Slow charge movement in mammalian skeletal muscle

Voltage-dependent charge movements were measured in the rat omohyoid muscle with the three-microelectrode voltage-clamp technique. Contraction was abolished with hypertonic sucrose. The standard (ON-OFF) protocol for eliciting charge movements was to depolarize the fiber from -90 mV to a variable test potential (V) and then repolarize the fiber to -90 mV. The quantity of charge moved saturated at test potentials of approximately 0 mV. The steady state dependence of the amount of charge that moves as a function of test potential could be well fitted by the Boltzmann relation: Q = Qmax/(1 + exp[-(V - V)/k]), where Qmax is the maximum charge that can be moved, V is the potential at which half the charge moves, and k is a constant. At 15 degrees C, these values were Qmax = 28.5 nC/microF, V = -34.2 mV, and k = 8.7 mV. Qmax, k, and V exhibited little temperature dependence over the range 7-25 degrees C. "Stepped OFF" charge movements were elicited by depolarizing the fiber from -90 mV to a fixed conditioning level that moved nearly all the mobile charge (0 mV), and then repolarizing the fiber to varying test potentials. The sum of the charge that moved when the fiber was depolarized directly from -90 mV to a given test potential and the stepped OFF charge that moved when the fiber was repolarized to the same test potential had at all test potentials a value close to Qmax for that fiber. In nearly all cases, the decay phase of ON, OFF, and stepped OFF charge movements could be well fitted with a single exponential. The time constant, tau decay, for an ON charge movement at a given test potential was comparable to tau decay for a stepped OFF charge movement at the same test potential. Tau decay had a bell-shaped dependence on membrane potential: it was slowest at a potential near V (the midpoint of the steady state charge distribution) and became symmetrically faster on either side of this potential. Raising the temperature from 7 to 15 degrees C caused tau decay to become faster by about the same proportion at all potentials, with a Q10 averaging 2.16. Raising the temperature from 15 to 25 degrees C caused tau decay to become faster at potentials near V, but not at potentials farther away.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of depolarization and low intracellular pH on charge movement currents of frog skeletal muscle fibers

The low intracellularpH and membrane depolarization associated with repeated skeletal musclestimulation could impair the function of the transverse tubular (ttubule) voltage sensor and result in a decreased sarcoplasmic reticulumCa²⁺ release and muscle fatigue. We therefore examined theeffects of membrane depolarization and low intracellular pH on thet-tubular charge movement. Fibers were voltage clamped in a doubleVaseline gap, at holding potential (HP) of 90 or 60 mV, and studiedat an internal pH of 7.0 and 6.2. Decreasing intracellular pH did notsignificantly alter the maximum amount of charge moved, transition voltage, or steepness factor at either HP. Depolarizing HPsignificantly decreased steepness factor and maximum charge moved andshifted the transition voltage to more positive potentials. Elevatedextracellular Ca²⁺ decreased the depolarization-inducedreduction in the charge movement. These results indicate that, althoughthe decrease in intracellular pH seen in fatigued muscle does notimpair the t-tubular charge movement, the membrane depolarizationassociated with muscle fatigue may be sufficient to inactivate asignificant fraction of the t-tubular charge. However, if t-tubularCa²⁺ increases, some of the charge may be stabilized in theactive state and remain available to initiate sarcoplasmic reticulum Ca²⁺ release.

     

The influence of transverse tubular delays on the kinetics of charge movement in mammalian skeletal muscle

A model was developed to describe the kinetics of slow, voltage-dependent charge movement in the rat omohyoid muscle. To represent the electrically distributed nature of the transverse tubular system (t-system), we followed an approach similar to that described by Adrian and Peachey (1973 J. Physiol. [Lond.]. 235:103), and approximated the fiber with 12 concentric cylindrical shells. Incorporated into each shell were capacitative and conductive elements that represented the passive electrical properties of the t-system, and an element representing the mobile charge. The charge was assumed to obey a two-state scheme, in which the redistribution of charge is governed by a first-order reaction, and the rate constants linking the two states were assumed to depend on potential according to the constant field expression. The predictions of this "distributed two-state model" were compared with charge movements experimentally measured in individual fibers. For this comparison, first, the passive electrical parameters of the model were adjusted to fit the experimental linear capacity transient. Next, the Boltzmann expression was fitted to the steady state Q vs. V data of the fiber, thereby constraining the voltage dependence of the rate constants, but not their absolute magnitude. The absolute magnitude was determined by fitting the theory to an experimental charge movement at a single test potential, which in turn constrained the fits at all other test potentials. The distributed two-state model well described the rising and falling phases of ON, OFF, and stepped OFF charge movements at temperatures ranging from 3 to 25 degrees C. We thus conclude that tubular delays are sufficient to account for the rounded rising phase of experimental charge movements, and that it is unnecessary to postulate higher-order reaction schemes for the underlying charge redistribution.     

