Freezing point and melting point of barnacle muscle fibers

The freezing point and the melting point of myoplasm were measured with two experimental models. In all samples, a supercooled stage was reached by lowering the temperature of the sample to approximately - 7 degrees C, and the freezing of the sample was mechanically induced. The freezing process was associated with a phase transition in the interstices between the contractile filaments. In intact muscle fibers, the freezing point showed a structural component (0.43 degrees C), and the melting point indicated that the intracellular and the extracellular compartments are isotonic. When the sample of myoplasm, previously inserted in a cylindrical cavity was incubated in an electrolyte solution, the freezing point showed a structural component similar to that of the intact muscle fiber, but the melting point was lower than the freezing and the melting points of the embedding solution. This was interpreted as evidence that the counterions around the contractile filaments occupied a nonnegligible fraction of the intracellular compartment.     

Calcium Binding and Tension Development in Detergent-Treated Muscle Fibers

The nonionic detergent Brij 58 eliminates irreversibly the capability of the sarcoplasmic reticulum (SR) of skinned crayfish muscle fibers to sequester Ca and to release it under appropriate stimulation. In contrast to deoxycholate (DOC) which causes an irreversible diminution of tension as well, Brij 58 does not affect the contractile proteins. Comparison of the time-course of tension development before and after Brij treatment demonstrates that Ca is accessible to the contractile proteins more rapidly after the SR is destroyed but, nevertheless, much more slowly than is predicted for free diffusion of Ca in the myoplasm. Slowing apparently results because of the presence of ca 1 mmol/kg fiber of myoplasmic Ca-binding sites that remain after Ca uptake of the SR is eliminated. A theoretical model is presented which allows for the effects of binding sites and of an unstirred layer in the vicinity of the fiber on Ca diffusion into the myoplasm.     

Myoplasmic fixed charges of the barnacle muscle fiber (author's transl)]

The experimental model used to study diffusion and electrical conduction in the cytoplasm of large muscle fibers was adapted to evaluate the myoplasmic density of fixed charges. Membranes of myoplasm were prepared and phi X, the myoplasmic thermodynamically effective charge density, was calculated from the membrane potential (Kamo, N., Toyoshima, Y. and Kobatake, Y. (1971) Kolloid Z.u.Z. Polymère 1061--1068) when these membranes were used as the partition between two electrolyte solutions. The dilution of KCl in the external solutions reduced phi X, which increases with the reduction of the water content in the membrane of myoplasm. With a water content of 73.0 ml/100 g KCl concentration in the external medium equal to 0.15 M, phi X was evaluated to 0.058 equiv/l. The substitution of KCl by NaCl introduces a reduction in phi X of 20--50% depending on E1KCl] in the external solutions. The addition of ATP, Mg2+, and Ca2+ also causes a reduction of phi B by 30--50% according to the experimental conditions. These results indicate that the fraction of counterions dissociated from the myoplasmic macromolecules is reduced when the concentration of the counterions is diminished or when CKl is replaced by Nal. It also suggests a reduction of phi X during muscular contraction.     

Mechanical Properties of the Sarcolemma and Myoplasm in Frog Muscle as a Function of Sarcomere Length

The elastimeter method was applied to the single muscle fiber of the frog semitendinosus to obtain the elastic moduli of the sarcolemma and myoplasm, as well as their relative contributions to resting fiber tension at different extensions. A bleb which was sucked into a flat-mouthed pipette at the fiber surface separated into an external sarcolemmal membrane and a thick inner myoplasmic region. Measurements showed that the sarcolemma does not contribute to intact fiber tension at sarcomere lengths below 3 µ. It was estimated that the sarcolemma contributed on the order of 10% to intact fiber tension at sarcomere lengths between 3 and 3.75 µ, and more so with further extension. Between these sarcomere lengths, the sarcolemma can be linearly extended and has a longitudinal elastic modulus of 5 x 10⁶ dyne/cm² (assuming a thickness of 0.1 µ). Resistance to deformation of the inner bleb region is due to myoplasmic elasticity. The myoplasmic elastic modulus was estimated by use of a model and was used to predict a fiber length-tension curve which agreed approximately with observations.     

