The Temperature Dependence of the Mechanical Characteristics of Demembranized Rabbit Slow Muscle Fibers

Biophysics - The temperature dependences of the tension and stiffness of actively contracting single fibers of the rabbit slow muscle (m. soleus) were studied using the Joule temperature jump....     

Effect of External Calcium and of Temperature on Contraction in Snake Muscle Fibers

The effect of external calcium and of temperature on the contractile responses has been studied in voltage clamped snake twitch muscle fibers. Increasing [Ca⁺⁺]_o from 0.2 to 7.0 mM raised contractile threshold by 15–20 mV, the latter coinciding with the appearance of delayed rectification. The duration of contracture, the rates of rise and decay of tension depended on the level of depolarization and [Ca⁺⁺]_o. The minimum duration of repolarization necessary to restore the contractile response was much shorter in high [Ca⁺⁺]_o. When the bathing solution was cooled to 10 from 20°C the time-course of contracture was markedly prolonged and the outward current was reduced without significant change in maximum tension. The threshold for contraction tended to be somewhat lower at the lower temperature. The contractile repriming was much slower at low temperature. However, reduction in temperature slowed the rate of recovery much less at low [Ca⁺⁺]_o than at normal [Ca⁺⁺]_o.     

The Role of Cell Calcium in the Contraction of Single Cannulated Muscle Fibers

Intracellular injections of the calcium-binding agent, EGTA,into single cannulated fibers of Balanus and Maia were ableto suppress, almost completely, the contractions induced byvarious contractile agents. The amount oE EGTA required in Maia fibers for the suppressionof the contractile response produced by caffeine and high-Ksaline, as has already been reported, was similar to the meanfiber calcium level. In Balanus fibers, however, although theamount of EGTA needed for the suppression of the caffeine-salineresponse was similar to the estimated level of fiber calcium,the amount required in the raised-K-saline experiments was considerablygreater. It has been suggested that membrane depolarizationunder these conditions allows calcium to enter the fiber fromthe external saline, the amount entering being related, at leastin part, to a large effective sarcolemmal surface area and thepresence of binding agent internally. The results of intracellularand plate-electrode stimulation of Belanus fibers also suggestedthat the fiber under certain conditions could utilize externalcalcium ions, while the results of plate-electrode stimulationof Maia fibers could be explained most easily in terms of mobilizationof mainly intracellular calcium for the process of contraction. Efflux of Sr⁸⁹ ions from Balanus fibers under various conditionssuggested that this ion is bound and mobilized internally ina manner similar to calcium. The results are seen not to contradict a chanelled-current theoryfor e-c coupling of the type proposed for crayfish fibers.     

The Role of the N-Terminus of the Myosin Essential Light Chain in Cardiac Muscle Contraction

To study the regulation of cardiac muscle contraction by the myosin essential light chain (ELC) and the physiological significance of its N-terminal extension, we generated transgenic (Tg) mice by partially replacing the endogenous mouse ventricular ELC with either the human ventricular ELC wild type (Tg-WT) or its 43-amino-acid N-terminal truncation mutant (Tg-Δ43) in the murine hearts. The mutant protein is similar in sequence to the short ELC variant present in skeletal muscle, and the ELC protein distribution in Tg-Δ43 ventricles resembles that of fast skeletal muscle. Cardiac muscle preparations from Tg-Δ43 mice demonstrate reduced force per cross-sectional area of muscle, which is likely caused by a reduced number of force-generating myosin cross-bridges and/or by decreased force per cross-bridge. As the mice grow older, the contractile force per cross-sectional area further decreases in Tg-Δ43 mice and the mutant hearts develop a phenotype of nonpathologic hypertrophy while still maintaining normal cardiac performance. The myocardium of older Tg-Δ43 mice also exhibits reduced myosin content. Our results suggest that the role of the N-terminal ELC extension is to maintain the integrity of myosin and to modulate force generation by decreasing myosin neck region compliance and promoting strong cross-bridge formation and/or by enhancing myosin attachment to actin.     