Calcium currents in embryonic and neonatal mammalian skeletal muscle

The whole-cell patch-clamp technique was used to study the properties of inward ionic currents found in primary cultures of rat and mouse skeletal myotubes and in freshly dissociated fibers of the flexor digitorum brevis muscle of rats. In each of these cell types, test depolarizations from the holding potential (-80 or -90 mV) elicited three distinct inward currents: a sodium current (INa) and two calcium currents. INa was the dominant inward current: under physiological conditions, the maximum inward INa was estimated to be at least 30-fold larger than either of the calcium currents. The two calcium currents have been termed Ifast and Islow, corresponding to their relative rates of activation. Ifast was activated by test depolarizations to around -40 mV and above, peaked in 10-20 ms, and decayed to baseline in 50-100 ms. Islow was activated by depolarizations to approximately 0 mV and above, peaked in 50-150 ms, and decayed little during a 200-ms test pulse. Ifast was inactivated by brief, moderate depolarizations; for a 1-s change in holding potential, half-inactivation occurred at -55 to -45 mV and complete inactivation occurred at -40 to -30 mV. Similar changes in holding potential had no effect on Islow. Islow was, however, inactivated by brief, strong depolarizations (e.g., 0 mV for 2 s) or maintained, moderate depolarizations (e.g., -40 mV for 60 s). Substitution of barium for calcium had little effect on the magnitude or time course of either Ifast or Islow. The same substitution shifted the activation curve for Islow approximately 10 mV in the hyperpolarizing direction without affecting the activation of Ifast. At low concentrations (50 microM), cadmium preferentially blocked Islow compared with Ifast, while at high concentrations (1 mM), it blocked both Ifast and Islow completely. The dihydropyridine calcium channel antagonist (+)-PN 200-110 (1 microM) caused a nearly complete block of Islow without affecting Ifast. At a holding potential of -80 mV, the half-maximal blocking concentration (K0.5) for the block of Islow by (+)-PN 200-110 was 182 nM. At depolarized holding potentials that inactivated Islow by 35-65%, K0.5 decreased to 5.5 nM.     

Effect of the calcium buffer EGTA on the "hump" component of charge movement in skeletal muscle.

Three manifestations of excitation-contraction (E-C) coupling were measured in cut skeletal muscle fibers of the frog, voltage clamped in a double Vaseline gap: intramembrane charge movements, myoplasmic Ca2+ transients, and changes in optical transparency. Pulsing patterns in the presence of high [EGTA] intracellularly, shown by García et al. (1989. J. Gen. Physiol. 94:973-986) to deplete Ca2+ in the sarcoplasmic reticulum, were found to change the above manifestations. With an intracellular solution containing 15 mM EGTA and 0 Ca, 10-15 pulses (100 ms) to -20 mV at a frequency of 2 min-1 reduced the "hump" component of charge movement current. This effect was reversible by 5 min of rest. The same effect was obtained in 62.5 mM EGTA and 0 Ca by pulsing at 0.2 min-1. This effect was reversible by adding calcium to the EGTA solution, for a nominal [Ca2+]i of 200 nM, and was prevented by adding calcium to the EGTA solution before pulsing. The suppression of the hump was accompanied by elimination of the optical manifestations of E-C coupling. The current suppressed was found by subtraction and had the following properties: delayed onset, a peak at a variable interval (10-20 ms) into the pulse, a negative phase (inward current) after the peak, and a variable OFF transient that could be multi-phasic and carried less charge than the ON transient. In the previous paper (Csernoch et al., 1991. J. Gen. Physiol. 97:845-884) it was shown that several interventions suppress a similar component of charge movement current, identified with the "hump" or Q gamma current (I gamma). Based on the similarity to that component, the charge movement suppressed by the depletion protocols can also be identified with I gamma. The fact that I gamma is suppressed by Ca2+ depletion and the kinetic properties of the charge suppressed is inconsistent with the existence of separate sets of voltage sensors underlying the two components of charge movement, Q beta and Q gamma. This is explicable if Q gamma is a consequence of calcium release from the sarcoplasmic reticulum.     

