Effects of shortening on stretch-induced force enhancement in single skeletal muscle fibers

The main purpose of this study was to evaluate the effects of shortening on the stretch-induced force enhancement in single muscle fibers, and indirectly test the hypothesis that force enhancement may be associated with the engagement of a passive element upon activation. Fibers were placed on the descending limb of the force-length relationship, and stretch and shortening contractions were performed. Fibers underwent two sets of shortening-stretch cycles. First, fibers were shortened by a fixed amplitude and speed (10% fiber length, and at 40% fiber length/s), and then were stretched (10% fiber length, and at 40% fiber length/s) immediately following shortening, or 500 or 1000 ms following the shortening. Second, fibers were shortened by varying amounts (5%, 10% and 15% fiber length) and at a constant speed (40% fiber length/s) immediately preceding a given fiber stretch (10% fiber length, and at 40% fiber length/s). When stretching was immediately preceded by shortening, force enhancement was decreased proportionally with the shortening magnitude. When intervals were introduced between shortening and stretch, the effects of shortening on the stretch-induced force enhancement became less prominent. We concluded that, in contrast to published suggestions, shortening affects the stretch-induced force enhancement in an amplitude-dependent manner in single fibers, as it does in whole muscles, but this effect is diminished by increasing the time period between the shortening and stretch phases.     

Passive interaction between sliding filaments in the osmotically compressed skinned muscle fibers of the frog.

Shortening and lengthening velocities, instantaneous stiffness, and tension transients after stretch were measured in compressed muscle fibers from the frog in the presence or absence of polyvinylpyrrolidone (PVP K30) or Dextran T70. Both shortening and lengthening velocities clearly decreased with the concentration of polymer. In the presence of polymer, "passive" stiffness was observed in relaxing solution depending on fiber diameter, and stiffness increased further by activation. This increase by activation above "passive" stiffness was nearly constant in the wide range of polymer concentrations. These active and "passive" stiffnesses were found to be dependent on sarcomere length. The stiffness of a compressed rigor fiber was indicated to be composed of constant rigor stiffness and a variable "passive" one. The tension transient after stretch in a compressed active or rigor fiber was also indicated to be composed of two kinds of transients. The above results suggest that (a) there exist two kinds of interactions in parallel in a compressed active or rigor fiber: one active or rigor and another "passive" between sliding filaments, and (b) the decrease in shortening velocity in a compressed fiber may be brought about by this "passive" interaction.     

Finite element modeling reveals complex strain mechanics in the aponeuroses of contracting skeletal muscle

A finite element model was used to investigate the counter-intuitive experimental observation that some regions of the aponeuroses of a loaded and contracting muscle may shorten rather than undergo an expected lengthening. The model confirms the experimental findings and suggests that pennation angle plays a significant role in determining whether regions of the aponeuroses stretch or shorten. A smaller pennation angles (25°) was accompanied by aponeurosis lengthening whereas a larger pennation angle (47°) was accompanied by mixed strain effects depending upon location along the length of the aponeurosis. This can be explained by the Poisson effect during muscle contraction and a Mohr’s circle analogy. Constant volume constraint requires that fiber cross sectional dimensions increase when a fiber shortens. The opposing influences of these two strains upon the aponeurosis combine in proportion to the pennation angle. Lower pennation angles emphasize the influence of fiber shortening upon the aponeurosis and thus favor aponeurosis compression, whereas higher pennation angles increase the influence of cross sectional changes and therefore favor aponeurosis stretch. The distance separating the aponeuroses was also found to depend upon pennation angle during simulated contractions. Smaller pennation angles favored increased aponeurosis separation larger pennation angles favored decreased separation. These findings caution that measures of the mechanical properties of aponeuroses in intact muscle may be affected by contributions from adjacent muscle fibers and that the influence of muscle fibers on aponeurosis strain will depend upon the fiber pennation angle.     

