged incubation with caldesmon (5 microM), the rate of relaxation is approximately 1.6 times greater than in untreated controls, with half-times of relaxation being 1.3 and 2.1 min, respectively. In contrast, preloading the fibres with calponin does not accelerate relaxation. Preloading the fibres with caldesmon also accelerates the relaxation of skinned fibres from the state of rigor contraction when the latter is terminated by immersion into an ATP-containing relaxing solution or, in the presence of inorganic phosphate (Pi), also by flash-photolytic release of ATP from caged-ATP. Even in the latter case, relaxation is comparatively slow, possibly because of cooperative reattachment of dephosphorylated crossbridges which delays net crossbridge detachment and hence relaxation. We propose that by inhibition of cooperative reattachment caldesmon accelerates relaxation, even in the presence of Pi, and that the latch-like state of skinned fibres is supported by dephosphorylated cooperatively attaching crossbridges and may be regulated by the activity of caldesmon in the smooth muscle cell.

We determined the primary structures of the three acclimation-temperature-associated isoforms of myosin subfragment-1 heavy chain from fast skeletal muscle of thermally acclimated carp. These isoforms were cloned by extending 5'-regions of cDNAs that encode the rod part of myosin heavy chain specifically expressed in 10 degrees C- and 30 degrees C-acclimated carp, together with the region that encodes an intermediate structure [Imai, J., Hirayama, Y., Kikuchi, K., Kakinuma, M.Watabe, S. (1997) J. Exp. Biol. 200, 27-34]. These three isoforms generally resembled each other in primary structure, showing 94.8, 90.9, and 92% similarity between the 10 degrees C- and intermediate-type, between the 10 degrees C- and 30 degrees C-type, and between the intermediate- and 30 degrees C-type myosin heavy chains, respectively. However, isoform-specific differences were clearly observed between the 10 degrees C- and 30 degrees C-type heavy chains in the first 60 amino acid residues from the N-terminus, where the intermediate-type showed an intermediate feature in its sequence compared to the 10 degrees C- and 30 degrees C-type isoforms. Other striking differences were observed in two surface loops between the 10 degrees C- and 30 degrees C-type isoform. Five amino acid residues out of sixteen were different in loop 1 near the ATP-binding pocket, and six out of twenty were different in loop 2 on the actin-binding site. The loops connecting beta-sheets that are known to surround the ATP-binding pocket were highly conserved in primary structure for the three types. In northern blot analysis, the accumulated mRNA levels of the 10 degrees C- and intermediate-type isoforms were significantly higher in carp acclimated to 10 degrees C and 20 degrees C than carp acclimated to 30 degrees C, whereas the level of the 30 degrees C-type isoform was significantly higher in carp acclimated to 30 degrees C than those acclimated to 10 degrees C and 20 degrees C.

Maximum lift production and the thermal sensitivity of lift production increase dramatically during adult maturation of Libellula pulchella dragonflies. Here, we report that the mechanistic basis for this transition appears to involve a developmental change in protein expression, which alters the Ca2+-sensitivity of muscle activation and twitch contraction kinetics. The alternatively spliced Ca2+ regulatory protein troponin T (TnT) undergoes an isoform shift during adult maturation. Skinned (demembranated) fibers of mature flight muscle are up to 13 times more sensitive to activation by Ca2+ than skinned fibers from teneral (newly emerged adult) flight muscle, and their Ca2+-sensitivity is more strongly affected by temperature. Intact muscle from mature individuals has a shorter time to peak tension and longer time to half-relaxation during twitch contractions, which is consistent with a greater Ca2+-sensitivity of mature muscle. Because it becomes activated more quickly and relaxes more slowly, mature flight muscle is able to generate, with each twitch, more force per unit area than teneral muscle; this difference in force becomes greater at high temperatures. There do not appear to be any age-related differences in actomyosin crossbridge properties, since teneral and mature flight muscles do not differ in shortening velocity, tetanic tension or instantaneous power output during isotonic contraction. Thus, variation in TnT expression appears to affect the temperature-dependent Ca2+-sensitivity of muscle activation, which in turn affects the kinetics and force production of the twitch contractions used by dragonflies during flight. This cascade of effects suggests that maturational changes in the expression of TnT isoforms may be a key determinant of overall muscle and organismal performance.

The expression of fast and slow myosin isoforms in individual cells is associated with differences in shortening velocities and power output in fully differentiated vertebrate striated muscle. This paradigm in which shortening velocity is determined by the myosin isoform (and load) is inappropriate for smooth muscle. Smooth muscle tissues express multiple myosin heavy and light chain isoforms, and it is not currently possible to separate and identify chemically distinct native myosin hexamers (i.e., isoforms). It is not known if different isoforms are localized in subpopulations of cells or in specific cellular domains nor whether they combine preferentially to form a small number of native myosin hexamer isoforms. Potentially, thick filaments are aggregates of many different combinations of heavy and light chain isoforms that may or may not exhibit different kinetics. Shortening velocities in smooth muscle are regulated by Ca++-dependent crossbridge phosphorylation of the myosin regulatory light chains. Much of the observed diversity in power output in smooth muscle may be attributed to regulatory mechanisms modulating crossbridge cycling rates rather than contractile protein isoform expression.