Absorbance signals from resting frog skeletal muscle fibers injected with the pH indicator dye, phenol red

Singly dissected twitch fibers from frog muscle were studied on an optical bench apparatus after micro-injection with the pH indicator dye, phenol red. Dye-related absorbances in myoplasm, denoted by A0(lambda) and A90(lambda), were estimated as a function of wavelength lambda (450 nm less than or equal to lambda less than or equal to 640 nm) with light polarized parallel (0 degrees) and perpendicular (90 degrees) to the fiber axis respectively. At all lambda, A0(lambda) was slightly greater than A90(lambda), indicating that some of the phenol red molecules were bound to oriented structures accessible to myoplasm. The phenol red "isotropic" signal, [A0(lambda) + 2A90(lambda)]/3, a quantity equal to the average absorbance of all the dye molecules independent of their orientation, had a spectral shape that was red-shifted by approximately 10 nm in comparison with in vitro dye calibration curves measured in 140 mM KCl. The red-shifted spectrum also indicates that some phenol red molecules were bound in myoplasm. A quantitative estimate of indicator binding was obtained from measurements of the dye's apparent diffusion constant in myoplasm, denoted by Dapp. The small value of Dapp, 0.37 x 10(-6) cm2 s-1 (at 16 degrees C), can be explained if approximately 80% of the dye was bound to myoplasmic sites of low mobility. To estimate the apparent myoplasmic pH, denoted by pHapp, the isotropic absorbance of phenol red was fitted by in vitro calibration spectra. pHapp was found to be independent of dye concentration (0.2-2 mM), but varied widely (range, 6.8-7.5; mean value, 7.17) among fibers judged from functional characteristics to be normal. When fibers were subjected to acid or alkaline loads by exposure to Ringer's solution containing, respectively, dissolved CO2 or NH3, the changes in pHapp were in agreement with those expected from pH micro-electrode studies. It is concluded that in spite of the several indications for the presence of bound phenol red inside muscle cells, the pHapp signal from the indicator is useful for monitoring changes in myoplasmic pH in response to physiological and pharmacological manipulations.     

Impedance of Membrane and Myoplasm during the Action Potential of Frog Muscle

The transient imbalance of a Wheatstone bridge was used to estimate the changes in both membrane and myoplasmic impedance that occurred as an action potential propagated over a 1 mm length of a frog muscle fiber. It was also used to estimate the changes in the membrane impedance alone. Alterations in myoplasmic impedance that might have been predicted were not found.     

Use of fura red as an intracellular calcium indicator in frog skeletal muscle fibers.

Fura red, a fluorescent Ca2+ indicator with absorbance bands at visible wavelengths, was injected into intact single muscle fibers that had been stretched to a long sarcomere length (approximately 3.8 microns) and bathed in a 'high-Ca2+' Ringer ([Ca2+] = 11.8 mM). From fura red's slow diffusion coefficient in myoplasm, 0.16 (+/- 0.01, SEM) x 10(-6) cm2 s-1 (N = 5; 16 degrees C), it is estimated that approximately 85% of the indicator molecules are bound to muscle constituents of large molecular weight. Binding appears to elevate, by 3- to 4-fold, the indicator's apparent dissociation constant for Ca2+ (KD), which is estimated to be 1.1-1.6 microM in myoplasm. Fura red's myoplasmic absorbance spectrum was used to estimate fr, the fraction of fura red molecules in the Ca2+-bound form at rest. In 3 fibers thought to be minimally damaged by the micro-injection, fr was estimated to be 0.15 (+/- 0.01). Thus, resting myoplasmic free [Ca2+] ([Ca2+]r) is estimated to be 0.19-0.28 microM. For fibers in normal Ringer solution ([Ca2+] = 1.8 mM), at shorter sarcomere length (approximately 2.7 microns), and containing a nonperturbing concentration of indicator (< or = 0.2 mM), [Ca2+]r is estimated to be 0.18-0.27 microM. This range is higher than estimated previously in frog fibers with other techniques. In 6 fibers, R, the indicator's fluorescence ratio signal (equal to the emission intensity measured with 420 nm excitation divided by that measured with 480 nm excitation), was measured at rest and following electrical stimulation and compared with absorbance measurements made from the same fiber region. The analysis implies that RMIN and RMAX (the values of R that would be measured if all indicator molecules were in the Ca(2+)-free and Ca(2+)-bound states, respectively) were substantially smaller in myoplasm than in calibration solutions lacking muscle proteins. Several methods for estimation of [Ca2+]r from R are analyzed and discussed.     