南海中南部鸢乌贼中型群群体结构

根据2012和2013年在南海中南部采集到的3177尾鸢乌贼中型群样品,对其胴长(ML)、右第一腕长(AL1)、右第二腕长(AL2)、右第三腕长(AL3)、右第四腕长(AL4)、右触腕长(TL)、鳍长(FL)和鳍宽(FW)等形态参数进行测定,并运用主成分分析等方法对其个体的形态差异进行研究.结果表明: 研究海域的鸢乌贼中型群可分为Ⅰ和Ⅱ 2个群体,分别占总数的69%和31%.2个群体的雌性比例均随胴长的增大呈先减少后增加的趋势,直至达到100%;群体Ⅰ雌性和雄性的初次性成熟胴长分别为164和112 mm,群体Ⅱ雌性和雄性初次性成熟胴长分别为157和108 mm.群体Ⅰ在西沙海域和南沙海域均有独立分布区域,而群体Ⅱ密集区主要分布于南沙海域.非参数检验表明:2个群体雌性和雄性个体的全部形态参数均存在显著性差异(P<0.05).在雌性鸢乌贼中,群体Ⅰ和群体Ⅱ的ML、AL1、AL2、AL4、TL和FW存在显著性差异(P<0.05);在雄性鸢乌贼中,群体Ⅰ和群体Ⅱ的ML、AL1、AL2、AL3、AL4和FL存在显著性差异(P<0.01).通过计算差异系数表明,2个群体的形态差异处于种内水平.     

Limited multiple ovulation in heifers fed high plane of nutrition before PMSG stimulation and steroid hormone concentrations in single, twin and multiple ovulators

Limited multiple ovulation (2-4 CL's) in heifers was attempted by feeding a low or a high plane of nutrition during an estrous cycle and injecting a low dose of PMSG at day 16 of that cycle. Multiple ovulation was achieved in 52% of the 19 dairy x beef crossbred heifers that received 1200 I.U. of PMSG. The ratio of the number of heifers which ovulated between 2-4 follicles to those ovulating 1 and more than 4 was higher (P<0.05) in heifers which were fed a high plane of nutrition than in those which were fed a low plane of nutrition. Thirty-six hours after the PMSG injection and prior to estrus, the concentration of E(2)-17beta was less in heifers with 1 growing follicle developing into 1 CL than in heifers with 2 or >2 growing follicles developing into CL's (P<0.01). Heifers with >2 growing follicles developing into CL's had more E(2)-17beta than those which eventually formed 2 CL's (P<0.01). Moreover, in heifers with >2 CL, E(2)-17beta concentration increased regularly until at least 96 hrs after the PMSG injection, while in heifers with 1 CL or 2 CL's, the concentration plateaued at 60 hrs after the PMSG injection. Progesterone concentration during proestrus was lower in heifers that developed >2 CL's than in those with 1 or 2 CL's (P<0.05).     

Sarcomere length dependence of the force-velocity relation in single frog muscle fibers.

The force-velocity relation of single frog fibers was measured at sarcomere lengths of 2.15, 2.65, and 3.15 microns. Sarcomere length was obtained on-line with a system that measures the distance between two markers attached to the surface of the fiber, approximately 800 microns apart. Maximal shortening velocity, determined by extrapolating the Hill equation, was similar at the three sarcomere lengths: 6.5, 6.0, and 5.7 microns/s at sarcomere lengths of 2.15, 2.65, and 3.15 microns, respectively. For loads not close to zero the shortening velocity decreased with increasing sarcomere length. This was the case when force was expressed as a percentage of the maximal force at optimal fiber length or as a percentage of the sarcomere-isometric force at the respective sarcomere lengths. The force-velocity relation was discontinuous around zero velocity: load clamps above the level that kept sarcomeres isometric resulted in stretch that was much slower than when the load was decreased below isometric by a similar amount. We fitted the force-velocity relation for slow shortening (less than 600 nm/s) and for slow stretch (less than 200 nm/s) with linear regression lines. At a sarcomere length of 2.15 microns the slopes of these lines was 8.6 times higher for shortening than for stretch. At 2.65 and 3.15 microns the values were 21.8 and 14.1, respectively. At a sarcomere length of 2.15 microm, the velocity of stretch abruptly increased at loads that were 160-170% of the sarcomere isometric load, i.e., the muscle yielded. However, at a sarcomere length of 2.65 and 3.15 microm yield was absent at such loads. Even the highest loads tested (260%) resulted in only slow stretch.(ABSTRACT TRUNCATED AT 250 WORDS)     

