Time-resolved X-ray diffraction by skinned skeletal muscle fibers during activation and shortening

Force, sarcomere length, and equatorial x-ray reflections (using synchrotron radiation) were studied in chemically skinned bundles of fibers from Rana temporaria sartorius muscle, activated by UV flash photolysis of a new photolabile calcium chelator, NP-EGTA. Experiments were performed with or without compression by 3% dextran at 4 degrees C. Isometric tension developed at a similar rate (t(1/2) = 40 +/- 5 ms) to the development of tetanic tension measured in other studies (Cecchi et al., 1991). Changes in intensity of equatorial reflections (I(11) t(1/2), 15-19 ms; I(10) t(1/2), 24-26 ms) led isometric tension development and were faster than for tetanus. During shortening at 0.14P(o), I(10) and I(11) changes were partially reversed (18% and 30%, respectively, compressed lattice), in agreement with intact cell data. In zero dextran, activation caused a compression of A-band lattice spacing by 0.7 nm. In 3% dextran, activation caused an expansion of 1.4 nm, consistent with an equilibrium spacing of 45 nm. But, in both cases, discharge of isometric tension by shortening caused a rapid lattice expansion of 1.0-1.1 nm, suggesting discharge of a compressive cross-bridge force, with or without compression by dextran, and the development of an additional expansive force during activation. In contrast to I(10) and I(11) data, these findings for lattice spacing did not resemble intact fiber data.     

Measurement of sarcomere shortening in skinned fibers from frog muscle by white light diffraction.

A new optical-electronic method has been developed to detect striation spacing of single muscle fibers. The technique avoids Bragg-angle and interference-fringe effects associated with laser light diffraction by using polychromatic (white) light. The light is diffracted once by an acousto-optical device and then diffracted again by the muscle fiber. The double diffraction reverses the chromatic dispersion normally obtained with polychromatic light. In frog skinned muscle fibers, active and passive sarcomere shortening were smooth when observed by white light diffraction, whereas steps and pauses occurred in the striation spacing signals obtained with laser illumination. During active contractions skinned fibers shortened at high rates (3-5 microns/s per half sarcomere, 0-5 degrees C) at loads below 5% of isometric tension. Compression of the myofibrillar lateral filament spacing using osmotic agents reduced the shortening velocity at low loads. A hypothesis is presented that high shortening velocities are observed with skinned muscle fibers because the cross-bridges cannot support compressive loads when the filament lattice is swollen.     

Block of contracture in skinned frog skeletal muscle fibers by calcium antagonists

The ability of a number of calcium antagonistic drugs including nitrendipine, D600, and D890 to block contractures in single skinned (sarcolemma removed) muscle fibers of the frog Rana pipiens has been characterized. Contractures were initiated by ionic substitution, which is thought to depolarize resealed transverse tubules in this preparation. Depolarization of the transverse tubules is the physiological trigger for the release of calcium ion from the sarcoplasmic reticulum and thus of contractile protein activation. Since the transverse tubular membrane potential cannot be measured in this preparation, tension development is used as a measure of activation. Once stimulated, fibers become inactivated and do not respond to a second stimulus unless allowed to recover or reprime (Fill and Best, 1988). Fibers exposed to calcium antagonists while fully inactivated do not recover from inactivation (became blocked or paralyzed). The extent of drug-induced block was quantified by comparing the height of individual contractures. Reprimed fibers were significantly less sensitive to block by both nitrendipine (10 degrees C) and D600 (10 and 22 degrees C) than were inactivated fibers. Addition of D600 to fibers recovering from inactivation stopped further recovery, confirming preferential interaction of the drug with the inactivated state. A concerted model that assumed coupled transitions of independent drug-binding sites from the reprimed to the inactivated state adequately described the data obtained from reprimed fibers. Photoreversal of drug action left fibers inactivated even though the drug was initially added to fibers in the reprimed state. This result is consistent with the prediction from the model. The estimated KI for D600 (at 10 degrees and 22 degrees C) and for D890 (at 10 degrees C) was approximately 10 microM. The estimated KI for nitrendipine paralysis of inactivated fibers at 10 degrees C was 16 nM. The sensitivity of reprimed fibers to paralysis by D600 and D890 was similar. However, inactivated fibers were significantly less sensitive to the membrane-impermeant derivative (D890) than to the permeant species (D600), which suggests a change in the drug-binding site or its environment during the inactivation process. The enantomeric dihydropyridines (+) and (-) 202-791, reported to be calcium channel agonists and antagonists, respectively, both caused paralysis, which suggests that blockade of a transverse tubular membrane calcium flux is not the mechanism responsible for antagonist-induced paralysis. The data support a model of excitation-contraction coupling involving transverse tubular proteins that bind calcium antagonists.     

