1H-NMR studies of the intracellular water of skeletal muscle fibers under various physiological conditions.

Intracellular water of frog skeletal muscle fibers has been studied under various physiological conditions by use of the 1H-nuclear magnetic resonance (NMR) technique. 1H-NMR spectra of muscle fibers had single peaks derived from the intracellular water. The spectra changed in a characteristic fashion when the fiber axis was aligned at various angles relative to the magnetic field of the NMR magnet. Further, the relaxation rates of the 1H-NMR spectra changed depending on the water content of muscle fibers, and in association with contraction and rigor formation of muscle fibers. The obtained results indicate that the intracellular water of muscle fibers is structured and aligned along the myofilaments, and further that the state of the intracellular water changes with physiological conditions.     

Effect of viscosity on mechanics of single, skinned fibers from rabbit psoas muscle.

Muscle contraction is highly dynamic and thus may be influenced by viscosity of the medium surrounding the myofilaments. Single, skinned fibers from rabbit psoas muscle were used to test this hypothesis. Viscosity within the myofilament lattice was increased by adding to solutions low molecular weight sugars (disaccharides sucrose or maltose or monosaccharides glucose or fructose). At maximal Ca2+ activation, isometric force (Fi) was inhibited at the highest solute concentrations studied, but this inhibition was not directly related to viscosity. Solutes readily permeated the filament lattice, as fiber diameter was unaffected by added solutes (except for an increased diameter with Fi < 30% of control). In contrast, there was a linear dependence upon 1/viscosity for both unloaded shortening velocity and also the kinetics of isometric tension redevelopment; these effects were unrelated to either variation in solution osmolarity or inhibition of force. All effects of added solute were reversible. Inhibition of both isometric as well as isotonic kinetics demonstrates that viscous resistance to filament sliding was not the predominant factor affected by viscosity. This was corroborated by measurements in relaxed fibers, which showed no significant change in the strain-rate dependence of elastic modulus when viscosity was increased more than twofold. Our results implicate cross-bridge diffusion as a significant limiting factor in cross-bridge kinetics and, more generally, demonstrate that viscosity is a useful probe of actomyosin dynamics.     

Regulation by magnesium of intracellular calcium movement in skinned muscle fibers

The effect of Mg on Ca movement between the sarcoplasmic reticulum (SR) and myofilament space (MFS) was studied in skinned muscle fibers by using isometric force as an indicator of MFS Ca. In Ca-loaded fibers at 20 degrees C, the large force spike induced by Ca in 1 mM Mg (5 mM ATP) was strongly inhibited in 3 mM Mg, and force development was extremely slow. After a brief Ca stimulus in 1 mM Mg, relaxation in Ca-free solution was significantly faster in 3 mM Mg. These changes were due to altered Ca movements, since the effect of 3 mM Mg on steady force in CaEGTA solutions was small. Changes in Mg alone induced force transients apparently due to altered Ca movement. In relaxed fibers, decreasing the Mg to 0.25 mM caused phasic force development. In contracting fibers in Ca solutions, increasing the Mg caused a large transient relaxation. The effects of increased Mg were antagonized by 0.5 mM Cd, an inhibitor of the SR Ca transport system. The results indicate that active Ca uptake by the SR in situ is stimulated by Mg, and that it can affect local MFS [Ca++] in the presence of a substantial Ca source. These results provide evidence that an increased rate of Ca uptake in 3 mM Mg could account for inhibition of the large force spike associated with Ca-induced Ca release in skinned fibers.     

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.     

Optical diffraction study of muscle fibers. II. Electro-optical properties of muscle fibers.

When an electric field is applied along the fiber axis, the intensities of all observable optical diffraction lines of skeletal muscle fibers increase. This electro-optical effect was extensively studied and it was confirmed that the effect is due to the interaction between electric dipole moments of thin filaments and the applied field. From the present study on the intensity modulation due to applied field in sinusoidal and square forms, we confirmed that (1) the thin filament is a semiflexible rod, (2) the second order mode of the bending motion of thin filaments contributes to the electro-optical effect of muscle fibers at higher frequencies of a sinusodidal field or shorter durations of a square field, (3) the induced moment has no appreciable effect, and (4) the estimated value of the flexural rigidity of thin filaments strongly depends on the concentrations of free calcium ions in the myofibrillar space.     

