Electrotonic Interaction between Muscle Fibers in the Rabbit Ventricle

Transmembrane potentials were recorded simultaneously from pairs of ventricular fibers in an isolated, regularly beating preparation. A double-barrelled microelectrode was used to record the potentials from, and to polarize, one fiber. A single microelectrode was used to record from a distant fiber. The existence of two systems of fibers, termed P and V, was confirmed. Histological evidence for the existence of two types of fibers is also presented. Electrotonic current spread was observed within both systems, electrotonic interaction between the two systems was rare and always weak. In the case of those pairs of fibers showing electrotonic interaction, the distance for an e-fold decrease in magnitude of the electrotonic potentials was found to be from 300 to 600 µ in P fibers and from 100 to 300 µ in V fibers. However, no electrotonic interaction could be observed in the majority of V fiber pairs. Moreover, the magnitude of the electrotonic potential did not decay monotonically with distance in any one direction. It is concluded that the rabbit ventricle cannot be regarded as a single freely interconnected syncytium.     

The Fraction of Myosin Motors That Participate in Isometric Contraction of Rabbit Muscle Fibers at Near-Physiological Temperature

The duty ratio, or the part of the working cycle in which a myosin molecule is strongly attached to actin, determines motor processivity and is required to evaluate the force generated by each molecule. In muscle, it is equal to the fraction of myosin heads that are strongly, or stereospecifically, bound to the thin filaments. Estimates of this fraction during isometric contraction based on stiffness measurements or the intensities of the equatorial or meridional x-ray reflections vary significantly. Here, we determined this value using the intensity of the first actin layer line, A1, in the low-angle x-ray diffraction patterns of permeable fibers from rabbit skeletal muscle. We calibrated the A1 intensity by considering that the intensity in the relaxed and rigor states corresponds to 0% and 100% of myosin heads bound to actin, respectively. The fibers maximally activated with Ca²⁺ at 4°C were heated to 31–34°C with a Joule temperature jump (T-jump). Rigor and relaxed-state measurements were obtained on the same fibers. The intensity of the inner part of A1 during isometric contraction compared with that in rigor corresponds to 41–43% stereospecifically bound myosin heads at near-physiological temperature, or an average force produced by a head of ∼6.3 pN.     

Diffusion Coefficients of Endogenous Cytosolic Proteins from Rabbit Skinned Muscle Fibers

Efflux time courses of endogenous cytosolic proteins were obtained from rabbit psoas muscle fibers skinned in oil and transferred to physiological salt solution. Proteins were separated by gel electrophoresis and compared to load-matched standards for quantitative analysis. A radial diffusion model incorporating the dissociation and dissipation of supramolecular complexes accounts for an initial lag and subsequent efflux of glycolytic and glycogenolytic enzymes. The model includes terms representing protein crowding, myofilament lattice hindrance, and binding to the cytomatrix. Optimization algorithms returned estimates of the apparent diffusion coefficients, D(r,t), that were very low at the onset of diffusion (∼10⁻¹⁰ cm² s⁻¹) but increased with time as cytosolic protein density, which was initially high, decreased. D(r,t) at later times ranged from 2.11 × 10⁻⁷ cm² s⁻¹ (parvalbumin) to 0.20 × 10⁻⁷ cm² s⁻¹ (phosphofructose kinase), values that are 3.6- to 12.3-fold lower than those predicted in bulk water. The low initial values are consistent with the presence of complexes in situ; the higher later values are consistent with molecular sieving and transient binding of dissociated proteins. Channeling of metabolic intermediates via enzyme complexes may enhance production of adenosine triphosphate at rates beyond that possible with randomly and/or sparsely distributed enzymes, thereby matching supply with demand.     

Diffusion Coefficients of Endogenous Cytosolic Proteins from Rabbit Skinned Muscle Fibers