Connectin filaments in stretched skinned fibers of frog skeletal muscle

Indirect immunofluorescence microscopy of highly stretched skinned frog semi-tendinous muscle fibers revealed that connectin, an elastic protein of muscle, is located in the gap between actin and myosin filaments and also in the region of myosin filaments except in their centers. Electron microscopic observations showed that there were easily recognizable filaments extending from the myosin filaments to the I band region and to Z lines in the myofibrils treated with antiserum against connectin. In thin sections prepared with tannic acid, very thin filaments connected myosin filaments to actin filaments. These filaments were also observed in myofibrils extracted with a modified Hasselbach-Schneider solution (0.6 M KCl, 0.1 M phosphate buffer, pH 6.5, 2 mM ATP, 2 mM MgCl2, and 1 mM EGTA) and with 0.6 M Kl. SDS PAGE revealed that connectin (also called titin) remained in extracted myofibrils. We suggest that connectin filaments play an important role in the generation of tension upon passive stretch. A scheme of the cytoskeletal structure of myofibrils of vertebrate skeletal muscle is presented on the basis of our present information of connectin and intermediate filaments.     

Determinants of relaxation rate in skinned frog skeletal muscle fibers

The influences of sarcomere uniformity andCa²⁺ concentration on the kineticsof relaxation were examined in skinned frog skeletal muscle fibersinduced to relax by rapid sequestration ofCa²⁺ by the photolysis of theCa²⁺ chelator, diazo-2, at10°C. Compared with an intact fiber, diazo-2-induced relaxationexhibited a faster and shorter initial slow phase and a fast phase witha longer tail. Stabilization of the sarcomeres by repeated releases andrestretches during force development increased the duration of the slowphase and slowed its kinetics. When force of contraction was decreasedby lowering the Ca²⁺concentration, the overall kinetics of relaxation was accelerated, withthe slow phase being the most sensitive toCa²⁺ concentration. Twitchlikecontractions were induced by photorelease ofCa²⁺ from a cagedCa²⁺ (DM-Nitrophen), withsubsequent Ca²⁺ sequestration byintact sarcoplasmic reticulum orCa²⁺ rebinding to cagedCa²⁺. These twitchlike responsesexhibited relaxation kinetics that were about twofold slower than thoseobserved in intact fibers. Results suggest that the slow phase ofrelaxation is influenced by the degree of sarcomere homogeneity andrate of Ca²⁺ dissociation fromthin filaments. The fast phase of relaxation is in part determined bythe level of Ca²⁺ activation.

     

Time-resolved X-ray diffraction studies of frog skeletal muscle isometrically twitched by two successive stimuli using synchrotron radiation

In order to clarify the delay between muscular structural changes and mechanical responses, the intensity changes of the equatorial and myosin layer-line reflections were studied by a time-resolved X-ray diffraction technique using synchrotron radiation. The muscle was stimulated at 12-13 degrees C by two successive stimuli at an interval (80-100 ms) during which the second twitch started while tension was still being exerted by the muscle. At the first twitch, the intensity changes of the 1.0 and 1.1 equatorial reflections reached 65 and 200% of the resting values, and further changes to 55 and 220% were seen at the second twitch, respectively. Although the second twitch decreased not only the time to peak tension but also that to the maximum intensity changes of the equatorial reflections (in both cases, about 15 ms), the delay (about 20 ms) between the intensity changes and the development of tension at the first twitch were still observed at the second twitch. On the other hand, the intensities of the 42.9 nm off-meridional and the 21.5 nm meridional myosin reflections decreased at the first twitch to the levels found when a muscle was isometrically tetanized, and no further decrease in their intensities was observed at the second twitch. These results indicate that a certain period of time is necessary for myosin heads to contribute to tension development after their arrival in the vicinity of the thin filaments during contraction.     

Effect of stretch and release on equatorial X-ray diffraction during a twitch contraction of frog skeletal muscle.