The myofilament lattice: studies on isolated fibers. IV. Lattice equilibria in striated muscle.

Accounts of similarities between the thick filament lattice of striated muscle and smectic liquid-crystalline structures have focused upon an equilibrium between electrostatic (repulsive) and van der Waal's (attractive) forces. In living, intact muscle the fiber volume constitutes an additional important parameter which influences the amount of interaxial separation between the filaments. This is demonstrable by comparison of the lattice behavior of living fibers with that of fibers from which the sarcolemma has either been removed or made leaky by glycerination. These comparisons were made mainly by low-angle X-ray diffraction under conditions of changes in sarcomere length, ionic strength or osmolarity, and pH. Single fibers with the sarcolemma removed and glycerinated muscle have lattices which behave in accord with equilibrium liquid-crystalline systems in which the thick filament spacing is determined by the balance between electrostatic and van der Waal's forces. Conversely, osmotic and shortening studies demonstrate that the living, intact muscle has a lattice which behaves in accord with the so-called non-equilibrium (volume-constrained) liquid-crystalline condition in which the interaxial separation between the thick filaments is solely due to the amount of volume available as determined by the Donnan steady-state across the sarcolemma.     

Permeability of barnacle muscle fibers to water and nonelectrolytes

Summary The permeability of isolated muscle fibers of the giant barnacleMegabalanus psittacus to water and nonelectrolytes was studied by determining their reflection and permeability coefficients. Reflection coefficients were obtained by comparing the osmotic fluxes produced by a test molecule to that produced by the impermeant sucrose molecule. Permeability coefficients were determined for measurements of tracer uptake.The results indicate that, in these fibers, nonelectrolyte permeability is closely related to lipid solubility and molecular size.A value of 2.16×10^–12 cm³/sec dyne for the hydraulic conductivity and a value of 10.45×10^–4 cm/sec for³HHO permeability coefficient were obtained.The effect of membrane surface invaginations and clefts on the determination of permeability coefficients is discussed.     

Excitation of skinned muscle fibers by imposed ion gradients. IV. Effects of stretch and perchlorate ion

Depolarizing ion gradients stimulate 45Ca release in skeletal muscle fibers skinned by microdissection. Several lines of indirect evidence suggest that sealed transverse (T) tubules rather than sarcoplasmic reticulum (SR) are the locus of such stimulatory depolarization. Two implications of this hypothesis were tested. (a) A requirement for signal transmission was evaluated from the stimulation of 45Ca efflux in fibers that had been highly stretched, an intervention that can impair the electrical stimulation of intact fibers. Length was increased over approximately 95-115 s, after loading with 45Ca and rinsing at normal length; prestimulus 45Ca loss due to stretch itself was very small. In the first study, stimulation of 45Ca release by KCl replacement of K propionate was inhibited completely in fibers stretched to twice slack length, compared with fibers at 1.05-1.1 times slack length. Identical protocols did not alter 45Ca release stimulated by caffeine or Mg2+ reduction, implying that SR Ca release per se was fully functional and inhibition was selective for a preceding step in ionic stimulation. In a second study, stimulation by choline Cl replacement of K methanesulfonate, at constant [K+] [Cl-] product, was inhibited strongly; total 45Ca release decreased 69%, and stimulation above control loss decreased 78%, in segments stretched to twice the length at which sarcomere spacing had been 2.2 micron, compared with paired controls from the same fibers kept at 2.3 micron. (b) Perchlorate potentiation of T tubule activation was evaluated in fibers stimulated at constant [K+] [Cl-] at normal length (2.3 micron); this anion shifts the voltage dependence of intramembrane charge movement and contractile activation in intact fibers. Perchlorate (8 mM) potentiated both submaximal stimulation of Ca2+-dependent 45Ca release by partial choline Cl replacement of K methanesulfonate and the small Ca2+-insensitive 45Ca efflux component stimulated by nearly full replacement in the presence of 5 mM EGTA. These results provide independent support for the hypothesis that the T tubules are the locus of stimulation by depolarizing ion gradients, with junctional transmission of this signal causing SR 45Ca release.