An electrostatic model with weak actin-myosin attachment resolves problems with the lattice stability of skeletal muscle.

The stability of the filament lattice in relaxed striated muscle can be viewed as a balance of electrostatic and van der Waals forces. The simplest electrostatic model, where actin and myosin filaments are treated as charged cylinders, generates reasonable lattice spacings for skinned fibers. However, this model predicts excessive radial stiffness under osmotic pressure and cannot account for the initial pressure (∼1 kPa) required for significant compression. Good agreement with frog compression data is obtained with an extended model, in which S1 heads are weakly attached to actin when the lattice spacing is reduced below a critical value; further compression moves fixed negative charges on the heads closer to the myofilament backbone as they attach at a more acute angle to actin. The model predicts pH data in which the lattice shrinks as pH is lowered and protons bind to filaments. Electrostatic screening implies that the lattice shrinks with increasing ionic strength, but the observed expansion of the frog lattice at ionic strengths above 0.1 M with KCl might be explained if Cl(-) binds to sites on the motor domain of S1. With myosin-myosin and actin-actin interactions, the predicted lattice spacing decreases slightly with sarcomere length, with a more rapid decrease when actin-myosin filament overlap is very small.

The mechanism of spontaneous oscillatory contractions in skeletal muscle.

Most striated muscles generate steady contractile tension when activated, but some preparations, notably cardiac myocytes and slow-twitch fibers, may show spontaneous oscillatory contractions (SPOC) at low levels of activation. We have provided what we believe is new evidence that SPOC is a property of the contractile system at low actin-myosin affinity, whether caused by a thin-filament regulatory system or by other means. We present a quantitative single-sarcomere model for isotonic SPOC in skeletal muscle with three basic ingredients: i), actin and myosin filaments initially in partial overlap, ii), stretch activation by length-dependent changes in the lattice spacing, and iii), viscoelastic passive tension. Modeling examples are given for slow-twitch and fast-twitch fibers, with periods of 10 s and 4 s respectively. Isotonic SPOC occurs in a narrow domain of parameter values, with small minimum and maximum values for actin-myosin affinity, a minimum amount of passive tension, and a maximum transient response rate that explains why SPOC is favored in slow-twitch fibers. The model also predicts the contractile, relaxed and SPOC phases as a function of phosphate and ADP levels. The single-sarcomere model can also be applied to a whole fiber under auxotonic and fixed-end conditions if the remaining sarcomeres are treated as a viscoelastic load. Here the model predicts an upper limit for the load stiffness that leads to SPOC; this limit lies above the equivalent loads expected from the rest of the fiber.

Morphological and biochemical alterations of skeletal muscles from the genetically obese (ob/ob) mouse.

BACKGROUND: Knowledge of the morphological and biochemical alterations occurring in skeletal muscles of obese animals is relatively limited, particularly with respect to non-limb muscles and relationship to fibre type. OBJECTIVE: Sternomastoid (SM; fast-twitch), extensor digitorum longus (EDL; fast-twitch), and soleus (SOL; mixed) muscles of ob/ob mouse (18-22 weeks) were examined with respect to size (mass, muscle mass-to-body mass ratio, cross-sectional area (CSA)), fibre CSA, protein content, myosin heavy chain (MHC) content, MHC isoform (MHC(i)) composition, MHC(i)-based fibre type composition, and lactate dehydrogenase isoenzyme (LDH(iso)) composition. RESULTS: Compared with (control) muscles from lean mice, all the three muscles from ob/ob mice were smaller in size (by 13-30%), with SM and EDL being the most affected. The CSA of IIB and IIB+IID fibres (the predominant fibre types in SM and EDL muscles) was markedly smaller (by approximately 30%) in ob/ob mice, consistent with differences in muscle size. Total protein content (normalised to muscle mass) was significantly lower in EDL (-9.7%) and SOL (-14.1%) muscles of ob/ob mice, but there were no differences between SM, EDL, and SOL muscles from the two animal groups with respect to MHC content (also normalised to muscle mass). Electrophoretic analyses of MHC(i) composition in whole muscle homogenates and single muscle fibres showed a shift towards slower MHC(i) content, slower MHC(i) containing fibres, and a greater proportion of hybrid fibres in all the three muscles of ob/ob mice, with a shift towards a more aerobic-oxidative phenotype also observed with respect to LDH(iso) composition. CONCLUSION: This study showed that SM, EDL, and SOL muscles of ob/ob mice display size reductions to an extent that seems to be largely related to fibre type composition, and a shift in fibre type composition that may result from a process of structural remodelling, as suggested by the increased proportion of hybrid fibres in muscles of ob/ob mice.

