Frog Skeletal Muscle Research Articles

Intracellular [H+]: a determinant of cell volume in skeletal muscle

Intracellular [H⁺]: a determinant of cell volume in skeletal muscle

Calcium sparks in skeletal muscle fibers

Ca 2+ sparks monitor transient local releases of Ca 2+ from the sarcoplasmic reticulum (SR) into the myoplasm. The release takes place through ryanodine receptors (RYRs), the Ca 2+-release channels of the SR. In intact fibers from frog skeletal muscle, the temporal and spatial properties of voltage-activated Ca 2+ sparks are well simulated by a model that assumes that the Ca 2+ flux underlying a spark is 2.5 pA (units of Ca 2+ current) for 4.6 ms (18 °C). This flux amplitude suggests that 1–5 active RYRs participate in the generation of a typical voltage-activated spark under physiological conditions. A major goal of future experiments is to estimate this number more precisely and, if it is two or more, to investigate the communication mechanism that allows multiple RYRs to be co-activated in a rapid but self-limited fashion.

Association of the Iγ and Iδ 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 20mM EGTA plus added calcium and antipyrylazo III. When the nominal free [Ca2+]i was 10nM, the shape of the broad Iγ 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 50nM, which was close to the physiological level, the Iγ humps were accelerated and a slow calcium-dependent Iδ component (or state) was generated. The peak of ON Iδ synchronized perfectly with the peak of the calcium release rate whereas the slow decay of ON Iδ followed the same time course as the decay of calcium release rate. Suppression of calcium release by TMB-8 reduced the amount of Qδ 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 Qδ needs to be primed by a physiological level of resting myoplasmic Ca2+. Once the priming is completed, more Iδ is mobilized by the released Ca2+ during depolarization.

The influence of hypothermia on P2 receptor-mediated responses of frog skeletal muscle

The contractile responses of isolated Rana ridibunda frog sartorius muscle contractions evoked by electrical field stimulation (EFS) were studied at three temperature conditions of 17, 22 and 27 °C. Temperature-dependent increase of muscle contractility was found. ATP (10–100 μM) concentration dependently inhibited the electrical field stimulation-evoked contractions of sartorius muscle at all three temperatures; this effect was significantly more prominent at a temperature of 17 °C than at other two temperatures. Adenosine (100 μM) also caused inhibition of electrical field stimulation-evoked contractions which was statistically identical at all three temperature conditions tested. A P2 receptor antagonist, pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS, 10 μM) reduced the inhibitory effect of ATP at all three temperatures but did not affect inhibitory action of adenosine. In contrast, 8-( p-sulfophenyl)theophylline (8-SPT, 100 μM), a nonselective P1 receptor antagonist, abolished inhibitory effects of adenosine at all three temperature conditions but did not antagonize inhibition caused by ATP. In electrophysiological experiments, ATP (100 μM) and adenosine (100 μM) temperature dependently reduced end-plate currents recorded in sartorius neuromuscular junction by voltage-clamp technique. The inhibitory effects of both agonists were enhanced with the decrease of temperature. 8-SPT (100 μM) abolished the inhibitory effect of adenosine but not ATP on end-plate currents. Suramin (100 μM), a nonselective P2 receptor antagonist, inhibited the action of ATP but not adenosine, while PPADS (10 μM) had no influence on the effects of either ATP or adenosine. It is concluded from this study that the effectiveness of P2 receptor-mediated inhibition of frog skeletal muscle contraction in contrast to that of adenosine is dependent on the temperature conditions.

Reciprocal communication between L-type Ca2+ channel and ryanodine receptor in frog skeletal muscle

Reciprocal communication between L-type Ca2+ channel and ryanodine receptor in frog skeletal muscle

In vivo expression of myosin essential light chain using plasmid expression vectors in regenerating frog skeletal muscle

