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

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

The origin of passive force enhancement in skeletal muscle

The aim of the present study was to test whether titin is a calcium-dependent spring and whether it is the source of the passive force enhancement observed in muscle and single fiber preparations. We measured passive force enhancement in troponin C (TnC)-depleted myofibrils in which active force production was completely eliminated. The TnC-depleted construct allowed for the investigation of the effect of calcium concentration on passive force, without the confounding effects of actin-myosin cross-bridge formation and active force production. Passive forces in TnC-depleted myofibrils (n = 6) were 35.0 +/- 2.9 nN/ microm(2) when stretched to an average sarcomere length of 3.4 microm in a solution with low calcium concentration (pCa 8.0). Passive forces in the same myofibrils increased by 25% to 30% when stretches were performed in a solution with high calcium concentration (pCa 3.5). Since it is well accepted that titin is the primary source for passive force in rabbit psoas myofibrils and since the increase in passive force in TnC-depleted myofibrils was abolished after trypsin treatment, our results suggest that increasing calcium concentration is associated with increased titin stiffness. However, this calcium-induced titin stiffness accounted for only approximately 25% of the passive force enhancement observed in intact myofibrils. Therefore, approximately 75% of the normally occurring passive force enhancement remains unexplained. The findings of the present study suggest that passive force enhancement is partly caused by a calcium-induced increase in titin stiffness but also requires cross-bridge formation and/or active force production for full manifestation.     

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

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

Protein kinase A changes calcium sensitivity but not crossbridge kinetics in human cardiac myofibrils

We investigated the effect of PKA treatment (1 U/ml) on the mechanical properties of isolated human cardiac myofibrils. PKA treatment was associated with significant incorporation of radiolabeled phosphate into several sarcomeric proteins including troponin I and myosin binding protein C and was also associated with a right shift in the tension-pCa relation (ΔpCa(50) = 0.2 ± 0.1). PKA treatment also caused right shifts in the pCa dependence of the rate of tension development, tension redevelopment, and the linear and exponential phases of myofibril relaxation. However, there was no change in the same measures of crossbridge turnover when expressed as a function of tension. We conclude that the changes in crossbridge kinetics as a function of calcium concentration reflect a reduced tension due to a lower calcium sensitivity and that the relationship between crossbridge kinetics and tension was unchanged, indicating no direct effect of PKA treatment on crossbridge cycling.     

Myogenic contractility is more dependent on myofilament calcium sensitization in term fetal than adult ovine cerebral arteries

Regulation of cytosolic calcium and myofilament calcium sensitivity varies considerably with postnatal age in cerebral arteries. Because these mechanisms also govern myogenic tone, the present study used graded stretch to examine the hypothesis that myogenic tone is less dependent on calcium influx and more dependent on myofilament calcium sensitization in term fetal compared with adult cerebral arteries. Term fetal and adult posterior communicating cerebral arteries exhibited similar myogenic responses, with peak tensions averaging 24 and 26% of maximum contractile force produced in any given tissue in response to an isotonic Krebs buffer containing 122 mM K(+) (K(max)) at optimum stretch ratios (working diameter/unstressed diameter) of 2.19 and 2.23, respectively. Graded stretch increased cytosolic Ca(2+) concentration at stretch ratios >2.0 in adult arteries, but increased Ca(2+) concentration only at stretch ratios >2.3 in fetal arteries. In permeabilized arteries, myogenic tone peaked at a stretch ratio of 2.1 in both fetal and adult arteries. The fetal %K(max) values at peak myogenic tone were not significantly different at either pCa 7.0 (23%) or pCa 5.5 (25%) but were significantly less at pCa 8.0 (8.4 +/- 2.3%). Conversely, adult %K(max) values at peak myogenic tone were significantly less at both pCa 8.0 (10.4 +/- 1.8%) and pCa 7.0 (16%) than at pCa 5.5 (27%). The maximal extents of stretch-induced increases in myosin light chain phosphorylation in intact fetal (20%) and adult (17%) arteries were similar. The data demonstrate that the cerebrovascular myogenic response is highly conserved during postnatal maturation but is mediated differently in fetal and adult cerebral arteries.     