Remodeling of calcium handling in skeletal muscle through PGC-1α: impact on force, fatigability, and fiber type

Regular endurance exercise remodels skeletal muscle, largely through the peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α). PGC-1α promotes fiber type switching and resistance to fatigue. Intracellular calcium levels might play a role in both adaptive phenomena, yet a role for PGC-1α in the adaptation of calcium handling in skeletal muscle remains unknown. Using mice with transgenic overexpression of PGC-1α, we now investigated the effect of PGC-1α on calcium handling in skeletal muscle. We demonstrate that PGC-1α induces a quantitative reduction in calcium release from the sarcoplasmic reticulum by diminishing the expression of calcium-releasing molecules. Concomitantly, maximal muscle force is reduced in vivo and ex vivo. In addition, PGC-1α overexpression delays calcium clearance from the myoplasm by interfering with multiple mechanisms involved in calcium removal, leading to higher myoplasmic calcium levels following contraction. During prolonged muscle activity, the delayed calcium clearance might facilitate force production in mice overexpressing PGC-1α. Our results reveal a novel role of PGC-1α in altering the contractile properties of skeletal muscle by modulating calcium handling. Importantly, our findings indicate PGC-1α to be both down- as well as upstream of calcium signaling in this tissue. Overall, our findings suggest that in the adaptation to chronic exercise, PGC-1α reduces maximal force, increases resistance to fatigue, and drives fiber type switching partly through remodeling of calcium transients, in addition to promoting slow-type myofibrillar protein expression and adequate energy supply.     

The myoplasm of ascidian eggs: a localized cytoskeletal domain with multiple roles in embryonic development

The myoplasm of ascidian eggs is a localized cytoplasmic region containing a unique cytoskeletal domain. During ooplasmic segregation, the myoplasm moves first to the vegetal pole and then to the future posterior region of the fertilized egg, where it subsequently enters the muscle cell lineage during cleavage. In the vegetal pole region, the myoplasm defines a developmental center which later controls gastrulation and embryonic axis formation. In the posterior region, the myoplasm defines another developmental center, which specifies muscle cell development. Evidence is described suggesting that the integrity of the myoplasmic cytoskeletal domain is required for normal embryonic functions of the myoplasm.     

Molecular characterization of myoplasmin-C1: a cytoskeletal component localized in the myoplasm of the ascidian egg

 Myoplasmin-C1 is a polypeptide detected by a monoclonal antibody, which is localized in the myoplasm of ascidian eggs. Since microinjection of the antibody blocks larval muscle development, myoplasmin-C1 may play a role in muscle cell differentiation (Nishikata et al. 1987). Isolation and characterization of myoplasmin-C1 cDNA clones revealed that the predicted amino acid sequence of myoplasmin-C1 had no similarity to any known protein. However, the deduced protein contains heptad repeats similar to those in myosin heavy chain, tropomyosin and the Drosophila Bicaudal D gene product, suggesting that it is a filamentous component of the myoplasmic cytoskeleton. The predicted amino acid sequence also showed several possible phosphorylation sites. Consistent with the prediction that myoplasmin-C1 is a cytoskeletal component, the protein remained in the myoplasmic cytoskeletal domain after detergent extraction. These results suggest that myoplasmin-C1 is a cytoskeletal component of the myoplasm and that it plays a role in anchoring and segregating muscle determinants. Received: 6 October 1995 / Accepted in revised form: 7 December 1995     

Charges fixes du protoplasme des fibres musculaires de balaneMyoplasmic fixed charges of the barnacle muscle fiber

The experimental model used to study diffusion and electrical conduction in the cytoplasm of large muscle fibers was adapted to evaluate the myoplasmic density of fixed charges. Membranes of myoplasm were prepared and øX, the myoplasmic thermodynamically effective charge density, was calculated from the membrane potential (Kamo, N., Toyoshima, Y. and Kobatake, Y. (1971) Kolloid Z.u.Z. Polymère 1061–1068) when these membranes were used as the partition between two electrolyte solutions. The dilution of KCl in the external solutions reduced øX, which increases with the reduction of the water content in the membrane of myoplasm. With a water content of 73.0 ml/100g KCl concentration in the external medium equal to 0.15 M, øX was evaluated to 0.058 equiv/l. The substitution of KCl by NaCl introduces a reduction in øX of 20–50% depending on [KCl] in the external solutions. The addition of ATP, Mg²⁺ and Ca²⁺ also causes a reduction of øX by 30–50% according to the experimental conditions. These results indicate that the fraction of counterions dissociated from the myoplasmic macromolecules is reduced when the concentration of the counterions is diminished or when KCl is replaced by NaCl. It also suggests a reduction of øX during muscular contraction.     

