Skinned Frog Skeletal Muscle Fibers Research Articles

Determinants of relaxation rate in skinned frog skeletal muscle fibers.

The influences of sarcomere uniformity and Ca2+ concentration on the kinetics of relaxation were examined in skinned frog skeletal muscle fibers induced to relax by rapid sequestration of Ca2+ by the photolysis of the Ca2+ chelator, diazo-2, at 10 degreesC. Compared with an intact fiber, diazo-2-induced relaxation exhibited a faster and shorter initial slow phase and a fast phase with a longer tail. Stabilization of the sarcomeres by repeated releases and restretches during force development increased the duration of the slow phase and slowed its kinetics. When force of contraction was decreased by lowering the Ca2+ concentration, the overall kinetics of relaxation was accelerated, with the slow phase being the most sensitive to Ca2+ concentration. Twitchlike contractions were induced by photorelease of Ca2+ from a caged Ca2+ (DM-Nitrophen), with subsequent Ca2+ sequestration by intact sarcoplasmic reticulum or Ca2+ rebinding to caged Ca2+. These twitchlike responses exhibited relaxation kinetics that were about twofold slower than those observed in intact fibers. Results suggest that the slow phase of relaxation is influenced by the degree of sarcomere homogeneity and rate of Ca2+ dissociation from thin filaments. The fast phase of relaxation is in part determined by the level of Ca2+ activation.

Effect of Luminal Calcium on Ca 2+ Release Channel Activity of Sarcoplasmic Reticulum in Situ

Ca 2+ influx into empty SR in the absence of Ca 2+ pump activity was determined in skinned frog skeletal muscle fibers and compared with Ca 2+ efflux from loaded SR (i.e., Ca 2+ release) to deepen our understanding of the properties of the Ca 2+ release channel (CRC). Calcium content in SR increased approximately in a first-order kinetics and finally reached the equilibrium level determined by cytoplasmic Ca 2+ ([Ca 2+] C). Because AMP caused an increase in the rate of Ca 2+ influx, and procaine, Mg 2+, and high concentrations of Ca 2+ caused a characteristic decrease, the major Ca 2+ influx pathway was concluded to be the CRC, as is true of Ca 2+ release. The apparent rate constant ( k app) of Ca 2+ efflux did not significantly change when the loading level was decreased to one-third. At a given [Ca 2+] C, the same equilibrium level of calcium in SR was attained with a similar k app by both Ca 2+ influx and Ca 2+ efflux. The relationship between [Ca 2+] C and calcium in SR indicated the Ca 2+ binding sites in SR. These results, together with the anticipated effects of these Ca 2+ buffer sites on kinetics, are consistent with the idea that luminal Ca 2+ inhibits the CRC.

The molecular origin of birefringence in skeletal muscle. Contribution of myosin subfragment S-1

The state of optical polarization of He-Ne laser light diffracted by single skinned frog skeletal muscle fibers has been determined after decoration of the thin filaments of rigor fibers with exogenous S-1. Light on the first diffraction order was analyzed using optical ellipsometry for changes occurring in total birefringence (delta nT) and total differential field ratio (rT) and the experimental results compared with theoretical predictions. Fibers were examined with SDS-gel electrophoresis and electron microscopy as independent assays of S-1 binding. The binding of S-1 to the thin filaments caused a significant increase in rT and a small but significant decrease in delta nT. Release of bound exogenous S-1 with magnesium pyrophosphate demonstrated that the effect of S-1 on the optical parameters was reversible and both electrophoresis and electron microscopy demonstrated the presence of S-1 specifically bound to the thin filaments. Model simulations based on the theory of Yeh, Y., and R. Baskin (1988. Biophys. J. 54:205-218) showed that the values of delta nT and rT were sensitive to the axial bonding angle of exogenous S-1 as well as to the volume fraction of added S-1. Analysis of the data in light of the model showed that an average axial S-1 binding angle of 68 degrees +/- 7 degrees best fit the data.

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.

Physiological role and selectivity of the in situ potassium channel of the sarcoplasmic reticulum in skinned frog skeletal muscle fibers.

The role of K+ as a counterion during Ca2+ release from the sarcoplasmic reticulum (SR) has been investigated. An optical technique using the Ca2+-sensitive dye antipyrylazo III monitored Ca2+ release from skinned (sarcolemma removed) muscle fibers of the frog. Skinned fibers were used since the removal of the sarcolemma allows direct access to the SR membrane. Releases were stimulated by caffeine, which activates Ca2+ release directly by binding to a receptor on the SR. Two different methods were used to decrease the SR K+ conductance so that its effect on Ca2+ release could be assessed: (a) the SR K+ channel blocker, 1,10-bis-quanidino-n-decane (bisG10) was used to eliminate current pathways and (b) substitution of the impermeant ion choline for K+ was used to decrease charge carriers. Both bisG10 and choline substitution caused a concentration-dependent decrease in the Ca2+ release rate. Therefore we conclude that K+ is an important counterion for Ca2+ during its release from the SR. The selectivity of the in situ SR K+ channel to several monovalent cations was determined by substituting them for K+ and comparing their effect on Ca2+ release. The substituted ions were expected to affect Ca2+ release in proportion to their ability to support a counterion flux, which is, in turn, a function of their relative conductance through the SR K+ channel. The selectivity sequence determined by these experiments was K+ = Rb+ = Na+ greater than Cs+ greater than Li+ greater than choline.

Muscle force and stiffness during activation and relaxation. Implications for the actomyosin ATPase.