Behaviour of the crossbridges in stretched or compressed muscle fibres

The maximum chord of the myosin heads is comparable to the closest surface-to-surface spacing between the myofilaments in a muscle at the slack length. Therefore, when the sarcomere length increases or when the fibre is compressed, the surface-to-surface myofilament spacing becomes lower than the head long axis. We conclude that, in stretched or compressed fibres, some crossbridges cannot attach, owing to steric hindrance. When the amount of compression is limited, this hindrance may be overcome by a tilting of the heads in the plane perpendicular to the filament axes; in this case, there is no consequence as concerns the crossbridge properties. In highly compressed fibres, the crossbridges become progressively hindered and all the crossbridges are hindered for an axis-to-axis spacing representing about 60% of the spacing observed under zero external osmotic pressure. In this case, both the isometric tension and the ATPase activity of the fibre are zero. In fibres stretched up to 3.77 microns (sarcomere length corresponding to the disappearance of the overlap between the thick and the thin filaments), the ratio of hindered crossbridges over the functional crossbridges may be estimated at about 55%. In stretched fibres, a noticeable proportion of crossbridges are sterically hindered and the crossbridges performance (e.g. constants of attachment and detachment) depends on filament spacing, i.e. on sarcomere length. Therefore, we think it is probably impossible to consider the crossbridges as independent force converters, since this idea requires that the crossbridge properties are independent of sarcomere length. In this connection, all the experiments performed on osmotically compressed fibres are of major importance for the understanding of the true mechanisms of muscle contraction.     

Titin-induced force enhancement and force depression: A ‘sticky-spring’ mechanism in muscle contractions?

The sliding filament and crossbridge theories do not suffice to explain a number of muscle experiments. For example, from the entire muscle to myofibrils, predictions of these theories were shown to underestimate the force output during and after active tissue stretch. The converse applies to active tissue shortening.In addition to the crossbridge cycle, we propose that another molecular mechanism is effective in sarcomere force generation. We suggest that, when due to activation, myosin binding sites are available on actin, the giant protein titin's PEVK region attaches itself to the actin filament at those sites. As a result, the molecular spring length is dramatically reduced. This leads to increased passive force when the sarcomere is stretched and to decreased or even negative passive force when the sarcomere shortens. Moreover, during shortening, the proposed mechanism interferes with active-force production by inhibiting crossbridges.Incorporation of a simple ‘sticky-spring’ mechanism model into a Hill-type model of sarcomere dynamics offers explanations for several force-enhancement and force-depression effects. For example, the increase of the sarcomere force compared to the force predicted solely by the sliding filament and crossbridge theories depends on the stretch amplitude and on the working range. The same applies to the decrease of sarcomere force during and after shortening. Using only literature data for its parameterization, the model predicts forces similar to experimental results.     

Magnitude of length-dependent changes in contractile properties varies with titin isoform in rat ventricles

The effects of differential expression of titin isoforms on sarcomere length (SL)-dependent changes in passive force, maximum Ca(2+)-activated force, apparent cooperativity in activation of force (n(H)), Ca(2+) sensitivity of force (pCa(50)), and rate of force redevelopment (k(tr)) were investigated in rat cardiac muscle. Skinned right ventricular trabeculae were isolated from wild-type (WT) and mutant homozygote (Ho) hearts expressing predominantly a smaller N2B isoform (2,970 kDa) and a giant N2BA-G isoform (3,830 kDa), respectively. Stretching WT and Ho trabeculae from SL 2.0 to 2.35 μm increased passive force, maximum Ca(2+)-activated force, and pCa(50), and it decreased n(H) and k(tr). Compared with WT trabeculae, the magnitude of SL-dependent changes in passive force, maximum Ca(2+)-activated force, pCa(50), and n(H) was significantly smaller in Ho trabeculae. These results suggests that, at least in rat ventricle, the magnitude of SL-dependent changes in passive force, maximum Ca(2+)-activated force, pCa(50), n(H), and k(tr) is defined by the titin isoform.     

Titin determines the Frank-Starling relation in early diastole

Titin, a giant protein spanning half the sarcomere, is responsible for passive and restoring forces in cardiac myofilaments during sarcomere elongation and compression, respectively. In addition, titin has been implicated in the length-dependent activation that occurs in the stretched sarcomere, during the transition from diastole to systole. The purpose of this study was to investigate the role of titin in the length-dependent deactivation that occurs during early diastole, when the myocyte is shortened below slack length. We developed a novel in vitro assay to assess myocyte restoring force (RF) by measuring the velocity of recoil in Triton-permeabilized, unloaded rat cardiomyocytes after rigor-induced sarcomere length (SL) contractions. We compared rigor-induced SL shortening to that following calcium-induced (pCa) contractions. The RF-SL relationship was linearly correlated, and the SL-pCa curve displayed a characteristic sigmoidal curve. The role of titin was defined by treating myocytes with a low concentration of trypsin, which we show selectively degrades titin using mass spectroscopic analysis. Trypsin treatment reduced myocyte RF as shown by a decrease in the slope of the RF-SL relationship, and this was accompanied by a downward and leftward shift of the SL-pCa curve, indicative of sensitization of the myofilaments to calcium. In addition, trypsin digestion did not alter the relationship between SL and interfilament spacing (assessed by cell width) after calcium activation. These data suggest that as the sarcomere shortens below slack length, titin-based restoring forces act to desensitize the myofilaments. Furthermore, in contrast to length-dependent activation at long SLs, length-dependent deactivation does not depend on interfilament spacing. This study demonstrates for the first time the importance of titin-based restoring force in length-dependent deactivation during the early phase of diastole.     