Association of the Igamma and Idelta charge movement with calcium release in frog skeletal muscle

Charge movement and calcium transient were measured simultaneously in stretched frog cut twitch fibers under voltage clamp, with the internal solution containing 20 mM EGTA plus added calcium and antipyrylazo III. When the nominal free [Ca2+]i was 10 nM, the shape of the broad I(gamma) hump in the ON segments of charge movement traces remained invariant when the calcium release rate was greatly diminished. When the nominal free [Ca2+]i was 50 nM, which was close to the physiological level, the I(gamma) humps were accelerated and a slow calcium-dependent I(delta) component (or state) was generated. The peak of ON I(delta) synchronized perfectly with the peak of the calcium release rate whereas the slow decay of ON I(delta) followed the same time course as the decay of calcium release rate. Suppression of calcium release by TMB-8 reduced the amount of Q(delta) concomitantly but not completely, and the effects were partially reversible. The same simultaneous suppression effects were achieved by depleting the sarcoplasmic reticulum calcium store with repetitive stimulation. The results suggest that the mobility of Q(delta) needs to be primed by a physiological level of resting myoplasmic Ca2+. Once the priming is completed, more I(delta) is mobilized by the released Ca2+ during depolarization.     

Relationship between myoplasmic calcium transients and calcium currents in frog skeletal muscle

Ca2+ currents (ICa) and myoplasmic Ca2+ transients were simultaneously recorded in single muscle fibers from the semitendinosus muscle of Rana pipiens. The vaseline-gap voltage-clamp technique was used. Ca2+ transients were recorded with the metallochromic indicator dye antipyrylazo III. Ca2+ transients consisted of an early fast rising phase followed by a late slower one. The second phase was increased by experimental maneuvers that enlarged ICa, such as augmenting [Ca2+]o (from 2 to 10 mM) or adding (-)-Bay K 8644 (2 microM). When [Ca2+]o was increased, the second phase of the Ca2+ transients and ICa showed an average increase at 0 mV of 2 +/- 0.9 microM (4) and 1.4 +/- 0.3 mA/ml (4), respectively. (-)-Bay K 8644 increased the late phase of the Ca2+ transients and ICa at 0 mV by 0.8 +/- 0.3 microM (3) and 6.7 +/- 2.0 mA/ml (4), respectively. The initial fast rising phase of the Ca2+ transients was not modified. (-)-Bay K 8644 slowed the time constant of decay of the transients by 57 +/- 6 ms. In other experimental conditions, Ca2+ release from the sarcoplasmic reticulum (SR) was impaired with repetitive stimulation in 1 mM [EGTA]i-containing fibers. Under those circumstances, Ca2+ transients directly followed the time integral of ICa. Pulses to 0 mV caused a large Ca2+ transient that became suppressed when large pulses to 100 mV were applied. In fibers with functioning SR, pulses to 100 mV elicited somewhat smaller or similar amplitude Ca2+ transients when compared with those elicited by pulses to 0 mV. The increase in ICa after raising [Ca2+]o or adding (-)-Bay K 8644 cannot directly explain the change in Ca2+ transients in fibers with functioning SR. On the other hand, when Ca2+ release from the SR is impaired Ca2+ transients depend on ICa.     

