Step In Excitation-contraction Coupling Research Articles

Measurement of force and calcium release using mechanically skinned fibers from mammalian skeletal muscle.

The mechanically skinned (or "peeled") skeletal muscle fiber technique is a highly versatile procedure that allows controlled examination of each of the steps in the excitation-contraction (EC)-coupling sequence in skeletal muscle fibers, starting with excitation/depolarization of the transverse tubular (T)-system through to Ca2+ release from sarcoplasmic reticulum (SR) and finally force development by the contractile apparatus. It can also show the overall response of the whole EC-coupling sequence together, such as in twitch and tetanic force responses. A major advantage over intact muscle fiber preparations is that it is possible to set and rapidly manipulate the "intracellular" conditions, allowing examination of the effects of key variables (e.g., intracellular pH, ATP levels, redox state, etc.) on each individual step in EC coupling. This Cores of Reproducibility in Physiology (CORP) article describes the rationale, procedures, and experimental details of the various ways in which the mechanically skinned fiber technique is used in our laboratory to examine the physiological mechanisms controlling Ca2+ release and contraction in skeletal muscle fibers and the aberrations and dysfunction occurring with exercise and disease.

Hypermuscular mice with mutation in the myostatin gene display altered calcium signalling

Myostatin, a member of the transforming growth factor β family, is a potent negative regulator of skeletal muscle growth, as myostatin-deficient mice show a great increase in muscle mass. Yet the physical performance of these animals is reduced. As an explanation for this, alterations in the steps in excitation-contraction coupling were hypothesized and tested for in mice with the 12 bp deletion in the propeptide region of the myostatin precursor (Mstn(Cmpt-dl1Abc) or Cmpt). In voluntary wheel running, control C57BL/6 mice performed better than the mutant animals in both maximal speed and total distance covered. Despite the previously described lower specific force of Cmpt animals, the pCa-force relationship, determined on chemically permeabilized fibre segments, did not show any significant difference between the two mouse strains. While resting intracellular Ca(2+) concentration ([Ca(2+)]i) measured on single intact flexor digitorum brevis (FDB) muscle fibres using Fura-2 AM was similar to control (72.0 ± 1.7 vs. 78.1 ± 2.9 nM, n = 38 and 45), the amplitude of KCl-evoked calcium transients was smaller (360 ± 49 vs. 222 ± 45 nM, n = 22) in the mutant strain. Similar results were obtained using tetanic stimulation and Rhod-2 AM, which gave calcium transients that were smaller (2.42 ± 0.11 vs. 2.06 ± 0.10 ΔF/F0, n = 14 and 13, respectively) on Cmpt mice. Sarcoplasmic reticulum (SR) calcium release flux calculated from these transients showed a reduced peak (23.7 ± 3.0 vs. 15.8 ± 2.1 mM s(-1)) and steady level (5.7 ± 0.7 vs. 3.7 ± 0.5 mM s(-1)) with no change in the peak-to-steady ratio. The amplitude and spatial spread of calcium release events detected on permeabilized FDB fibres were also significantly smaller in mutant mice. These results suggest that reduced SR calcium release underlies the reduced muscle force in Cmpt animals.

S100A1: a physiological modulator of RYR1, Ca2+ release, and contractility in skeletal muscle. Focus on “S100A1 promotes action potential-initiated calcium release flux and force production in skeletal muscle”

SKELETAL MUSCLES adjust their contractile output over an exceptionally wide dynamic range, as needed to produce movements ranging from sitting and standing, to athletics and dance. At the single fiber level, the force generated by skeletal muscles is determined, ultimately, by the formation of cycling cross bridges between actin and myosin. This interaction requires Ca 2 and is governed by the simple relationship: force is proportional to intracellular ionized Ca 2 . Cross-bridge formation is inhibited in resting muscle when Ca 2 is sequestered within the sarcoplasmic reticulum (SR); it is activated when stored Ca 2 is released into the cytosol, allowing actin-myosin interactions to proceed. Contraction stops as Ca 2 is actively resequestered by the Ca 2 -ATPase of the SR (SERCA1). Thus Ca 2 is the central messenger in muscle activation, and the amplitude and time course of the intracellular Ca 2 change is the major determinant of contractile output. The key mechanisms that activate Ca 2 release from the SR are now well defined. This sequence, termed excitation-contraction coupling, begins when an excitatory input from the motor nerve triggers a propagating action potential on the muscle membrane. The action potential rapidly depolarizes the entire sarcolemma, including transverse tubules, which invaginate from the surface membrane and form specialized junctions (triads) with the SR along much of their length. The triad junction serves as a platform for assembling sarcolemmal calcium

