Model of calcium movements during activation in the sarcomere of frog skeletal muscle

A model of calcium movement during activation of frog skeletal muscle is described. The model was based on the half sarcomere of a myofibril and included compartments representing the terminal cisternae, the longitudinal sarcoplasmic reticulum, the extramyofibrillar space, and the myofibrillar space. The calcium-binding proteins troponin, parvalbumin, and calsequestrin were present in appropriate locations and with realistic binding kinetics. During activation a time-dependent permeability in the terminal cisternal wall led to calcium release into the myoplasm and its diffusion through the myoplasm longitudinally and radially was computed. After adjustment of three parameters, the model produced a myoplasmic free-calcium concentration that was very similar to those recorded experimentally with calcium indicators. The model has been used to demonstrate the importance of parvalbumin in the relaxation of skeletal muscle, to describe the time course and magnitude of calcium gradients associated with diffusion across the sarcomere, and to estimate the errors associated with the use of aequorin as an intracellular calcium indicator in muscle.     

The Intracellular Site of Calcium Activation of Contraction in Frog Skeletal Muscle

Radioautography has been used to localize ⁴⁵Ca in isotopically labeled frog skeletal muscle fibers which had been quickly frozen during a maintained tetanus, a declining tetanus, or during the period immediately following a tetanus or a contracture. During a tetanus almost all of the myofibrillar ⁴⁵Ca is localized in the region of the sarcomere occupied by the thin filaments. The amount varies with the tension being developed by the muscle. The movement of calcium within the reticulum from the tubular portion to the terminal cisternae during the posttetanic period has a half-time of about 9 sec at room temperature and a Q₁₀ of about 1.7. Repolarization is not necessary for this movement. Evidence is given to support the notion that most calcium efflux from the cell occurs from the terminal cisternae into the transverse tubules.     

Is a guanine nucleotide-binding protein involved in excitation-contraction coupling in skeletal muscle?

Plasma membrane depolarization causes skeletal muscle contraction by triggering Ca2+ release from an intracellular membrane network, the sarcoplasmic reticulum. A specialized portion of the sarcoplasmic reticulum, the terminal cisternae, is junctionally associated with sarcolemmal invaginations called the transverse tubules, but the mechanism by which the action potential at the level of the transverse tubules is coupled to Ca2+ release from the terminal cisternae is still mysterious. Here we show that: (i) GTP gamma S, a non-hydrolyzable analog of GTP, elicits isometric force development in skinned muscle fibre; (ii) GTP gamma S is unable to release CA2+ from isolated sarcoplasmic reticulum fractions; (iii) the threshold for tension development is shifted to higher GTP gamma S concentrations by pre-incubation with pertussis toxin. These results suggest that a GTP-binding protein is involved in coupling the action potential of transverse tubules to Ca2+ release from the terminal cisternae.     

Voltage dependence of depolarization-contraction coupling processes in skeletal muscle cells

Changes of contraction and optical anisotropy of isolated skeletal muscle cells from Rana temporaria and Astacus fluviatilis were compared under voltage clamp conditions. The time course of the optical signal from frog muscle was shown to consist of two components which could be attributed to calcium binding both on the sarcoplasmic reticulum Ca-ATPase and troponin C. The optical signal from the crayfish muscle has no distinguishable components, and its onset probably reflects the start of Ca-ATPase activity. This hypothesis is supported by the analysis of effects of some pharmacological agents and conditioning depolarization on tension and optical signal.     

