The Relaxation Properties of Myofibrils Are Compromised by Amino Acids that Stabilize α-Tropomyosin.

We investigated the functional impact of α-tropomyosin (Tm) substituted with one (D137L) or two (D137L/G126R) stabilizing amino acid substitutions on the mechanical behavior of rabbit psoas skeletal myofibrils by replacing endogenous Tm and troponin (Tn) with recombinant Tm mutants and purified skeletal Tn. Force recordings from myofibrils (15°C) at saturating [Ca2+] showed that Tm-stabilizing substitutions did not significantly affect the maximal isometric tension and the rates of force activation (kACT) and redevelopment (kTR). However, a clear effect was observed on force relaxation: myofibrils with D137L/G126R or D137L Tm showed prolonged durations of the slow phase of relaxation and decreased rates of the fast phase. Both Tm-stabilizing substitutions strongly decreased the slack sarcomere length (SL) at submaximal activating [Ca2+] and increased the steepness of the SL-passive tension relation. These effects were reversed by addition of 10 mM 2,3-butanedione 2-monoxime. Myofibrils also showed an apparent increase in Ca2+ sensitivity. Measurements of myofibrillar ATPase activity in the absence of Ca2+ showed a significant increase in the presence of these Tms, indicating that single and double stabilizing substitutions compromise the full inhibition of contraction in the relaxed state. These data can be understood with the three-state (blocked-closed-open) theory of muscle regulation, according to which the mutations increase the contribution of the active open state in the absence of Ca2+ (M-). Force measurements on myofibrils substituted with C-terminal truncated TnI showed similar compromised relaxation effects, indicating the importance of TnI-Tm interactions in maintaining the blocked state. It appears that reducing the flexibility of native Tm coiled-coil structure decreases the optimum interactions of the central part of Tm with the C-terminal region of TnI. This results in a shift away from the blocked state, allowing myosin binding and activity in the absence of Ca2+. This work provides a basis for understanding the effects of disease-producing mutations in muscle proteins.

Segmental fibre type composition of the rat iliopsoas muscle.

The iliopsoas of the rat is composed of two muscles - the psoas major muscle and the iliacus muscle. The psoas major muscle arises from all the lumbar vertebrae and the iliacus muscle from the fifth and sixth lumbar vertebrae and ilium. Their common insertion point is the lesser trochanter of the femur, and their common action is the lateral rotation of the femur and flexion of the hip joint. Unlike humans, the rat is a quadruped and only occasionally rises up on its hind legs. Therefore, it is expected that the fibre type composition of the rat iliopsoas muscle will be different than that of humans. The iliopsoas muscle of the rat is generally considered to be a fast muscle. However, previous studies of the fibre type composition of the rat psoas muscle showed different results. Moreover, very little is known about the composition of the rat iliacus muscle. The aim of our study was to examine the fibre type composition of the rat iliopsoas muscle in order to better understand the complex function of the listed muscle. The psoas major muscle was examined segmentally at four different levels of its origin. Type I, IIA, IIB and IIX muscle fibres were typed using monoclonal antibodies for myosin heavy chain identification. The percentage of muscle fibre types and muscle fibre cross-sectional areas were calculated. In our study we showed that in the rat iliopsoas muscle both the iliacus and the psoas major muscles had a predominance of fast muscle fibre types, with the highest percentage of the fastest IIB muscle fibres. Also, the IIB muscle fibres showed the largest cross-sectional area (CSA) in both muscles. As well, the psoas major muscle showed segmental differences of fibre type composition. Our results showed changes in percentages, as well as the CSAs of muscle fibre types in cranio-caudal direction. The most significant changes were visible in type IIB muscle fibres, where there was a decrease of percentages and the CSAs from the cranial towards the caudal part of the muscle. From our results it is evident that the rat iliopsoas muscle has a heterogeneous composition and is composed of all four muscle fibre types. Primarily, it is a fast, dynamic muscle with a predominance of fast type IIB muscle fibres with the largest CSAs. The composition of the rat psoas major muscles changes in a cranio-caudal direction, thus pointing to a more postural role of the caudal part of the muscle.

The increase in non-cross-bridge forces after stretch of activated striated muscle is related to titin isoforms.