It is well established that mutations in specific structural elements of the motor protein myosin are directly linked to debilitating diseases involving malfunctioning striated muscle cells. A potential way to study the relationship between myosin structure and function is to express exogenous myosin in vivo and determine contractile properties of the transgenic muscle cells. However, in vivo expression of functional levels of contractile proteins using transient transgenesis in skeletal muscle has not been demonstrated. Presently, we used in vivo gene transfer to express high levels of full-length myosin light chain (MLC) in skeletal muscle fibers of Rana pipiens. Anterior tibialis (AT) muscles were injected with cardiotoxin to cause degeneration and then injected at various stages of regeneration with plasmid expression vectors encoding full-length MLC1(f). In fibers from the most robustly transfected muscles 3 weeks after plasmid injections, trans-MLC1(f) expression averaged 22-43% of the endogenous MLC1(f). Trans-MLC1(f) expression was the same whether a small epitope tag was placed on the C- or N-terminus and was highly variable along individual fibers. Confocal microscopy of skinned fibers showed correct sarcomeric incorporation of trans-MLC1(f). The expression profile of myosin heavy chain isoforms 21 days after transfection was similar to normal AT muscle. These data demonstrate the feasibility of using in vivo gene transfer to probe the structural basis of contractile protein function in skeletal muscle. Based on these promising results, we discuss how further improvements in the level and consistency of myosin transgene expression may be achieved in future studies, and the therapeutic potential of plasmid gene transfer in regenerating muscle.

Sarcomere strain and heterogeneity correlate with injury to frog skeletal muscle fiber bundles.

Sarcomere length and first-order diffraction line width were measured by laser diffraction during elongation of activated frog tibialis anterior muscle fiber bundles (i.e., eccentric contraction) at nominal fiber strains of 10, 25, or 35% (n = 18) for 10 successive contractions. Tetanic tension, measured just before each eccentric contraction, differed significantly among strain groups and changed dramatically during the 10-contraction treatment (P < 0.01). Average maximum tetanic tension for the three groups measured before any treatment was 203.7 +/- 6.8 kN/m2, but after the 10-eccentric contraction sequence decreased to 180.3 +/- 3.8, 125.1 +/- 7.8, and 78.3 +/- 5.1 kN/m2 for the 10, 25, and 35% strain groups, respectively (P < 0.0001). Addition of 10 mM caffeine to the bathing medium decreased the loss of tetanic tension in the 10% strain group but had only a minimal effect on either the 25 or 35% strain groups. Diffraction pattern line width, a measure of sarcomere length heterogeneity, increased significantly with muscle activation and then continued to increase with successive stretches of the activated muscle. Line width increase after each stretch was significantly correlated with the lower yield tension of the successive contractile record. These data demonstrate a direct association and, perhaps, a causal relationship between sarcomere strain and fiber bundle injury. They also demonstrate that muscle injury is accompanied by a progressive increase in sarcomere length heterogeneity, yielding lower yield tension as injury progresses.

Regulation of muscle Cav1.1 channels by long-term depolarization involves proteolysis of the alpha1s subunit.

The effects of long-term depolarization on frog skeletal muscle Cav1.1 channels were assessed. Voltage-clamp and Western-blot experiments revealed that long-term depolarization brings about a drastic reduction in the amplitude of currents flowing through Cav1.1 channels and in the levels of the alpha1s subunit, the main subunit of muscle L-type channels. The decline of both phenomena was prevented by the action of the protease inhibitors E64 (50 microM) and leupeptin (50 microM). In contrast, long-term depolarization had no effect on beta1, the auxiliary subunit of alpha1s. The levels of mRNAs coding the alpha1s and the beta1 subunits were measured by RNase protection assays. Neither the content of the alpha1s nor the beta1 subunit mRNAs were affected by long-term depolarization, indicating that the synthesis of Cav1.1 channels remained unaffected. Taken together, our experiments suggest that the reduction in the amplitude of membrane currents and in the alpha1s subunit levels is caused by increased degradation of this subunit by a Ca2+-dependent protease.

RyR1 exhibits lower gain of CICR activity than RyR3 in the SR: evidence for selective stabilization of RyR1 channel.

We showed that frog alpha-ryanodine receptor (alpha-RyR) had a lower gain of Ca(2+)-induced Ca(2+) release (CICR) activity than beta-RyR in sarcoplasmic reticulum (SR) vesicles, indicating selective "stabilization" of the former isoform (Murayama T and Ogawa Y. J Biol Chem 276: 2953-2960, 2001). To know whether this is also the case with mammalian RyR1, we determined [(3)H]ryanodine binding of RyR1 and RyR3 in bovine diaphragm SR vesicles. The value of [(3)H]ryanodine binding (B) was normalized by the number of maximal binding sites (B(max)), whereby the specific activity of each isoform was expressed. This B/B(max) expression demonstrated that ryanodine binding of individual channels for RyR1 was <15% that for RyR3. Responses to Ca(2+), Mg(2+), adenine nucleotides, and caffeine were not substantially different between in situ and purified isoforms. These results suggest that the gain of CICR activity of RyR1 is markedly lower than that of RyR3 in mammalian skeletal muscle, indicating selective stabilization of RyR1 as is true of frog alpha-RyR. The stabilization was partly eliminated by FK506 and partly by solubilization of the vesicles with CHAPS, each of which was additive to the other. In contrast, high salt, which greatly enhances [(3)H]ryanodine binding, caused only a minor effect on the stabilization of RyR1. None of the T-tubule components, coexisting RyR3, or calmodulin was the cause. The CHAPS-sensitive intra- and intermolecular interactions that are common between mammalian and frog skeletal muscles and the isoform-specific inhibition by FKBP12, which is characteristic of mammals, are likely to be the underlying mechanisms.