Myoplasmic calcium transients in intact frog skeletal muscle fibers monitored with the fluorescent indicator furaptra

Furaptra (Raju, B., E. Murphy, L. A. Levy, R. D. Hall, and R. E. London. 1989. Am. J. Physiol. 256:C540-C548) is a "tri-carboxylate" fluorescent indicator with a chromophore group similar to that of fura-2 (Grynkiewicz, G., M. Poenie, and R. Y. Tsien. 1985. J. Biol. Chem. 260:3440-3450). In vitro calibrations indicate that furaptra reacts with Ca2+ and Mg2+ with 1:1 stoichiometry, with dissociation constants of 44 microM and 5.3 mM, respectively (16-17 degrees C; ionic strength, 0.15 M; pH, 7.0). Thus, in a frog skeletal muscle fiber stimulated electrically, the indicator is expected to respond to the change in myoplasmic free [Ca2+] (delta[Ca2+]) with little interference from changes in myoplasmic free [Mg2+]. The apparent longitudinal diffusion constant of furaptra in myoplasm was found to be 0.68 (+/- 0.02, SEM) x 10(-6) cm2 s-1 (16-16.5 degrees C), a value which suggests that about half of the indicator was bound to myoplasmic constituents of large molecular weight. Muscle membranes (surface and/or transverse-tubular) appear to have some permeability to furaptra, as the total quantity of indicator contained within a fiber decreased after injection; the average time constant of the loss was 302 (+/- 145, SEM) min. In fibers containing less than 0.5 mM furaptra and stimulated by a single action potential, the calibrated peak value of delta[Ca2+] averaged 5.1 (+/- 0.3, SEM) microM. This value is about half that reported in the preceding paper (9.4 microM; Konishi, M., and S. M. Baylor. 1991. J. Gen. Physiol. 97:245-270) for fibers injected with purpurate-diacetic acid (PDAA). The latter difference may be explained, at least in part, by the likelihood that the effective dissociation constant of furaptra for Ca2+ is larger in vivo than in vitro, owing to the binding of the indicator to myoplasmic constituents. The time course of furaptra's delta[Ca2+], with average values (+/- SEM) for time to peak and half-width of 6.3 (+/- 0.1) and 9.5 (+/- 0.4) ms, respectively, is very similar to that of delta[Ca2+] recorded with PDAA. Since furaptra's delta[Ca2+] can be recorded at a single excitation wavelength (e.g., 420 nm) with little interference from fiber intrinsic changes, movement artifacts, or delta[Mg2+], furaptra represents a useful myoplasmic Ca2+ indicator, with properties complementary to those of other available indicators.     

Some quantitative aspects of myoplasmic ATPMg and total internal ATP and ArP levels in resting barnacle muscle fibres

1. Flash height recorded following the injection of firefly into the external calibration medium depends on the concentration of K-glutamate used, e.g. 120 mM K glutamate reduces flash height by approximately 20 percent. 2. Suspending the fibre in air instead of artificial sea water (ASW) or replacement of NaCl in the bathing medium with Na-glutamate fails to alter flash height. 3. Firefly preparations from DuPont, Packard and SAI give similar myoplasmic ATPMg values viz. 1.1 mM. 4. Analysis of 36 fibres shows the following: myoplasmic ATP = 1.03 +/- 0.06 mM; total ATP (firefly method) = 5.26 +/- 0.12 mmol/kg water; total ATP (enzymic fluorimetry) = 6.27 +/- 0.13 mmol/kg water and ArP = 20.76 +/- 0.59 mmol/kg water. 5. Measurement of ATPMg in samples of myoplasmic aspirate gives a value that is greater than that obtained in situ. 6. Iodoacetate, whether applied externally or internally, reduces resting luminescence in a dose-dependent manner. It also reduces myoplasmic ATP and total ATP. 7. 2-Deoxy-d-glucose fails to reduce myoplasmic ATP but reduces total ATP. 8. Diethylpyrocarbonate, whether applied externally or internally, reduces myoplasmic ATP. It also causes a slow decline in ArP but little change in total ATP. 9. Injection of L-arginine causes a fall in resting luminescence in some fibres while in others it causes a prompt transitory rise. Injection of L-arginine also causes a fall in total ATP. 10. Collectively, these results suggest that the immediate buffering system in the myoplasm is ArP and that ATP supplied by glycolysis lies in a compartment, presumably the interfibrillar space, which is inaccessible to injected firefly.     