Skeletal muscles present a non-cross-bridge increase in sarcomere stiffness and tension on Ca(2+) activation, referred to as static stiffness and static tension, respectively. It has been hypothesized that this increase in tension is caused by Ca(2+)-dependent changes in the properties of titin molecules. To verify this hypothesis, we investigated the static tension in muscles containing different titin isoforms. Permeabilized myofibrils were isolated from the psoas, soleus, and heart ventricle from the rabbit, and tested in pCa 9.0 and pCa 4.5, before and after extraction of troponin C, thin filaments, and treatment with the actomyosin inhibitor blebbistatin. The myofibrils were tested with stretches of different amplitudes in sarcomere lengths varying between 1.93 and 3.37 µm for the psoas, 2.68 and 4.21 µm for the soleus, and 1.51 and 2.86 µm for the ventricle. Using gel electrophoresis, we confirmed that the three muscles tested have different titin isoforms. The static tension was present in psoas and soleus myofibrils, but not in ventricle myofibrils, and higher in psoas myofibrils than in soleus myofibrils. These results suggest that the increase in the static tension is directly associated with Ca(2+)-dependent change in titin properties and not associated with changes in titin-actin interactions.

Force produced by isolated sarcomeres and half-sarcomeres after an imposed stretch.

When a stretch is imposed to activated muscles, there is a residual force enhancement that persists after the stretch; the force is higher than that produced during an isometric contraction in the corresponding length. The mechanisms behind the force enhancement remain elusive, and there is disagreement if it represents a sarcomeric property, or if it is associated with length nonuniformities among sarcomeres and half-sarcomeres. The purpose of this study was to investigate the effects of stretch on single sarcomeres and myofibrils with predetermined numbers of sarcomeres (n = 2, 3. . . , 8) isolated from the rabbit psoas muscle. Sarcomeres were attached between two precalibrated microneedles for force measurements, and images of the preparations were projected onto a linear photodiode array for measurements of half-sarcomere length (SL). Fully activated sarcomeres were subjected to a stretch (5-10% of initial SL, at a speed of 0.3 µm·s(-1)·SL(-1)) after which they were maintained isometric for at least 5 s before deactivation. Single sarcomeres showed two patterns: 31 sarcomeres showed a small level of force enhancement after stretch (10.46 ± 0.78%), and 28 sarcomeres did not show force enhancement (-0.54 ± 0.17%). In these preparations, there was not a strong correlation between the force enhancement and half-sarcomere length nonuniformities. When three or more sarcomeres arranged in series were stretched, force enhancement was always observed, and it increased linearly with the degree of half-sarcomere length nonuniformities. The results show that the residual force enhancement has two mechanisms: 1) stretch-induced changes in sarcomeric structure(s); we suggest that titin is responsible for this component, and 2) stretch-induced nonuniformities of half-sarcomere lengths, which significantly increases the level of force enhancement.

Thick-filament strain and interfilament spacing in passive muscle: effect of titin-based passive tension.

We studied the effect of titin-based passive tension on sarcomere structure by simultaneously measuring passive tension and low-angle x-ray diffraction patterns on passive fiber bundles from rabbit skinned psoas muscle. We used a stretch-hold-release protocol with measurement of x-ray diffraction patterns at various passive tension levels during the hold phase before and after passive stress relaxation. Measurements were performed in relaxing solution without and with dextran T-500 to compress the lattice toward physiological levels. The myofilament lattice spacing was measured in the A-band (d(1,0)) and Z-disk (d(Z)) regions of the sarcomere. The axial spacing of the thick-filament backbone was determined from the sixth myosin meridional reflection (M6) and the equilibrium positions of myosin heads from the fourth myosin layer line peak position and the I(1,1)/I(1,0) intensity ratio. Total passive tension was measured during the x-ray experiments, and a differential extraction technique was used to determine the relations between collagen- and titin-based passive tension and sarcomere length. Within the employed range of sarcomere lengths (∼2.2-3.4 µm), titin accounted for >80% of passive tension. X-ray results indicate that titin compresses both the A-band and Z-disk lattice spacing with viscoelastic behavior when fibers are swollen after skinning, and elastic behavior when the lattice is reduced with dextran. Titin also increases the axial thick-filament spacing, M6, in an elastic manner in both the presence and absence of dextran. No changes were detected in either I(1,1)/I(1,0) or the position of peaks on the fourth myosin layer line during passive stress relaxation. Passive tension and M6 measurements were converted to thick-filament compliance, yielding a value of ∼85 m/N, which is several-fold larger than the thick-filament compliance determined by others during the tetanic tension plateau of activated intact muscle. This difference can be explained by the fact that thick filaments are more compliant at low tension (passive muscle) than at high tension (tetanic tension). The implications of our findings are discussed.