Structure and function of skeletal muscle in zebrafish early larvae

Zebrafish muscles were examined at an early developmental stage (larvae 5-7 d). Using aluminum clips, preparations (approximately 1.5 mm length, 150 microm diameter) were mounted for force registration and small angle x-ray diffraction. Sarcomeres were oriented mainly in parallel with the preparation long axis. Electrical stimulation elicited fast and reproducible single twitch contractions. Length-force relations showed an optimal sarcomere length of 2.15 microm. X-ray diffraction revealed clear equatorial 1.1/1.0 reflections, showing that myofilaments are predominantly arranged along the preparation long axis. In contrast, reflections from older (2 mo) zebrafish showed two main filament orientations each at an approximately 25 degrees angle relative to the preparation long axis. Electrical stimulation of larvae muscles increased the 1.1/1.0 intensity ratio, reflecting mass transfer to thin filaments during contraction. The apparent lattice volume was 3.42 x 10(-3) microm(3), which is smaller than that of mammalian striated muscle and more similar to that of frog muscles. The relation between force and stimulation frequency showed fusion of responses at a comparatively high frequency (approximately 186 Hz), reflecting a fast muscle phenotype. Inhibition of fast myosin with N-benzyl-p-toluene sulphonamide (BTS) showed that the later phase of the tetanus was less affected than the initial peak. This suggests that, although the main contractile phenotype is fast, slow twitch fibers can contribute to sustained contraction. A fatigue stimulation protocol with repeated 220 ms/186 Hz tetani showed that tetanic force decreased to 50% at a train rate of 0.1 s(-1). In conclusion, zebrafish larvae muscles can be examined in vitro using mechanical and x-ray methods. The muscles and myofilaments are mainly orientated in parallel with the larvae long axis and exhibit a significant fast contractile component. Sustained contractions can also involve a small contribution from slower muscle types.     

Influence of ADP on cross-bridge-dependent activation of myofibrillar thin filaments

Contraction of skeletal muscle is regulated by calcium at the level of the thin filament via troponin and tropomyosin. Studies have indicated that strong cross-bridge binding is also involved in activation of the thin filament. To further test this, myofibrils were incubated with a wide range of fluorescent myosin subfragment 1(fS1) at pCa 9 or pCa 4 with or without ADP. Sarcomere fluorescence intensity and the fluorescence intensity ratio (non-overlap region/overlap region) were measured to determine the amount and location of bound fS1 in the myofibril. There was lower sarcomere fluorescence intensity with ADP compared to without ADP for both calcium levels. Similar data were obtained from biochemical measures of bound fS1, validating the fluorescence microscopy measurements. The intensity ratio, which is related to activation of the thin filament, increased with increasing [fS1] with or without ADP. At pCa 9, the fluorescence intensity ratio was constant until 80-160 nM fS1 without ADP conditions, then it went up dramatically and finally attained saturation. The dramatic shift of the ratio demonstrated the cooperative character of strong cross-bridge binding, and this was not observed at high calcium. A similar pattern was observed with ADP in that the ratio was right-shifted with respect to total [fS1]. Saturation was obtained with both the fluorescence intensity and ratio data. Plots of intensity ratio as a function of normalized sarcomere intensity (bound fS1) showed little difference between with and without ADP. This suggests that the amount of strongly bound fS1, not fS1 state (with or without ADP) is related to activation of the thin filament.     

X-ray diffraction and electron microscopy from Lethocerus flight muscle partially relaxed by adenylylimidodiphosphate and ethylene glycol