Myoplasmic calcium transients monitored with purpurate indicator dyes injected into intact frog skeletal muscle fibers

Intact single twitch fibers from frog muscle were studied on an optical bench apparatus after microinjection with tetramethylmurexide (TMX) or purpurate-3,3' diacetic acid (PDAA), two compounds from the purpurate family of absorbance Ca2+ indicators previously used in cut muscle fibers (Maylie, J., M. Irving, N. L. Sizto, G. Boyarsky, and W. K. Chandler. 1987. J. Gen. Physiol. 89:145-176; Hirota, A., W. K. Chandler, P. L. Southwick, and A. S. Waggoner. 1989. J. Gen. Physiol. 94:597-631.) The apparent longitudinal diffusion constant of PDAA (mol wt 380) in myoplasm was 0.99 (+/- 0.04, SEM) x 10(-6) cm2 s-1 (16-17 degrees C), a value which suggests that 24-43% of the PDAA molecules were bound to myoplasmic constituents of large molecular weight. The corresponding values for TMX (mol wt 322) were 0.98 (+/- 0.05) x 10(-6) cm2 s-1 and 44-50%, respectively. Muscle membranes (surface and/or transverse-tubular) appear to be permeable to TMX and, to a lesser extent, to PDAA, since the total amount of indicator contained within a fiber decreased with time after injection. The average time constants for disappearance of indicator were 46 (+/- 7, SEM) min for TMX and 338 (+/- 82) min for PDAA. The fraction of indicator in the Ca2(+)-bound state in resting fibers was significantly different from zero for TMX (0.070 +/- 0.008) but not for PDAA (0.026 +/- 0.009). In in vitro calibrations PDAA but not TMX appeared to react with Ca2+ with 1:1 stoichiometry. In agreement with Hirota et al. (Hirota, A., W. K. Chandler, P. L. Southwick, and A. S. Waggoner. 1989. J. Gen. Physiol. 94:597-631), we conclude that PDAA is probably a more reliable myoplasmic Ca2+ indicator than TMX. In fibers that contained PDAA and were stimulated by a single action potential, the calibrated peak value of the myoplasmic free [Ca2+] transient (delta[Ca2+]) averaged 9.4 (+/- 0.6) microM, a value about fivefold larger than that calibrated with antipyrylazo III under otherwise identical conditions (Baylor, S. M., and S. Hollingworth. 1988. J. Physiol. 403:151-192). The fivefold difference is similar to that previously reported in cut fibers with antipyrylazo III and PDAA. Since in both intact and cut fibers the percentage of PDAA bound to myoplasmic constituents is considerably smaller than that found for antipyrylazo III, the PDAA calibration of delta[Ca2+] is likely to be more accurate. Interestingly, in intact fibers the peak value of delta[Ca2+] calibrated with either PDAA or antipyrylazo III is about half that calibrated in cut fibers.(ABSTRACT TRUNCATED AT 400 WORDS)     

Improved electrical coupling in uterine smooth muscle is associated with increased numbers of gap junctions at parturition