A slow component of intramembranous charge movement during sarcoplasmic reticulum calcium release in frog cut muscle fibers

Cut muscle fibers from Rana temporaria were mounted in a double Vaseline-gap chamber and equilibrated with an end-pool solution that contained 20 mM EGTA and 1.76 mM Ca (sarcomere length, 3.3-3.8 microns; temperature, 14-16 degrees C). Sarcoplasmic reticulum (SR) Ca release, delta[CaT], was estimated from changes in myoplasmic pH (Pape, P.C., D.- S. Jong, and W.K. Chandler. 1995. J. Gen. Physiol. 106:259-336). The maximal value of delta[CaT] obtained during a depleting depolarization was assumed to equal the SR Ca content before stimulation, [CaSR]R (expressed as myoplasmic concentration). After a depolarization to -55 to -40 mV in fibers with [CaSR]R = 1,000-3,000 microM, currents from intramembranous charge movement, Icm, showed an early I beta component. This was followed by an I gamma hump, which decayed within 50 ms to a small current that was maintained for as long as 500 ms. This slow current was probably a component of Icm because the amount of OFF charge, measured after depolarizations of different durations, increased according to the amount of ON charge. Icm was also measured after the SR had been depleted of most of its Ca, either by a depleting conditioning depolarization or by Ca removal from the end pools followed by a series of depleting depolarizations. The early I beta component was essentially unchanged by Ca depletion, the I gamma hump was increased (for [CaSR]R > 200 microM), the slow component was eliminated, and the total amount of OFF charge was essentially unchanged. These results suggest that the slow component of ON Icm is not movement of a new species of charge but is probably movement of Q gamma that is slowed by SR Ca release or some associated event such as the accompanying increase in myoplasmic free [Ca] that is expected to occur near the Ca release sites. The peak value of the apparent rate constant associated with this current, 2-4%/ms at pulse potentials between -48 and -40 mV, is decreased by half when [CaSR]R approximately equal to 500-1,000 microM, which gives a peak rate of SR Ca release of approximately 5-10 microM/ms.     

A damped oscillation in the intramembranous charge movement and calcium release flux of frog skeletal muscle fibers

Asymmetric membrane currents and calcium transients were recorded simultaneously from cut segments of frog skeletal muscle fibers voltage clamped in a double Vaseline-gap chamber in the presence of high concentration of EGTA intracellularly. An inward phase of asymmetric currents following the hump component was observed in all fibers during the depolarization pulse to selected voltages (congruent to -45 mV). The average value of the peak inward current was 0.1 A/F (SEM = 0.01, n = 18), and the time at which it occurred was 34 ms (SEM = 1.8, n = 18). A second delayed outward phase of asymmetric current was observed after the inward phase, in those experiments in which hump component and inward phase were large. It peaked at more variable time (between 60 and 130 ms) with amplitude 0.02 A/F (SEM = 0.003, n = 11). The transmembrane voltage during a pulse, measured with a glass microelectrode, reached its steady value in less than 10 ms and showed no oscillations. The potential was steady at the time when the delayed component of asymmetric current occurred. ON and OFF charge transfers were equal for all pulse durations. The inward phase moved 1.4 nC/microF charge (SEM = 0.8, n = 6), or about one third of the final value of charge mobilized by these small pulses, and the second outward phase moved 0.7 nC/microF (SEM = 0.8, n = 6), bringing back about half of the charge moved during the inward phase. When repolarization intersected the peak of the inward phase, the OFF charge transfer was independent of the repolarization voltage in the range -60 to -90 mV. When both pre- and post-pulse voltages were changed between -120 mV and -60 mV, the equality of ON and OFF transfers of charge persisted, although they changed from 113 to 81% of their value at -90 mV. The three delayed phases in asymmetric current were also observed in experiments in which the extracellular solution contained Cd2+, La3+ and no Ca2+. Large increases in intracellular [Cl-] were imposed, and had no major effect on the delayed components of the asymmetric current. The Ca2+ transients measured optically and the calculated Ca2+ release fluxes had three phases whenever a visible outward phase followed the inward phase in the asymmetric current. Several interventions intended to interfere with Ca release, reduced or eliminated the three delayed phases of the asymmetric current.(ABSTRACT TRUNCATED AT 400 WORDS)     

Intramembrane charge movement and L-type calcium current in skeletal muscle fibers isolated from control and mdx mice