Abstract Previous studies have shown that cardiac endothelial cells release substances that influence myocardial contraction. Since P o 2 is an important stimulus that modulates endothelial function, we investigated the effects of acute moderate hypoxia and reoxygenation on the release of cardioactive factors by endothelial cells. Endothelial cells cultured from several vascular beds were superfused with normoxic (equilibrated with room air; P o 2 , ≈160 mm Hg) or hypoxic (P o 2 , 40 to 50 mm Hg) physiological buffer solution, and the superfusates were reequilibrated to a P o 2 of ≈160 mm Hg and then tested for their effects on various myocardial assays. Endothelial cell viability and buffer ionic composition were unaltered after the superfusion procedures. The superfusates of hypoxic endothelial cells induced rapid, potent, reversible inhibition of isolated cardiac myocyte contraction without reducing cytosolic Ca 2+ transients. This activity was not lost after heating (95°C) and was present in low molecular weight ( M r , 2+ or other second messenger signaling pathways. Such a mechanism could have important implications for the regulation of oxygen supply-demand balance in the heart and be relevant to conditions such as myocardial hibernation.

The limits of flight performance have been estimated in tethered Drosophila melanogaster by modulating power requirements in a ‘virtual reality’ flight arena. At peak capacity, the flight muscles can sustain a mechanical power output of nearly 80 W kg−1 muscle mass at 24 °C, which is sufficient to generate forces of approximately 150 % of the animal’s weight. The increase in flight force above that required to support body weight is accompanied by a rise in wing velocity, brought about by an increase in stroke amplitude and a decrease in stroke frequency. Inertial costs, although greater than either profile or induced power, would be minimal with even modest amounts of elastic storage, and total mechanical power energy should be equivalent to aerodynamic power alone. Because of the large profile drag expected at low Reynolds numbers, the profile power was approximately twice the induced power at all levels of force generation. Thus, it is the cost of overcoming drag, and not the production of lift, that is the primary requirement for flight in Drosophila melanogaster. By comparing the estimated mechanical power output with respirometrically measured total power input, we determined that muscle efficiency rises with increasing force production to a maximum of 10 %. This change in efficiency may reflect either increased crossbridge activation or a favorable strain regime during the production of peak forces.

This paper investigates mathematical relations between models of calcium activation kinetics and Huxley-type models of cross-bridge dynamics in muscle. It is found that different calcium-activation schemes lead to the same form of generalized Huxley rate equation with calcium activation (∂n/∂t)−v(∂n/∂x)=rf(α−n)−gn if it is assumed that calcium–troponin interaction rates are fast compared to the rates of transition associated with force-generating cross-bridge states. Calcium affects cross-bridge dynamics by modifying the bonding rate f, but does not affect the number of interacting cross bridges α or the unbonding rate g; this occurs through the appearance in the equation of an activation factor, r, which is a pure function of sarcoplasmic free calcium concentration. In particular, it is shown that both the “tight-coupling” and “loose-coupling” calcium-activation schemes introduced by Zahalak and Ma [1] lead to the same rate equation with the same activation factor; the difference between them appears in the calcium mass-balance equation. While both of these activation models can be made to fit simple twitch and force-velocity data equally well, experimentally observed load-dependent shifts in the free calcium concentration are compatible with the tight-coupling scheme, but not with loose coupling.

Crossbridge properties of cardiomyopathic Syrian hamster (CSH) diaphragm from the dilated Bio 53-58 strain were analyzed after 5-mo of treatment with the angiotensin converting enzyme (ACE) inhibitor perindopril (1 mg/kg/d by oral gavage). Three groups were studied: control F1B hamsters (C; n = 14); CSH given placebo (PL; n = 11 ); and perindopril-treated CSH (PE; n = 11). Peak isometric tension was lower in PL than in C, in both twitch (21.4 +/- 1.5 versus 46.9 +/- 1.5 mN/mm2; p0.001) and tetanus (41.0 +/- 2.7 versus 90.5 +/- 3.3 mN/mm2; p0.001). In PE, peak isometric tension was intermediate between C and PL, and was significantly lower than in C and higher than in PL. The single force of one crossbridge (pi), the number (m) of crossbridges, the turnover rate of myosin adenosine triphosphatase (ATPase) (kcat), and peak mechanical efficiency (Effmax) were calculated from A.F. Huxley's equations; m was lower in PL than in C, in both twitch (3.4 +/- 0.2 versus 4.9 +/- 0.2 10(9)/mm2; p0.001) and tetanus (4.0 +/- 0.3 versus 8.9 +/- 0.7 10(9)/mm2; p0.001); m was higher in PE than in PL, in both twitch 4.3 +/- 0.5 versus 3.4 +/- 0.2 10(9)/mm2; NS) and tetanus (6.2 +/- 0.4 versus 4.0 +/- 0.3 10(9)/mm2; p0.01), with no change in pi. In the three groups, Effmax correlated linearly with kcat (r = 0.93; p = 0.001) and showed a negative linear correlation with pi (r = 0.996; p = 0.001). In conclusion, our results show that in experimental cardiomyopathy, ACE inhibitor mainly helps to prevent a decrease in the number of diaphragm muscle crossbridges, resulting in preserved peak isometric tension.