The Effect of Myofilament Compliance on Kinetics of Force Generation by Myosin Motors in Muscle

We use the inhibitor of isometric force of skeletal muscle N-benzyl-p-toluene sulfonamide (BTS) to decrease, in a dose dependent way, the number of myosin motors attached to actin during the steady isometric contraction of single fibers from frog skeletal muscle (4°C, 2.1 μm sarcomere length). In this way we can reduce the strain in the myofilament compliance during the isometric tetanus (T₀) from 3.54 nm in the control solution (T_0,NR) to ∼0.5 nm in 1 μM BTS, where T₀ is reduced to ∼0.15 T_0,NR. The quick force recovery after a step release (1-3 nm per half-sarcomere) becomes faster with the increase of BTS concentration and the decrease of T₀. The simulation of quick force recovery with a multistate model of force generation, that adapts Huxley and Simmons model to account for both the high stiffness of the myosin motor (∼3 pN/nm) and the myofilament compliance, shows that the increase in the rate of quick force recovery by BTS is explained by the reduced strain in the myofilaments, consequent to the decrease in half-sarcomere force. The model estimates that i), for the same half-sarcomere release the state transition kinetics in the myosin motor are five times faster in the absence of filament compliance than in the control; and ii), the rate of force recovery from zero to T₀ is ∼6000/s in the absence of filament compliance.     

The Role of Sodium Current in the Radial Spread of Contraction in Frog Muscle Fibers

The membrane potential of isolated muscle fibers was controlled with a two-electrode voltage clamp, and the radial extent of contraction elicited by depolarizing pulses of increasing magnitude was observed microscopically. Depolarizations of the fiber surface only 1–2 mv greater than the contraction threshold produced shortening throughout the entire cross-section of the muscle fiber. The radial spread of contraction was less effective in fibers exposed to tetrodotoxin or to a bathing medium with a greatly reduced sodium concentration. The results provide evidence that depolarization of a muscle fiber produces an increase in sodium conductance in the T tubule membrane and that the resultant sodium current contributes to the spread of depolarization along the T system.     

The Mode of Transverse Spread of Contraction Initiated by Local Activation in Single Crayfish Muscle Fibers

Isolated single crayfish muscle fibers were locally activated by applying negative current pulses to a pipette whose tip was in contact with the fiber surface. The contraction initiated by a moderate depolarization spread inwards in a graded manner according to the magnitude and duration of depolarization. Increase of the depolarized area increased the distance of the inward spread for a given amount of depolarization. If a large area of the surface membrane was depolarized with a large pipette for a sufficiently long time, the contraction spread not only inwards, but further transversely passing through the center of the fiber. Successive brief depolarizations given at an appropriate interval could produce contraction more effectively for a given amount of total current than did a prolonged depolarization. On the other hand, the contraction initiated by a strong negative current was observed to spread around the whole perimeter but not through the center of the fiber. Each type of local contraction always spread along the striation pattern and not longitudinally. Possible mechanisms of these responses are discussed in connection with the transverse tubular system of the muscle fibers.     

Myosin Heads Contribute to the Maintenance of Filament Order in Relaxed Rabbit Muscle

Raising the temperature of rabbit skeletal muscle from ∼0°C to ∼20°C has been shown to enhance the helical organization of the myosin heads and to change the intensities of the 10 and 11 equatorial reflections. We show here by time-resolved x-ray diffraction combined with temperature jump that the movement of the heads to enhance the organized myosin helix occurs at the same fast rate as the change in the intensities of the equatorial reflections. However, model calculations indicate that the change in the equatorials cannot be explained simply in terms of the movement of myosin heads. Analysis of electron micrographs of transverse sections of relaxed muscle fibers cryofixed at ∼5°C and ∼35°C shows that in addition to the reorganization of the heads the thin and thick filaments are less constrained to their positions in the hexagonal filament lattice in the warm muscle than in the cold. Incorporating the changes in filament order in model calculations reconciles these with the observed changes in equatorial reflections. We suggest the thin filaments in the cold muscle are boxed into their positions by the thermal movement of the disordered myosin heads. In the warmer muscle, the packed-down heads leave the thin filaments more room to diffuse laterally.     

The Mode of Transverse Spread of Contraction Initiated by Local Activation in Single Frog Muscle Fibers

Isolated single frog muscle fibers were locally activated by applying negative current pulses to a pipette whose tip was in contact with the fiber surface. In contrast to the graded inward spread of contraction initiated by a moderate depolarization, the contraction in response to a strong negative current was observed to spread transversely around the whole perimeter but not through the center of the fiber. This response was elicited only with pipettes of more than 6 µ diameter. The response was still present if the sodium of the Ringer solution was replaced by choline, or the chloride was replaced by nitrate or propionate. The duration of the response appeared to be independent of the duration of stimulating current in fresh fibers, while the contraction lasted as long as the current went on in deteriorated fibers. The contraction was first initiated at the area of fiber surface covered by the pipette, and spread around the perimeter of the fiber with a velocity of 0.8–6 cm/sec. Possible mechanisms of the response are discussed in connection with the properties of the transverse tubular system, the possibility of some self-propagating process along the walls of the tubules being suggested.     