Morphology of isolated triads

The triad is the junctional association of transverse tubule with sarcoplasmic reticulum terminal cisternae. A procedure for the isolation of highly enriched triads from skeletal muscle has been described in the previous paper. In the present study, the structural features of isolated triads have been examined by thin-section, negative-staining, and freeze-fracture electron microscopy. In isolated triads, key features of the structure observed in situ have been retained, including the osmiophilic "feet," junctional structures between the transverse tubule and terminal cisternae. New insight into triad structure is obtained by negative staining, which also enables visualization of feet at the junctional face of the terminal cisternae, whereas smaller surface particles, characteristic of calcium pump protein, are not visualized there. Therefore, the junctional face is different from the remainder of the sarcoplasmic reticulum membrane. Junctional feet as viewed by thin section or negative staining have similar periodicity and extend approximately 100 A from the surface of the membrane. Freeze-fracture of isolated triads reveals blocklike structures associated with the membrane of the terminal cisternae at the junctional face, interjunctional connections between the terminal cisternae and t-tubule, and intragap particles. The intragap particles can be observed to be closely associated with the t-tubule. The structure of isolated triads is susceptible to osmotic and salt perturbation, and examples are given regarding differential effects on transverse tubules and terminal cisternae. Conditions that adversely affect morphology must be considered in experimentation with triads as well as in their preparation and handling.     

Simultaneous measurements of ionic currents, tension and optical properties of voltage clamped skeletal muscle fibres

The paper presents the voltage clamp method for isolated skeletal muscle cells, which allows simultaneous measurements of ionic currents, tension and changes in their optical properties. Experimental results illustrate the range of possible applications. Measurements with two types of preparations, frog and crayfish, exhibiting differences in EC coupling are compared. Basic characteristics of the voltage dependence of tension and birefringence during activation of contraction are described.     

Electrical models of excitation-contraction coupling and charge movement in skeletal muscle

The consequences of ionic current flow from the T system to the sarcoplasmic reticulum (SR) of skeletal muscle are examined. The Appendix analyzes a simple model in which the conductance gx, linking T system and SR, is in series with a parallel resistor and capacitor having fixed values. The conductance gx is supposed to increase rapidly with depolarization and to decrease slowly with repolarization. Nonlinear transient currents computed from this model have some of the properties of gating currents produced by intramembrane charge movement. In particular, the integral of the transient current upon depolarization approximates that upon repolarization. Thus, equality of nonlinear charge movement can occur without intramembrane charge movement. A more complicated model is used in the text to fit the structure of skeletal muscle and other properties of its charge movement. Rectification is introduced into gx and the membrane conductance of the terminal cisternae to give asymmetry in the time- course of the transient currents and saturation in the curve relating charge movement to depolarization, respectively. The more complex model fits experimental data quite well if the longitudinal tubules of the sarcoplasmic reticulum are isolated from the terminal cisternae by a substantial resistance and if calcium release from the terminal cisternae is, for the most part, electrically silent. Specific experimental tests of the model are proposed, and the implications for excitation-contraction coupling are discussed.     

Doxorubicin induces calcium release from terminal cisternae of skeletal muscle. A study on isolated sarcoplasmic reticulum and chemically skinned fibers

In this study, we investigated the effect of the anticancer drug doxorubicin on Ca2+ fluxes of isolated highly purified sarcoplasmic reticulum fractions (longitudinal tubules and terminal cisternae (Saito, A., Seiler, S., Chu, A., and Fleischer, S. (1984) J. Cell Biol. 99, 875-885] and of chemically skinned skeletal muscle fibers of the rabbit. In terminal cisternae, doxorubicin inhibits Ca2+ uptake (IC50 at 0.5 microM) and increases 2.6-fold Ca2+-dependent ATPase rate (half-maximal activation at 3 microM) and unidirectional Ca2+ efflux (8-fold stimulation at 25 microM). On the contrary, doxorubicin is without effect on longitudinal tubules. In skinned muscle fibers, doxorubicin induces rapid and transient Ca2+ release, as measured by tension development (half-maximal stimulation at 6 microM), which is completely and reversibly inhibited by ruthenium red, a known inhibitor of Ca2+ release from isolated terminal cisternae. Doxorubicin has no effect on the sarcoplasmic reticulum Ca2+ pump and on the contractile apparatus of skinned muscle fibers. It is concluded that doxorubicin activates Ca2+ release from sarcoplasmic reticulum and opens a Ca2+ efflux pathway (Ca2+ channel) selectively localized in terminal cisternae. Doxorubicin might interact with Ca2+ channels involved in physiological Ca2+ release.     