Fatigue and recovery of dynamic and steady-state performance in frog skeletal muscle.

Muscle fatigue reflects alterations of both activation and cross-bridge function, which will have markedly different affects on steady-state vs. dynamic performance. Such differences offer insight into the specific origins of fatigue, its mechanical manifestation, and its consequences for animal movement. These were inferred using dynamic contractions (twitches and cyclic work as might occur during locomotion) and steady-state performance with maximal, sustained activation (tetani, stiffness, and isokinetic force) during fatigue and then recovery of frog (Rana pipiens) anterior tibialis muscle. Stiffness remained unaltered during early fatigue of force and then declined only 25% as force dropped 50%, suggesting a decline with fatigue in first the force-generating ability and then the number of cross bridges. The relationship between stiffness and force was different during fatigue and recovery; thus the number of cross bridges and force per cross bridge are not intimately linked. Twitch duration increased with fatigue and then recovered, with trajectories that were remarkably similar to and linear with changes in tetanic force, perhaps belying a common mechanism. Twitch force increased and then returned to resting levels during fatigue, reflecting a slowing of activation kinetics and a decline in cross-bridge number and force. Net cyclic work fatigued to the degree of becoming negative when tetanic force had declined only 15%. Steady-state isokinetic force (i.e., shortening work) declined by 75%, while cyclic shortening work declined only 30%. Slowed activation kinetics were again responsible, augmenting cyclic shortening work but greatly augmenting lengthening work (reducing net work). Steady-state measures can thus seriously mislead regarding muscle performance in an animal during fatigue.

Peptide and protein modulation of local Ca2+ release events in permeabilized skeletal muscle fibers

Local discrete elevations in myoplasmic Ca2+ (Ca2+ sparks) arise from the opening of a small group of RyRs. Summation of a large number of Ca2+ sparks gives rise to the whole cell Ca2+ transient necessary for muscle contraction, Unlike sarcoplasmic reticulum vesicle preparations and isolated single channels in artificial membranes, the study of Ca2+ sparks provides a means to understand the regulation of a small group of RyRs in the environment of a functionally intact triad and in the presence of endogenous regulatory proteins. To gain insight into the mechanisms that regulate the gating of RyRs we have utilized laser scanning confocal microscopy to measure Ca2+ sparks in permeabilized frog skeletal muscle fibers. This review summarizes our recent studies using both exogenous (ImperatoxinA and domain peptides) and endogenous (calmodulin) modulators of RyR to gain insight into the number of RyR Ca2+ release channels underlying a Ca2+ spark, how domain-domain interactions within RyR regulate the functional state of the channel as well as gating mechanisms of RyR in living muscle fibers.

1P184 収縮中の筋に長さ変化を加えたときのx-線反射像の変化(筋肉 : 筋蛋白・収縮)

1P184 収縮中の筋に長さ変化を加えたときのx-線反射像の変化(筋肉 : 筋蛋白・収縮)

Stereospecific synthesis of “para‐hydroxymexiletine” and sodium channel blocking activity evaluation

Both enantiomers of "para-hydroxymexiletine" (PHM), one of the main metabolites of mexiletine, were synthesized and fully characterized. Properties of (R)- and (S)-PHM, in terms of blocking potency and stereoselectivity on frog skeletal muscle Na(+) channels, were evaluated. The presence of a hydroxy group on the aryloxy moiety in the 4-position, as in PHM, reduced potency with respect to mexiletine in reducing I(Na max). However, PHM showed clear use-dependent behavior similar to that of mexiletine and, in contrast with what is observed with the parent compound, maintained its stereoselectivity during the use-dependent block. Chirality 16:72-78, 2004.