The energetic cost of activation was measured during an isometric tetanus of white muscle fibres from the dogfish Scyliorhinus canicula. The total heat production by the fibres was taken as a measure of the total energetic cost. This energy consists of two parts. One is due to crossbridge interaction which produces isometric force, and this part varies linearly with the degree of filament overlap in the fibres. The other part of the energy is that associated with activation of the crossbridges by Ca2+, mainly with uptake of Ca2+ into the sarcoplasmic reticulum by the ATP-driven Ca2+ pump. Total heat production was measured at various degrees of filament overlap beyond the optimum for force development. Extrapolation of heat versus force production data to evaluate the heat remaining at zero force gave a value of 34±5 % (mean ± S.E.M., N=24) for activation heat as a percentage of total heat production in a 2.0 s isometric tetanus. Values for 0.4 and 1.0 s of stimulation were similar. Comparison with values in the literature shows that the energetic cost of activation in dogfish muscle is very similar to that of frog skeletal muscle and it cannot explain the lower maximum efficiency of dogfish muscle compared with frog muscle. The proportion of energy for activation (Ca2+ turnover) is similar to that expected from a simple model in which Ca2+ turnover was varied to minimize the total energy cost for a contraction plus relaxation cycle.

Force responses to fast ramp stretches at various velocities were recorded from single muscle fibres isolated from either lumbricalis digiti IV or tibialis anterior muscle of the frog (Rana esculenta) at sarcomere length between 2.15 and 3.25 μm at 15° C. Stretches were applied at rest, at tetanus plateau and during the tetanus rise. Stretches with the same velocity but different accelerations were imposed to the fibre to evaluate the effect of fibre inertia on the force responses. Length changes were measured at sarcomere level with either a laser diffractometer or a striation follower apparatus. The force response to a fast ramp stretch could be divided into two phases. The initial fast one (phase 1) lasts for the acceleration period during which the stretching velocity rises up to the steady state. The second slower phase (phase 2) lasts for the remainder of the stretch and corresponds to the well-known elastic response of the fibre. Most of this paper is concerned with phase 1. The amplitude of the initial fast phase was proportional to the stretching velocity as expected from a viscous response. This viscosity was associated with a very short (about 10 μs) relaxation time. The amplitude of the fast phase increased progressively with tension during the tetanus rise and scaled down with sarcomere length approximately in the same way as tetanic tension and fibre stiffness. These data suggest that activated fibres have a significant internal viscosity which may arise from crossbridge interaction

Intracellular accumulation of inorganic phosphate (Pi) and intracellular acidosis, which occur in ischemic and hypoxic hearts, reduce the force of contraction by decreasing the responsiveness of contractile system to Ca2+. In the present study we investigated the effects of MCI-154, a Ca2+ sensitizer that can enhance crossbridge interaction, on the decline in maximal Ca2+-activated force by Pi or acidic pH in skinned fiber bundles of guinea pig hearts. MCI-154 can concentration-dependently reverse the depression in maximal Ca2+-activated force (pCa 4.4) by 20mM Pi, which was not recovered by a higher concentration of Ca2+ ion (pCa 4.0). The effects of MCI-154 were observed even at a concentration (0.01 M) at which the drug has no effect on the pCa4.4–induced maximal force in the absence of 20 mM Pi when given alone. MCI-154 inhibited the rightward shift of the pCa–tension relationships, with a marked decrease of maximal force produced by 20 mM Pior acidic pH (decrease in pH from 7.0 to 6.6). MCI-154 also improved the decline in maximal Ca2+-activated force by 20 mM Pi under acidic pH, but the acidosis did not further decrease the effect of 20 mM Pi. Milrinone, a cyclic AMP–dependent phosphodiesterase inhibitor, and pimobendan, another Ca2+ sensitizer, did not improve the Pi-induced contractile failure. These results indicate that the Ca2+ sensitizer MCI-154 could reverse the contractile failure induced by Pi and/or acidic pH in a skinned fiber preparation via modulation of the strong crossbridge reaction with myosin. MCI-154 may be a promising agent for myocardial contractile failure, in which Pi and H+ progressively increase.