History-dependent properties of skeletal muscle myofibrils contracting along the ascending limb of the force–length relationship

There is a history dependence of skeletal muscle contraction: stretching activated muscles induces a long-lasting force enhancement, while shortening activated muscles induces a long-lasting force depression. These history-dependent properties cannot be explained by the current model of muscle contraction, and its mechanism is unknown. The purposes of this study were (i) to evaluate if force enhancement and force depression are present at short lengths (the ascending limb of the force–length (FL) relationship), (ii) to evaluate if the history-dependent properties are associated with sarcomere length (SL) non-uniformity and (iii) to determine the effects of cross-bridge (de)activation on force depression. Rabbit psoas myofibrils were isolated and attached between two microneedles for force measurements. Images of the myofibrils were projected onto a linear photodiode array for measurements of SL. Myofibrils were activated by either Ca²⁺ or MgADP; the latter induces cross-bridge attachment to actin independently of Ca²⁺. Activated myofibrils were subjected to three stretches or shortenings (approx. 4% SL at approx. 0.07 µm s⁻¹ sarcomere⁻¹) along the ascending limb of the FL relationship separated by periods (approx. 5 s) of isometric contraction. Force after stretch was higher than force after shortening at similar SLs. The differences in force could not be explained by SL non-uniformity. The FL relationship produced by Ca²⁺- and MgADP-activated myofibrils were similar in stretch experiments, but after shortening MgADP activation produced forces that were higher than Ca²⁺ activation. Since MgADP induces the formation of strongly bound cross-bridges, this result suggests that force depression following shortening is associated with cross-bridge deactivation.     

Dependence of cyclic stretch-induced stress fiber reorientation on stretch waveform

Cyclic uniaxial stretching of adherent nonmuscle cells induces the gradual reorientation of their actin stress fibers perpendicular to the stretch direction to an extent dependent on stretch frequency. By subjecting cells to various temporal waveforms of cyclic stretch, we revealed that stress fibers are much more sensitive to strain rate than strain frequency. By applying asymmetric waveforms, stress fibers were clearly much more responsive to the rate of lengthening than the rate of shortening during the stretch cycle. These observations were interpreted using a theoretical model of networks of stress fibers with sarcomeric structure. The model predicts that stretch waveforms with fast lengthening rates generate greater average stress fiber tension than that generated by fast shortening. This integrated approach of experiment and theory provides new insight into the mechanisms by which cells respond to matrix stretching to maintain tensional homeostasis.     

Efficiency of flexible sorting grids to improve size selectivity of the bottom trawl in the Balearic Islands (western Mediterranean), with comparison to a change in mesh cod-end geometry