Chicken breast muscle connectin: passive tension and I-band region primary structure

We performed cDNA cloning of chicken breast muscle connectin. Together with previous results, our analysis elucidated a 24.2 kb sequence encoding the amino terminus of the protein. This corresponded to the I-band region of the skeletal muscle sarcomere, which is involved in extension and contraction between the Z-line and the A-I junction. There were fewer middle immunoglobulin domains and amino acid residues in the PEVK segment of chicken breast muscle connectin than in human skeletal muscle connectin, but more than in human cardiac muscle connectin. We measured passive tension generation by stretching mechanically skinned myofibril bundles. This revealed that appreciable tension development in chicken breast muscle began at longer sarcomere spacings than in rabbit cardiac muscle, but at shorter spacings than in rabbit psoas and soleus muscles. We suggest that the chicken breast muscle sarcomere remains in a relatively extended state even in unstrained sarcomeres. This would explain why chicken breast muscle does not extend under force to the same degree as rabbit psoas and soleus muscles.     

Passive and active tension in single cardiac myofibrils.

Single myofibrils were isolated from chemically skinned rabbit heart and mounted in an apparatus described previously (Fearn et al., 1993; Linke et al., 1993). We measured the passive length-tension relation and active isometric force, both normalized to cross sectional area. Myofibrillar cross sectional area was calculated based on measurements of myofibril diameter from both phase-contrast images and electron micrographs. Passive tension values up to sarcomere lengths of approximately 2.2 microns were similar to those reported in larger cardiac muscle specimens. Thus, the element responsible for most, if not all, passive force of cardiac muscle at physiological sarcomere lengths appears to reside within the myofibrils. Above 2.2 microns, passive tension continued to rise, but not as steeply as reported in multicellular preparations. Apparently, structures other than the myofibrils become increasingly important in determining the magnitude of passive tension at these stretched lengths. Knowing the myofibrillar component of passive tension allowed us to infer the stress-strain relation of titin, the polypeptide thought to support passive force in the sarcomere. The elastic modulus of titin is 3.5 x 10(6) dyn cm-2, a value similar to that reported for elastin. Maximum active isometric tension in the single myofibril at sarcomere lengths of 2.1-2.3 microns was 145 +/- 35 mN/mm2 (mean +/- SD; n = 15). This value is comparable with that measured in fixed-end contractions of larger cardiac specimens, when the amount of nonmyofibrillar space in those preparations is considered. However, it is about 4 times lower than the maximum active tension previously measured in single skeletal myofibrils under similar conditions (Bartoo et al., 1993).     

Model of calcium diffusion, binding and membrane transport in the sarcomere of frog skeletal muscle

A model of calcium distribution in the sarcomere during activation of contraction was developed. It allows for diffusion and binding of calcium ions to various sarcoplasmic binding sites in the three dimensional spatial coordinate system. The model was used to analyze the influence of kinetic characteristic of binding processes on the temporal and spatial distribution of calcium in the sarcomere during activation of contraction by the action potential and by rectangular depolarizing pulses. The hypothesis concerning the calcium release control in the membrane of terminal cisternae was tested.     

Relation of muscle cell contraction and calcium distribution in sarcomere

A model of activation of muscle contraction has been proposed. It is based on calcium diffusion and binding to specific regulatory sites in a sarcomere. Calcium ions activate interactions of contractile proteins and thus the generation of force. The model quantifies the relation between calcium released from intracellular stores and the elicited force.     

