Donnan potentials from striated muscle liquid crystals. Sarcomere length dependence.

Donnan potentials from A-bands and I-bands were measured as a function of sarcomere length in skinned long-tonic muscle fibers of the crayfish. These measurements were made using standard electrophysiological technique. Simultaneously, the relative cross-sectional area of the fibers was determined. Lattice plane spacings and hence unit-cell volumes were determined by low-angle x-ray diffraction. At a sarcomere length at which the myosin filaments and actin filaments nominally do not overlap, measurements of potential, relative cross-sectional area, and unit-cell volume were used in conjunction with Donnan equilibrium theory to calculate the effective linear charge densities along the myosin filament (6.6 X 10(4) e-/mu) and actin filament (6.8 X 10(3) e-/mu). Using these linear charge densities, unit-cell volumes and Donnan equilibrium theory, an algorithm was developed to predict A-band and I-band potentials at any sarcomere length. Over the range of sarcomere lengths investigated, the predicted values coincide with the experimental data. The ability of the model to predict the data demonstrates the applicability of Donnan equilibrium theory to measurements of electrochemical potential from liquid-crystalline systems.     

Donnan potentials from striated muscle liquid crystals. Lattice spacing dependence.

Electrochemical potentials were measured as a function of myofilament packing density in crayfish striated muscle. The A-band striations are supramolecular smectic B1 lattice assemblies of myosin filaments and the I-band striations are nematic liquid crystals of actin filaments. Both A- and I-bands generate potentials derived from the fixed charge that is associated with structural proteins. In the reported experiments, filament packing density was varied by osmotically reducing lattice volume. The electrochemical potentials were measured from the A- and I-bands in the relaxed condition over a range of lattice volumes. From the measurements of relative cross-sectional area, unit-cell volume (obtained by low-angle x-ray diffraction) and previously determined effective linear charge densities (Aldoroty, R.A., N.B. Garty, and E.W. April, 1985, Biophys. J., 47:89-96), Donnan potentials can be predicted for any amount of compression. In the relaxed condition, the predicted Donnan potentials correspond to the measured electrochemical potentials. In the rigor condition, however, a net increase in negative charge associated with the myosin filament is observed. The predictability of the data demonstrates the applicability of Donnan equilibrium theory to the measurement of electrochemical potentials from liquid-crystalline systems. Moreover, the relationship between filament spacing and the Donnan potential is consistent with the concept that surface charge provides the necessary electrostatic force to stabilize the myofilament lattice.     

The effect of temperature on the Donnan potentials in biological polyelectrolyte gels: cornea and striated muscle

The effect of temperature on the fixed electric charge in biological polyelectrolyte gels was studied between 10 and 35 degrees C using the Donnan microelectrode technique. Two tissues; cornea and striated muscle were used. In cornea, there is a gentle and uniform decrease in fixed charge over the temperature range. In rigor muscle, there is a dramatic step-function decrease in charge at around 28 degrees C. There is a charge decrease in relaxed muscle at around the same temperature, but the step function is less distinct. The significance of these different experimental relationships is discussed in relation to the Saroff model for ion binding to proteins, linked to the possible disordering effects of excess electric charge. The diverse effects in these systems are important for the physiological functions of the different tissues.     

Donnan potentials in rabbit psoas muscle in rigor.

Collins and Edwards (1971, Am. J. Physiol., 221:1130-1133) have shown that a tissue potential can be measured with microelectrodes in glycerinated muscle and that this potential is consistent with a Donnan equilibrium of small ions due to the concentration of net fixed electric charge on the contractile proteins. This approach has been combined with x-ray and light diffraction measurements of the muscle lattice dimensions, and the data are used to determine the thick filament charge and thin filament charge under a variety of different conditions. The thick filament charge is a function of the bathing solution, in particular its pH and ionic composition. These parameters are important in determining the volume of the equilibrium lattice and possibly are involved in the contraction mechanism itself.     

Donnan potential measurements in extended hexagonal polyelectrolyte gels such as muscle.