We have studied some passive electrical properties of uterine smooth muscle to determine whether a change in electrical parameters accompanies gap junction formation at delivery. The length constant of the longitudinal myometrium increased from 2.6 +/- 0.8 mm (X +/- SD) before term to 3.7 +/- 1 mm in tissues from delivering animals. The basis of the change was a 33% decrease in internal resistance and a 46% increase in membrane resistance. Axial current flow in an electrical syncytium such as myometrium is impeded by the cytoplasm of individual cells plus the junctions between cells. Measurement of the longitudinal impedance indicated that the specific resistance of the myoplasmic component was constant at 319 +/- 113 omega . cm before term and 340 +/- 93 omega . cm at delivery. However, a decrease in junctional resistance was apparent from 323 +/- 161 omega . cm to 134 +/- 64 omega . cm at delivery. 1.5-2 d after delivery, the junctional resistance was increased, as was the myoplasmic resistance. Thin-section electron microscopy of some of the same muscle samples showed that gap junctions were present in significantly greater numbers in the delivering tissues. Therefore, our results support the hypothesis that gap junction formation at delivery is associated with improved electrical coupling of uterine smooth muscle.     

Direct observation of failing fibers in muscles of dystrophic mice provides mechanistic insight into muscular dystrophy

Duchenne muscular dystrophy is caused by the absence of the protein dystrophin. Dystrophin's function is not known, but its cellular location and associations with both the force-generating contractile core and membrane-spanning entities suggest a role in mechanically coupling force from its intracellular origins to the fiber membrane and beyond. We report here the presence of destructive contractile activity in lumbrical muscles from dystrophin-deficient (mdx) mice during nominally quiescent periods following exposure to mechanical stress. The ectopic activity, which was observable microscopically, resulted in longitudinal separation and clotting of fiber myoplasm and was absent when calcium (Ca(2+)) was removed from the bathing medium. Separation and clotting of myoplasm were also produced in dystrophin-deficient muscles by local application of a Ca(2+) ionophore to create membrane breaches in the absence of mechanical stress, whereas muscles from control mice tolerated ionophore-induced entry of Ca(2+) without damage. These observations suggest a failure cascade in dystrophin-deficient fibers that 1) is initiated by a stress-induced influx of extracellular Ca(2+), causing localized activation to continue after cessation of stimulation, and 2) proceeds as the persistent local activation, combined with reduced lateral mechanical coupling between the contractile core and the extracellular matrix, results in longitudinal separation of myoplasm in nonactivated regions of the fiber. This mechanism invokes both the membrane stabilization and the mechanical coupling functions frequently proposed for dystrophin and suggests that, whereas the absence of either function alone is not sufficient to cause fiber failure, their combined absence is catastrophic.     

Characterization of Antigenic Polypeptides Recognized by Monoclonal Antibodies Specific to the Myoplasm of Eggs of the Ascidian Ciona intestinalis

The determinants responsible for the differentiation of ascidian larval muscle cells are thought to be contained within the egg myoplasm. To analyze the macromolecules composing the myoplasm, several hybridoma cell lines which secrete monoclonal antibodies specific to myoplasmic components of Ciona eggs have been established (17). In the present investigation, seven of these myoplasm-specific antigens were characterized according to their molecular features and distribution patterns within the egg cytoplasm. Four of the seven antigenic polypeptides were shown to be components of the cortical cytoplasm, two were related to mitochondria, and one is likely to be a yolk protein. An antigen recognized by IIG6B2 antibody, which inhibited muscle development when injected into fertilized eggs, was a single polypeptide of relative molecular mass about 40,000 and isoelectric point about 5. The antigen was designated myoplasmin-C1 after its characteristic localization. The IIF9E9 antigen was a single 35-kDa polypeptide related to mitochondria and was thus designated myoplasmin-M1. The other five antibodies recognized two or more spots by immunoblotting analysis using two-dimensional gel electrophoresis. All of these myoplasm-specific antigens, except for the IIH10D6 antigen, are likely to be produced by the oocyte itself. Synthesis of IIH10D6 antigen seems to be associated with test cells.     