Chloride conductance in the transverse tubular system of rat skeletal muscle fibres: importance in excitation-contraction coupling and fatigue.

Contraction in skeletal muscle fibres is governed by excitation of the transverse-tubular (t-) system, but the properties of the t-system and their importance in normal excitability are not well defined. Here we investigate the properties of the t-system chloride conductance using rat skinned muscle fibres in which the sarcolemma has been mechanically removed but the normal excitation-contraction coupling mechanism kept functional. When the t-system chloride conductance was eliminated, either by removal of all Cl(-) or by block of the chloride channels with 9-anthracene carboxylic acid (9-AC) or by treating muscles with phorbol 12,13-dibutyrate, there was a marked reduction in the threshold electric field intensity required to elicit a t-system action potential (AP) and twitch response. Calculations of the t-system chloride conductance indicated that it constitutes a large proportion of the total chloride conductance observed in intact fibres. Blocking the chloride conductance increased the size of the twitch response and was indicative that Cl(-) normally carries part of the repolarizing current across the t-system membrane on each AP. Block of the t-system chloride conductance also reduced tetanic force responses at higher frequency stimulation (100 Hz) and greatly reduced twitch responses in the period shortly after a brief tetanus, owing to rapid loss of t-system excitability during the AP train. Blocking activity of the Na(+)-K(+) pump in the t-system membrane caused loss of excitability owing to K(+) build-up in the sealed t-system, and this occurred approximately 3-4 times faster when the chloride conductance was blocked. These findings show that the t-system chloride conductance plays a vital role during normal activity by countering the effects of K(+) accumulation in the t-system and maintaining muscle excitability.

Different isometric force - [Ca2+] relationships in slow and fast twitch skinned muscle fibres of the rat.

Skinned muscle fibres prepared from fast and slow twitch muscles of rat have been activated in Ca2+-buffered solutions using a new activation procedure (Moisescu, D.G. and Thieleczek, R. (1978) J. Physiol. 275, 241--262). The results indicate that (i) the Ca2+ activation curve is less steep for slow fibres, (ii) physiologically relevant force levels are attained considerably faster at constant [Ca2+] in fast fibres, and (iii) active force becomes noticeable at lower [Ca2+], but reaches saturation at higher [Ca2+] for slow fibres.

Studies on the luminescent response of the Ca2+-activated photoprotein, obelin.

1. The kinetics and stoicheiometry of the Ca2+-activated luminescent reaction of the photoprotein obelin were studied at different temperatures and in the presence of various substances, including the physiologically occurring cations K+, Na+, Ca+, Mg2+ and H+. 2. The results suggest Ca2+-independent rates of rise and fall in obelin luminescence following sudden changes in [Ca2+] and indicate that changes in [Ca2+] over the range 1 x 10(-6) - 3 x 10(-4) M are followed significantly faster by the obelin response (approx. 3 ms delay at 20 degrees C) than by the aequorin response (approx. 10 ms delay at 20 degrees C). 3. Obelin was found to emit low-intensity light (less than 10(-6) of the maximum Ca2+-activated response), which was independent of Ca2+ at concentrations below about 10(-7) M. The level of this Ca2+-independent light emission is sensitive to temperature and the ionic composition of the solution. 4. The log-log plot of light intensity against ionized Ca indicates a maximum slope of 2.5, suggesting the involvement of three Ca ions in the luminescent reaction. 5. Increase in the concentration of K+, Na+, Mg2+ and H+ generally shift the Ca2+ activation curve for obelin toward higher Ca2+ concentrations. These cations can also affect the maximum rate of obelin utilization at more extreme concentrations. 6. The maximal rate of obelin utilization was also affected to varying degrees by the presence of uncharged substances such as glucose, sucrose and polyvinylpyrrolidone. However, neither the sensitivity of obelin to Ca2+ nor the quantum yield were modified by the substances. 7. Caffeine (less than 20 mM), procaine (less than 20 mM) and sodium dantrolene (saturated solution), substances known to modify cellular Ca2+ movements, had little effect on the Ca2+-induced luminescent reaction. The general anaesthetics chlorpromazine and halothane appeared to lower greatly the quantum yield without, however, modifying the maximum rate of obelin utilization. 8. A scheme of reaction for obelin activation by Ca2+ is presented which adequately explains the experimental observations and allows one to make accurate predictions regarding the relative obelin response under a variety of ionic conditions at room temperature.