Electron microscopy and x-ray diffraction evidence for two Z-band structural states.

In vertebrate muscles, Z-bands connect adjacent sarcomeres, incorporate several cell signaling proteins, and may act as strain sensors. Previous electron microscopy (EM) showed Z-bands reversibly switch between a relaxed, "small-square" structure, and an active, "basketweave" structure, but the mechanism of this transition is unknown. Here, we found the ratio of small-square to basketweave in relaxed rabbit psoas muscle varied with temperature, osmotic pressure, or ionic strength, independent of activation. By EM, the A-band and both Z-band lattice spacings varied with temperature and pressure, not ionic strength; however, the basketweave spacing was consistently 10% larger than small-square. We next sought evidence for the two Z-band structures in unfixed muscles using x-ray diffraction, which indicated two Z-reflections whose intensity ratios and spacings correspond closely to the EM measurements for small-square and basketweave if the EM spacings are adjusted for 20% shrinkage due to EM processing. We conclude that the two Z-reflections arise from the small-square and basketweave forms of the Z-band as seen by EM. Regarding the mechanism of transition during activation, the effects of Ca(2+) in the presence of force inhibitors suggested that the interconversion of Z-band forms was correlated with tropomyosin movement on actin.

Immunohistochemical analysis of the human psoas major muscle with regards to the body side and aging.

The aim of our study was to explore the age related changes of the fibre type composition of the human psoas major muscle. Moreover, we wanted to compare the fibre type composition of the left and right muscle. Muscle samples were collected from 15 young and 15 old males. Type I, IIA and IIX muscle fibres were typed using myosin heavy chain identification. The serial transverse sections were analysed using a light microscope. Results of our study showed that the age-related atrophy affected all three fibre types. Type IIA fibres were affected most profoundly while type I fibres were affected most weakly. The percentage of the different fibre types did not change during aging. There were no differences in the fibre type composition between the left and right muscle. Human psoas major muscle undergoes normal aging changes with the atrophy of all three fibre types, whereas atrophy most profoundly affects type IIA fibres. No differences in the fibre type composition between the left and right muscle point to the equal engagement of both legs in normal everyday activities of human.

A novel mutant cardiac troponin C disrupts molecular motions critical for calcium binding affinity and cardiomyocyte contractility.

Troponin C (TnC) belongs to the superfamily of EF-hand (helix-loop-helix) Ca(2+)-binding proteins and is an essential component of the regulatory thin filament complex. In a patient diagnosed with idiopathic dilated cardiomyopathy, we identified two novel missense mutations localized in the regulatory Ca(2+)-binding Site II of TnC, TnC((E59D,D75Y)). Expression of recombinant TnC((E59D,D75Y)) in isolated rat cardiomyocytes induced a marked decrease in contractility despite normal intracellular calcium homeostasis in intact cardiomyocytes and resulted in impaired myofilament calcium responsiveness in Triton-permeabilized cardiomyocytes. Expression of the individual mutants in cardiomyocytes showed that TnC(D75Y) was able to recapitulate the TnC((E59D,D75Y)) phenotype, whereas TnC(E59D) was functionally benign. Force-pCa relationships in TnC((E59D,D75Y)) reconstituted rabbit psoas fibers and fluorescence spectroscopy of TnC((E59D,D75Y)) labeled with 2-[(4'-iodoacetamide)-aniline]naphthalene-6-sulfonic acid showed a decrease in myofilament Ca(2+) sensitivity and Ca(2+) binding affinity, respectively. Furthermore, computational analysis of TnC showed the Ca(2+)-binding pocket as an active region of concerted motions, which are decreased markedly by mutation D75Y. We conclude that D75Y interferes with proper concerted motions within the regulatory Ca(2+)-binding pocket of TnC that hinders the relay of the thin filament calcium signal, thereby providing a primary stimulus for impaired cardiomyocyte contractility. This in turn may trigger pathways leading to aberrant ventricular remodeling and ultimately a dilated cardiomyopathy phenotype.