Efflux time courses of endogenous cytosolic proteins were obtained from rabbit psoas muscle fibers skinned in oil and transferred to physiological salt solution. Proteins were separated by gel electrophoresis and compared to load-matched standards for quantitative analysis. A radial diffusion model incorporating the dissociation and dissipation of supramolecular complexes accounts for an initial lag and subsequent efflux of glycolytic and glycogenolytic enzymes. The model includes terms representing protein crowding, myofilament lattice hindrance, and binding to the cytomatrix. Optimization algorithms returned estimates of the apparent diffusion coefficients, D(r,t), that were very low at the onset of diffusion (∼10⁻¹⁰ cm² s⁻¹) but increased with time as cytosolic protein density, which was initially high, decreased. D(r,t) at later times ranged from 2.11 × 10⁻⁷ cm² s⁻¹ (parvalbumin) to 0.20 × 10⁻⁷ cm² s⁻¹ (phosphofructose kinase), values that are 3.6- to 12.3-fold lower than those predicted in bulk water. The low initial values are consistent with the presence of complexes in situ; the higher later values are consistent with molecular sieving and transient binding of dissociated proteins. Channeling of metabolic intermediates via enzyme complexes may enhance production of adenosine triphosphate at rates beyond that possible with randomly and/or sparsely distributed enzymes, thereby matching supply with demand.     

Calcium Efflux from Barnacle Muscle Fibers : Dependence on External Cations

Calcium-45 was injected into single giant barnacle muscle fibers, and the rate of efflux was measured under a variety of conditions. The rate constant (k) for ⁴⁵Ca efflux into standard seawater averaged 17 x 10^–4 min^–1 which corresponds to an efflux of about 1–2 pmol/cm²·s. Removal of external Ca (Ca_o) reduced the efflux by 50%. In most fibers about 40% of the ⁴⁵Ca efflux into Ca-free seawater was dependent on external Na (Na_o); treatment with 3.5 mM caffeine increased the magnitude of the Na_o-dependent efflux. In a few fibers removal of Na_o, in the absence of Ca_o, either had no effect or increased k; caffeine (2–3.5 mM) unmasked an Na_o-dependent efflux in these fibers. The Na_o-dependent Ca efflux had a Q₁₀ of about 3.7. The data are consistent with the idea that a large fraction of the Ca efflux may be carrier-mediated, and may involve both Ca-Ca and Na-Ca counterflow. The relation between the Na_o-dependent Ca efflux and the external Na concentration is sigmoid, and suggests that two, or more likely three, external Na⁺ ions may activate the efflux of one Ca⁺². With a three-for-one Na-Ca exchange, the Na electrochemical gradient may be able to supply sufficient energy to maintain the Ca gradient in these fibers. Other, more complex models are not excluded, however, and may be required to explain some puzzling features of the Ca efflux such as the variable Na_o-dependence.     

Effects of Cannabinoids on Caffeine Contractures in Slow and Fast Skeletal Muscle Fibers of the Frog

The effect of cannabinoids on caffeine contractures was investigated in slow and fast skeletal muscle fibers using isometric tension recording. In slow muscle fibers, WIN 55,212-2 (10 and 5 μM) caused a decrease in tension. These doses reduced maximum tension to 67.43 ± 8.07% (P = 0.02, n = 5) and 79.4 ± 14.11% (P = 0.007, n = 5) compared to control, respectively. Tension-time integral was reduced to 58.37 ± 7.17% and 75.10 ± 3.60% (P = 0.002, n = 5), respectively. Using the CB₁ cannabinoid receptor agonist ACPA (1 μM) reduced the maximum tension of caffeine contractures by 68.70 ± 11.63% (P = 0.01, n = 5); tension-time integral was reduced by 66.82 ± 6.89% (P = 0.02, n = 5) compared to controls. When the CB₁ receptor antagonist AM281 was coapplied with ACPA, it reversed the effect of ACPA on caffeine-evoked tension. In slow and fast muscle fibers incubated with the pertussis toxin, ACPA had no effect on tension evoked by caffeine. In fast muscle fibers, ACPA (1 μM) also decreased tension; the maximum tension was reduced by 56.48 ± 3.4% (P = 0.001, n = 4), and tension-time integral was reduced by 57.81 ± 2.6% (P = 0.006, n = 4). This ACPA effect was not statistically significant with respect to the reduction in tension in slow muscle fibers. Moreover, we detected the presence of mRNA for the cannabinoid CB₁ receptor on fast and slow skeletal muscle fibers, which was significantly higher in fast compared to slow muscle fiber expression. In conclusion, our results suggest that in the slow and fast muscle fibers of the frog cannabinoids diminish caffeine-evoked tension through a receptor-mediated mechanism.     