Differences in the Charge Distribution of Glycerol-Extracted Muscle Fibers in Rigor, Relaxation, and Contraction

Glycerol-extracted rabbit psoas muscle fibers were impaled with KCl-filled glass microelectrodes. For fibers at rest-length, the potentials were significantly more negative in solutions producing relaxation than in solutions producing either rigor or contraction; further the potentials in the latter two cases were not significantly different. For stretched fibers, with no overlap between thick and thin filaments, the potentials did not differ in the rigor, the relaxation, or the contraction solutions. The potentials measured from fibers in rigor did not vary significantly with the sarcomere length. For relaxed fibers, however, the potential magnitude decreased with increasing sarcomere length. The difference between the potentials measured for rigor and relaxed fibers exhibited a nonlinear relationship with sarcomere length. The potentials from calcium-insensitive fibers were less negative in both the rigor and the relaxation solutions than those from normal fibers. When calcium-insensitive fibers had been incubated in Hasselbach and Schneider's solution plus MgCl₂ or Guba-Straub's solution plus MgATP the potentials recorded upon impalement were similar in the rigor and the relaxation solution to those obtained from normal fibers in the relaxed state. It is concluded that the increase in the negative potential as the glycerinated fiber goes from rigor to relaxation may be due to an alteration in the conformation of the contractile proteins in the relaxed state.     

Initial Heat Production in Isometric Frog Muscle at 15°C

An infrared radiation-detecting system was used to measure initial heat production in bull frog sartorius muscle at 15°C. Numerous tests with the system showed that thermal artifacts were not noticeable. Many previous measurements with myothermic thermopiles were corroborated with this method. In addition, a cooling phase as large as 0.39 of peak exothermicity was found during and after relaxation. Cooling diminished with both increasing sarcomere length and increasing duration of mechanical activity. No large rapid increase in heat rate accompanied a 0.6 reactivation at the peak of twitch tension. Above rest length, initial heat rate and the heat produced up to the peak of tension decreased nearly proportionally with overlap of myofilaments, while the total twitch initial heat decreased slightly.     

Dynamics of Thin-Filament Activation in Rabbit Skeletal Muscle Fibers Examined by Time-Resolved X-Ray Diffraction

By using skinned-rabbit skeletal muscle fibers, the time courses of changes of thin filament-based x-ray reflections were followed at a 3.4-ms time resolution during thin-filament activation. To discriminate between the effects of calcium binding and myosin binding on thin-filament activity, measurements were performed after caged-calcium photolysis in fibers with full-filament or no-filament overlap, or during force recovery after a quick release. All three reflections examined, i.e., the second actin layer line (second ALL, reporting the tropomyosin movement), the sixth ALL (reporting actin structural change), and the meridional troponin reflections, exhibited calcium-induced and myosin-induced components, but their rate constants and polarities were different. Generally, calcium-induced components exhibited fast rate constants (>100 s⁻¹). The myosin-induced components of the second ALL had a rate constant similar to that of the force (7-10 s⁻¹), but that of the sixth ALL was apparently faster. The myosin-induced component of troponin reflection was the only one with negative polarity, and was too slow to be analyzed with this protocol. The results suggest that the three regulation-related proteins change their structures with different rate constants, and the significance of these findings is discussed in the context of a cooperative thin-filament activation mechanism.     

Expression of Masticatory-specific Isoforms of Myosin Heavy-chain,Myosin-binding Protein-C and Tropomyosin in Muscle Fibers and Satellite Cell Cultures of Cat Masticatory Muscle