Dystrophin-deficient muscle fibers from mdx mice are believed to suffer from increased calcium entry and elevated submembranous calcium level, the actual source and functional consequences of which remain obscure. Here we compare the properties of the dihydropyridine receptor as voltage sensor and calcium channel in control and mdx muscle fibers, using the silicone-voltage clamp technique. In control fibers charge movement followed a two-state Boltzmann distribution with values for maximal charge, midpoint voltage, and steepness of 23 +/- 2 nC/ micro F, -37 +/- 3 mV, and 13 +/- 1 mV (n = 7). Essentially identical values were obtained in mdx fibers and the time course of charge recovery from inactivation was also similar in the two populations (tau approximately 6 s). In control fibers the voltage dependence of the slow calcium current elicited by 100-ms-long pulses gave values for maximal conductance, apparent reversal potential, half-activation potential, and steepness factor of 156 +/- 15 S/F, 65.5 +/- 2.9 mV, -0.76 +/- 1.2 mV, and 6.2 +/- 0.5 mV (n = 17). In mdx fibers, the half-activation potential of the calcium current was slightly more negative (-6.2 +/- 1.2 mV, n = 16). Also, when using longer pulses, the time constant of calcium current decay was found to be significantly larger (by a factor of 1.5-2) in mdx than in control fibers. These changes in calcium current properties are unlikely to be primarily responsible for a dramatic alteration of intracellular calcium homeostasis. They may be speculated to result, at least in part, from remodeling of the submembranous cytoskeleton network due to the absence of dystrophin.     

Electrical models of excitation-contraction coupling and charge movement in skeletal muscle

The consequences of ionic current flow from the T system to the sarcoplasmic reticulum (SR) of skeletal muscle are examined. The Appendix analyzes a simple model in which the conductance gx, linking T system and SR, is in series with a parallel resistor and capacitor having fixed values. The conductance gx is supposed to increase rapidly with depolarization and to decrease slowly with repolarization. Nonlinear transient currents computed from this model have some of the properties of gating currents produced by intramembrane charge movement. In particular, the integral of the transient current upon depolarization approximates that upon repolarization. Thus, equality of nonlinear charge movement can occur without intramembrane charge movement. A more complicated model is used in the text to fit the structure of skeletal muscle and other properties of its charge movement. Rectification is introduced into gx and the membrane conductance of the terminal cisternae to give asymmetry in the time- course of the transient currents and saturation in the curve relating charge movement to depolarization, respectively. The more complex model fits experimental data quite well if the longitudinal tubules of the sarcoplasmic reticulum are isolated from the terminal cisternae by a substantial resistance and if calcium release from the terminal cisternae is, for the most part, electrically silent. Specific experimental tests of the model are proposed, and the implications for excitation-contraction coupling are discussed.     

Effect of denervation on pigeon slow skeletal muscle

Summary The slow anterior latissimus dorsi muscle (ALD) of the pigeon was denervated surgically and examined after varying post-operative intervals. Muscles were studied with respect to changes in weight, histological and ultrastructural alterations, and changes in size and number of fibers. The weights of the denervated muscles increased over the contralateral control, reaching a maximum hypertrophy in the first 18 days, but the hypertrophy persisted for several months. The fibers of the denervated muscle did not hypertrophy. They showed a gradation in size from the posterior to the anterior border, with the fibers in the anterior third of the muscle being the smallest. After measuring cross-sectional sizes from the anterior, middle, and posterior thirds of the muscle, the overall fiber change was one of atrophy.Morphologically, the fibers showed various signs of pathological changes, including nuclear proliferation, swelling and migration away from the sarcolemmal position, vacuolation, myofibril degeneration, connective-tissue infiltration and replacement of the fibers, and regenerative activities in the form of budding and myoblast formation. A condition termed a peripheral rim of degeneration is described. Although many abnormal conditions were found in these denervated muscles, much of the muscle appeared normal; the neurotrophic relationship of slow muscle is discussed.This investigation was supported in part by a Public Health Service Fellowship, 2 F 2 NB 35, 582, from the National Institute of Neurological Diseases and Stroke, and by an Ohio University Research Grant to R. Hikida; and a grant 5 RO 1 AN 10856 from the National Institute of Arthritis and Metabolic Diseases to W. Bock.The authors wish to acknowledge gratefully the skillful technical assistance of Mr. Lawrence Mezza and Miss Sally Mitchell.     