Further Study of the Electrical and Mechanical Responses of Slow Fibers in Cat Extraocular Muscles

Electrical and mechanical responses have been obtained in situ and in vitro from the superior oblique muscle stimulated by single and repetitive electrical pulses, applied to the trochlear nerve. Two different types of muscle fibers are described, the twitch and the slow. The slow type is characterized electrically by the presence of junctional potentials, which have reversal potentials between -10 and -20 mv, and do not show propagated responses or spikes, during nerve stimulation. When the slow muscle fibers are repetitively stimulated in situ, a prolonged contraction is maintained during stimulation. At the time, the recorded electrical activity is produced locally, at the level of the neuromuscular junctions of the slow fibers. These results indicate that the contractile mechanism of the slow muscle fibers is activated locally and segmentally.     

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.     

The Ultrastructure of Myosin-Extracted Striated Muscle Fibers

Electron microscope examination of myosin-extracted psoas muscleof the rabbit reveals a number of thin filaments, about 25 Äin diameter, bridging the gap between the ends of the actinsand the remaining myosin of the M band. Extraction of actinfrom the region of the Z disc shows similar thin filaments nearthe Z disc. Myosin-extraction of several invertebrate musclesreveals a remaining network of thin filaments in the regionof the A band. These thin filaments are probably elastic, andcould contribute to the functioning of these muscles.     

In Vivo Studies on Fast and Slow Muscle Fibers in Cat Extraocular Muscles

In anesthetized in vivo preparations, responses of two types of extraocular muscle fibers have been studied. The small, multiply innervated slow fibers have been shown to be capable of producing propagated impulses, and thus have been labeled slow multi-innervated twitch fibers. Fast and slow multi-innervated twitch fibers are distinguished by impulse conduction velocities, by ranges of membrane potentials, by amplitudes and frequencies of the miniature end plate potentials, by responses to the intravenous administration of succinylcholine, by the frequency of stimulation required for fused tetanus, and by the velocities of conduction of the nerve fibers innervating each of the muscle fiber types.     

Time Course and Strain Dependence of ADP Release during Contraction of Permeabilized Skeletal Muscle Fibers

A phosphorylated, single cysteine mutant of nucleoside diphosphate kinase, labeled with N-[2-(iodoacetamido)ethyl]-7-diethylaminocoumarin-3-carboxamide (P∼NDPK-IDCC), was used as a fluorescence probe for time-resolved measurement of changes in [MgADP] during contraction of single permeabilized rabbit psoas fibers. The dephosphorylation of the phosphorylated protein by MgADP occurs within the lattice environment of permeabilized fibers with a second-order rate constant at 12°C of 10⁵ M⁻¹ s⁻¹. This dephosphorylation is accompanied by a change in coumarin fluorescence. We report the time course of P∼NDPK-IDCC dephosphorylation during the period of active isometric force redevelopment after quick release of fiber strain at pCa²⁺ of 4.5. After a rapid length decrease of 0.5% was applied to the fiber, the extra NDPK-IDCC produced during force recovery, above the value during the approximately steady state of isometric contraction, was 2.7 ± 0.6 μM and 4.7 ± 1.5 μM at 12 and 20°C, respectively. The rates of P∼NDPK-IDCC dephosphorylation during force recovery were 28 and 50 s⁻¹ at 12 and 20°C, respectively. The time courses of isometric force and P∼NDPK-IDCC dephosphorylation were simulated using a seven-state reaction scheme. Relative isometric force was modeled by changes in the occupancy of strongly bound A.M.ADP.P_i and A.M.ADP states. A strain-sensitive A.M.ADP isomerization step was rate-limiting (3-6 s⁻¹) in the cross-bridge turnover during isometric contraction. At 12°C, the A.M.ADP.P_i and the pre- and postisomerization A.M.ADP states comprised 56%, 38%, and 7% of the isometric force-bearing AM states, respectively. At 20°C, the force-bearing A.M.ADP.P_i state was a lower proportion of the total force-bearing states (37%), whereas the proportion of postisomerization A.M.ADP states was higher (19%). The simulations suggested that release of cross-bridge strain caused rapid depopulation of the preisomerization A.M.ADP state and transient accumulation of MgADP in the postisomerization A.M.ADP state. Hence, the strain-sensitive isomerization of A.M.ADP seems to explain the rate of change of P∼NDPK-IDCC dephosphorylation during force recovery. The temperature-dependent isometric distribution of myosin states is consistent with the previous observation of a small decrease in amplitude of the P_i transient during force recovery at 20°C and the current observation of an increase in amplitude of the ADP-sensitive NDPK-IDCC transient.     