Effect of inorganic phosphate on the force and number of myosin cross-bridges during the isometric contraction of permeabilized muscle fibers from rabbit psoas.

The relation between the chemical and mechanical steps of the myosin-actin ATPase reaction that leads to generation of isometric force in fast skeletal muscle was investigated in demembranated fibers of rabbit psoas muscle by determining the effect of the concentration of inorganic phosphate (Pi) on the stiffness of the half-sarcomere (hs) during transient and steady-state conditions of the isometric contraction (temperature 12 degrees C, sarcomere length 2.5 mum). Changes in the hs strain were measured by imposing length steps or small 4 kHz oscillations on the fibers in control solution (without added Pi) and in solution with 3-20 mM added Pi. At the plateau of the isometric contraction in control solution, the hs stiffness is 22.8 +/- 1.1 kPa nm(-1). Taking the filament compliance into account, the total stiffness of the array of myosin cross-bridges in the hs (e) is 40.7 +/- 3.7 kPa nm(-1). An increase in [Pi] decreases the stiffness of the cross-bridge array in proportion to the isometric force, indicating that the force of the cross-bridge remains constant independently of [Pi]. The rate constant of isometric force development after a period of unloaded shortening (r(F)) is 23.5 +/- 1.0 s(-1) in control solution and increases monotonically with [Pi], attaining a maximum value of 48.6 +/- 0.9 s(-1) at 20 mM [Pi], in agreement with the idea that Pi release is a relatively fast step after force generation by the myosin cross-bridge. During isometric force development at any [Pi], e and thus the number of attached cross-bridges increase in proportion to the force, indicating that, independently of the speed of the process that leads to myosin attachment to actin, there is no significant (>1 ms) delay between generation of stiffness and generation of force by the cross-bridges.

A Cryosectioning Technique for the Observation of Intracellular Structures and Immunocytochemistry of Tissues in Atomic Force Microscopy (AFM).

The use of cryosectioning facilitates the morphological analysis and immunocytochemistry of cells in tissues in atomic force microscopy (AFM). The cantilever can access all parts of a tissue sample in cryosections after the embedding medium (sucrose) has been replaced with phosphate-buffered saline (PBS), and this approach has enabled the production of a type of high-resolution image. The images resembled those obtained from freeze-etching replica electron microscopy (EM) rather than from thin-section EM. The AFM images showed disks stacked and enveloped by the cell membrane in rod photoreceptor outer segments (ROS) at EM resolution. In addition, ciliary necklaces on the surface of connecting cilium, three-dimensional architecture of synaptic ribbons, and the surface of the post-synaptic membrane facing the active site were revealed, which were not apparent using thin-section EM. AFM could depict the molecular binding of anti-opsin antibodies conjugated to a secondary fluorescent antibody bound to the disk membrane. The specific localization of the anti-opsin binding sites was verified through correlation with immunofluorescence signals in AFM combined with confocal fluorescence microscope. To prove reproducibility in other tissues besides retina, cryosectioning-AFM was also applied to elucidate molecular organization of sarcomere in a rabbit psoas muscle.

Detection and quantification of the age-related point mutation A189G in the human mitochondrial DNA.

Mutation analysis in the mitochondrial DNA (mtDNA) control region is widely used in population genetic studies as well as in forensic medicine. Among the difficulties linked to the mtDNA analysis, one can find the detection of heteroplasmy, which can be inherited or somatic. Recently, age-related point mutation A189G was described in mtDNA and shown to accumulate with age in muscles. We carried out the detection of this 189 heteroplasmic point mutation using three technologies: automated DNA sequencing, Southern blot hybridization using a digoxigenin-labeled oligonucleotide probe, and peptide nucleic acid (PNA)/real-time PCR combined method on different biological samples. Our results give additional information on the increase in mutation frequency with age in muscle tissue and revealed that the PNA/real-time PCR is a largely more sensitive method than DNA sequencing for heteroplasmy detection. These investigations could be of interest in the detection and interpretation of mtDNA heteroplasmy in anthropological and forensic studies.