In recent years several studies have been developed to improve the selectivity of the Mediterranean bottom trawl fisheries, which exert high fishing pressure on young individuals. The focus has been mainly on increasing the mesh size in the cod‐end or changing its mesh geometry; few studies have assessed the efficiency of the sorting grid systems. Analysis of 21 trawl hauls carried out at depths of 117–697 m off the Balearic Islands (western Mediterranean) in October–November 2005, studied size selectivity of the trawl using flexible sorting grids with 15 (SG15) and 20 (SG20) mm bar spacing. A divided polyamide bottom trawl designed for commercial purposes demonstrated high efficiency for the simultaneous use of both sorting grid sizes at different depth intervals (DS: deep shelf; US: upper slope; MS: middle slope). The results were also compared with those from the authors’ previous studies in the area where 40 mm diamond (DM) and square (SM) mesh cod‐ends were used. A saturation effect was detected on the DS for both sorting grids, due to the large amount of biomass captured at this depth interval. Size selectivity of 11 species was modelled, which showed an increase in length at first capture (L₅₀) from SG15 to SG20. Values of L₅₀ estimated for the main target species were: 10.9 cm total length (TL) with SG15, and 18.9 cm TL with SG20 for Merluccius merluccius; 21.1 mm carapace length (CL) with SG15, and 23.8 mm CL with SG20 for Nephrops norvegicus; and 15.9 mm CL with SG15, and 19.6 mm CL with SG20 for Aristeus antennatus. Comparison of size selectivity parameters between the sorting grids and cod‐ends of different geometry showed clear differences. For most species, the highest value of L₅₀ was obtained with SM (e.g. 26.6 mm CL for N. norvegicus and 22.1 mm CL for A. antennatus). Exceptions were the round‐fish M. merluccius, the flatfish Lepidorhombus boscii, and the crustacean Parapenaeus longirostris, with values of SG20 (18.9 cm TL, 12.1 cm TL, and 25.7 mm CL, respectively) higher than with SM (15.2 cm TL, 10.2 cm TL, and 20.2 mm CL, respectively). Sorting grid selection ranges were broader than those estimated for DM and SM. Experience shows that the exchange of the DM for the SM and/or the introduction of SG20 can improve selectivity in the Mediterranean trawl, SM being more efficient than SG20 in the Balearic Islands.     

In vitro induction of tumor-specific immunity. IV. Specific adoptive immunotherapy with cytotoxic T cells induced in vitro to plasmacytoma antigens

Summary Specifically activated cytotoxic T cells (CL's) were raised by cocultivation in vitro of normal spleen cells with syngeneic plasmacytoma cells. These CL's were fully active both in vitro in lysing ⁵¹Cr-labelled tumor cells and in in vivo Winn assays, in inhibiting the growth of tumor cells in syngeneic mice when inoculated admixed with the tumor cells, even at ratios of 2 CL's to 1 tumor cell. However the same CL populations were only marginally effective in an immunotherapy model in which CL's were injected intravenously and tumor cells subcutaneously or intramuscularly (at a ratio of up to 100 CL/1 tumor cell). It is suggested that the in vitro conditions alter cell surface properties, thereby interfering with normal cell traffic, and that if this problem can be overcome, in vitro educated CL's may constitute a potential source of activated cells for human tumor immunotherapy.National Health and Medical Research Council postgraduate research scholar     

Age does not influence muscle fiber length adaptation to increased excursion.

Muscle fiber length adaptation to static stretch or shortening depends on age, with sarcomere addition in young muscle being dependent on mobility. Series sarcomere number can also increase in young animals in response to increased muscle excursion, but it is not clear whether adult muscles respond similarly. The ankle flexor retinaculum was transected in neonatal and adult rats to increase tibialis anterior muscle excursion. Sarcomere number in tibialis anterior was determined after 8 wk of adaptation. Muscle moment arm and excursion were increased 30% (P < 0.01) in both age groups. Muscle cross-sectional area was reduced by 12% (P < 0.01) in response to the increased mechanical advantage, and this reduction was unaffected by age. Fiber length change was also unaffected by age, with both groups showing a trend (P < 0.10) for slightly (6%) increased fiber length. Retinaculum transection results in shorter muscle length in all joint configurations, so this trend opposes the fiber length decrease predicted by an adaptation to muscle length and indicates that fiber length is influenced by dynamic mechanical signals in addition to static length.     

Voluntary activation level and muscle fiber recruitment of human quadriceps during lengthening contractions.