Effect of sarcoplasmic reticulum calcium depletion on intramembranous charge movement in frog cut muscle fibers

Cut muscle fibers from Rana temporaria (sarcomere length, 3.3-3.5 microns; temperature, 13-16 degrees C) were mounted in a double Vaseline-gap chamber and equilibrated for at least an hour with an internal solution that contained 20 mM EGTA and phenol red and an external solution that contained predominantly TEA-gluconate; both solutions were nominally Ca-free. The increase in total myoplasmic concentration of Ca (delta[CaT]) produced by sarcoplasmic reticulum (SR) Ca release was estimated from the change in pH produced when the released Ca was complexed by EGTA (Pape, P.C., D.-S. Jong, and W.K. Chandler. 1995. Journal of General Physiology. 106:259-336). The resting value of SR Ca content, [CaSR]R (expressed as myoplasmic concentration), was taken to be equal to the value of delta[CaT] obtained during a step depolarization (usually to -50 to -40 mV) that was sufficiently long (200-750 ms) to release all of the readily releasable Ca from the SR. In ten fibers, the first depolarization gave [CaSR]R = 839-1,698 microM. Progressively smaller values were obtained with subsequent depolarizations until, after 30-40 depolarizations, the value of [CaSR]R had usually been reduced to < 10 microM. Measurements of intramembranous charge movement, Icm, showed that, as the value of [CaSR]R decreased, ON-OFF charge equality held and the amount of charge moved remained constant. ON Icm showed brief initial I beta components and prominent I gamma "humps", even after the value of [CaSR]R was < 10 microM. Although the amplitude of the hump component decreased during depletion, its duration increased in a manner that preserved the constancy of ON charge. In the depleted state, charge movement was steeply voltage dependent, with a mean value of 7.2 mV for the Boltzmann factor k. These and other results are not consistent with the idea that there is one type of charge, Q beta, and that I gamma is a movement of Q beta caused by SR Ca release, as proposed by Pizarro, Csernoch, Uribe, Rodriguez, and Rios (1991. Journal of General Physiology. 97:913-947). Rather, our results imply that Q beta and Q gamma represent either two distinct species of charge or two transitions with different properties of a single species of charge, and that SR Ca content or release or some related event alters the kinetics, but not the amount of Q gamma. Many of the properties of Q gamma, as well as the voltage dependence of the rate of SR Ca release for small depolarizations, are consistent with predictions from a simple model in which the voltage sensor for SR Ca release consists of four interacting charge movement particles.     

Development of ionic currents of zebrafish slow and fast skeletal muscle fibers

Voltage-gated Na+ and K+ channels play key roles in the excitability of skeletal muscle fibers. In this study we investigated the steady-state and kinetic properties of voltage-gated Na+ and K+ currents of slow and fast skeletal muscle fibers in zebrafish ranging in age from 1 day postfertilization (dpf) to 4-6 dpf. The inner white (fast) fibers possess an A-type inactivating K+ current that increases in peak current density and accelerates its rise and decay times during development. As the muscle matured, the V50s of activation and inactivation of the A-type current became more depolarized, and then hyperpolarized again in older animals. The activation kinetics of the delayed outward K+ current in red (slow) fibers accelerated within the first week of development. The tail currents of the outward K+ currents were too small to allow an accurate determination of the V50s of activation. Red fibers did not show any evidence of inward Na+ currents; however, white fibers expressed Na+ currents that increased their peak current density, accelerated their inactivation kinetics, and hyperpolarized their V50 of inactivation during development. The action potentials of white fibers exhibited significant changes in the threshold voltage and the half width. These findings indicate that there are significant differences in the ionic current profiles between the red and white fibers and that a number of changes occur in the steady-state and kinetic properties of Na+ and K+ currents of developing zebrafish skeletal muscle fibers, with the most dramatic changes occurring around the end of the first day following egg fertilization.     