Alterations in the calcium homeostasis of skeletal muscle from postmyocardial infarcted rats

In chronic heart failure, skeletal muscles develop a weakness that is not associated to an impaired circulatory function but rather to alterations in the skeletal muscle fibers themselves. To understand these changes, the steps in excitation-contraction coupling of rats that underwent a left anterior coronary artery occlusion were studied. About 24 weeks after the myocardial infarction, neither the total amount nor the voltage dependence of intramembrane charge were altered. In contrast, calcium release from the sarcoplasmic reticulum was considerably suppressed, and its voltage dependence shifted toward more positive voltages. Elementary calcium-release events showed altered morphology as the relative proportion of embers increased. Calcium sparks were smaller in amplitude and had larger time-to-peak values. Isolated ryanodine receptors (RyR) displayed an unusual rectification with increased single-channel conductance at positive (cis vs trans) voltages. In addition, the bell-shaped calcium dependence of channel activity was broader, with a slight shift of activation to lower and a larger shift in inactivation to higher calcium concentrations. These data indicate that the number of channels that open during a calcium-release event is decreased and that RyR function is altered; thus, calcium-release is suppressed after a myocardial infarction. These observations give an explanation for the impaired skeletal muscle function in these animals.

A Novel Role of VIP in Colonic Motility Function: Induction of Excitation–Transcription Coupling in Smooth Muscle Cells

Vasoactive intestinal polypeptide (VIP) relaxes smooth muscle by generation of cAMP and activation of protein kinase A (PKA). However, PKA activation also phosphorylates the transcription factor CREB. The aim of this study was to investigate whether the phosphorylation of CREB induces gene expression of the pore-forming alpha(1C) subunit of Ca(v)1.2 channels (L-type calcium channels), whose promoter has 2 binding sites for CREB. The experiments were performed on primary cultures of human colonic circular smooth muscle cells and freshly obtained human and rat colonic circular muscle strips. The incubation of human colonic circular smooth muscle cells or muscle strips with VIP for 24 hours enhanced the expression of alpha(1C) protein and mRNA as well as the contractile response to acetylcholine and KCl. On the contrary, incubation of the muscle strips with VIP antagonist for 24 hours suppressed cell contractility. The incubation of the cells with VIP caused sustained generation of cAMP for 24 hours, but PKA activation and CREB phosphorylation were transient. The inhibition of PKA by H-89 or silencing of CREB gene with targeted RNAi blocked the transcription of alpha(1C). Progressive 5' deletions of halpha(1C)1b promoter and site-directed mutations of the 2 CREB binding cis-elements indicated that most of alpha(1C) transcription was mediated by the 5' cAMP response element. The excitation-transcription coupling stimulated by VIP induces expression of the Ca(v)1.2 channels. The influx of calcium through these channels is a critical step in excitation-contraction coupling in smooth muscle cells.

Altered Elementary Calcium Release Events and Enhanced Calcium Release by Thymol in Rat Skeletal Muscle

The effects of thymol on steps of excitation-contraction coupling were studied on fast-twitch muscles of rodents. Thymol was found to increase the depolarization-induced release of calcium from the sarcoplasmic reticulum, which could not be attributed to a decreased calcium-dependent inactivation of calcium release channels/ryanodine receptors or altered intramembrane charge movement, but rather to a more efficient coupling of depolarization to channel opening. Thymol increased ryanodine binding to heavy sarcoplasmic reticulum vesicles, with a half-activating concentration of 144 μM and a Hill coefficient of 1.89, and the open probability of the isolated and reconstituted ryanodine receptors, from 0.09 ± 0.03 to 0.22 ± 0.04 at 30 μM. At higher concentrations the drug induced long-lasting open events on a full conducting state. Elementary calcium release events imaged using laser scanning confocal microscopy in the line-scan mode were reduced in size, 0.92 ± 0.01 vs. 0.70 ± 0.01, but increased in duration, 56 ± 1 vs. 79 ± 1 ms, by 30 μM thymol, with an increase in the relative proportion of lone embers. Higher concentrations favored long events, resembling embers in control, with duration often exceeding 500 ms. These findings provide direct experimental evidence that the opening of a single release channel will generate an ember, rather than a spark, in mammalian skeletal muscle.