Cooperative mechanisms in the activation dependence of the rate of force development in rabbit skinned skeletal muscle fibers.

Regulation of contraction in skeletal muscle is a highly cooperative process involving Ca(2+) binding to troponin C (TnC) and strong binding of myosin cross-bridges to actin. To further investigate the role(s) of cooperation in activating the kinetics of cross-bridge cycling, we measured the Ca(2+) dependence of the rate constant of force redevelopment (k(tr)) in skinned single fibers in which cross-bridge and Ca(2+) binding were also perturbed. Ca(2+) sensitivity of tension, the steepness of the force-pCa relationship, and Ca(2+) dependence of k(tr) were measured in skinned fibers that were (1) treated with NEM-S1, a strong-binding, non-force-generating derivative of myosin subfragment 1, to promote cooperative strong binding of endogenous cross-bridges to actin; (2) subjected to partial extraction of TnC to disrupt the spread of activation along the thin filament; or (3) both, partial extraction of TnC and treatment with NEM-S1. The steepness of the force-pCa relationship was consistently reduced by treatment with NEM-S1, by partial extraction of TnC, or by a combination of TnC extraction and NEM-S1, indicating a decrease in the apparent cooperativity of activation. Partial extraction of TnC or NEM-S1 treatment accelerated the rate of force redevelopment at each submaximal force, but had no effect on kinetics of force development in maximally activated preparations. At low levels of Ca(2+), 3 microM NEM-S1 increased k(tr) to maximal values, and higher concentrations of NEM-S1 (6 or 10 microM) increased k(tr) to greater than maximal values. NEM-S1 also accelerated k(tr) at intermediate levels of activation, but to values that were submaximal. However, the combination of partial TnC extraction and 6 microM NEM-S1 increased k(tr) to virtually identical supramaximal values at all levels of activation, thus, completely eliminating the activation dependence of k(tr). These results show that k(tr) is not maximal in control fibers, even at saturating [Ca(2+)], and suggest that activation dependence of k(tr) is due to the combined activating effects of Ca(2+) binding to TnC and cross-bridge binding to actin.

Morphometric analysis of somatotropic cells of the adenohypophysis and muscle fibers of the psoas muscle in the process of aging in humans.

The aim of this research was to quantify changes of the adenohypophyseal somatotropes and types 1 and 2 muscle fibers with aging, as well as to establish mutual interactions and correlations with age. Material was samples of hypophysis and psoas major muscle of 27 cadavers of both genders, aged from 30 to 90 years. Adenohypophyseal and psoas major tissue sections were immunohistochemically processed and stained by anti-human growth hormone and anti-fast myosin antibodies, respectively. Morphometric analysis was performed by ImageJ. Results of morphometric analysis showed a significant increase in the somatotrope area, and significant decrease in somatotrope volume density and nucleocytoplasmic ratio with age. Cross-sectional areas of types 1 and 2, and volume density of type 2 muscle fibers decreased significantly with age. One Way ANOVA showed that the latter cited changes in the somatotropes and types 1 and 2 muscle fibers mostly become significant after the age of 70. Significant positive correlation was observed between the area of the somatotropes and volume density of type 2 muscle fibers. A significant negative correlation was detected between the nucleocytoplasmic ratio of the somatotropes and cross-sectional areas of types 1 and 2 muscle fibers. So, it can be concluded that after the age of 70, there is significant loss of the anterior pituitary's somatotropes associated with hypertrophy and possible functional decline of the remained cells. Age-related changes in the somatotropes are correlated with the simultaneous atrophy of type 1, as well as with the atrophy and loss of type 2 muscle fibers.

Response of compressed skinned skeletal muscle fibers to conditions that simulate fatigue.