We test the hypothesis that cat jaw satellite cells belong to a distinct lineage preprogrammed to express masticatory-specific isoforms of myosin heavy-chain (m-MyHC), myosin-binding protein-C (m-MBP-C), and tropomyosin (m-Tm) during myogenesis in vitro. A monoclonal antibody (MAb) against m-MyHC and MAbs raised here against cat m-MBP-C and m-Tm were used to stain cryostat sections of cat masseter muscle and cultured myotubes derived from satellite cells of cat temporalis and limb muscles, using peroxidase immunohistochemistry. MAbs against m-MBP-C bound purified m-MBP-C in Western blots. MAbs against m-Tm failed to react with m-Tm in Western blots, but reacted with native m-Tm in gel electrophoresis–derived ELISA. In cat masseter sections, MAbs against m-MyHC, m-MBP-C, and m-Tm stained all masticatory fibers, but not the jaw-slow fibers. Cat jaw and limb muscle cultures mature significantly more slowly relative to rodent cultures. However, at 3 weeks, all three MAbs extensively stained temporalis myotubes, whereas they apparently stained isolated myotubes weakly in cat limb and rat jaw cultures. We conclude that satellite cells of masticatory fibers are preprogrammed to express these isoforms during myogenesis in vitro. These results consolidate the notion that masticatory and limb muscle allotypes are distinct. (J Histochem Cytochem 58:623–634, 2010)     

Surgical Sympathetic Denervation Increases α1Adrenoceptor-Mediated Accumulation of myo-Inositol Trisphosphate and Muscle Contraction in Rabbit Iris Dilator Smooth Muscle

Sympathetic denervation of the iris muscle produces increases in both the breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2) and in muscle contraction in response to norepinephrine (NE). To shed more light on the biochemical basis underlying this supersensitivity we investigated: the effects of NE on PIP2 breakdown, measured as myo-inositol trisphosphate (IP3) accumulation, and on muscle contraction in normal and denervated rabbit iris dilator; and the effects of denervation on selected biochemical properties of this muscle. The data obtained from these studies can be summarized as follows: The EC50 values (microM) for NE-induced IP3 accumulation in normal and denervated dilators were 14 and 3, respectively. This accumulation of IP3 was blocked by prazosin (1 microM). The EC50 values (microM) for NE-induced contraction for the normal and denervated muscles were 10 and 0.6, respectively. The NE-induced muscle contraction was blocked by prazosin (1 microM). The t1/2 values (s) for IP3 accumulation in normal and denervated muscles were 31 and 11, respectively, and for contraction the values were 19 and 9, respectively. Denervation increased significantly (15-18%) the basal labelling of phosphoinositides from myo-[3H]inositol, but not from 32P or [14C]arachidonic acid. Denervation had little effect on the activities of the enzymes involved in phosphoinositide metabolism. However, the activities of protein kinase C and Ca2+-ATPase increased in the denervated muscle. It is concluded that sympathetic denervation of the iris dilator renders the coupling between alpha1 receptors and PIP2 breakdown into IP3 and 1,2-diacylglycerol (DG) more efficient.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Effects of Temperature upon Contraction and Ionic Exchange in Rabbit Ventricular Myocardium : Relation to control of active state

The mechanical responses (active and resting tension, dP/dt, TPT) and ionic exchange characteristics (Ca⁺⁺, K⁺, Na⁺) which follow upon a variation in temperature, rate, and [K⁺]₀ were studied in the rabbit papillary muscle and arterially perfused rabbit interventricular setpum. Abrupt changes in temperature provided a means of separating the contributions of rate of development (intensity) of active state and duration of active state to total active tension development (approximated by isometric tension). Threefold changes in duration of active state with proportional changes in active tension can be induced without evidence for alteration of Ca⁺⁺, K⁺, or Na⁺ exchange. Abrupt cooling produced a moderate (~15%) increase of dP/dt which suggests an augmentation of active state intensity. Evidence is presented to suggest that this increase of dP/dt is based upon an increase in membrane Ca⁺⁺ concentration which occurs secondary to inhibition of active Na⁺ transport. The alterations in ionic exchange and active state produced by variation of temperature are discussed in terms of a five-component control system.     