In this paper we reconsider the theoretical and practical aspects of using KCl-filled microelectrodes in extended polyelectrolyte gels such as muscle to measure Donnan potentials, and then calculate protein fixed-charge concentrations. An analytical calculation of the electrical potential function between muscle filaments shows that whether the microelectrode averages the ionic concentration or the local potentials the results are indistinguishable in the practical regime. After consideration of this and other possible sources of error, we conclude that the charge-concentrations measurements that have appeared in the literature are legitimate.     

Average electrostatic potential between the filaments in striated muscle and its relation to a simple Donnan potential.

An approximate analytical solution to the Poisson-Boltzmann equation for a cylindrical particle was used to calculate the relationship between the charge on the filaments and the average electrostatic potential. Both thick and then filaments were considered in the muscle lattice with a filament charge ratio of 4 to 1. Comparing this with a similar relationship obtained using simple Donnan theory showed a discrepancy at high charge where the Poisson-Boltzmann equation leads to saturation of the average potential. However, using two separate experiments from the literature, we have shown that at pH 7.0 muscle must not be close to saturation and thus is in a region of the curve where the two approaches agree.     

Filament lattice of frog striated muscle. Radial forces, lattice stability, and filament compression in the A-band of relaxed and rigor muscle.

Repulsive pressure in the A-band filament lattice of relaxed frog skeletal muscle has been measured as a function of interfilament spacing using an osmotic shrinking technique. Much improved chemical skinning was obtained when the muscles were equilibrated in the presence of EGTA before skinning. The lattice shrank with increasing external osmotic pressure. At any specific pressure, the lattice spacing in relaxed muscle was smaller than that of muscle in rigor, except at low pressures where the reverse was found. The lattice spacing was the same in the two states at a spacing close to that found in vivo. The data were consistent with an electrostatic repulsion over most of the pressure range. For relaxed muscle, the data lay close to electrostatic pressure curves for a thick filament charge diameter of approximately 26 nm, suggesting that charges stabilizing the lattice are situated about midway along the thick filament projections (HMM-S1). At low pressures, observed spacings were larger than calculated, consistent with the idea that thick filament projections move away from the filament backbone. Under all conditions studied, relaxed and rigor, at short and very long sarcomere lengths, the filament lattice could be modeled by assuming a repulsive electrostatic pressure, a weak attractive pressure, and a radial stiffness of the thick filaments (projections) that differed between relaxed and rigor conditions. Each thick filament projection could be compressed by approximately 5 or 2.6 nm requiring a force of 1.3 or 80 pN for relaxed and rigor conditions respectively.     

Membrane potentials in the Donnan equilibrium. II

Measurements were made of electromotive force in the Donnan equilibrium of systems containing dilute solutions of protein and acid. Removal of the membrane produced a decrease of no more than 2 to 4 mv. in electromotive force, while the membrane potentials, as estimated by the usual arbitrary assumption, were of the order of 12 to 34 mv. Ion ratios, as calculated from analyses for total chloride, were definitely greater than those calculated from the electromotive force of cells with salt bridges, as if there had been combination of some of the chloride ion with protein.     

Amphioxus connectin exhibits merged structure as invertebrate connectin in I-band region and vertebrate connectin in A-band region

Connectin is an elastic protein found in vertebrate striated muscle and in some invertebrates as connectin-like proteins. In this study, we determined the structure of the amphioxus connectin gene and analyzed its sequence based on its genomic information. Amphioxus is not a vertebrate but, phylogenetically, the lowest chordate. Analysis of gene structure revealed that the amphioxus gene is approximately 430 kb in length and consists of regions with exons of repeatedly aligned immunoglobulin (Ig) domains and regions with exons of fibronectin type 3 and Ig domain repeats. With regard to this sequence, although the region corresponding to the I-band is homologous to that of invertebrate connectin-like proteins and has an Ig-PEVK region similar to that of the Neanthes sp. 4000K protein, the region corresponding to the A-band has a super-repeat structure of Ig and fibronectin type 3 domains and a kinase domain near the C-terminus, which is similar to the structure of vertebrate connectin. These findings revealed that amphioxus connectin has the domain structure of invertebrate connectin-like proteins at its N-terminus and that of vertebrate connectin at its C-terminus. Thus, amphioxus connectin has a novel structure among known connectin-like proteins. This finding suggests that the formation and maintenance of the sarcomeric structure of amphioxus striated muscle are similar to those of vertebrates; however, its elasticity is different from that of vertebrates, being more similar to that of invertebrates.     