The low-angle X-ray diffraction pattern from Lethocerus flight muscle fibres was recorded in rigor or under two conditions that modify crossbridge structure and behaviour, aqueous adenylylimidodiphosphate (AMPPNP) and AMPPNP + calcium in an ethylene glycol-water mixture. The effects on the 38.7 nm layer-line peaks (hk.6) of the diffraction patterns were studied in detail. In aqueous AMPPNP at room temperature, a condition in which rigor tension drops to half without loss of stiffness, the peaks remained nearly as intense as in rigor except for the 10.6, which dropped to half. In 20% (v/v) ethylene glycol-AMPPNP + 100 microM-Ca2+ at 23 degrees C (gly + pnp + Ca), a condition which removed muscle tension but left stiffness close to the rigor value, the 10.6 and 11.6 peaks greatly decreased but the 31.6 remained relatively high. The 14.5 nm meridional peak (00.16) became stronger on addition of AMPPNP and again on adding glycol + calcium. Considered in terms of constructively interfering filaments and crossbridges, the X-ray data indicated a transfer of diffracting crossbridge mass towards the thick filament as relaxation proceeds. We compared the X-ray diffraction patterns and crossbridge structure seen with electron microscopy (EM) under the same chemical conditions. EM and X-ray observations were mutually quite consistent overall. However, X-ray data indicated that more crossbridge mass was stereospecifically related to actin before fixation in the partially relaxed state (gly + pnp + Ca) than was suggested by the disordered crossbridge profiles seen by EM. We conclude that myosin heads at the start of the power stroke may both be closely related to their thick filament origins and form actin-determined attachments to the thin filament.     

Does thin filament compliance diminish the cross-bridge kinetics? A study in rabbit psoas fibers.

The effect of thin filament compliance on our ability to detect the cross-bridge kinetics was examined. Our experiment is based on the facts that in rabbit psoas the thin filament (1.12 micrometer) is longer than half the thick filament length (0.82 micrometer) and that the thick filament has a central bare zone (0.16 micrometer). Consequently, when sarcomere length is increased from 2.1 to 2.4 micrometer, the same number of cross-bridges is involved in force generation but extra series compliance is introduced in the I-band. Three apparent rate constants (2pia, 2pib, and 2pic) were characterized by sinusoidal analysis at pCa 4.66. Our results demonstrate that 2pia and 2pib increased 13-16% when sarcomere length was increased from 2.0 to 2.5 micrometer, and 2pic decreased slightly (9%). This slight decrease can be explained by compression of the lattice spacing. These observations are at variance with the expectation based on increased series compliance, which predicts that the rate constants will decrease. We also determined compliance of the I-band during rigor. I-band compliance during rigor induction was 35% of sarcomere compliance at sarcomere length 2.4 micrometer, and 24% at sarcomere length 2.1 micrometer. We conclude that the presence of thin filament compliance does not seriously interfere with our ability to detect cross-bridge kinetics using sinusoidal analysis.     

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

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

Effect of Ultrasound and Calcium Chloride on the Ultrastructure and the Warner-Bratzler Shear Force Value of Beef Shank Muscle Fibers

Meat tenderness is a major factor in consumer satisfaction. Tenderness may be quantified by changes in muscle sarcomere length, roughness (ultrastructure) and shear force. The ultrastructure and the Warner-Bratzler shear force value of beef shank muscle fibers were experimentally measured in controlled applications of ultrasound and calcium chloride. Uncooked beef samples were treated with ultrasound at 40 kHz at different intensities for 6 min, and then immersed in a range of calcium chloride solutions for 24 h. Warner–Bratzler shear analysis and atomic force microscopy (AFM) data collection were performed for each sample. AFM height derived data (the sarcomere length and roughness values) and the Warner–Bratzler shear force values indicate optimum ranges of ultrasound intensity and calcium chloride concentration for meat tenderization. While the effect of low intensity ultrasound alone was not significant, calcium chloride treatment increased the sarcomere length and decreased shear force values. Furthermore, the combination of ultrasound and calcium chloride increased the sarcomere length significantly and simultaneously reduced the Warner–Bratzler shear force tolerance of the sample. These data suggested that low intensity and frequency ultrasound alongside Calcium Chloride immersion can promote the transmembrane transport of calcium ions via the calpain system to improve meat tenderness.     

The effect of sarcomere non-uniformity on the sarcomere length-tension relationship of skinned fibers

It has proved difficult to activate skinned muscle fibers to produce high tension (3 kg/cm² level) without loss of clear striations. A new method was developed which permits high tension production in skinned muscle fibers while retaining clear striations. Clear striations allow reliable measurement of the sarcomere lengths during contraction by microscopy and diffractometry. The method is to increase the Ca⁺⁺ concentration of the bathing solution very gradually over a time period of 5 to 10 minutes. Once the skinned fiber is conditioned by this slow activation, subsequent contractions can be elicited by ordinary quick activations without loss of striations. When the experiments are carried out with careful controls for the uniformity of the sarcomere length distribution along the entire length of the fiber, contractions are highly repeatable. Using the new method and stringent quality control of fibers, the sarcomere length-isometric tension relationship of skinned rabbit soleus fibers was obtained. The results differ from those previously obtained by conventional activation methods in that tension increases with sarcomere length not only at low (pCa = 5.8), but also at high (pCa = 5.2), calcium concentration.     