Local Control Model of Excitation–Contraction Coupling in Skeletal Muscle

The voltage-activated H⁺ selective conductance of rat alveolar epithelial cells was studied using whole-cell and excised-patch voltage-clamp techniques. The effects of substituting deuterium oxide, D₂O, for water, H₂O, on both the conductance and the pH dependence of gating were explored. D⁺ was able to permeate proton channels, but with a conductance only about 50% that of H⁺. The conductance in D₂O was reduced more than could be accounted for by bulk solvent isotope effects (i.e., the lower mobility of D⁺ than H⁺), suggesting that D⁺ interacts specifically with the channel during permeation. Evidently the H⁺ or D⁺ current is not diffusion limited, and the H⁺ channel does not behave like a water-filled pore. This result indirectly strengthens the hypothesis that H⁺ (or D⁺) and not OH⁻ is the ionic species carrying current. The voltage dependence of H⁺ channel gating characteristically is sensitive to pH_o and pH_i and was regulated by pD_o and pD_i in an analogous manner, shifting 40 mV/U change in the pD gradient. The time constant of H⁺ current activation was about three times slower (τ_act was larger) in D₂O than in H₂O. The size of the isotope effect is consistent with deuterium isotope effects for proton abstraction reactions, suggesting that H⁺ channel activation requires deprotonation of the channel. In contrast, deactivation (τ_tail) was slowed only by a factor ≤1.5 in D₂O. The results are interpreted within the context of a model for the regulation of H⁺ channel gating by mutually exclusive protonation at internal and external sites (Cherny, V.V., V.S. Markin, and T.E. DeCoursey. 1995. J. Gen. Physiol. 105:861–896). Most of the kinetic effects of D₂O can be explained if the pK _a of the external regulatory site is ∼0.5 pH U higher in D₂O.     

In Vivo Orientation of Single Myosin Lever Arms in Zebrafish Skeletal Muscle

Cardiac and skeletal myosin assembled in the muscle lattice power contraction by transducing ATP free energy into the mechanical work of moving actin. Myosin catalytic/lever-arm domains comprise the transduction/mechanical coupling machinery that move actin by lever-arm rotation. In vivo, myosin is crowded and constrained by the fiber lattice as side chains are mutated and otherwise modified under normal, diseased, or aging conditions that collectively define the native myosin environment. Single-myosin detection uniquely defines bottom-up characterization of myosin functionality. The marriage of in vivo and single-myosin detection to study zebrafish embryo models of human muscle disease is a multiscaled technology that allows one-to-one registration of a selected myosin molecular alteration with muscle filament-sarcomere-cell-fiber-tissue-organ- and organism level phenotypes. In vivo single-myosin lever-arm orientation was observed at superresolution using a photoactivatable-green-fluorescent-protein (PAGFP)-tagged myosin light chain expressed in zebrafish skeletal muscle. By simultaneous observation of multiphoton excitation fluorescence emission and second harmonic generation from myosin, we demonstrated tag specificity for the lever arm. Single-molecule detection used highly inclined parallel beam illumination and was verified by quantized photoactivation and photobleaching. Single-molecule emission patterns from relaxed muscle in vivo provided extensive superresolved dipole orientation constraints that were modeled using docking scenarios generated for the myosin (S1) and GFP crystal structures. The dipole orientation data provided sufficient constraints to estimate S1/GFP coordination. The S1/GFP coordination in vivo is rigid and the lever-arm orientation distribution is well-ordered in relaxed muscle. For comparison, single myosins in relaxed permeabilized porcine papillary muscle fibers indicated slightly differently oriented lever arms and rigid S1/GFP coordination. Lever arms in both muscles indicated one preferred spherical polar orientation and widely distributed azimuthal orientations relative to the fiber symmetry axis. Cardiac myosin is more radially displaced from the fiber axis. Probe rigidity implies the PAGFP tag reliably indicates cross-bridge orientation in situ and in vivo.     

The Ionic Mechanisms of Hyperpolarizing Responses in Lobster Muscle Fibers

Lobster muscle fibers develop hyperpolarizing responses when subjected to sufficiently strong hyperpolarizing currents. In contrast to axons of frog, toad, and squid, the muscle fibers produce their responses without the need for prior depolarization in high external K⁺. Responses begin at a threshold polarization (50 to 70 mv), the potential reaching 150 to 200 mv hyperpolarization while the current remains constant. The increased polarization develops at first slowly, then becomes rapid. It usually subsides from its peak spontaneously, falling temporarily to a potential less hyperpolarized than at threshold for the response. As long as current is applied there can be oscillatory behavior with sequential rise and subsidence of the polarization, repeating a number of times. Withdrawal of current leads to rapid return of the potential to the resting level and a small, brief depolarization. Associated with the latter, but of longer duration, is an increased conductance whose magnitude and duration increase with the antecedent current. Hyperpolarizing responses of lobster muscle fibers are due to increased membrane resistance caused by hyperpolarizing K inactivation. The oscillatory characteristic of the response is due to a delayed superimposed and prolonged increase in membrane permeability, probably for Na⁺ and for either K⁺ or Cl^-. The hyperpolarizing responses of other tissues also appear to result from hyperpolarizing K inactivation, on which is superimposed an increased conductance for some other ion or ions.