Voluntary activation levels during lengthening, isometric, and shortening contractions (angular velocity 60 degrees/s) were investigated by using electrical stimulation of the femoral nerve (triplet, 300 Hz) superimposed on maximal efforts. Recruitment of fiber populations was investigated by using the phosphocreatine-to-creatine ratio (PCr/Cr) of single characterized muscle fibers obtained from needle biopsies at rest and immediately after a series of 10 lengthening, isometric, and shortening contractions (1 s on/1 s off). Maximal voluntary torque was significantly higher during lengthening (270 +/- 55 N.m) compared with shortening contractions (199 +/- 47 N.m, P < 0.05) but was not different from isometric contractions (252 +/- 47 N.m). Isometric torque was higher than torque during shortening (P < 0.05). Voluntary activation level during maximal attempted lengthening contractions (79 +/- 8%) was significantly lower compared with isometric (93 +/- 5%) and shortening contractions (92 +/- 3%, P < 0.05). Mean PCr/Cr values of all fibers from all subjects at rest were 2.5 +/- 0.6, 2.0 +/- 0.7, and 2.0 +/- 0.7, respectively, for type I, IIa, and IIax fibers. After 10 contractions, the mean PCr/Cr values for grouped fiber populations (regardless of fiber type) were all significantly different from rest (1.3 +/- 0.2, 0.7 +/- 0.3, and 0.8 +/- 0.6 for lengthening, isometric, and shortening contractions, respectively; P < 0.05). The cumulative distributions of individual fiber populations after either contraction mode were significantly different from rest (P < 0.05). Curves after lengthening contractions were less shifted compared with curves from isometric and shortening contractions (P < 0.05), with a smaller shift for the type IIax compared with type I fibers in the lengthening contractions. The results indicate a reduced voluntary drive during lengthening contractions. PCr/Cr values of single fibers indicated a hierarchical order of recruitment of all fiber populations during maximal attempted lengthening contractions.     

Breeding biology of the Louisiana red swamp crayfish Procambarus clarkii Girard in Lake Naivasha,Kenya

This paper describes the breeding biology of the Louisiana red swamp crayfish (Procambarus clarkii Girard) that has been introduced in a tropical lake, Naivasha. Notable differences in crayfish biology are found between the place of origin in Louisiana and lake Naivasha.In Naivasha sexual maturity in females is reached at a larger size (42 mm carapace length (CL), 76 mm full length (FL)) than in males (40 mm CL, 71 mm FL). The corresponding figures for Chalmette area of Louisiana are 31–32 mm CL for both sexes and at Ben Hur Research Centre, Louisiana, 33 mm CL. The females weigh between 15 and 20 g at the onset of egg deposition. The corresponding Louisiana values are from 5 to 10 g. Crayfish in Naivasha as in Louisiana breed both inside burrows which are 10–70 cm deep, and on the sediment in shallow water at a depth of between 0.5 and 4 m. These observations suggest that reproduction is very much a lake edge activity. The number of breeding individuals in Naivasha varies with the trend of the water surface level. Sexually active females (form I) as judged from secondary sexual characters (appearance of basal hooks at the base of 3rd and 4th walking legs) persist throughout the year at no less than 60% of the adult male population. This is confirmed by the appearance of recently hatched juveniles (3.4 to 6.5 mm CL) in the water throughout the year. Similarly berried females in Naivasha are found throughout the year, though with peaks at irregular intervals. In Louisiana breeding takes place only from August through November with a single peak in October. The fecundity of crayfish in Naivasha is 433 (SE ± 21, N = 133) and there is a positive correlation between body length and egg number.     

X-ray interference studies of crossbridge action in muscle contraction: evidence from muscles during steady shortening

During normal muscle shortening, the myosin heads must undergo many cycles of interaction with the actin filaments sliding past them. It is important to determine what range of configurations is found under these circumstances, and, in terms of the tilting lever arm model, what range of orientations the lever arms undergo. We have studied this using the X-ray interference technique described in the previous article, focusing mainly on the changes in the first order meridional reflection (M3) as compared to isometric. The change in ratio of the heights of the interference peaks indicates how far the mean lever arm angle has moved towards the end of the working stroke; the total intensity change depends on the angle change, on the number of heads now attached at any one time, and on the dispersion of lever arm angles. The latter provides a measure of the distance over which myosin heads remain attached to actin as they go through their working strokes. Surprisingly, the mean position of the attached heads moves only about 1 nm inwards (towards the center of the A-band) at low velocity shortening (around 0.9 T0): their dispersion changes very little. This shows that they must be detaching very early in the working stroke. However, at loads around 0.5 T0, the mean lever arm angle is about half way towards the end of the working stroke, and the dispersion of lever arm angles (with a uniform dispersion) is such as to distribute the heads throughout the whole of the working stroke. At higher velocities of shortening (at 0.3 T0), the mean position shifts further towards the end of the stroke, and the dispersion increases further. The details of the measurements, together with other data on muscle indicate that the force-generating mechanism within the myosin heads must have some unexpected properties.     