Time-resolved intensity measurements of the x-ray equatorial reflections were made during twitch contractions of frog skeletal muscles, to which stretches or releases were applied at various times. A ramp stretch applied at the onset of a twitch (duration, 15 ms; amplitude, approximately 3% of muscle length) caused a faster and larger development of contractile force than in an isometric twitch. The stretch accelerated the decrease of the 1.0 reflection intensity (I1,0). The magnitude of increase of the 1,1 reflection intensity (I1,1) was reduced by the stretch, but its time course was also accelerated. A release applied at the peak of a twitch or later (duration, 5 ms; amplitude, approximately 1.5%) caused only a partial redevelopment of tension. The release produced clear reciprocal changes of reflections toward their relaxed levels, i.e., the I1,0 increased and the I1,1 decreased. A release applied earlier than the twitch peak had smaller effects on the reflection intensities. The results suggest that a strength applied at the onset of a twitch causes a faster radial movement of the myosin heads toward actin, whereas a release applied at or later than the peak of a twitch accelerates their return to the thick filament backbone. The results are discussed in the context of the regulation of the myosin head attachment by calcium.     

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.     

Structural changes during activation of frog muscle studied by time-resolved X-ray diffraction

The pattern given by contracting frog muscle can be followed with high time resolution using synchrotron radiation as a high-intensity X-ray source. We have studied the behaviour of the second actin layer-line (axial spacing of approximately 179 A) at an off-meridional spacing of approximately 0.023 A-1, a region of the diagram that is sensitive to the position of tropomyosin in the thin filaments. In confirmation of earlier work, we find that there is a substantial increase in the intensity of this part of the pattern during contraction. We find that the reflection reaches half its final intensity about 17 milliseconds after the stimulus at 6 degrees C. The changes in the equatorial reflections, which arise from movement of crossbridges towards the thin filaments, occur with a delay of about 12 to 17 milliseconds relative to this change in the actin pattern. In over-stretched muscle, where thick and thin filaments no longer overlap, the changes in the actin second layer-line still take place upon stimulation with a time course and intensity similar to that observed at full overlap. This indicates that tropomyosin movement, in response to calcium binding to troponin, is the first structural step in muscular contraction, and is the prerequisite for myosin binding. A change in intensity similar to that found in contracting muscle is seen in rigor, where tropomyosin is probably locked in the active position. During relaxation the earlier stages in the decrease in intensity of the second actin layer-line take place significantly sooner after the last stimulus than tension decay. In over-stretched muscles the intensity decay is appreciably faster than in the same muscles at rest length, where attached crossbridges may interfere with the return of tropomyosin to its resting position.     

Hg2+-induced contracture in mechanically skinned fibers of frog skeletal muscle

In mechanically skinned fibers of the semitendinosus muscle of bullfrogs, we examined the role of membrane sulfhydryl groups on Ca2+ release from the sarcoplasmic reticulum (SR). Hg2+, a sulfhydryl reagent (20-100 microM), induced a repetitive contracture of skinned fibers, and this contracture did not occur in skinned fibers in which the SR had been disrupted by treatment with a detergent (Brij 58). Procaine (10 mM), Mg2+ (5 mM), or dithiothreitol (1 mM) blocked the Hg2+-induced contracture. Ag+ or p-chloromercuribenzenesulfonic acid produced similar contractures to that induced by Hg2+. We conclude that Hg2+ releases Ca2+ from SR of a skinned fiber by modifying sulfhydryl groups on the SR membrane, and suggest that the Ca2+ released by Hg2+ may trigger a greater release of Ca2+ from SR to develop tension.     

X-ray study of myosin heads in contracting frog skeletal muscle

Using synchrotron radiation, the behaviour of the diffuse X-ray scatter was investigated in the relaxed and active phases of auxotonic and isometric contractions. Muscles were stimulated tetanically for 0.75 of a second, leaving intervals of three minutes between successive contractions. In isometric contractions the scatter is very asymmetric, which means that the myosin heads have a strongly preferred orientation. During tension rise the scatter expands in the meridional direction and contracts in the equatorial direction, the maximal local intensity change being about 20%. The shape change indicates that on average the myosin heads become oriented more perpendicularly to the fibre axis. The distribution of orientations at peak tension is quite different from that we found previously in X-ray scattering data from rigor muscles. In auxotonic contractions where muscles shorten against an increasing tension the scatter is practically circularly symmetrical. This suggests that during shortening the myosin heads go evenly through a wide range of orientations. It is concluded that the results from both the auxotonic and isometric experiments provide strong support for the rotating myosin head model. In isometric contractions the transition between the relaxed phase and peak tension is accompanied by an overall increase in scattering intensity of about 10%: this corresponds to a relative increase in the fraction of disordered myosin heads by almost 30%.     