Identification and purification of a transverse tubule coupling protein which binds to the ryanodine receptor of terminal cisternae at the triad junction in skeletal muscle

In fast twitch skeletal muscle, the signal for excitation-contraction coupling is transferred from transverse tubule across the triad junction; calcium is thereby released from the terminal cisternae of sarcoplasmic reticulum triggering muscle contraction. Recently, the feet structures of terminal cisternae, which bridge the gap at the triad junction, have been identified as the ryanodine receptor and in turn with the calcium release channels of sarcoplasmic reticulum. The latter consists of an oligomer of a single high molecular weight polypeptide (Mr 360,000). This study attempts to identify the component in the transverse tubule which ligands with the foot structure to form the triad junction. The purified ryanodine receptor, derivatized with sulfosuccinimidyl-2-(p-azidosalicylimido)-1,3'-dithiopropionate (SASD), a thiol-cleavable, 125I-iodinatable, and photoactive probe, was shown to selectively cross-link to a protein with Mr of 71,000 in isolated transverse tubules. This coupling protein was purified from transverse tubule by solubilization with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) and then purified by sequential column chromatography. In the absence of sulfhydryl agents, the purified polypeptide has an Mr of 61,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A complementary approach using SASD was employed to confirm association of the coupling protein with the ryanodine receptor of terminal cisternae. We conclude that the transverse tubule coupling protein together with the ryanodine receptor (foot structure) is involved in the liganding between transverse tubule and terminal cisternae of sacroplasmic reticulum.     

Indentations in the terminal cisternae of amphibian and mammalian skeletal muscle fibers

Indentations (hillocks and dimples) in the terminal cisternae of mammalian and amphibian skeletal muscle fibers were studied using freeze-fracture and serial thin-section techniques. The structures were seen in all muscles and had a regular separation from each other and from the T-tubule. Indentations were smaller than fenestrations and formed concavities in, but not macromolecular pores through, the terminal cisternae. The average numbers of indentations in rat muscles (measured along the length of the terminal cisternae, within 150 nm of the triadic junction) varied from 0.9 per micrometer in soleus fibers to 9.6 per micrometer in posterior cricoarytenoid fibers. The average numbers in amphibian sartorius fibers varied from 1.6 to 3.6 per micrometer in muscles from different species. The regular alignment of the indentations along the triad, as well as a close correlation between their numbers and the contractile properties of the muscle, suggest that they function in contractile activation and may represent sites of calcium release from the terminal cisternae.     

The effect of altered temperature on Ca2(+)-sensitive force in permeabilized myocardium and skeletal muscle. Evidence for force dependence of thin filament activation

The effect of changes in temperature on the calcium sensitivity of tension development was examined in permeabilized cellular preparations of rat ventricle and rabbit psoas muscle. Maximum force and Ca2+ sensitivity of force development increased with temperature in both muscle types. Cardiac muscle was more sensitive to changes in temperature than skeletal muscle in the range 10-15 degrees C. It was postulated that the level of thin filament activation may be decreased by cooling. To investigate this possibility, troponin C (TnC) was partially extracted from both muscle types, thus decreasing the level of thin filament activation independent of temperature and, at least in skeletal muscle fibers, decreasing cooperative activation of the thin filament as well. TnC extraction from cardiac muscle reduced the calcium sensitivity of tension less than did extraction of TnC from skeletal muscle. In skeletal muscle the midpoint shift of the tension-pCa curve with altered temperature was greater after TnC extraction than in control fibers. Calcium sensitivity of tension development was proportional to the maximum tension generated in cardiac or skeletal muscle under all conditions studied. Based on these results, we conclude that (a) maximum tension-generating capability and calcium sensitivity of tension development are related, perhaps causally, in fast skeletal and cardiac muscles, and (b) thin filament activation is less cooperative in cardiac muscle than in skeletal muscle, which explains the differential sensitivity of the two fiber types to temperature and TnC extraction. Reducing thin filament cooperativity in skeletal muscle by TnC extraction results in a response to temperature similar to that of control cardiac cells. This study provides evidence that force levels in striated muscle influence the calcium binding affinity of TnC.     