Force generation induced by rapid temperature jumps in intact mammalian (rat) skeletal muscle fibres

We examined the tension (force) responses induced by rapid temperature jumps (T-jumps) in electrically stimulated, intact fibre bundles (5–10 fibres, fibre length ∼2 mm) isolated from a foot muscle (flexor hallucis brevis) of the rat; the muscle contains ∼90 % type 2 fast fibres. In steady state experiments, the temperature dependence of the twitch tension was basically similar to that previously described from other fast muscles; the tetanic tension increased 3- to 4-fold in raising the temperature from ∼2 to 35 °C and the relation between the tetanic tension and the reciprocal absolute temperature was sigmoidal with half-maximal tension at 9.5 °C. A rapid T-jump of 3–5 °C was induced during a contraction by applying an infrared laser pulse (λ= 1.32 μ, 0.2 ms) to the 50 μl trough containing the fibre bundle immersed in physiological saline. At ∼10 °C, a T-jump induced a large transient tension rise when applied during the rising phase of a twitch contraction, the amplitude of which decreased when the T-jump was delayed with respect to the stimulus; a T-jump probably perturbs an early step in excitation-contraction coupling. No transient increase was seen when a T-jump was applied during twitch relaxation. When applied during the plateau of a tetanic contraction a T-jump induced a tension rise to a higher steady tension level; the tension rise after a T-jump was 2–3 times faster than the corresponding phase of the initial tension rise in a tetanus. The approach to a new steady tension level after a T-jump was biphasic with a fast (phase 2b, ∼35 s−1 at 10 °C) and a slow component (phase 3, < 10 s−1). The rates of both components increased (Q10∼3) but their amplitudes decreased with increase of the steady temperature. These results from tetanised intact fibres are consistent with the thesis previously proposed from studies on Ca2+-activated skinned fibres, that the elementary force generation step in muscle is enhanced by increased temperature; the findings indicate that an endothermic molecular step underlies muscle force generation.

Force generation induced by rapid temperature jumps in intact mammalian (rat) skeletal muscle fibres.

We examined the tension (force) responses induced by rapid temperature jumps (T-jumps) in electrically stimulated, intact fibre bundles (5-10 fibres, fibre length approximately 2 mm) isolated from a foot muscle (flexor hallucis brevis) of the rat; the muscle contains approximately 90 % type 2 fast fibres. In steady state experiments, the temperature dependence of the twitch tension was basically similar to that previously described from other fast muscles; the tetanic tension increased 3- to 4-fold in raising the temperature from approximately 2 to 35 degrees C and the relation between the tetanic tension and the reciprocal absolute temperature was sigmoidal with half-maximal tension at 9.5 degrees C. A rapid T-jump of 3-5 degrees C was induced during a contraction by applying an infrared laser pulse (lambda = 1.32 micro, 0.2 ms) to the 50 microl trough containing the fibre bundle immersed in physiological saline. At approximately 10 degrees C, a T-jump induced a large transient tension rise when applied during the rising phase of a twitch contraction, the amplitude of which decreased when the T-jump was delayed with respect to the stimulus; a T-jump probably perturbs an early step in excitation-contraction coupling. No transient increase was seen when a T-jump was applied during twitch relaxation. When applied during the plateau of a tetanic contraction a T-jump induced a tension rise to a higher steady tension level; the tension rise after a T-jump was 2-3 times faster than the corresponding phase of the initial tension rise in a tetanus. The approach to a new steady tension level after a T-jump was biphasic with a fast (phase 2b, approximately 35 s-1 at 10 degrees C) and a slow component (phase 3, < 10 s-1). The rates of both components increased (Q10 approximately 3) but their amplitudes decreased with increase of the steady temperature. These results from tetanized intact fibres are consistent with the thesis previously proposed from studies on Ca2+-activated skinned fibres, that the elementary force generation step in muscle is enhanced by increased temperature; the findings indicate that an endothermic molecular step underlies muscle force generation.

Targeting calcium cycling proteins in heart failure through gene transfer.