The Conformation of Myosin Head Domains in Rigor Muscle Determined by X-Ray Interference

In the absence of adenosine triphosphate, the head domains of myosin cross-bridges in muscle bind to actin filaments in a rigor conformation that is expected to mimic that following the working stroke during active contraction. We used x-ray interference between the two head arrays in opposite halves of each myosin filament to determine the rigor head conformation in single fibers from frog skeletal muscle. During isometric contraction (force T 0), the interference effect splits the M3 x-ray reflection from the axial repeat of the heads into two peaks with relative intensity (higher angle/lower angle peak) 0.76. In demembranated fibers in rigor at low force (<0.05 T 0), the relative intensity was 4.0, showing that the center of mass of the heads had moved 4.5 nm closer to the midpoint of the myosin filament. When rigor fibers were stretched, increasing the force to 0.55 T 0, the heads’ center of mass moved back by 1.1–1.6 nm. These motions can be explained by tilting of the light chain domain of the head so that the mean angle between the Cys 707–Lys 843 vector and the filament axis increases by ∼36° between isometric contraction and low-force rigor, and decreases by 7–10° when the rigor fiber is stretched to 0.55 T 0.

Calmodulin Modulates Initiation but Not Termination of Spontaneous Ca 2+ Sparks in Frog Skeletal Muscle

Calmodulin is a ubiquitous Ca 2+ sensing protein that binds to and modulates the sarcoplasmic reticulum Ca 2+ release channel, ryanodine receptor (RYR). Here we assessed the effects of calmodulin on the local Ca 2+ release properties of RYR in permeabilized frog skeletal muscle fibers. Fluorescently labeled recombinant calmodulin in the internal solution localized at the Z-line/triad region. Calmodulin (0.05–5.0 μM) in the internal solution (free [Ca 2+] i ∼50–100 nM) initiated a highly cooperative dose-dependent increase in Ca 2+ spark frequency, with a half-maximal activation ( K) of 1.1 μM, a Hill coefficient ( n) of 4.2 and a fractional maximal increase in frequency ( R) of 17-fold. A non-Ca 2+ binding mutant of calmodulin elicited a similar highly cooperative dose-dependent increase in spark frequency ( K = 1.0 μM; n = 3.7; R = 12-fold). Spatiotemporal properties of Ca 2+ sparks were essentially unaffected by either wild-type or mutant calmodulin. An N-terminal extension of calmodulin, (N+3)calmodulin, that binds to but does not activate RYR at nM [Ca 2+] in sarcoplasmic reticulum vesicles, prevented the calmodulin-induced increase in spark frequency. These data suggest that exogenous Ca 2+-free calmodulin cooperatively sensitizes the Ca 2+ release channel to open, but that Ca 2+ binding to the added calmodulin does not play a significant role in the termination of Ca 2+ sparks.

Differential Effects of Voltage-Dependent Inactivation and Local Anesthetics on Kinetic Phases of Ca 2+ Release in Frog Skeletal Muscle

In voltage-clamped frog skeletal muscle fibers, Ca 2+ release rises rapidly to a peak, then decays to a nearly steady state. The voltage dependence of the ratio of amplitudes of these two phases ( p/s) shows a maximum at low voltages and declines with further depolarization. The peak phase has been attributed to a component of Ca 2+ release induced by Ca 2+, which is proportionally greater at low voltages. We compared the effects of two interventions that inhibit Ca 2+ release: inactivation of voltage sensors, and local anesthetics reputed to block Ca 2+ release induced by Ca 2+. Holding the cells partially depolarized strongly reduced the peak and steady levels of Ca 2+ release elicited by a test pulse and suppressed the maximum of the p/s ratio at low voltages. The p/s ratio increased monotonically with test voltage, eventually reaching a value similar to the maximum found in noninactivated fibers. This implies that the marked peak of Ca 2+ release is a property of a cooperating collection of voltage sensors rather than individual ones. Local anesthetics reduced the peak of release flux at every test voltage, and the steady phase to a lesser degree. At variance with sustained depolarization, they made p/s low at all voltages. These observations were well-reproduced by the “couplon” model of dual control, which assumes that depolarization and anesthetics respectively, and selectively, disable its Ca 2+-dependent or its voltage-operated channels. This duality of effects and their simulation under such hypotheses are consistent with the operation of a dual, two-stage control of Ca 2+ release in muscle, whereby Ca 2+ released through multiple directly voltage-activated channels builds up at junctions to secondarily open Ca 2+-operated channels.