Single-fiber study of contractile and biochemical properties of skeletal muscles in streptozotocin-induced diabetic rats.

Mechanically skinned, single muscle fibers, isometrically activated in pH and Ca2+ (Sr2+) buffered solutions were used to examine the function of the contractile apparatus in slow- and fast-twitch fibers from soleus (SOL, predominantly slow-twitch) and extensor digitorum longus (EDL, predominantly fast-twitch) muscles of streptozotocin (STZ)-induced diabetic rats and age-matched controls. Three and 14 days after STZ administration, the contractile properties of muscle fibers from diabetic rats did not differ significantly from those of controls with respect to several mechanical parameters, such as maximum Ca-activated tension, activation threshold, and sensitivity to Ca2+ and Sr2+. In contrast, 28 days after STZ administration, 37.5% of the fast-twitch EDL fibers developed maximum activated tensions (77.1 +/- 10.4 kN/m2), which were significantly lower than those developed by controls (244.0 +/- 14.3 kN/m2), and the slow-twitch SOL fibers displayed a significantly higher sensitivity to Ca2+ (and Sr2+) than the controls. All fibers from diabetic rats, including the low-tension EDL fibers and higher Ca sensitivity SOL fibers displayed control-like electrophoretic profiles of the major myofibrillar proteins. Taken together with data from earlier studies on the effects of long-term diabetes on whole skeletal muscle contractility, these results strongly suggest that 1) the decrease in tetanic tension output of EDL muscles induced by diabetes is caused mainly by direct effects of the diabetic condition on the contractile/regulatory system of a subpopulation of fast-twitch fibers, which develop little force, and 2) the diabetes-induced slowing of twitch times of SOL muscles is caused in part by the increased sensitivity to Ca2+ of the contractile apparatus in the slow-twitch fibers.

Time course of calcium transients derived from Fura-2 fluorescence measurements in single fast twitch fibres of adult mice and rat myotubes developing in primary culture.

In this study, we applied a method to correct for the altered binding kinetics of Fura-2 for Ca2+ in vivo, on Ca2+ fluorescence transients (Ca2+F) measured using Fura-2 in single adult fast twitch skeletal muscle fibres of the mouse, which exhibit very fast [Ca2+] responses, and rat myotubes developing in culture which exhibit slower [Ca2+] responses (rise time [20-80% of peak] of Ca2+F transients: 1.81 +/- 0.17 ms and 16.14 +/- 2.60 ms, respectively). After correction, the [Ca2+] transients (Ca2+C) measured in both the adult mouse fibres and the myotubes rose more rapidly (mean rise time of Ca2+C transients: adult mouse fibres, 0.76 +/- 0.12 ms; rat myotubes, 8.25 +/- 2.83 ms) and often exhibited a Ca2+ spike which exceeded the peak of the Ca2+F transient. In the adult mouse fibres, correction increased the mean peak [Ca2+] of the Ca2+F transients by a factor of 7 from 0.53 +/- 0.08 microM to 3.76 +/- 0.71 microM. The accuracy of the time course of the corrected Ca2+ transients was confirmed by comparison to the time course of Ca2+ transients measured with Mag-Fura-5, which had a similar mean rise time (0.94 +/- 0.10 ms, t-test, P = 0.80). The more slowly rising Ca2+ transients measured in the rat myotubes were less affected by the correction process, increasing in mean peak [Ca2+] by a factor of only 1.2 from 0.82 +/- 0.17 microM to 0.97 +/- 0.15 microM. During the decay phase of the Ca2+ transients elicited in the adult mouse fibres and the myotubes, the corrected Ca2+C signal largely followed the unmodified Ca2+F transient. The correction process was found to have little effect on Ca2+ transients with rise time values greater than 10 ms, which included most of the Ca2+ transients measured in the myotubes.

Measurement of membrane potential and myoplasmic [Ca2+] in developing rat myotubes at rest and in response to stimulation.