Our understanding of cardiac excitation-contraction coupling has improved significantly over the last 10 years. Furthermore, defects in the various steps of excitation-contraction coupling that characterize cardiac dysfunction have been identified in human and experimental models of heart failure. The various abnormalities in ionic channels, transporters, kinases and various signalling pathways collectively contribute to the 'failing phenotype.' However, deciphering the causative changes continues to be a challenge. An important tool in dissecting the importance of the various changes in heart failure has been the use of cardiac gene transfer. To achieve effective cardiac gene transfer a number of obstacles remain, including appropriate vectors for gene delivery, appropriate delivery systems, and a better understanding of the biology of the disease. In this review, we will examine our current understanding of these various factors. Gene transfer provides not only a potential therapeutic modality but also an approach to identifying and validating molecular targets.

Effects of dantrolene on steps of excitation-contraction coupling in mammalian skeletal muscle fibers.

The effects of the muscle relaxant dantrolene on steps of excitation-contraction coupling were studied on fast twitch muscles of rodents. To identify the site of action of the drug, single fibers for voltage-clamp measurements, heavy SR vesicles for calcium efflux studies and solubilized SR calcium release channels/RYRs for lipid bilayer studies were isolated. Using the double Vaseline-gap or the silicone-clamp technique, dantrolene was found to suppress the depolarization-induced elevation in intracellular calcium concentration ([Ca2+]i) by inhibiting the release of calcium from the SR. The suppression of [Ca2+]i was dose-dependent, with no effect at or below 1 microM and a 53 +/- 8% (mean +/- SEM, n = 9, cut fibers) attenuation at 0 mV with 25 microM of extracellularly applied dantrolene. The drug was not found to be more effective if injected than if applied extracellularly. Calculating the SR calcium release revealed an equal suppression of the steady (53 +/- 8%) and of the early peak component (46 +/- 6%). The drug did not interfere with the activation of the voltage sensor in as much as the voltage dependence of both intramembrane charge movements and the L-type calcium currents (I(Ca)) were left, essentially, unaltered. However, the inactivation of I(Ca) was slowed fourfold, and the conductance was reduced from 200 +/- 16 to 143 +/- 8 SF(-1) (n = 10). Dantrolene was found to inhibit thymol-stimulated calcium efflux from heavy SR vesicles by 44 +/- 10% (n = 3) at 12 microM. On the other hand, dantrolene failed to affect the isolated RYR incorporated into lipid bilayers. The channel displayed a constant open probability for as long as 30-50 min after the application of the drug. These data locate the binding site for dantrolene to be on the SR membrane, but be distinct from the purified RYR itself.

Molecular basis of Ca(2)+ activation of the mouse cardiac Ca(2)+ release channel (ryanodine receptor).

Activation of the cardiac ryanodine receptor (RyR2) by Ca(2)+ is an essential step in excitation-contraction coupling in heart muscle. However, little is known about the molecular basis of activation of RyR2 by Ca(2)+. In this study, we investigated the role in Ca(2)+ sensing of the conserved glutamate 3987 located in the predicted transmembrane segment M2 of the mouse RyR2. Single point mutation of this conserved glutamate to alanine (E3987A) reduced markedly the sensitivity of the channel to activation by Ca(2)+, as measured by using single-channel recordings in planar lipid bilayers and by [(3)H]ryanodine binding assay. However, this mutation did not alter the affinity of [(3)H]ryanodine binding and the single-channel conductance. In addition, the E3987A mutant channel was activated by caffeine and ATP, was inhibited by Mg(2)+, and was modified by ryanodine in a fashion similar to that of the wild-type channel. Coexpression of the wild-type and mutant E3987A RyR2 proteins in HEK293 cells produced individual single channels with intermediate sensitivities to activating Ca(2)+. These results are consistent with the view that glutamate 3987 is a major determinant of Ca(2)+ sensitivity to activation of the mouse RyR2 channel, and that Ca(2)+ sensing by RyR2 involves the cooperative action between ryanodine receptor monomers. The results of this study also provide initial insights into the structural and functional properties of the mouse RyR2, which should be useful for studying RyR2 function and regulation in genetically modified mouse models.

Stimulus-dependent modulation of smooth muscle intracellular calcium and force by altered intracellular pH.