Functional characterization of junctional terminal cisternae from mammalian fast skeletal muscle sarcoplasmic reticulum

Junctional terminal cisternae are a recently isolated sarcoplasmic reticulum fraction containing two types of membranes, the junctional face membrane with morphologically intact "feet" structures and the calcium pump membrane [Saito, A., Seiler, S., Chu, A., & Fleischer, S. (1984) J. Cell Biol. 99, 875-885]. In this study, the Ca2+ fluxes of junctional terminal cisternae are characterized and compared with three other well-defined fractions derived from the sarcotubular system of fast-twitch skeletal muscle, including light and heavy sarcoplasmic reticulum, corresponding to longitudinal and terminal cisternae regions of the sarcoplasmic reticulum, and isolated triads. Functionally, junctional terminal cisternae have low net energized Ca2+ transport measured in the presence or absence of a Ca2+-trapping anion, as compared to light and heavy sarcoplasmic reticulum and triads. Ca2+ transport and Ca2+ pumping efficiency can be restored to values similar to those of light sarcoplasmic reticulum with ruthenium red or high [Mg2+]. In contrast to junctional terminal cisternae, heavy sarcoplasmic reticulum and triads have higher Ca2+ transport and are stimulated less by ruthenium red. Heavy sarcoplasmic reticulum appears to be derived from the nonjunctional portion of the terminal cisternae. Our studies indicate that the decreased Ca2+ transport is referable to the enhanced permeability to Ca2+, reflecting the predominant localization of Ca2+ release channels in junctional terminal cisternae. This conclusion is based on the following observations: The Ca2+, -Mg2+ -dependent ATPase activity of junctional terminal cisternae in the presence of a Ca2+ ionophore is comparable to that of light sarcoplasmic reticulum when normalized for the calcium pump protein content; i.e., the enhanced Ca2+ transport cannot be explained by a faster turnover of the pump. Ruthenium red or elevated [Mg2+] enhances energized Ca2+ transport and Ca2+ pumping efficiency in junctional terminal cisternae so that values approaching those of light sarcoplasmic reticulum are obtained. Rapid Ca2+ efflux in junctional terminal cisternae can be directly measured and is blocked by ruthenium red or high [Mg2+]. Ryanodine at pharmacologically significant concentrations blocks the ruthenium red stimulation of Ca2+ loading. Ryanodine binding in junctional terminal cisternae, which appears to titrate Ca2+ release channels, is 2 orders of magnitude lower than the concentration of the calcium pump protein. By contrast, light sarcoplasmic reticulum has a high Ca2+ loading rate and slow Ca2+ efflux that are not modulated by ruthenium red, ryanodine, or Mg2+.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of DHP binding protein in crayfish striated muscle

The dihydropyridine calcium channel blocker, [3H]PN 200-110, binds specifically also to crayfish muscle membranes, though with a binding capacity smaller than that measured with rabbit or human skeletal muscle membranes. [3H]PN 200-110 binding proteins from the crayfish T-tubules were solubilized and purified on WGA Sepharose or extracted from gel. The purified protein has a molecular mass of approximately 190 kDa under nonreducing conditions and was able to transport calcium after reconstitution. Polyclonal antibodies against crayfish T-tubules enriched with purified DHP-binding protein were shown to bind to DHP-binding protein from both the crayfish and the rabbit skeletal muscle, although not with the same intensity. Electron microscopy showed the presence of ovoid particles. Our results suggest that a voltage-dependent calcium channel may be present in crayfish skeletal muscle, which is homological with the L-type calcium channel in rabbit skeletal muscle.     

Passive tension in cardiac muscle: contribution of collagen, titin, microtubules, and intermediate filaments.

