Stiffness and tension during and after sudden length changes of glycerinated single insect fibrillar muscle fibres

Rapid length changes were applied (within 0.2 ms or 0.4 ms) to single isometrically contracted glycerol extracted muscle fibres of the dorsal longitudinal muscle ofLethocerus maximus suspended in an Ca²⁺ and ATP containing solution at 20–23‡ C. Force transients and the fibre stiffness were measured during and after rapid length changes. At length changesbelow 0.5% of the initial fibre length (∼ 2.4 Μm sarcomere length) the mechanical transients were characterized as follows: (1) After stretch and after release the force regains at least partly the value of tension before the length change within a quick phase of tension recovery. The quick phase induced by stretch was nearly completed within 1–2 ms. (2) A pulse in length of 1.5 ms duration, i.e., a stretch followed by a release to the initial length or a release followed by a stretch to the initial length, was applied to the fibre. The force transient induced by this procedure regains after the second length change the value of the isometric tension before the procedure. (3) The stiffness was constant during each length change of the “pulse” and was equal during the first and the second length changes. These findings are predicted by the muscle contraction model of Huxley and Simmons (1971): The identical force before and after a length pulse may indicate that the rotation of cross bridges after the first length change is followed by a rotation into the original position after the second length change. The constancy of the stiffness during the length changes may indicate a Hookean elastic element of the cross bridge. The similarity of the stiffness during the first and the second length changes, i.e., before and after the quick phase, gives evidence that the quick phases after stretch and after release are not accompanied by a change in the net number of attached cross bridges. If stretches ofmore than 0.5% of the initial length were applied, the mechanical transient of the muscle fibre changed as follows: (1) An ultra fast tension decay phase (duration < 0.4 ms) was observed in addition to the slower decay phase induced by the smaller stretches. (2) If the initial stretch was followed by a release to the initial length, no fast recovery phase was observed, which returns the force to the value before the stretch. The reduced tension value persists for a longer period in time than 10 ms. (3) If the muscle was stretched and released repetitively an ultra fast quick phase was induced only by the first stretch. (4) The stiffness increased during stretch, but was found to be the same in the isometrically contracting muscle and after the quick tension decay phase following a large stretch. These findings indicate that the contraction model of Huxley and Simmons has to be extended by a further process additional to cross bridge rotation in case of large stretches (> 0.5%L ini). The findings are taken to indicate a rapid detachment and reattachment of overstrained cross bridges, i.e., a cross bridge slippage induced by large stretches.     

Effect of ATP concentration and pH on rigor tension development and dissociation of rigor complex in glycerinated rabbit psoas muscle fiber

Isometric rigor tension development of glycerinated rabbit psoas muscle fibers in a medium, due to the formation of rigor complexes, was estimated at varying ATP concentrations from 0 to 2.5 mM and pH values from 6.75 to 8.20. The dissociation of rigor complexes was also estimated under the same conditions. When muscle fibers developed rigor tension from the relaxed and rigor states, the magnitude of rigor tension increased with increasing concentration of ATP. Transition between rigor and relaxation in single fibers occurred discontinuously at constant levels (critical levels) of ATP which were determined by pH. The critical concentrations of ATP necessary for inducing the transitions between rigor and relaxed states were also increased exponentially with increased pH. Incomplete repetition of tension development by the same fiber was also observed. This incomplete reversibility was divided into two types: one which showed a decay in rigor tension and another which showed no decay. The reason for the incomplete reversibility was discussed.     

Cross bridge slippage induced by the ATP analogue AMP-PNP and stretch in glycerol-extracted fibrillar muscle fibres

Glycerol-extracted insect fibrillar muscle fibres in rigor exhibited both an elastic and a plastic phase in the length-tension diagram. The transition between these phases took place at a critical tension, the yield point or elastic limit. In the plastic phase the apparent static elastic modulus became zero, whereas the immediate elastic modulus (measured by rapid length changes completed within 4 ms) exhibited no abrupt change at the yield point. The tension value of the yield point (but not immediate stiffness) was lowered by addition of AMP-PNP and was partially restored by washing out AMP-PNP. The dependence of the critical tension at which plastic flow begins on cooperative cross bridge behaviour is discussed in terms of breaking and reforming acto-myosin linkages. Evidence is presented that addition of AMP-PNP induces slippage of cross bridges on the actin filament by affecting the interaction between myosin and actin.     