MicroRNA调控哺乳动物骨骼肌发育

MicroRNA (miRNA)是一类长度大约为22 bp的小分子非编码RNA,广泛存在于哺乳动物中,部分miRNA表达具有时空和组织特异性。哺乳动物中miRNA主要通过与靶基因3° UTR区结合抑制其翻译,调控机体生物学功能。miRNA在哺乳动物骨骼肌发育中发挥重要调节作用。哺乳动物骨骼肌发育是一个复杂的生物学过程,包括骨骼肌干细胞增殖、迁移、分化,成肌细胞增殖、分化、肌管融合,肌纤维肥大,能量代谢,纤维类型转换等。miRNA参与骨骼肌发育的各个环节,通过靶向各个时期的关键因子调控骨骼肌发育。本文对miRNA在骨骼肌发育中的调控作用进行了综述,以期为深入理解骨骼肌发育规律提供参考。     

Fiber-type proportions in mammalian soleus muscle during postnatal development

We analyzed the fiber-type composition of the soleus muscle in rats and mice to determine whether the adult proportion of fiber types is fixed soon after birth or whether it changes during postnatal maturation. We examined muscles from animals varying in age from 1 week to 1 year using monoclonal antibodies that distinguish between fast and slow isoforms of myosin heavy chains. In cross sections of unfixed muscle containing profiles of all myofibers in the muscle, we counted the fibers that stained with antibodies to fast myosin, and in adjacent sections, those that stained positive with an antibody to slow myosin. We also counted the total number of fibers in each section. Rat soleus contained about 2500 myofibers, and mouse about 1000 at all ages studied, suggesting that myogenesis ceases in soleus by 1 week after birth or sooner. In mouse soleus, the relative proportions of fibers staining positive with fast and slow myosin antibodies were similar at all ages studied, about 60%–70% being fast and 30%–40% slow. In rat soleus, however, the proportions of fast antibody-positive and slow antibody-positive fibers changed dramatically during postnatal maturation. At 1 week after birth, about 50% of rat soleus fibers stained with fast myosin antibodies, whereas between 1 and 2 months this value fell to about 10%. In mouse, about 10% of fibers at 1 week, but none at 1 year, reacted with both fast and slow antibodies, whereas in rat, fewer than 3% bound both antibodies to a significant degree at 1 week. It is puzzling why, in rat soleus, the majority of apparently fast fibers present at 1 week is converted to a slow phenotype, whereas in mouse soleus the predominant change appears to be the suppression of fast myosin expression in a subset of fibers that expresses both myosin types at 1 week. It is possible that this may be related to differences in size and the amount of body growth between these two species.     

Dissociation of charge movement from calcium release and calcium current in skeletal myotubes by gabapentin

The skeletal muscle L-type calcium channel or dihydropyridine receptor (DHPR) plays an integral role in excitation-contraction (E-C) coupling. Its activation initiates three sequential events: charge movement (Q(r)), calcium release, and calcium current (I(Ca,L)). This relationship suggests that changes in Q(r) might affect release and I(Ca,L). Here we studied the effect of gabapentin (GBP) on the three events generated by DHPRs in skeletal myotubes in culture. GBP specifically binds to the alpha(2)/delta(1) subunit of the brain and skeletal muscle DHPR. Myotubes were stimulated with a protocol that included a depolarizing prepulse to inactivate voltage-dependent proteins other than DHPRs. Gabapentin (50 microM) significantly increased Q(r) while decreasing the rate of rise of calcium transients. Gabapentin also reduced the maximum amplitude of the I(Ca,L) (as we previously reported) without modifying the kinetics of activation. Exposure of GBP-treated myotubes to 10 microM nifedipine prevented the increase of Q(r) promoted by this drug, indicating that the extra charge recorded originated from DHPRs. Our data suggest that GBP dissociates the functions of the DHPR from the initial voltage-sensing step and implicates a role for the alpha(2)/delta(1) subunit in E-C coupling.     

Pharmacological dissection of charge movement in frog skeletal muscle fibers.

When charge movement is measured from muscle fibers bathed in a moderately hypertonic solution, a secondary hump appears in the decay phase of the signal during the "on" of the test pulse. The hump can be suppressed by the application of dantrolene sodium or tetracaine. The amount of charge associated with the hump is approximately 20-25% of the total charge. All the observed properties of the hump charge are consistent with the possibility that it is more closely associated with calcium release from the sarcoplasmic reticulum, and thus more relevant to excitation-contraction coupling, than the rest of the charge.