Measurements of simultaneous force and intracellular Ca2+ concentration ([Ca2+]i) in rat uterine smooth muscle have been made to elucidate the mechanisms involved when force produced spontaneously, by high-K+ depolarization or carbachol is altered by a change of intracellular pH (pHi). Rises in force and [Ca2+]i were closely correlated for all forms of contraction, with the Ca2+ transient peaking before force. In spontaneously active preparations, alkalinization significantly increased, and acidification decreased, force and [Ca2+]i. Inhibition of the sarcoplasmic reticulum ATPase (cyclopiazonic acid) did not affect these changes, whereas removal of external Ca2+ abolished both responses, suggesting that the effect of pHi is on Ca2+ entry. Alkalinization caused a prolongation of the action potential complex, associated with a potentiation of contractile activity. Acidification produced hyperpolarization and abolition of action potentials and spontaneous activity, but did not prevent brief applications of carbachol or high-K+ from producing depolarization and increasing force, suggesting no impairment of the mechanism of generation of the action potential. For depolarized preparations, acidification increased tonic force and [Ca2+]i; the increase in the calcium signal persisted in zero-external calcium. In the presence of carbachol, acidification transiently increased force and [Ca2+]i, followed by a reduction in both. It is concluded that changes in pHi act at more than one step in excitation-contraction coupling and that changes in [Ca2+]i can account for most of the changes in uterine force.

Effects of Fatigue of Rat EDL In Situ on Metabolism of Phosphoinositides

The study was conducted to determine the effect of persistent fatigue in situ on the inositol phosphate second messenger system proposed to constitute a step in excitation-contraction coupling. Rat EDL, after stimulation in situ for 1 hr (100 Hz for 330 ms, 1/s), showed increased incorporation of myo-[3H]inositol into membrane phosphoinositides during a subsequent 4-h incubation period. The rate of hydrolysis of this pool resulting from 10 sec of tetanic stimulation, as well as the rate of production of inositol phosphates InsP, InsP2, and InsP3, were significantly reduced in fatigued muscles. These results suggest that the metabolic changes that parallel the alteration in contractile response with fatigue reflect a disruption in E-C coupling.

Corbular (CSR) and extended junctional SR (EJSR) in skeletal muscle

Excitation-calcium release (ECR) is one of the first steps in excitation-contraction coupling in striated muscle. In skeletal muscle, there is some evidence that ECR depends on physical interaction between electrical activity at the plasmalemma (PL) and intracellular calcium stores (junctional sarcoplasmic reticulum; JSR). In cardiac muscle, there is very strong evidence for signal transmission from PL through the release of a diffusible transmitter substance, initially at couplings (= JSR tightly apposed to PL). Avian cardiac muscle has no transverse tubules (TT) but JSR, both as part of peripheral couplings (25%) and as extended JSR (EJSR) unattached to PL (75%). A subset of EJSR, corbular SR (CSR), occurs in small numbers in mammalian hearts and, topographically, fills in wherever TT and, thus, interior couplings (i.e. JSR) are missing. The total volume fraction of avian EJSR (75%) + JSR (25%) is identical to murine JSR (&gt;90%) + CSR (&lt; 10%). JSR-total (JSR, EJSR, CSR) establishes calcium release sites at about equidistant locations throughout the volume of a myocyte. Whereas, in avian hearts a signal for calcium release initiated at a peripheral coupling must be propagated transversely for up to 4 μm (cell radius) by quasi “saltatory” propagation of calcium-induced calcium release (CICR) along the EJSR’s junctional processes (JPs = ryanodine sites) eliciting calcium release for contraction at each contact, in mammalian cardiac and in skeletal muscle calcium release is effected in a one-step process through a propagated action potential kickstarting calcium release at each of a string of peripheral and interior couplings located along TT.

Excitation-contraction coupling in pigs heterozygous for malignant hyperthermia.

A defect in the skeletal muscle sarcoplasmic reticulum (SR) calcium release channel of malignant hyperthermia-susceptible (MHS) pigs greatly enhances SR calcium release in pigs homozygous for the malignant hyperthermia (MH) gene. In pigs heterozygous at this locus, rates of calcium release from isolated SR stimulated by Ca2+, ATP, or caffeine are intermediate to those of both MHS and normal SR [Mickelson et al. Am. J. Physiol. 257 (Cell Physiol. 26): C787-C794, 1989]. In this study bundles of intact muscle cells dissected from pigs of various genotypes were used to examine the effects of the MH gene on contractile responses to caffeine (direct stimulation of the SR) or to surface membrane (sarcolemma) depolarization (i.e., stimulation by way of the steps in excitation-contraction coupling). The caffeine threshold for contractures in the heterozygous muscles (5 mM) was intermediate to both types of homozygous muscles (2 mM for MHS and 10 mM for normal) as is the case with direct stimulation of calcium release from SR vesicles [Mickelson et al. Am. J. Physiol. 257 (Cell Physiol. 26): C787-C794, 1989]. Sarcolemmal depolarization was elicited by electrical stimuli or elevated extracellular potassium. Control twitch tension for MHS and heterozygous muscles did not differ and was significantly greater in both than in homozygous normal muscles. Potassium-induced contractures were significantly larger in MHS and heterozygous than in normal muscles. Thus, in heterozygous muscles, force production via sarcolemmal depolarization (twitches and potassium contractures) was enhanced as much as in homozygous MHS muscles. This could be the result of feedback from abnormal SR calcium channels producing altered (enhanced) transverse tubule to SR signal transduction.