Parallel measurements of bound calcium and force in glycerinated rabbit psoas muscle fibers

A simple double-isotope procedure has been developed for making simultaneous measurements of bound Ca²⁺ and relative force in glycerinated rabbit psoas bundles containing two fibers. With this preparation it is possible to study Ca²⁺-troponin interactions coincident with MgATP-induced force development. Over the free [Ca²⁺] range 6 · 10^?8–1.2 · 10^?5 M the bound Ca²⁺ varied from 0.25 to 1.65 μmol/g protein. The free [Ca²⁺] at half-maximal Ca²⁺ saturation was 2 · 10^?7 M while that a half-maximal force was 5 · 10^?7 M. Half-maximal Ca²⁺ saturation was associated with 20% maximal force. The force-[Ca²⁺] saturation curve showed a steep rise in slope at greater than half saturation. The observed relationship was consistent with a model in which multiple occupancy of troponin Ca²⁺-binding sites is essential for initiation of cross-bridge cycling.     

Temperature adaptation and the contractile properties of live muscle fibres from teleost fish

Summary The contractile properties of swimming muscles have been investigated in marine teleosts from Antarctic (Trematomus lepidorhinus, Pseudochaenichthys georgianus), temperate (Pollachius virens, Limanda limanda, Agonis cataphractus, Callionymus lyra), and tropical (Abudefduf abdominalis, Thalassoma duperreyi) latitudes. Small bundles of fast twitch fibres were isolated from anterior myotomes and/or the pectoral fin adductor profundis muscle (m. add. p). Live fibre preparations were viable for several days at in vivo temperatures, but became progressively inexcitable at higher or lower temperatures. The stimulation frequency required to produce fused isometric tetani increased from 50 Hz in Antarctic species at 0°C to around 400 Hz in tropical species at 25°C. Maximum isometric tension (P_o) was produced at the normal body temperature (NBT) of each species (Antarctic, 0–2°C; North Sea and Atlantic, 8–10°C; Indo-West Pacific, 23–25°C). P₀ values at physiological temperatures (200–300 kN·m^–2) were similar for Antarctic, temperate, and tropical species. A temperature induced tension hysteresis was observed in muscle fibres from some species. Exposure to <0°C in Antarctic and <2°C in temperate fish resulted in the temporary depression of tension over the whole experimental range, an effect reversed by incubation at higher temperatures. At normal body temperatures the half-times for activation and relaxation of twitch and tetanic tension increased in the order Antarctic>temperate>tropical species. Relaxation was generally much slower at temperatures <10°C in fibres from tropical than temperate fish. Q₁₀ values for these parameters at NBTs were 1.3 2.1 for tropical species, 1.7–2.6 for temperate species, and 1.6–3.5 for Antarctic species. The forcevelocity (P-V) relationship was studied in selected species using iso-velocity releases and the data below 0.8 P₀ iteratively fitted to Hill's equation. The P-V relation at NBT was found to be significantly less curved in Antarctic than temperate species. The unloaded contraction velocity (V_max) of fibres was positively correlated with NBT increasing from about 1 muscle fibre length·s^–;1 in an Antarctic fish (Trematomus lepidorhinus) at 1°C to around 16 muscle fibre lengths·s^–1 in a tropical species (Thalassoma duperreyi) at 24°C. It is concluded that although muscle contraction in Antarctic fish shows adaptations for low temperature function, the degree of compensation achieved in shortening speed and twitch kinetics is relatively modest.Abbreviations ET environmental temperature - m. add. p major adductor profundis - m. add. s. major adductor superficialis - NBT normal body temperature - P ₀ maximum isometric tension - P-V force velocity - SR sarcoplasmic reticulum - T 1/2 a half activation time - T 1/2 r half relaxation time - V _max unloaded contraction     

Effect of deuterium oxide on contraction characteristics and ATPase activity in glycerinated single rabbit skeletal muscle fibers

We studied the effect of deuterium oxide (D₂O) on contraction characteristics and ATPase activity of single glycerinated muscle fibers of rabbit psoas. D₂O increased the maximum isometric force P₀ by about 20%, while the force versus stiffness relation did not change appreciably. The maximum shortening velocity under zero load V_max did not change appreciably in D₂O, so that the force-velocity (P-V) curve was scaled depending on the value of P₀. The Mg-ATPase activity of the fibers during generation of steady isometric force P₀ was reduced by about 50% in D₂O. Based on the Huxley contraction model, these results can be accounted for in terms of D₂O-induced changes in the rate constants f₁ and g₁ for making and breaking actin-myosin linkages in the isometric condition, in such a way that f₁/(f₁+g₁) increases by about 20%, while (f₁+g₁) remains unchanged. The D₂O effect at the molecular level is discussed in connection with biochemical studies on actomyosin ATPase.     