Chemically skinned mammalian skeletal muscle. I. The structure of skinned rabbit psoas.

We studied the morphology of rabbit psoas muscle fixed at increasing intervals of time in a chemical skinning solution (Wood et al., 1975), or after skinning and storage for times up to 1 week. The storage solution, in which the chemically skinned muscled fibers were kept at -20 degrees C, had the same ionic composition as the skinning solution but was made with 50% (v/v) glycerol. Progressive structural changes occurred in fibers exposed to skinning solution. The structural changes were essentially complete after 24-48 hr in skinning solution and no further changes were detected in fibers stored for periods up to 1 week. Structural changes were: (i) holes or gaps in the plasma membrane; (ii) swelling of mitochondria and disorganization of their internal structure; (iii) slight swelling of the sarcoplasmic reticulum; (iv) disappearance of sarcoplasmic reticulum (SR) feet from triadic gaps. Other changes included loss of glycogen between fibrils and extraction of myoplasm, or the change of its staining properties. All architectural elements of the SR, except "feet", remained during skinning and storage, and the SR remained able to accumulate calcium. The morphology of the myofilaments during chemical skinning and during storage did not differ from control fibers. We conclude that chemical skinning alters the gross structure of the plasma membrane and mitochondria, but produces minimal changes in the sarcoplasmic reticulum and contractile proteins.     

Histochemical and molecular determination of fiber types in chemically skinned single equine skeletal muscle fibers

Until now, there has been no reliable method for histochemical determination of fiber types of single skinned muscle fibers. The major problem arises from the fact that most histochemical techniques use cross-sections of a large group of fibers and compare a given fiber with those surrounding it. This is not possible with a single skinned fiber which has been separated from a bundle to be used for mechanical analysis. A further problem is that the skinning procedure itself may alter the staining pattern. We have developed a procedure by which multiple cross-sections of single skinned fibers can be exposed to various histochemical reactions and the staining patterns compared on the same slide to those of frozen muscle and skinned bundles. By this procedure, three fiber types were distinguished by both Ca2+-ATPase and SDH reactions. The fiber typings determined from both enzyme systems correlated well with each other. Although we were able to differentiate only between slow and fast fibers by SDS-PAGE, these results corroborated the histochemical classification. This procedure will clearly be useful in skinned single muscle fiber mechanics experiments performed to determine functional differences among fiber types.     

Direct modeling of X-ray diffraction pattern from contracting skeletal muscle

A direct modeling approach was used to quantitatively interpret the two-dimensional x-ray diffraction patterns obtained from contracting mammalian skeletal muscle. The dependence of the calculated layer line intensities on the number of myosin heads bound to the thin filaments, on the conformation of these heads and on their mode of attachment to actin, was studied systematically. Results of modeling are compared to experimental data collected from permeabilized fibers from rabbit skeletal muscle contracting at 5°C and 30°C and developing low and high isometric tension, respectively. The results of the modeling show that: i), the intensity of the first actin layer line is independent of the tilt of the light chain domains of myosin heads and can be used as a measure of the fraction of myosin heads stereospecifically attached to actin; ii), during isometric contraction at near physiological temperature, the fraction of these heads is ∼40% and the light chain domains of the majority of them are more perpendicular to the filament axis than in rigor; and iii), at low temperature, when isometric tension is low, a majority of the attached myosin heads are bound to actin nonstereospecifically whereas at high temperature and tension they are bound stereospecifically.     

5'-nucleotidase: an ecto-enzyme of frog skeletal muscle.

Using 1-4C-labeled AMP and IMP as substrates, 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) activity was detected at the external surface of frog skeletal muscle with the active site facing toward the extracellular space. The enzyme was firmly bound to the muscle membrane. Its activity was dependent on Ca2+ or Mg2+ and was inhibited by non-radioactive ribonucleoside 5'-monophosphates, or theophylline, while adenosine 3'-monophosphate and p-nitrophenylphosphate had little or no effect. 5'-Nucleotidase with similar properties was also found in the isolated plasma membrane fraction of the muscle.     