In this study, the membrane potential and cytosolic [Ca2+] were measured in rat myotubes developing in culture from days 6-14. It was found that as the myotubes developed in culture, the resting membrane potential (RMP) became more negative during days 6-8, and then did not significantly change until after day 13, when it started to become less negative. The mean RMP measured at days 8-13 was -59 +/- 1 mV (n = 70). The amplitude of action potentials elicited in the myotubes by anode break stimulation increased in size during development (range: 47.5-119 mV) and this closely correlated with the development of a more negative RMP. Cytosolic [Ca2+] was measured in the rat myotubes using the Ca2+ indicator Fura-2, and no significant change in the resting [Ca2+] was observed during development (days 6-14). Ca2+ responses triggered by action potentials varied from small slow increases in [Ca2+] that failed to return to the baseline to rapid [Ca2+] transients. The size of the [Ca2+] transients positively correlated with both the observed increase in the RMP during development and the size of the action potential. Larger [Ca2+] transients also had more rapid rates of [Ca2+] decay, indicating a tandem increase in the ability of the sarcoplasmic reticulum to release and resequester Ca2+ during development of rat myotubes. Repetitive stimulation (10 Hz) of the myotubes exhibiting small [Ca2+] transients produced a step-like rise in [Ca2+]. Many myotubes exhibiting larger [Ca2+]transients could not be stimulated at 10 Hz by anode break stimulation due to the presence of action potentials with large hyperpolarisations. However, when these myotubes were depolarised at 10 Hz, they produced a tetanic Ca2+ response similar to that seen in adult skeletal muscle.

Abnormalities in structure and function of limb skeletal muscle fibres of dystrophic mdx mice.

In this study we have shown that the skeletal muscle fibres from adult (older than 26 weeks) mdx mice have gross structural deformities. We have characterized the onset and age dependence of this feature in mdx mice. The three dimensional structure of these deformities has been visualized in isolated fibres and the orientation of these deformities was determined within the muscle by confocal laser scanning microscopy. We have also shown that the occurrence of morphologically abnormal fibres is greater in muscles with longer fibres (extensor digitorum longus (EDL) and soleus, 6-7.3 mm long), than in muscles with shorter fibres (flexor digitorum brevis (FDB), 0.3-0.4 mm long). A population of post-degenerative fibres, with both central and peripheral nuclei coexistent along the length of the fibre, has also been identified in the muscles studied. We showed that a mild protocol of lengthening (eccentric) contractions (the muscle was stretched by 12% during a tetanic contraction) caused a major reduction in the maximal tetanic force subsequently produced by mdx EDL muscle. In contrast, maximal tetanic force production in normal soleus, normal EDL and mdx soleus muscles was not altered by this protocol. We suggest that the deformed fast glycolytic fibres which are found in adult mdx EDL but not in adult mdx soleus muscles are the population of fibres damaged by the lengthening protocol.

Effects of chlorpromazine on excitation-contraction coupling events in fast-twitch skeletal muscle fibres of the rat.

1. Single mechanically skinned fibres from the rat extensor digitorum longus muscle, which allow access to intracellular compartments, were used to examine the effects of 0.5-100 microM chlorpromazine hydrochloride (CPZ) on the major steps of the excitation-contraction (E-C) coupling to elucidate the involvement of skeletal muscle in the neuroleptic malignant syndrome (NMS). 2. At 1 microM, CPZ caused a 20-30% increase in the force response induced by t-system depolarisation and a marked increase in the rate of caffeine-induced SR Ca(2+) release. At [CPZ]> or =2.5 microM, there was an initial increase followed by a marked decrease of the t-system depolarisation-induced force responses, while the potentiating effect on the caffeine-induced SR Ca(2+) release remained. These effects were reversible. 3. CPZ had no effect on the maximum Ca(2+)-activated force, but caused reversible, concentration-dependent increases in the Ca(2+) sensitivity of the contractile apparatus at [CPZ] > or =10 microM, with a 50% predicted shift of 0.11 pCa (-log [Ca(2+)]) units at 82.3 microM CPZ. 4. CPZ did not alter the rate of SR-Ca(2+) loading at 1 and 10 microM, but reversibly reduced it by approximately 40% at 100 microM by reducing the SR Ca(2+) pump. Nevertheless, the SR Ca(2+) content was greater when fibres became unresponsive to t-system-induced depolarisation in the presence than in the absence of 100 microM CPZ. 5. The results show that CPZ has concentration-dependent stimulatory and inhibitory effects on various steps of the E-C coupling, which can explain the involvement of skeletal muscle in NMS and reconcile previous divergent data on CPZ effects on muscle.