Effects of Cannabinoids on Tension Induced by Acetylcholine and Choline in Slow Skeletal Muscle Fibers of the Frog

We investigated the effects of cannabinoids on acetylcholine (ACh) or choline contractures in slow skeletal muscle fibers from Rana pipiens. Bundles of cruralis muscle fibers were incubated with the cannabinoid receptor 1 (CB₁) agonist, arachidonylcyclopropylamide (ACPA), which diminished the maximum isometric tension by 10 % and the total tension by 5 % of the ACh contracture, and 40 and 22 % of the choline contracture, respectively. Preincubation with the CB₁ antagonist, AM281, or with pertussis toxin (PTX) completely blocked the effect of ACPA on the ACh contracture. On the other hand, the decrease in choline contracture by ACPA was only partially blocked by AM281 (~16 % decrease), PTX (20 %), or by dantrolene (~46 %). Our results show that ACPA modulates ACh and choline contractures, and suggest that this effect involves the participation of CB₁, the ACh receptor, and ?RyR in ACh contractures. For choline contractures, ACPA may also be acting through cannabinoid receptor-independent mechanisms.     

The Complete Amino Acid Sequence of Actins from Bovine Aorta, Bovine Heart, Bovine Fast Skeletal Muscle, and Rabbit, Slow Skeletal Muscle

Complete amino acid sequences for four mammalian muscle actins are reported: bovine skeletal muscle actin, bovine cardiac actin, the major component of bovine aorta actin, and rabbit slow skeletal muscle actin. The number of different actins in a higher mammal for which full amino acid sequences are now available is therefore increased from two to five. Screening of different smooth muscle tissues revealed in addition to the aorta type actin a second smooth muscle actin, which appears very similar if not identical to chicken gizzard actin. Since the sequence of chicken gizzard actin is known, six different actins are presently characterized in a higher mammal.
The two smooth muscle actins—bovine aorta actin and chicken gizzard actin—differ by only three amino acid substitutions, all located in the amino-terminal end. In the rest of their sequences both smooth muscle actins share the same four amino acid substitutions, which distinguish them from skeletal muscle actin. Cardiac muscle actin differs from skeletal muscle actin by only four amino acid exchanges. No amino acid substitutions were found when actins from rabbit fast and slow skeletal muscle were compared.
In addition we summarize the amino acid substitution patterns of the six different mammalian actins and discuss their tissue specificity. The results show a very close relationship between the four muscle actins in comparison to the nonmuscle actins. The amino substitution patterns indicate that skeletal muscle actin is the highest differentiated actin form, whereas smooth muscle actins show a noticeably closer relation to nonmuscle actins. By these criteria cardiac muscle actin lies between skeletal muscle actin and smooth muscle actins.     

鳜慢肌胚后发育与生长特征

鱼类快肌和慢肌分别占据骨骼肌的不同位置,表现不同的生长发育特征。为了解鳜(Sinipercachuatsi)慢肌纤维的胚后发育特征,本研究通过制作孵化后1～33日龄鳜个体的石蜡切片,采用慢肌特异抗体的免疫组织化学染色,观察了背鳍起点处躯干横切面慢肌的发育变化特征,并利用图像分析软件统计慢肌纤维的数目和面积。结果表明,孵化后鳜仔鱼慢肌位于水平肌隔附近,呈楔形,向背、腹两侧生长。孵化后1～9日龄为单层肌纤维,11日龄发育为多层肌纤维,19日龄覆盖侧线附近,33日龄延伸至背侧第2背肌节、腹侧腹部肌肉2/3处,并在水平肌隔和侧线处分别形成两个肌群。位于骨骼肌最外层的扁平状表层细胞,可能为慢肌增生生长的主要来源。躯干单侧慢肌肌纤维数目由孵化后6个增加至315个,总面积从13.18μm2增加到7 839.58μm2,孵化后13日龄的增生生长占优势,其他发育阶段,肥大生长一直占主导优势。     

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.     