The passive tension-sarcomere length relation of rat cardiac muscle was investigated by studying passive (or not activated) single myocytes and trabeculae. The contribution of collagen, titin, microtubules, and intermediate filaments to tension and stiffness was investigated by measuring (1) the effects of KCl/KI extraction on both trabeculae and single myocytes, (2) the effect of trypsin digestion on single myocytes, and (3) the effect of colchicine on single myocytes. It was found that over the working range of sarcomeres in the heart (lengths approximately 1.9-2.2 microns), collagen and titin are the most important contributors to passive tension with titin dominating at the shorter end of the working range and collagen at longer lengths. Microtubules made a modest contribution to passive tension in some cells, but on average their contribution was not significant. Finally, intermediate filaments contributed about 10% to passive tension of trabeculae at sarcomere lengths from approximately 1.9 to 2.1 microns, and their contribution dropped to only a few percent at longer lengths. At physiological sarcomere lengths of the heart, cardiac titin developed much higher tensions (> 20-fold) than did skeletal muscle titin at comparable lengths. This might be related to the finding that cardiac titin has a molecular mass of 2.5 MDa, 0.3-0.5 MDa smaller than titin of mammalian skeletal muscle, which is predicted to result in a much shorter extensible titin segment in the I-band of cardiac muscle. Passive stress plotted versus the strain of the extensible titin segment showed that the stress-strain relationships are similar in cardiac and skeletal muscle. The difference in passive stress between cardiac and skeletal muscle at the sarcomere level predominantly resulted from much higher strains of the I-segment of cardiac titin at a given sarcomere length. By expressing a smaller titin isoform, without changing the properties of the molecule itself, cardiac muscle is able to develop significant levels of passive tension at physiological sarcomere lengths.     

Model of muscle-tendon interaction during frog semitendinosis fixed-end contractions.

A structural model was developed to explain sarcomere shortening at the expense of tendon lengthening in the frog semitendinosis (ST) muscle-tendon system. The model was based on the data of Lieber et al. [Am. J. Physiol. 261, C86-C92 (1991)], who determined the relationship between the sarcomere length, tendon load (as a fraction of maximum isometric tension) and tendon, bone-tendon junction (BTJ), and aponeurosis strain. The model was generated assuming a finite time-course of cross-bridge attachment [Huxley, Prog. Biophys. 7,255-318 (1957)], an ideal sarcomere length-tension relationship [Gordon et al., J. Physiol. 184, 170-192 (1966)] and an ideal force-velocity relationship [Katz, J. Physiol. 96, 45-64 (1939); Edman, J. Physiol. 291, 143-159 (1979)]. Functionally, sarcomeres operated on three distinct regions of the length-tension curve: (1) regions where the muscle force decreased as sarcomeres shortened (the shallow and steep ascending limbs); (2) regions where the muscle force increased as sarcomeres shortened and there was little passive tension (descending limb, where sarcomere length greater than or equal to 3.0 microns); and (3) regions where the muscle force increased as sarcomeres shortened and there was a significant passive tension (descending limb where sarcomere length greater than 3.0 microns). Using such a physiological model, it was found that the effect of tendon compliance was to 'skew' the sarcomere length-tension curve to the right and to increase the operating range of the muscle-tendon unit.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of the calcium release channel of cardiac and skeletal muscle sarcoplasmic reticulum by target inactivation analysis

The calcium release channel of sarcoplasmic reticulum which triggers muscle contraction in excitation-contraction coupling has recently been isolated. The channel has been found to be morphologically identical with the feet structures of the junctional face membrane of terminal cisternae and consists of an oligomer of a unique high molecular weight polypeptide. In this study, we compare the target size of the calcium release channel from heart and skeletal muscle using target inactivation analysis. The target molecular weights of the calcium release channel estimated by measuring ryanodine binding after irradiation are similar for heart (139,000) and skeletal muscle (143,000) and are smaller than the monomeric unit (estimated to be about 360,000). The target size, estimated by measuring polypeptide remaining after irradiation, was essentially the same for heart and skeletal muscle, 1,061,000 and 1,070,000, respectively, indicating an oligomeric association of protomers. Thus, the calcium release channel of both cardiac and skeletal muscle reacts uniquely with regard to target inactivation analysis in that (1) the size by ryanodine binding is smaller than the monomeric unit and (2) a single hit leads to destruction of more than one polypeptide, by measuring polypeptide remaining. Our target inactivation analysis studies indicate that heart and skeletal muscle receptors are structurally very similar.