International Academy of Astronautics 5th cosmic study--preparing for a 21st century program of integrated, Lunar and Martian exploration and development (executive summary).

This report is an initial review of plans for a extensive program to survey and develop the Moon and to explore the planet Mars during the 21st century. It presents current typical plans for separate, associated and fully integrated programs of Lunar and Martian research, exploration and development, and concludes that detailed integrated plans must be prepared and be subject to formal criticism. Before responsible politicians approve a new thrust into space they will demand attractive, defensible, and detailed proposals that explain the WHEN, HOW and WHY of each stage of an expanded program of 21st century space research, development and exploration. In particular, the claims of daring, innovative, but untried systems must be compared with the known performance of existing technologies. The time has come to supersede the present haphazard approach to strategic space studies with a formal international structure to plan for future advanced space missions under the aegis of the world's national space agencies, and supported by governments and the corporate sector.

Effects of elevated physiological temperatures on sarcoplasmic reticulum function in mechanically skinned muscle fibers of the rat.

Properties of the sarcoplasmic reticulum (SR) with respect to Ca(2+) loading and release were measured in mechanically skinned fiber preparations from isolated extensor digitorum longus (EDL) muscles of the rat that were either kept at room temperature (23 degrees C) or exposed to temperatures in the upper physiological range for mammalian skeletal muscle (30 min at 40 or 43 degrees C). The ability of the SR to accumulate Ca(2+) was significantly reduced by a factor of 1.9-2.1 after the temperature treatments due to a marked increase in SR Ca(2+) leak, which persisted for at least 3 h after treatment. Results with blockers of Ca(2+) release channels (ruthenium red) and SR Ca(2+) pumps [2,5-di(tert-butyl)-1,4-hydroquinone] indicate that the increased Ca(2+) leak was not through the SR Ca(2+) release channel or the SR Ca(2+) pump, although it is possible that the leak pathway was via oligomerized Ca(2+) pump molecules. No significant change in the maximum SR Ca(2+)-ATPase activity was observed after the temperature treatment, although there was a tendency for a decrease in the SR Ca(2+)-ATPase. The observed changes in SR properties were fully prevented by the superoxide (O(2)(*-)) scavenger Tiron (20 mM), indicating that the production of O(2)(*-) at elevated temperatures is responsible for the increase in SR Ca(2+) leak. Results show that physiologically relevant elevated temperatures 1) induce lasting changes in SR properties with respect to Ca(2+) handling that contribute to a marked increase in the SR Ca(2+) leak and, consequently, to the reduction in the average coupling ratio between Ca(2+) transport and SR Ca(2+)-ATPase and muscle performance, and 2) that these changes are mediated by temperature-induced O(2)(*-) production.

Effect of ADP on slow-twitch muscle fibres of the rat: implications for muscle fatigue.

Slow-twitch mechanically skinned fibres from rat soleus muscle were bathed in solutions mimicking the myoplasmic environment but containing different [ADP] (0.1 microm to 1.0 mm). The effect of ADP on sarcoplasmic reticulum (SR) Ca2+-content was determined from the magnitude of caffeine-induced force responses, while temporal changes in SR Ca2+-content allowed determination of the effective rates of the SR Ca2+-pump and of the SR Ca2+-leak. The SR Ca2+-pump rate, estimated at pCa (-log10[Ca2+]) 7.8, was reduced by 20% as the [ADP] was increased from 0.1 to 40 microm, with no further alteration when the [ADP] was increased to 1.0 mm. The SR Ca2+-leak rate constant was not altered by increasing [ADP] from 0.1 to 40 microm, but was increased by 26% when the [ADP] was elevated to 1.0 mm. This ADP-induced SR Ca2+-leak was insensitive to ruthenium red but was abolished by 2,5-di(tert-butyl)-1,4-hydroquinone (TBQ), indicating that the leak pathway is via the SR Ca2+-pump and not the SR Ca2+-release channel. The decrease in SR Ca2+-pump rate and SR Ca2+-leak rate when [ADP] was increased led to a 40% decrease in SR Ca2+-loading capacity. Elevation of [ADP] had only minor direct effects on the contractile apparatus of slow-twitch fibres. These results suggest that ADP has only limited depressing effects on the contractility of slow-twitch muscle fibres. This is in contrast to the marked effects of ADP on force responses in fast-twitch muscle fibres and may contribute to the fatigue-resistant nature of slow-twitch muscle fibres.