During fatigue, muscles become weaker, slower, and more economical at producing tension. Studies of skinned muscle fibers can explain some but not all of these effects, and, in particular, they are less economical in conditions that simulate fatigue. We investigated three factors that may contribute to the different behavior of skinned fibers. 1) Skinned fibers have increased myofilament lattice spacing, which is reversible by osmotic compression. 2) A myosin subunit becomes phosphorylated during fatigue. 3) Inosine 5'-monophosphate (IMP) accumulates during fatigue. We tested the response of phosphorylated and unphosphorylated single skinned fibers (isometric tension, contraction velocity, and adenosinetriphosphatase activity) to changes in lattice spacing (0-5% dextran) and IMP (0-5 mM) in the presence of altered concentrations of P(i) (3-25 mM), H+ (pH 7-6.2), and ADP (0-5 mM). The response of maximally activated skinned fibers to the direct metabolites of ATP hydrolysis is not altered by osmotic compression, phosphorylating myosin subunits, or increasing IMP concentration. These factors, therefore, do not explain the discrepancy between intact and skinned fibers during fatigue.

Skeletal muscle force and actomyosin ATPase activity reduced by nitric oxide donor.

Nitric oxide (NO) may exert direct effects on actin-myosin cross-bridge cycling by modulating critical thiols on the myosin head. In the present study, the effects of the NO donor sodium nitroprusside (SNP; 100 microM to 10 mM) on mechanical properties and actomyosin adenosinetriphosphatase (ATPase) activity of single permeabilized muscle fibers from the rabbit psoas muscle were determined. The effects of N-ethylmaleimide (NEM; 5-250 microM), a thiol-specific alkylating reagent, on mechanical properties of single fibers were also evaluated. Both NEM (>/=25 microM) and SNP (>/=1 mM) significantly inhibited isometric force and actomyosin ATPase activity. The unloaded shortening velocity of SNP-treated single fibers was decreased, but to a lesser extent, suggesting that SNP effects on isometric force and actomyosin ATPase were largely due to decreased cross-bridge recruitment. The calcium sensitivity of SNP-treated single fibers was also decreased. The effects of SNP, but not NEM, on force and actomyosin ATPase activity were reversed by treatment with 10 mM DL-dithiothreitol, a thiol-reducing agent. We conclude that the NO donor SNP inhibits contractile function caused by reversible oxidation of contractile protein thiols.

Contractility of single myofibrils of rabbit skeletal muscle studied at various MgATP concentrations.

A novel experimental method was developed to study the contractility of single myofibrils of skeletal muscle. Single myofibrils (ca. 1 microm in diameter) prepared from glycerinated rabbit psoas muscle were suspended between rigid and flexible microneedles by the entwining method. The length changes of the preparations applied via the rigid microneedle by an actuator and the force produced were measured by photo-electrically detecting the nanometer deflections of the flexible microneedle. Single myofibril preparations maintained uniform sarcomere striations during contraction-relaxation cycles. The isometric force produced, the velocity of unloaded shortening, and the force-velocity relationship of single myofibrils were investigated at various MgATP concentrations. The contractility of single myofibrils thus obtained in the absence of ATP regenerative systems was essentially the same as that of skinned muscle fibers under comparable conditions in the presence of ATP regenerative systems. Thus, it was found that (1) the present experimental method is useful for studying the contractility of single myofibrils, and (2) in single myofibril preparations, the MgATP concentration at actomyosin sites is well equilibrated with that in bathing solutions.

Evidence for structural changes in crossbridges during force generation.

During muscle contraction, it is generally thought that myosin heads undergo large scale conformational changes, such as an oar-like rotation between 90 degrees and 45 degrees while attached to actin. However, evidence for conformational changes of the attached crossbridges associated with force generation has been ambiguous. In this study, we compared the conformations of attached crossbridges in (i) the pre-force generating state, (ii) force generating state, (ii) rigor state. High resolution equatorial X-ray diffraction patterns have been obtained from single chemically skinned rabbit psoas fibers under relaxed, fully Ca(2+)-activated and rigor conditions. The experimental condition was chosen (ionic strength = 50 mM and temperature = 5 degrees C) such that there are large fractions (80-100%) of crossbridges attached in all the three states, and the attached crossbridges in the relaxed muscle represent the pre-force generating state. Upon activation, changes in the two innermost intensities I10 and I11 did not follow the familiar reciprocal changes. Instead, there was almost no change in I11 while I10 decreased by 30%. Similarly, greater changes were found in I10 as the fiber goes into rigor from the activate state. Changes were also found in the higher order reflections suggesting that the structure of the force generating crossbridges is not a mixture of those found in the weakly bound and rigor crossbridges. Therefore, our data provides evidence that the average conformation of the force generating crossbridges is different from the weakly attached and from rigor crossbridges.