Perturbation of sarcoplasmic reticulum calcium release and phenol red absorbance transients by large concentrations of fura-2 injected into frog skeletal muscle fibers

Intact single twitch fibers from frog muscle were studied on an optical bench apparatus after micro-injection with two indicator dyes: phenol red, to monitor a previously described signal (denoted delta pHapp; Hollingworth and Baylor. 1990. J. Gen. Physiol. 96:473-491) possibly reflective of a myoplasmic pH change following action potential stimulation; and fura-2, to monitor the associated change in the myoplasmic free calcium concentration (delta[Ca2+]). Additionally, it was expected that large myoplasmic concentrations of fura-2 (0.5-1.5 mM) might alter delta pHapp, since it was previously found (Baylor and Hollingworth. 1988. J. Physiol. 403:151-192) that the Ca2(+)-buffering effects of large fura-2 concentrations: (a) increase the estimated total concentration of Ca2+ (denoted by delta[CaT]) released from the sarcoplasmic reticulum (SR), but (b) reduce and abbreviate delta[Ca2+]. The experiments show that delta pHapp was increased at the larger fura-2 concentrations; moreover, the increase in delta pHapp was approximately in proportion to the increase in delta[CaT]. At all fura-2 concentrations, the time course of delta pHapp, through time to peak, was closely similar to, although probably slightly slower than, that of delta[CaT]. These properties of delta pHapp are consistent with an hypothesis proposed by Meissner and Young (1980. J. Biol. Chem. 255:6814-6819) and Somlyo et al. (1981. J. Cell Biol. 90:577-594) that a proton flux from the myoplasm into the SR supplies a portion of the electrical charge balance required as Ca2+ is released from the SR into the myoplasm. A comparison of the amplitude of delta pHapp with that of delta[CaT] indicates that, in response to a single action potential, 10-15% of the charge balance required for Ca2+ release may be carried by protons.     

The excitation–contraction coupling mechanism in skeletal muscle

First coined by Alexander Sandow in 1952, the term excitation–contraction coupling (ECC) describes the rapid communication between electrical events occurring in the plasma membrane of skeletal muscle fibres and Ca²⁺ release from the SR, which leads to contraction. The sequence of events in twitch skeletal muscle involves: (1) initiation and propagation of an action potential along the plasma membrane, (2) spread of the potential throughout the transverse tubule system (T-tubule system), (3) dihydropyridine receptors (DHPR)-mediated detection of changes in membrane potential, (4) allosteric interaction between DHPR and sarcoplasmic reticulum (SR) ryanodine receptors (RyR), (5) release of Ca²⁺ from the SR and transient increase of Ca²⁺ concentration in the myoplasm, (6) activation of the myoplasmic Ca²⁺ buffering system and the contractile apparatus, followed by (7) Ca²⁺ disappearance from the myoplasm mediated mainly by its reuptake by the SR through the SR Ca²⁺ adenosine triphosphatase (SERCA), and under several conditions movement to the mitochondria and extrusion by the Na⁺/Ca²⁺ exchanger (NCX). In this text, we review the basics of ECC in skeletal muscle and the techniques used to study it. Moreover, we highlight some recent advances and point out gaps in knowledge on particular issues related to ECC such as (1) DHPR-RyR molecular interaction, (2) differences regarding fibre types, (3) its alteration during muscle fatigue, (4) the role of mitochondria and store-operated Ca²⁺ entry in the general ECC sequence, (5) contractile potentiators, and (6) Ca²⁺ sparks.     

Fluorescence signals from the Mg2+/Ca2+ indicator furaptra in frog skeletal muscle fibers.

The fluorescent Mg2+/Ca2+ indicator, furaptra, was injected into single frog skeletal muscle fibers, and the indicator's fluorescence signals were measured and analyzed with particular interest in the free Mg2+ concentration ([Mg2+]) in resting muscle. Based on the fluorescence excitation spectrum of furaptra, the calibrated myoplasmic [Mg2+] level averaged 0.54 mM, if the value of dissociation constant (KD) for Mg2+ obtained in vitro (5.5 mM) was used. However, if the indicator reacts with Mg2+ with a two-fold larger KD in myoplasm, as previously suggested for the furaptra-Ca2+ reaction (M. Konishi, S. Hollingworth, A.B. Harkins, S.M. Baylor. 1991. J. Gen. Physiol. 97:271-301), the calculated [Mg2+] would average 1.1 mM. Thus, the value 1.1 mM probably represents the best estimate from furaptra of [Mg2+] in resting muscle fibers. Extracellular perfusion of muscle fibers with high Mg2+ concentration solution or low Na+ concentration solution did not cause any detectable changes in the [Mg2+]-related furaptra fluorescence within 4 min. The results suggest that the myoplasmic [Mg2+] is highly regulated near the resting level of 1 mM, and that changes only occur with a very slow time course.