Temperature dependence of the force-generating process in single fibres from frog skeletal muscle.

Generation of force and shortening in striated muscle is due to the cyclic interactions of the globular portion (the head) of the myosin molecule, extending from the thick filament, with the actin filament. The work produced in each interaction is due to a conformational change (the working stroke) driven by the hydrolysis of ATP on the catalytic site of the myosin head. However, the precise mechanism and the size of the force and length step generated in one interaction are still under question. Here we reinvestigate the endothermic nature of the force-generating process by precisely determining, in tetanized intact frog muscle fibres under sarcomere length control, the effect of temperature on both isometric force and force response to length changes. We show that raising the temperature: (1) increases the force and the strain of the myosin heads attached in the isometric contraction by the same amount (approximately 70 %, from 2 to 17 degrees C); (2) increases the rate of quick force recovery following small length steps (range between -3 and 2 nm (half-sarcomere)-1) with a Q10 (between 2 and 12 degrees C) of 1.9 (releases) and 2.3 (stretches); (3) does not affect the maximum extent of filament sliding accounted for by the working stroke in the attached heads (10 nm (half-sarcomere)-1). These results indicate that in isometric conditions the structural change leading to force generation in the attached myosin heads can be modulated by temperature at the expense of the structural change responsible for the working stroke that drives filament sliding. The energy stored in the elasticity of the attached myosin heads at the plateau of the isometric tetanus increases with temperature, but even at high temperature this energy is only a fraction of the mechanical energy released by attached heads during filament sliding.

Ultra-small-angle X-ray diffraction and scattering experiments using medium-length beamlines at SPring-8

Ultra-small-angle X-ray diffraction experiments were made at the two so-called medium-length beamlines at SPring-8. One is a bending magnet beamline (BL20B2) and the other an undulator beamline (BL20XU). Both have two experimental stations separated by 140–150 m. Specimens were placed in the first hutch and the diffraction or scattering patterns were recorded in the second hutch with an X-ray image intensifier or an imaging plate. At an X-ray energy of 12–21 keV, a small-angle resolution of 2 µm was achieved. The exposure time required to record sarcomere diffraction from frog skeletal muscle was 10 min at BL20B2 and 2 s at BL20XU. These results demonstrate the capability of these beamlines in ultra-small angle diffraction/scattering experiments.

Hypotonic stimulation of the Na+ active transport in frog skeletal muscle: role of the cytoskeleton

Hypotonicity produces a marked activation of the Na+ pump in frog sartorius muscle. The increase in net Na+ efflux under hypotonic conditions occurs despite the reductions in [Na+]i that are due to fibre swelling and Na+ loss. The pump density (ouabain binding) increases not only upon reduction of the medium osmotic pressure (π) from its normal value (π= 1) to one-half (π= 0.5), but also in muscles that are returned to π= 1 after equilibration in π= 2 medium. The equilibration in π= 2 medium does not affect pump density. Ouabain-binding increments cannot be ascribed to a rise in the Na+–K+ exchange rate of a fixed number of pumps: they also occurred in the continued presence of a saturating concentration of ouabain (50 μm). Under those conditions, the π= 1 ★π= 0.5 transfer produced a 43 % increase in pump sites, while the π= 2 ★π= 1 transfer induced a rise of 46 %. Actinomycin D did not alter the stimulation of Na+ extrusion elicited by hypotonicity, suggesting that de novo synthesis of pumps was not involved in the increase of the apparent number of pump sites. Disruption of microtubules by colchicine (100 μm) and intermediate filaments by acrylamide (4 mm) did not alter the hypotonic effect. Likewise, genistein (100 μm), a specific inhibitor of tyrosine kinase, did not affect significantly the hypotonic response. Microfilament-disrupting agents like cytochalasin B (5 μm) and latrunculin B (10 μm) reduced the increase in Na+ efflux induced by π= 1 ★π= 0.5 transfer by about 35 % and 72 %, respectively. Latrunculin B reduced the increases in pump density generated by π= 1 ★π= 0.5 and π= 2 ★π= 1 transfers by about 79 % and 91 %, respectively. The results suggest that the membrane stretch due to hypotonic fibre volume increase would promote a microfilament-mediated insertion of submembranous spare Na+ pumps in the sarcolemma and, consequently, the rise in active Na+ transport.

Energy storage during stretch of active single fibres from frog skeletal muscle

Energy storage during stretch of active single fibres from frog skeletal muscle