Calcium-dependence of Donnan potentials in glycerinated rabbit psoas muscle in rigor, at and beyond filament overlap; a role for titin in the contractile process

In glycerinated rabbit psoas muscle, Donnan potential measurements demonstrated that the net electric charge on the actin-myosin matrix undergoes a sharp switch-like transition at pCa₅₀ = 6.8. The potentials are 2 mV less negative at the lower pCa²⁺ (P < 0.001). If ATP is present, the muscle contracts and breaks the microelectrode. Therefore the rigor state was studied. There is no reason to suppose a priori that a similar voltage switch does not occur during contraction, however.Calcium dependence is still apparent in muscles stretched beyond overlap (sarcomere length > 3.8 μm) and is also seen in the gap filaments between the A- and I-band ends; further stretching abolishes the dependence. These experiments strongly suggest that calcium dependence is controlled initially by the titin component, and that this control is lost when titin filaments break. We suppose that that effect is mediated by the titin kinase in the M-line region and may involve the extensible PEVK region of titin.There is great interest in the electric charge on proteins in muscle within the structural system. We suggest how changes in these charges may control the calcium activation process. We also suggest some simple experimental approaches that could clarify these effects.     

In vitro correlation of ultrastructural morphology and creatine phosphokinase release in L6 skeletal muscle cells after exposure to parenteral antibiotics

Summary Morphologic changes in a rat skeletal muscle cell line (L6) exposed for 1 h to the parenteral antibiotics amphotericin B (AMP), tetracycline-HCl (TET), erythromycin lactobionate (ERY), and cephaloridine (CEP) were characterized by transmission and scanning electron microscopy and compared to cellular release of creatine phosphokinase (CRK). AMP (0.05, 0.1, 0.5 mg/ml) caused a concentration-related swelling of nuclei, endoplasmic reticulum, and mitochondria. Loss of membrane integrity associated with AMP exposure was evident at the middle concentration and extensive at the high concentration, which correlated well with the 43 and 90% depletion of CPK from the muscle cells, respectively. TET (0.25, 1.0, 2.5 mg/ml) caused dilation of endoplasmic reticulum and cytoplasmic blebbing at the low concentration but had no effect on the cytoplasmic membrane or CPK. Cells exposed to the high concentration of TET had extensive damage to the cytoplasmic membrane, and CPK was completely depleted. ERY (2.5, 5.0, 25 mg/ml) caused a pattern of morphologic changes and CPK depletion similar to TET. CEP (4.0, 20, 50 mg/ml) had no effect on membrane integrity or CPK; however, membranous whorls were prominent in the cytoplasm. A good correlation between CPK release and cytoplasmic membrane integrity was evident and the ability of these agents to release CPK from muscle cells in culture correlated with the known irritancy potential of these parenteral antibiotics. Furthermore, CPK depletion seems to be a reliable indicator of muscle cell damage after cytoplasmic membrane perturbation and is therefore an appropriate index of toxicity in this in vitro muscle irritation model.     

Differential effects of muscle fibre length and insulin on muscle-specific mRNA content in isolated mature muscle fibres during long-term culture

The aims of this study were (1) to determine the relationship between muscle fibre cross-sectional area and cytoplasmic density of myonuclei in high- and low-oxidative Xenopus muscle fibres and (2) to test whether insulin and long-term high fibre length caused an increase in the number of myonuclei and in the expression of α-skeletal actin and of myogenic regulatory factors (myogenin and MyoD) in these muscle fibres. In high- and low-oxidative muscle fibres from freshly frozen iliofibularis muscles, the number of myonuclei per millimetre fibre length was proportional to muscle fibre cross-sectional area. The in vivo myonuclear density thus seemed to be strictly regulated, suggesting that the induction of hypertrophy required the activation of satellite cells. The effects of muscle fibre length and insulin on myonuclear density and myonuclear mRNA content were investigated on high-oxidative single muscle fibres cultured for 4–5 days. Muscle fibres were kept at a low length (~15% below passive slack length) in culture medium with a high insulin concentration (~6 nmol/l: “high insulin medium”) or without insulin, and at a high length (~5% above passive slack length) in high insulin medium. High fibre length and high insulin medium did not change the myonuclear density of isolated muscle fibres during culture. High insulin increased the myonuclear α-skeletal actin mRNA content, whereas fibre length had no effect on α-skeletal actin mRNA content. After culture at high fibre length in high insulin medium, the myonuclear myogenin mRNA content was 2.5-fold higher than that of fibres cultured at low length in high insulin medium or in medium without insulin. Myonuclear MyoD mRNA content was not affected by fibre length or insulin. These in vitro experiments indicate that high muscle fibre length and insulin enhance muscle gene expression but that other critical factors are required to induce adaptation of muscle fibre size and performance.This work was partially supported by a research grant from the Haak Bastiaanse Kuneman Stichting.     