Time-resolved equatorial X-ray diffraction studies of skinned muscle fibres during stretch and release.

Equatorial X-ray diffraction patterns were recorded from small bundles of one to three chemically skinned frog sartorius muscle fibres (time resolution 250 microseconds) during rapid stretch and subsequent release. In the relaxed state, the dynamic A-band lattice spacing change as a result of a 2 % step stretch (determined from the positions of the 10 and 11 reflections) resulted in a 21 % increase in lattice volume, while static studies of spacing and sarcomere length indicated than an increase in volume of >/=50 % for the same length change. In rigor, stretch caused a lattice volume decrease which was reversed by a subsequent release. In activated fibres (pCa 4.5) exposed to 10 mM 2,3-butanedione 2-monoxime (BDM), stretch was accompanied by a lattice compression exceeding that of constant volume behaviour, but during tension recovery, compression was partially reversed to leave a net spacing change close to that observed in the relaxed fibre. In the relaxed state, spacing changes were correlated with the amplitude of the length step, while in rigor and BDM states, spacing changes correlated more closely with axial force. This behaviour is explicable in terms of two components of radial force, one due to structural constraints as seen in the relaxed state, and an additional component arising from cross-bridge formation. The ratio of axial to radial force for a single thick filament resulting from a length step was four in rigor and BDM, but close to unity for the relaxed state.     

Length-tension relation in Limulus striated muscle

Laser diffraction techniques coupled with simultaneous tension measurements were used to determine the length-tension relation in intact, small (0.5-mm thick, 10-mm wide, 20-25-mm long) bundles of a Limulus (horseshoe crab) striated muscle, the telson levator muscle. This muscle differs from the model vertebrate systems in that the thick filaments are not of a constant length, but shorten from 4.9 to approximately 2.0 micrometers as the sarcomeres shorten from 7 to 3 micrometers. In the Limulus muscle, the length-tension relation plateaued to an average maximum tension of 0.34 N/mm2 at a sarcomere length of 6.5 micrometers (Lo) to 8.0 micrometers. In the sarcomere length range from 3.8 to 12.5 micrometers, the muscle developed 50% or more of the maximum tension. When the sarcomere lengths are normalized (expressed as L/Lo) and the Limulus data are compared to those from frog muscle, it is apparent that Limulus muscle develops tension over a relatively greater range of sarcomere lengths.     

Length-dependent activation and auto-oscillation in skeletal myofibrils at partial activation by Ca2+

The length-dependent activation of skeletal myofibrils was examined at the single-sarcomere level with phase-contrast microscopy at sarcomere length (SL) >2.2 μm. At the maximal activation by Ca²⁺ (pCa 4.5) the active force linearly decreased with increasing SL, while at partial activation by Ca²⁺ (pCa 6.1-6.5) the larger active force was generated at longer SL. Throughout these experiments, the distribution of SL was kept homogeneous upon activation. In addition, we found that the spontaneous oscillation of force and SL frequently occurs in the SL range 2.2-2.6 μm at pCa 6.1-6.2. Either changes in [Ca²⁺] or osmotic compression of the myofilament lattice induced by the addition of dextran T-500, affected both the length dependence of activation and the occurrence of auto-oscillation. These results suggest that the force-generating properties of sarcomeres in striated muscle are determined not only by [Ca²⁺], but also by the lattice spacing as a function of SL.     

Cross-bridge attachment and stiffness during isotonic shortening of intact single muscle fibers.

Equatorial x-ray diffraction pattern intensities (I10 and I11), fiber stiffness and sarcomere length were measured in single, intact muscle fibers under isometric conditions and during constant velocity (ramp) shortening. At the velocity of unloaded shortening (Vmax) the I10 change accompanying activation was reduced to 50.8% of its isometric value, I11 reduced to 60.7%. If the roughly linear relation between numbers of attached bridges and equatorial signals in the isometric state also applies during shortening, this would predict 51-61% attachment. Stiffness (measured using 4 kHz sinusoidal length oscillations), another putative measure of bridge attachment, was 30% of its isometric value at Vmax. When small step length changes were applied to the preparation (such as used for construction of T1 curves), no equatorial intensity changes could be detected with our present time resolution (5 ms). Therefore, unlike the isometric situation, stiffness and equatorial signals obtained during ramp shortening are not in agreement. This may be a result of a changed crossbridge spatial orientation during shortening, a different average stiffness per attached crossbridge, or a higher proportion of single headed crossbridges during shortening.