Effects of Verapamil and Gadolinium on Caffeine-Induced Contractures and Calcium Fluxes in Frog Slow Skeletal Muscle Fibers

In this work, we tested whether L-type Ca²⁺ channels are involved in the increase of caffeine-evoked tension in frog slow muscle fibers. Simultaneous net Ca²⁺ fluxes and changes in muscle tension were measured in the presence of caffeine. Isometric tension was recorded by a mechanoelectrical transducer, and net fluxes of Ca²⁺ were measured noninvasively using ion-selective vibrating microelectrodes. We show that the timing of changes in net fluxes and muscle tension coincided, suggesting interdependence of the two processes. The effects of Ca²⁺ channel blockers (verapamil and gadolinium) were explored using 6 mm caffeine; both significantly reduced the action of caffeine on tension and on calcium fluxes. Both caffeine-evoked Ca²⁺ leak and muscle tension were reduced by 75% in the presence of 100 μm GdCl₃, which also caused a 92% inhibition of net Ca²⁺ fluxes in the steady-state condition. Application of 10 μm verapamil to the bath led to 30% and 52% reductions in the Ca²⁺ leak caused by the presence of caffeine for the peak and steady-state values of net Ca²⁺ fluxes, respectively. Verapamil (10 μm) caused a 30% reduction in the maximum values of caffeine-evoked muscle tension. Gd³⁺ was a more potent inhibitor than verapamil. In conclusion, L-type Ca²⁺ channels appear to play the initial role of trigger in the rather complex mechanism of slow fiber contraction, the latter process being mediated by both positive Ca²⁺-induced Ca²⁺ release and negative (Ca²⁺ removal from cytosol) feedback loops. Lana Shabala and Xóchitl Trujillo contributed equally to this study.     

The Concentration Dependence of Sodium Efflux from Muscle

Frog sartorius muscles subjected to overnight loading with Na⁺ in K-free Ringer in the cold were subsequently labeled with Na²⁴ and then immersed in choline Ringer and the efflux of Na²⁴ followed for 4 hours. The initial efflux of Na⁺ appeared to be 17 pmole/cm² sec.; this value was maintained for 20 minutes and was followed by an abrupt decline to about 9 pmole/cm² sec. This latter rate was maintained for the next 20 minutes of efflux. The efflux then declined gradually with time and reached values of the order of 0.1 pmole/cm² sec. The back addition of counts lost from muscles enabled one to calculate the relationship between efflux and [Na]_i for muscle. This roughly approximates an S-shaped curve with a value at half-saturation of about 17 mmole Na per liter of fiber water. The efflux-concentration curve is closely described by assuming that 3 Na⁺ are transported per carrier cycle.     

The Photoactivated Relaxation of Smooth Muscle of Rabbit Aorta

Smooth muscle of strips of rabbit aorta, placed in a state of active tonic contraction by addition of a stimulating drug, relaxes during exposure to light. The relaxation is reversible. The extent of relaxation produced by a standard exposure depends on the preexposure level of active contraction but not on the nature of the stimulating drug used to produce contraction. With strips brought to an intermediate level of contraction, the degree of relaxation (steady state levels) is a rectangular hyperbolic function of radiation intensity. The kinetics of the relaxation process during irradiation and the recovery process following irradiation are consistent with the hypothesis that the primary photoactivated material initiates a reaction or reactions leading to a product which inhibits some process involved in the production of active contraction. The photorelaxation does not require the presence of oxygen. It is potentiated by reducing the temperature of the aortic strip. The action spectrum of the photorelaxation shows relatively low effectiveness at wavelengths above 450 mµ. The effectiveness increases markedly and progressively as the wavelength is lowered below 450 mµ, reaching a peak at 310 mµ. A deep trough occurs at 280 mµ. However, both peak and trough probably result from internal filtering due to absorption by proteins in the aortic strip. It is surmised that if a correction could be made for this internal filtering, the action spectrum would rise continuously down to wavelengths at least as low as 250 mµ.     

The Suppression of the Late After-Potential in Rubidium-Containing Frog Muscle Fibers

The late after-potential that follows trains of impulses in frog muscle fibers is virtually absent when most of the intracellular potassium is replaced by rubidium and the muscle is immersed in rubidium-containing Ringer's fluid. Its amplitude is also reduced in freshly dissected, potassium-containing muscle fibers that are immersed directly in Rb-Ringer's fluid. These findings are discussed in terms of the model for muscle membrane of Adrian and Freygang (1962 a, b) and in relation to the report of Adrian (1964) that Rb-containing muscle fibers do not exhibit the variations in potassium permeability as a function of membrane potential that are found in fibers with normal intracellular potassium concentration immersed in Ringer's fluid.