Myosin from striated adductor muscle of Chlamys nipponensis akazara.

Myosin was isolated from striated adductor muscle of Akazara shell-fish, and purified on DEAE-Sephadex A50. The sedimentation constant (s 20,2 0 W) and the intrinsic viscosity, [eta] of Akazara myosin thus purified were estimated to be 6.6 S and 2.10 dl/g, respectively. In many respects, Akazara myosin was similar to scallop myosin. (1) Only one size of light-chain component (17,000 daltons) was detectable in SDS-gel electrophoresis of Akazara myosin, but two types of light-chain component were seen in urea-gel electrophoresis; these were equivalent to EDTA-light chain and SH-light chain of scallop myosin. The molar ratio of heavy chain (206,000 daltons), EDTA-light chain, and SH-light chain in Akazara myosin was estimated, from the staining densities of gel-electrophoretic bands, to be approximately 1 : 1 : 1. (2) EDTA-washing procedure removed EDTA-light chain only, causing desensitization of Akazara myosin. EDTA-light chain isolated from Akazara myofibrils was able to resensitize EDTA-washed Akazara myosin. Akazara myosin, however, was found to be different from scallop myosin in two important properties: (1) complete removal of EDTA-light chains was required to achieve a complete loss of calcium sensitivity, and full resensitization was attained on recombination of EDTA-light chains with desensitized myosin prepared essentially free from EDTA-light chains. (2) EDTA-light chains isolated from Akazara myofibrils show a calcium-induced UV absorption difference spectrum.     

A-band shortening in single fibers of frog skeletal muscle.

The question of whether A-bands shorten during contraction was investigated using two methods: high-resolution polarization microscopy and electron microscopy. During shortening from extended sarcomere lengths in the passive state, sarcomere-length changes were essentially accounted for by I-band shortening. During active shortening under otherwise identical conditions, the sarcomere length change was taken up approximately equally by A- and I-bands. Several potential artifacts that could give rise to apparent A-band shortening were considered and judged unlikely. Results obtained with polarization microscopy were similar to those obtained with electron microscopy. Thus, modest but significant thick filament shortening appears to occur during active sarcomere shortening under physiological conditions.     

Inertial artifact in viscoelastic measurements of striated muscle: Modeling and experimental results

Viscoelastic properties of striated muscle are often measured using length perturbation analysis and quantified as a complex modulus, whose elastic and viscous components reflect the energy-storage and energy-absorbing properties of the tissue, respectively. The energy stored as inertia is commonly ignored due to the small size of samples examined, typically <1 mm. Considering recent advances in tissue engineering to generate muscle tissues of larger sizes, we questioned whether ignoring the inertial artifact was still reasonable in these samples. To answer this question, we derived and solved the one-dimensional wave equation that describes the propagation of strain along the length of a sample. The inertial artifact was predicted to contaminate the elastic modulus with (2πf)²L₀²ρ/6, where f is perturbation frequency, L₀ is muscle length, and ρ is muscle density. We then measured viscoelastic properties up to 500 Hz in mouse skeletal muscle fibers at long (4.8 mm) and short (<1 mm) lengths and up to 100 Hz in rat cardiac slices at long (10–12 mm) and short (<2 mm) lengths. We found the elastic modulus of long preparations was elevated as frequency increased and was about half the magnitude of that predicted by the model. While the prediction tended to overestimate the measured inertial artifact, these results provided some validity to the model. We used the predicted artifact as an overly conservative estimate of error that might arise in a mechanics assay of mammalian striated muscle, whose nominal resting stiffness is on the order 100 kN m^?2. We found that muscle lengths of <1 mm resulted in negligible inertial artifact (<0.5% error) for perturbation frequencies under 250 Hz. Muscle samples longer than 5 mm, on the other hand, would result in >5% error at frequencies of 200 Hz and higher.     