Effects of ionic strength on force transients induced by flash photolysis of caged ATP in covalently crosslinked rabbit psoas muscle fibers.

Single fibers from glycerinated rabbit psoas muscle were treated with 1-ethyl-3[3-(dimethylamino) propyl] carbodiimide (EDC), after rigor was induced, to crosslink myosin heads to actin. The optimally pre-stretched (approximately 1.8%), partially crosslinked fibers produce a large force when MgATP is depleted, and this force is abolished when MgATP is reintroduced, even in high ionic strength solution of 0.5 M (Tawada et al. 1989). We investigated the rate of force decay in the crosslinked, force-producing fibers using pulse photolysis of caged ATP (Goldman et al. 1984). The decay of force was fast, the rate of which depending both on the ionic strength and on the amount of ATP released (0.2-2.2 mM) with the second-order rate constant of 0.5-1 x 10(5) M-1s-1 at the ionic strength of 0.5 M. At high ionic strength (1-2M) force decayed at lower rate. At low ionic strength (0.1-0.2 M), however, force decayed more rapidly, but force redeveloped subsequently, which is probably caused by uncrosslinked myosin heads.

Kinetics of force generation and Pi release in rabbit soleus muscle fibers.

Recent studies have shown that the release of Pi from the AM.ADP.Pi crossbridge occurs in two steps: an initial force-generating isomerization which is followed by the release per se of Pi. These two steps have been shown to be little affected by the presence or absence of Ca2+ but both steps are significantly altered by temperature. In this study, the kinetics of the force generating and Pi release steps of the actomyosin ATPase cycle have been compared in Ca(2+)-activated skinned fibers of rabbit soleus (slow twitch) and psoas (fast twitch) muscle. Pi was rapidly photogenerated within the fiber lattice by laser flash photolysis of caged Pi (1-(2-nitro)phenylethyl phosphate). Pi reduces isometric tension in the steady state, but is less effective in slow muscle than in fast (eg. 14 mM Pi reduces tension by 29 +/- 4.6% in slow muscle and by 47 +/- 5.3% in fast). As in fast muscle, the tension response to a sudden increase in [Pi] in slow muscle has four phases. As in fast muscle, only phase II (an exponential decline in force) appears to be caused by Pi binding to cross-bridges, while the other three phases are probably indirect effects caused by caged Pi photolysis. The amplitude of phase II is consistent with the steady state reduction in force by Pi. The rate of phase II (kpi) is 3.9 +/- 0.33 s-1 at 20 degrees C and 0.28 +/- 0.02 s-1 at 10 degrees C (1 mM Pi).(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of lattice spacing change on cross-bridge kinetics in rabbit psoas fibers.

The effect of compression on the elementary steps of the cross-bridge cycle was investigated with the sinusoidal analysis technique and ATP hydrolysis rate measurement. The lattice spacing of rabbit psoas muscle fibers was osmotically compressed with a macromolecule, dextran T-500 (0-16%). The effects of MgATP, MgADP, Pi on exponential processes (B), (C), (D), and isometric tension were studied at different dextran concentrations. The experiments were performed at the saturating Ca concentration (pCa 4.5-4.8), 200 mM ionic strength, pH 7.0, and 20 degrees C. Our results show that the fiber width decreased linearly with an increase in the dextran concentration, and the width measurement was perfectly correlated with the lattice spacing measurement using equatorial x-ray diffraction studies. We find that the nucleotide binding steps, the ATP-isomerization step, and the cross-bridge detachment step were minimally affected by the compression. Our results indicate that the rate constant of the reverse power stroke step (k-4) decreases with mild compression (0-6.3% dextran), presumably because of the stabilization of the attached cross-bridges in the AM*DP state. We also found that the rate constant of the power stroke step (k4) decreases with higher compression (> 6.3% dextran), presumably because of increased difficulty in performing the power stroke reaction. Our results further show that the association constant (K5) of phosphate to cross-bridges is not changed with compression. The ATP hydrolysis rate declined almost linearly with an increase in the dextran concentration. This observation indicates that the rate limiting step is also affected by the lattice spacing change so that the associated rate constant (k6) becomes progressively less with compression.