Isolated skinned frog skeletal muscle fibers were activated (increasing [Ca2+]) and then relaxed (decreasing [Ca2+]) with solution changes, and muscle force and stiffness were recorded during the steady state. To investigate the actomyosin cycle, the biochemical species were changed (lowering [MgATP] and elevating [H2PO4-]) to populate different states in the actomyosin ATPase cycle. In solutions with 200 microM [MgATP], compared with physiological [MgATP], the slope of the plot of relative steady state muscle force vs. stiffness was decreased. At low [MgATP], cross-bridge dissociation from actin should be reduced, increasing the population of the last cross-bridge state before dissociation. These data imply that the last cross-bridge state before dissociation could be an attached low-force-producing or non-force-producing state. In solutions with 10 mM total Pi, compared to normal levels of MgATP, the maximally activated muscle force was reduced more than muscle stiffness, and the slope of the plot of relative steady state muscle force vs. stiffness was reduced. Assuming that in elevated Pi, Pi release from the cross-bridge is reversed, the state(s) before Pi release would be populated. These data are consistent with the conclusion that the cross-bridges are strongly bound to actin before Pi release. In addition, if Ca2+ activates the ATPase by allowing for the strong attachment of the myosin to actin in an A.M.ADP.Pi state, it could do so before Pi release. The calcium sensitivity of muscle force and stiffness in solutions with 4 mM [MgATP] was bracketed by that measured in solutions with 200 microM [MgATP], where muscle force and stiffness were more sensitive to calcium, and 10 mM total Pi, where muscle force and stiffness were less sensitive to calcium. The changes in calcium sensitivity were explained using a model in which force-producing and rigor cross-bridges can affect Ca2+ binding or promote the attachment of other cross-bridges to alter calcium sensitivity.

Isotonic contraction of skinned muscle fibers on a slow time base: effects of ionic strength and calcium.

The force development by calcium-activated skinned frog skeletal muscle fibers and the motion on a slow time base after a quick decrease in load were studied at 0-1 degrees C as a function of the ionic strength and the degree of activation. The ionic strength was varied between 50 and 190 mM by adding appropriate concentrations of KCl to the bathing solution. Under these conditions, the fibers could be maximally activated for several cycles at low ionic strength without developing residual tension. We found that the steady isometric force in fully activated fibers linearly decreased when the KCl concentration was increased from 0 to 140 mM. The steady isotonic motion at a given relative load in fully activated fibers was almost the same at KCl concentration greater than or equal to 50 mM. In 0 and 20 mM KCl, the isotonic velocity decreased continuously for more than 300 ms. At a given relative load, the initial velocity of the motion in 0 and 20 mM KCl was about 0.6 and 0.9 times, respectively, that in 140 mM KCl. The initial velocity decreased further when residual tension developed; this observation provides additional evidence that residual tension may reflect the presence of an internal load. The effect of calcium on the motion was examined at 70 mM KCl. In this solution, the motion during the velocity transient at a given relative load appeared to be the same at different levels of activation. The speed of the subsequent motion was almost steady at high calcium levels but decreased continuously in low calcium levels. These results support the idea that at low ionic strength the response of the fiber to calcium is switch-like, but that other factors also affect the contraction mechanism under these conditions.

A simple electrostatic model can explain the effect of pH upon the force-pCa relation of skinned frog skeletal muscle fibers

The relative force-pCa relation of skinned frog skeletal muscle fibers is shifted along the pCa axis by changes in pH. This shift has been interpreted as arising from competition between H+ and Ca2+ for a binding site on troponin. Unfortunately, binding studies have been unable to confirm such competition. Alternatively, however, the data fit a model where H+ influences the degree of dissociation of ionizable groups on the surface of the thin filaments, thus altering the electrostatic potential surrounding the filaments. Alterations in the potential will, in turn, change the concentration of Ca2+ near the troponin binding sites in accordance with the Boltzmann relation. A simple model, based upon the Gouy-Chapman relation between surface potential and charge density, provides a quantitative explanation for the shift of the relative force-pCa curve with pH, given a reasonable estimate of the surface charge density on the thin filament. A best fit is obtained when the ionizable groups giving rise to the potential have a log proton ionization constant (pKa) of 6.1, similar to that for the imidazole group on histidine, and when the density of these groups is near that estimated from amino acid analysis of thin filament proteins and from filament geometry. In preliminary experiments, reaction of skinned frog fibers with diethylpyrocarbonate (DEP) at pH 6 shifted the force-pCa curve toward lower Ca2+. This would be expected in the model since DEP at pH 6 is reported to specifically react with histidine imidazole groups and to irreversibly decrease their pKa, which would increase the net negative charge of the filaments.

The comparative effects of (Ca2+) and (Mg2+) on on tension generation in the fibers of skinned frog skeletal muscle and mechanically disrupted rat ventricular cardiac muscle.

The effects of intracellular Mg2+ on Ca2+-activated isometric tension generation in rat cardiac muscle fibers and frog skeletal muscle were compared. The membranous sarcolemmal barrier was removed from rat cardiac muscle fibers by mechanical disruption and from frog skeletal muscle by skinning. Tension was recorded in the fibers in bathing solutions of different Ca2+ concentrations and either 5 X 10(-5) M or 1 X 10(-3) M Mg2+ concentration (the same concentrations used in a previous study on single skinned frog skeletal muscle fibers [3]). The amount of Ca2+ required to activate the muscle increased with Mg2+ concentration for both rat ventricular muscle and frog skeletal muscle. These data indicate that intracellular Mg2+ concentration could strongly modulate Ca2+-activated tension in cardiac muscle and that very similar molecular mechanisms are responsible for Ca2+-activated tension in rat ventricular muscle and frog skeletal muscle. The possible sites of action of Mg2+ on Ca2+-activated tension are discussed.