Regulatory proteins of lobster striated muscle.

The regulatory proteins of lobster muscles consist of tropomyosin and of troponin. Troponin contains a 17,000 chain weight component, two closely related components of about 30,000 and a 52,000 chain weight component. In addition to troponin, tropomyosin is required for the inhibition of the magnesium activated actomyosin ATPase activity in the absence of calcium and for the reversal of this inhibition by calcium. Lobster tropomyosin interacts with rabbit actin and lobster troponin interacts with rabbit tropomyosin. The 30,000 doublet component corresponds to the troponin-I of rabbit and inhibits the ATPase activity of actomyosin both in the presence and in the absence of calcium. The 17,000 component corresponds to the troponin-C of rabbit; it binds calcium and reverses the inhibition of the ATPase activity by troponin-I in the presence of calcium. No more than 1 mol of calcium is bound by a mole of troponin-C or by troponin. The 52,000 component interacts with tropomyosin and has been tentatively identified as troponin-T; however, it has not been demonstrated as yet that this component had a role in the regulation of lobster actomyosin.     

Compositional studies of myofibrils from rabbit striated muscle

The localization of high-molecular-weight (80,000-200,000-daltons) proteins in the sarcomere of striated muscle has been studied by coordinated electron-microscopic and sodium dodecyl sulfate (SDS) gel electrophoretic analysis of native myofilaments and extracted and digested myofibrils. Methods were developed for the isolation of thick and thin filaments and of uncontracted myofibrils which are devoid of endoproteases and membrane fragments. Treatment of crude myofibrils with 0.5% Triton X-100 results in the release of a 110,000-dalton component without affecting the myofibrillar structure. Extraction of uncontracted myofibrils with a relaxing solution of high ionic strength results in the complete disappearance of the A band and M line. In this extract, five other protein bands in addition to myosin are resolved on SDS gels: bands M 1 (190,000 daltons) and M 2 (170,000 daltons), which are suggested to be components of the M line; M 3 (150,000 daltons), a degradation product; and a doublet M 4, M 5 (140,000 daltons), thick-filament protein having the same mobility as C protein. Extraction of myofibrils with 0.15% deoxycholate, previously shown to remove Z-line density, releases a doublet Z 1, Z 2 (90,000 daltons) with the same mobility as alpha-actinin, as well as proteins of 60,000 daltons and less, and small amounts of M 1, M 2, M 4, and M 5; these proteins were not extracted with 0.5% Triton X-100. The C, M-line, and Z-line proteins and/or their binding to myofibrils are very sensitive to tryptic digestion, whereas the M 3 (150,000 daltons) component and an additional band at 110,000 daltons are products of proteolysis. Gentle treatment of myofibrils with an ATP relaxing solution results in the release of thick and thin myofilaments which can be pelleted by 100,000-g centrifugation. These myofilaments lack M-and Z-line structure when examined with the electron microscope, and their electrophoretograms are devoid of the M 1, M 2, Z 1, and Z 2 bands. The M 4, M 5 (C-protein doublet), and M 3 bands, however, remain associated with the filaments.     

Transdifferentiation from striated muscle of medusae in vitro

We have established an in vitro transdifferentiation and regeneration system which is based entirely on mononucleated striated muscle cells. The muscle tissue is isolated from anthomedusae and activated by various means to undergo cell cycles and transdifferentiation to several new cell types. In all cases DNA-replication is initiated and the division products are smooth muscle cells, characterized by their ultrastructure and monoclonal antibodies, and nerve/sensory cells, characterized by their ultrastructure and FMRFamide-staining. Both cell types are found at a 1:1 ratio after the first division. The nerve cells stop to replicate, whereas the smooth muscle cells continue and keep producing in each successive division a smooth muscle cell and a nerve cell. The observed data indicate that smooth muscle cells behave like stem cells. Depending on the destabilization and culturing methods, some isolated muscle tissue will form a bilayered fragment and within only two cell cycles manubria (the feeding and sexual organ) or tentacles will regenerate. In this case six to eight new non-muscle cell types have been formed by transdifferentiation.