Inhibition by cocaine of excitation-contraction coupling in isolated cardiomyocytes

The effects of cocaine on the Ca2+ fluxes responsible for excitation-contraction coupling were studied in isolated ventricular rat heart cells loaded with the fluorescent Ca2+ indicator fura-2. Ca2+ transients in response to electrical field stimulation were followed using a fluorescence ratio method in which excitation light was alternated with 5-ms resolution. The cardiomyocytes maintained a basal cytosolic Ca2+ concentration of approximately 70 nM, which increased to a peak of 450 nM in response to each electrical stimulus. The addition of cocaine (10 microM) to cells stimulated at 0.4 Hz decreased the magnitude of the electrically induced Ca2+ transients by 30 +/- 4% within 5 s. This inhibitory effect of cocaine was dose dependent, with a 50% reduction in the Ca2+ transient occurring at 40 microM cocaine. The effects of cocaine were not associated with any permanent cell damage and could be reversed by washing the cells free of the drug. Cytosolic Ca2+ increases in response to K(+)-induced depolarization of the cardiomyocytes were much less sensitive to cocaine than the electrically induced Ca2+ transients. In this respect the effects of cocaine were similar to the actions of lidocaine and tetrodotoxin but distinct from the effects of nitrendipine and verapamil. Cocaine had no effect on the caffeine-releasable Ca2+ pool in cardiomyocytes. These data demonstrate that cocaine directly inhibits the Ca2(+)-dependent steps of excitation-contraction coupling in heart muscle cells. Sarcolemmal Na+ channels represent a possible locus for this action of cocaine.

Rapid activation by photolysis of nitr-5 in skinned fibres of the striated adductor muscle from the scallop

Photolysis of nitr-5, a caged calcium molecule, has been used for rapid activation of skinned fibre bundles of a myosin-regulated muscle, the striated adductor of the scallop, Pecten maximus. Chemically skinned fibre bundles (diameter 70–200 μm) were equilibrated in solutions containing 1–3 mM nitr-5 (p Ca 6.1) and then activated by ultraviolet laser pulse (25 ns). Pulse energies of 60–95 mJ gave contractions of over 90% maximum tension and a mean half-time for tension rise of 43 ms ( n = 4) at 12°C. Electrically stimulated bundles of intact fibres develop a tetanus with a rise half-time of 60.2 ms at 10°C ( n = 5) (Rall, J.A. (1981) J. Physiol. 321, 287–295, and personal communication). At lower pulse energies the skinned fibres gave smaller amplitude contractions with slower rates of rise (up to 260 ms half-time). In addition, a slower component of tension development (mean rise half-time 13.3 s) was often observed. In ATP-free solutions containing hexokinase and glucose, rigour tension developed with a delayed onset. Rapid release of ATP (0.47–0.59 mM) from photolysis of caged ATP (2 mM) at p Ca 4.5 then caused a rapid contraction with a mean half-time for tension development of 17 ms ( n = 4). The fast activation rates obtained by the photorelease of Ca 2+ from nitr-5 are similar to those obtained with skinned skeletal fibres of actin-regulated muscle. The results imply that the rate-limiting step in excitation-contraction coupling of the scallop muscle is not the increase in sarcoplasmic Ca 2+, but rather the activation of the muscle in response to this increase. The half-times of ATP-induced contractions at p Ca 4.5 suggest that in a contraction activated by a rapid Ca 2+ jump the process comprising ATP hydrolysis and cross-bridge cycling occurs at a somewhat faster rate than the Ca 2+-dependent activation process which preceds it.

Effects of hypoxia on high‐energy phosphagen content, energy metabolism and isometric force in guinea‐pig taenia caeci.