The telomere lengthening conundrum – it could be biology

Longitudinal studies of human leucocyte telomere length often report a percentage of individuals whose telomeres appear to lengthen. However, based on theoretical considerations and empirical data, Steenstrup et al. (Nucleic Acids Research, 2013, vol 41(13): e131) concluded that this lengthening is unlikely to be a real biological phenomenon and is more likely to be an artefact of measurement error. We dispute the logic underlying this claim. We argue that Steenstrup et al.'s analysis is incomplete because it failed to compare predictions derived from assuming a scenario with no true telomere lengthening with alternative scenarios in which true lengthening occurs. To address this deficit, we built a computational model of telomere dynamics that allowed us to compare the predicted percentage of observed telomere length gainers given differing assumptions about measurement error and the true underling dynamics. We modelled a set of scenarios, all assuming measurement error, but both with and without true telomere lengthening. We found a range of scenarios assuming some true telomere lengthening that yielded either similar or better quantitative fits to the empirical data on the percentage of individuals showing apparent telomere lengthening. We conclude that although measurement error contributes to the prevalence of apparent telomere lengthening, Steenstrup et al.'s conclusion was too strong, and current data do not allow us to reject the hypothesis that true telomere lengthening is a real biological phenomenon in epidemiological studies. Our analyses highlight the need for process‐level models in the analysis of telomere dynamics.     

Technique for interpretation of electromyography for concentric and eccentric contractions in gait

We present a technique to combine muscle shortening and lengthening velocity information with electromyographic (EMG) profiles during gait. A biomechanical model was developed so that each muscle's length could be readily calculated over time as a function of angles of the joints it crossed. The velocity of shortening and lengthening of the muscle fiber was then calculated, and with computer graphics this information was overlaid on the EMG profiles. Thus, researchers and clinicians were not only able to interpret the processed EMG signal as level of activity (tension) but also to gain insight as to the muscles' role as generators (muscle shortening) or absorbers (muscle lengthening) of energy. Six common muscles are documented, using database profiles; soleus (SOL), medial gastrocnemius (MG), tibialis anterior (TA), vastus lateralis (VL), rectus femoris (RF), and semitendinosus (ST). The protocol thus demonstrates a relatively simple technique for calculating muscle fiber velocity and for combining that velocity information with EMG activity profiles.     

CONTRACTILITY AND ULTRASTRUCTURE IN GLYCEROL-EXTRACTED MUSCLE FIBERS : I. The Relationship of Contractility to Sarcomere Length

This study was undertaken to determine whether glycerol-extracted rabbit psoas muscle fibers can develop tension and shorten after being stretched to such a length that the primary and secondary filaments no longer overlap. A method was devised to measure the initial sarcomere length and the ATP-induced isotonic shortening in prestretched isolated fibers subjected to a small preload (0.02 to 0.15 P₀). At all degrees of stretch, the fiber was able to shorten (60 to 75 per cent): to a sarcomere length of 0.7 µ when the initial length was 3.7 µ or less, and to an increasing length of 0.9 to 1.8 µ with increasing initial sarcomere length (3.8 to 4.4 µ). At sarcomere lengths of 3.8 to 4.5 µ, overlap of filaments was lost, as verified by electron microscopy. The variation in sarcomere length within individual fibers has been assessed by both light and electron microscopic measurements. In fibers up to 10 mm in length the stretch was evenly distributed along the fiber, and with sarcomere spacings greater than 4 µ there was only a slight chance of finding sarcomeres with filament overlap. These observations are in apparent contradiction to the assumption that an overlap of A and I filaments is necessary for tension generation and shortening.     