Identification and partial characterization of a latent ATP,Mg-dependent protein phosphatase in rabbit skeletal muscle cytosol

Summary Fractionation of rabbit skeletal muscle cytosol on Aminohexyl-Sepharose has resulted in the identification of a latent ATP, Mg-dependent protein phosphatase whose catalytic subunit is in the active conformation, but is inhibited by the presence of more than one modulator unit. The partially purified enzyme is converted to an inactive, kinase F_A-dependent form upon incubation at 30°C unless modulator-specific polyclonal antibodies are added to the preparation. The immunoglobulins also relieve the inhibition which is responsible for the low basal phosphatase activity of the enzyme, and they counteract all of the heat-stable inhibitor activity present in the preparation. Addition of free catalytic subunit abolishes the inhibition of the latent enzyme in a dose-dependent way, but cannot prevent the inactivation process. The inactivated phosphatase and the original latent enzyme exhibit the same apparent M _r in sucrose density-gradient centrifugation (70 000) and in gel filtration (110 000).Abbreviations PMSF Phenylmethanesulphonyl Fluoride - TLCK L-l-chloro-3-(4-tosylamido)-7-amino2-heptanone-hydrochloride - TPCK L-l-chloro-3-(4-tosvlamido)-4-phenyl-2-butanone     

Ultrastructural and cytological changes in the muscle fibers of the pectoralis of the giant Canada goose (Branta canadensis maxima) in disuse atrophy during molt

Summary Adult male Branta canadensis maxima were collected from a nonmigratory feral population during their premolt, molt and postmolt phases. Lean dry weight of the pectoralis muscle decreased significantly (p0.0001) during molt, as a result of disuse atrophy. Histochemical analysis revealed that the region of the pectoralis muscle sampled consisted of Red (fast-twitch oxidative-glycolytic) and White (fast-twitch glycolytic) muscle fiber types, in an approximate ratio of 9 to 1. There was no significant (p= 0.1238) difference in the relative percentages of the two fiber types during the three periods of study. There was, however, a significant decrease in mean cross-sectional area of both Red (p0.0194) and White (p0.0001) fibers during molt. Red and White fiber areas were strongly correlated with each other during molt (r ²=0.76, p=0.0010) and postmolt (r ²=0.70, p=0.0052), but not during premolt (r ²=0.02, p=0.7626). The latter finding may be related to fiber-type specific hypertrophy in premolt breeding males. Analysis of ultrastructure revealed that there was a significant (p=0.0003) decrease in the mean myofibrillar crosssectional area, and a significant increase in both the density (p=0.0227) and total number (p=0.0058) of myofibrils within the muscle fibers of the molting birds. These results indicate that the myofibrils split longitudinally during moltassociated disuse atrophy. A significant (p=0.0375) reduction in the amount of non-myofibrillar material (mitochondria) was also observed in the periphery of the muscle fibers of the molting birds. The changes observed during disuse atrophy are neither as pathological nor as extreme as those induced by experimental models of avian muscle atrophy.     

A fluid mechanical analysis of the velocity,adhesion, and destruction of cancer cells in capillaries during metastasis

Metastasis, a multistep process by which cancer disseminates through the body, mainly by intravascular routes, constitutes a major problem in cancer. When cancer cells are injected directly into the veins of animals, they are apparently arrested in the vascular bed of the first organ encountered and gradually released over the next 24 h. These interactions with the microvasculature are often associated in some manner with the death of many cancer cells, and are thought to contribute to the inefficiency of the metastatic process. We have made a theoretical analysis of cancer cells deformed into capillaries with respect to their intravascular velocity, adhesion to the vascular endothelium and intravascular destruction, in terms of the dynamics of the thin liquid film separating the surfaces of the blood vessels and cancer cells. Our calculations, which are based on previously reported experimental observations, indicate that the transit of cancer cells through the microvasculature is discontinuous, being interrupted by adhesions between the two. In addition, in some cases cell membrane rupture (and cell death) will occur when the critical membrane tension of the cancer cells is exceeded by the sum of their initial equilibrium membrane tension and the increased tension in the cancer cell membranes caused by friction generated as they move over the intraluminal surfaces of the capillaries. Our calculations on membrane rupture are consistent with previously unexplained observations by Sato and Suzuki relating cancer cell deformability to death on transpulmonary passage, and constitute a novel mechanism for “metastatic inefficiency” in terms of intravascular cancer cell death.