Calcium-induced structural changes in chemically skinned muscle fibers. Detection by optical diffractometry.

The intensity of the first order diffraction line produced by chemically skinned muscle fibers was detected by a self scanning photodiode array and minicomputer system. Line intensity was observed to decrease in fibers stretched to zero filament overlap when subjected to calcium-EGTA buffers in the physiological pCa range. Calcium dependent intensity decreases were not observed for myosin extracted fibers indicating that the thick filament proteins may be the source of the calcium effect seen in non-extracted fibers. These results can be interpreted in terms of calcium dependent effects on thick filament disordering which are not dependent upon cross bridge formation.     

Myosin-ATPase fibre typing of chemically skinned muscle fibres

Summary The efficacy of myosin (M)-ATPase fibre typing to differentiate fibre types in chemically (EGTA) skinned muscle fibres was investigated. Cryosections or single fibres from isolated bundles of chemically skinned rat extensor digitorum longus (EDL) and soleus (SOL) muscles were stained for M-ATPase activity. The results indicate that two major fibre types (type I and II, Brooke & Kaiser, 1970) can be indentified, as well as subgrouping of the type II fibres into types IIa and IIb. Thus, chemically skinning muscle fibres appears to have no detrimental effects on subsequent M-ATPase fibre typing.     

Potassium efflux from single skinned skeletal muscle fibers.

The efflux of 42K from single, skinned (sarcolemma removed) skeletal muscle fibers has been determined. Isotope washout curves are kinetically complex and can be fit as the sum of three exponentials, including a fast component (k = 0.25 s-1) with a pool size equivalent to 91% of the fiber volume, an intermediate component (k = 0.08 s-1) equivalent to 6% of the fiber volume, and a slow component (k = 0.008 s-1) equivalent to 0.5% of fiber volume. Only the intermediate kinetic component is significantly affected by pretreatment of fibers with detergent. Efflux curves from detergent-treated fibers could be fit as the sum of two exponentials with coefficients and rate constants comparable to those of the fast and slow component of washout of untreated controls. Similarly the washout of [14C]sucrose can be described as the sum of two exponentials. We conclude that the intermediate component of 42K washout results from the movement of ions from a membrane bound space within the skinned fiber. Because of its relative volume, the sarcoplasmic reticulum seems to be a reasonable choice as a structural correlate for this component. Our estimate of the potassium permeability for the sarcoplasmic reticulum (SR) based on the efflux data is 10(-7) cm/s. This value is less than previous estimates from isolated preparations.     

X-ray diffraction studies of cross-bridges weakly bound to actin in relaxed skinned fibers of rabbit psoas muscle.

X-ray diffraction patterns were obtained from skinned rabbit psoas muscle under relaxing and rigor conditions over a wide range of ionic strengths (50-170 mM) and temperatures (1 degree C-30 degrees C). For the first time, an intensification of the first actin-based layer line is observed in the relaxed muscle. The intensification, which increases with decreasing ionic strength at various temperatures, including 30 degrees C, parallels the formation of weakly attached cross-bridges in the relaxed muscle. However, the overall intensities of the actin-based layer lines are low. Furthermore, the level of diffuse scattering, presumably a measure of disorder among the cross-bridges, is little affected by changing ionic strength at a given temperature. The results suggest that the intensification of the first actin layer line is most likely due to the cross-bridges weakly bound to actin, and that the orientations of the weakly attached cross-bridges are hardly distinguishable from the detached cross-bridges. This suggests that the orientations of the weakly attached cross-bridges are not precisely defined with respect to the actin helix, i.e., nonstereospecific. Intensities of the myosin-based layer lines are only marginally affected by changing ionic strength, but markedly by temperature. The results could be explained if in a relaxed muscle the cross-bridges are distributed between a helically ordered and a disordered population with respect to myosin filament structure. Within the disordered population, some are weakly attached to actin and others are detached. The fraction of cross-bridges in the helically ordered assembly is primarily a function of temperature, while the distribution between the weakly attached and the detached within the disordered population is mainly affected by ionic strength. Some other notable features in the diffraction patterns include a approximately 1% decrease in the pitch of the myosin helix as the temperature is raised from 4 degrees C to 20 degrees C.