1. Previously, Ishida, Takagi & Urakawa (1984) reported that, in the presence of high K+ (45.4 mM) under hypoxia (95% N2-5% CO2 bubbling), tension and ATP content of the smooth muscle of the guinea-pig taenia caeci increased concomitantly when the glucose concentration was raised. Tension and energy metabolism of the taenia seemed to be closely correlated. In the present experiments, we investigated the metabolic changes during the relaxation phase after the taenia was exposed to hypoxia in the presence of high K+ by measuring the content of phosphagen, inorganic phosphate (Pi) and lactate in the tissue. Oxygen consumption and lactate release from the tissue were also determined to estimate the rate of ATP synthesis. 2. Under hypoxic conditions, high-K(+)-induced tension decreased to one-tenth of maximum in aerobic conditions (95% O2-5% CO2 bubbling); increasing the calcium concentration from 2.5 to 10 mM had no effect. To test receptor-associated stimuli, carbachol or histamine (both 5 microM) had little effect on tension in hypoxia. The calcium ionophore A23187 (10 microM) also did not produce any significant contraction in the presence of high K+ under hypoxia. 3. Hypoxia in the presence of high K+ elicited a rapid decrease in phosphocreatine, approximately in parallel with the decrease in tension. The ATP content decreased gradually while Pi content increased. Lactate content increased rapidly and then partially decreased. 4. The rate of ATP synthesis estimated from the oxygen consumption and lactate release of the taenia was linearly correlated with tension development under aerobic and hypoxic conditions. 5. These results suggest that the decrease in tension of the taenia observed under hypoxic conditions is due to an inhibition of energy metabolism, and not due to an oxygen-sensing step in excitation-contraction coupling.

Excitation contraction coupling in normal and mdx mice

The protein dystrophin is absent from patients with Duchenne muscular dystrophy and from the muscles of mdx mice. Recent studies have shown that dystrophin is located at the surface membrane and at the triadic junction, where it is associated with the transverse tubular membrane. Since the triadic junction is the site of excitation-contraction (EC) coupling, we have investigated whether intramembrane charge movement, a step in EC coupling, is modified by the absence of dystrophin. Charge movements are thought to arise from the transverse tubular membrane and to underlie the dependence of sarcoplasmic reticulum Ca2+ release on transverse tubular membrane potential. We find no differences between intramembrane charge movements or passive membrane electrical properties measured in muscles from mdx mice compared with normal mice. If dystrophin does play a role in EC coupling, that role is likely to be subsequent to the charge movement step.

Anatomical distribution of voltage-dependent membrane capacitance in frog skeletal muscle fibers.

Components of nonlinear capacitance, or charge movement, were localized in the membranes of frog skeletal muscle fibers by studying the effect of 'detubulation' resulting from sudden withdrawal of glycerol from a glycerol-hypertonic solution in which the muscles had been immersed. Linear capacitance was evaluated from the integral of the transient current elicited by imposed voltage clamp steps near the holding potential using bathing solutions that minimized tubular voltage attenuation. The dependence of linear membrane capacitance on fiber diameter in intact fibers was consistent with surface and tubular capacitances and a term attributable to the capacitance of the fiber end. A reduction in this dependence in detubulated fibers suggested that sudden glycerol withdrawal isolated between 75 and 100% of the transverse tubules from the fiber surface. Glycerol withdrawal in two stages did not cause appreciable detubulation. Such glycerol-treated but not detubulated fibers were used as controls. Detubulation reduced delayed (q gamma) charging currents to an extent not explicable simply in terms of tubular conduction delays. Nonlinear membrane capacitance measured at different voltages was expressed normalized to accessible linear fiber membrane capacitance. In control fibers it was strongly voltage dependent. Both the magnitude and steepness of the function were markedly reduced by adding tetracaine, which removed a component in agreement with earlier reports for q gamma charge. In contrast, detubulated fibers had nonlinear capacitances resembling those of q beta charge, and were not affected by adding tetracaine. These findings are discussed in terms of a preferential localization of tetracaine-sensitive (q gamma) charge in transverse tubule membrane, in contrast to a more even distribution of the tetracaine-resistant (q beta) charge in both transverse tubule and surface membranes. These results suggest that q beta and q gamma are due to different molecules and that the movement of q gamma in the transverse tubule membrane is the voltage-sensing step in excitation-contraction coupling.