Residual force enhancement after stretch of contracting frog single muscle fibers

Single fibers from the tibialis anterior muscle of Rana temporaria at 0.8-3.8 degrees C were subjected to long tetani lasting up to 8 s. Stretch of the fiber early in the tetanus caused an enhancement of force above the isometric control level which decayed only slowly and stayed higher throughout the contraction. This residual enhancement was uninfluenced by velocity of stretch and occurred only on the descending limb of the length-tension curve. The absolute magnitude of the effect increased with sarcomere length to a maximum at approximately 2.9 micrometers and then declined. The phenomenon was further characterized by its dependence on the amplitude of stretch. The final force level reached after stretch was usually higher than the isometric force level corresponding to the starting length of the stretch. The possibility that the phenomenon was caused by nonuniformity of sarcomere length along the fiber was examined by (a) laser diffraction studies that showed sarcomere stretch at all locations and (b) studies of 9-10 segments of approximately 0.6-0.7 mm along the entire fiber, which all elongated during stretch. Length-clamped segments showed residual force enhancement after stretch when compared with the tetanus produced by the same segment held at the short length as well as at the long length. It is concluded that residual force enhancement after stretch is a property shown by all individual segments along the fiber.     

The Lever Arm Effects a Mechanical Asymmetry of the Myosin-V-Actin Bond

Myosin-V is a two-headed molecular motor taking multiple ATP-dependent steps toward the plus end (forward) of actin filaments. At high mechanical loads, the motor processively steps toward the minus end (backward) even in the absence of ATP, whereas analogous forward steps cannot be induced. The detailed mechanism underlying this mechanical asymmetry is not known. We investigate the effect of force on individual single headed myosin-V constructs bound to actin in the absence of ATP. If pulled forward, the myosin-V head dissociates at forces twice as high than if pulled backward. Moreover, backward but not forward distances to the unbinding barrier are dependent on the lever arm length. This asymmetry of unbinding force distributions in a single headed myosin forms the basis of the two-headed asymmetry. Under load, the lever arm functions as a true lever in a mechanical sense.     

Evidence for increased low force cross-bridge population in shortening skinned skeletal muscle fibers: implications for actomyosin kinetics.

The dynamic characteristics of the low force myosin cross-bridges were determined in fully calcium-activated skinned rabbit psoas muscle fibers shortening under constant loads (0.04-0.7 x full isometric tension Po). The shortening was interrupted at various times by a ramp stretch (duration, 10 ms; amplitude, up to 1.8% fiber length) and the resulting tension response was recorded. Except for the earlier period of velocity transients, the tension response showed nonlinear dependence on stretch amplitude; i.e., the magnitude of the tension response started to rise disproportionately as the stretch exceeded a critical amplitude, as in the presence of inorganic phosphate (Pi). This result, as well as the result of stiffness measurement, suggests that the low force cross-bridges similar to those observed in the presence of Pi (presumably A.M.ADP.Pi) are significantly populated during shortening. The critical amplitude of the shortening fibers was greater than that of isometrically contracting fibers, suggesting that the low force cross-bridges are more negatively strained during shortening. As the load was reduced from 0.3 to 0.04 P0, the shortening velocity increased more than twofold, but the amount of the negative strain stayed remarkably constant (approximately 3 nm). This This insensitiveness of the negative strain to velocity is best explained if the dissociation of the low force cross-bridges is accelerated approximately in proportion to velocity. Along with previous reports, the results suggest that the actomyosin ATPase cycle in muscle fibers has at least two key reaction steps in which rate constants are sensitively regulated by shortening velocity and that one of them is the dissociation of the low force A.M.ADP.Pi cross-bridges. This step may virtually limit the rate of actomyosin ATPase turnover and help increase efficiency in fibers shortening at high velocities.