Intracellular calcium movements during excitation-contraction coupling in mammalian slow-twitch and fast-twitch muscle fibers

In skeletal muscle fibers, action potentials elicit contractions by releasing calcium ions (Ca(2+)) from the sarcoplasmic reticulum. Experiments on individual mouse muscle fibers micro-injected with a rapidly responding fluorescent Ca(2+) indicator dye reveal that the amount of Ca(2+) released is three- to fourfold larger in fast-twitch fibers than in slow-twitch fibers, and the proportion of the released Ca(2+) that binds to troponin to activate contraction is substantially smaller.     

How perchlorate improves excitation-contraction coupling in skeletal muscle fibers.

The effect of the "chaotropic" anion, perchlorate, on the activation of contraction has been studied in voltage clamped frog skeletal muscle fibers. It was found that the voltage dependence of either the contractile force or the intramembrane charge movement was shifted towards more negative membrane potentials. The maximum values of force or charge movement attained with large depolarizing pulses did not change significantly. It is concluded that a specific perchlorate effect on the movement of charged particles can explain the potentiating effect of perchlorate anions on contractile force, strengthening the view that these charged particles serve as voltage sensors regulating Ca2+ release from the sarcoplasmic reticulum.     

The role of calcium in excitation-contraction coupling of lobster muscle

Potassium contractures were induced in lobster muscle bundles under conditions which produced varying KCl fluxes into the fibers. The presence or absence of chloride fluxes during depolarization by high concentrations of potassium, had no effect on the tensions developed. The curve relating tension to the membrane potential had a typical sigmoid shape with an apparent "threshold" for tension at -60 mv. Soaking the muscles in low (0.1 mM) calcium salines for 30 min completely eliminated the potassium contractures but the caffeine contractures were only slightly reduced under these conditions. The potassium contracture could be completely restored in less than 2 min by return of the calcium ions to the saline. Evidence is presented for independent, superficial, and deep calcium sites; the superficial sites appear to be involved in the coupling mechanisms associated with potassium contractures. These sites are highly selective for Ca⁺⁺, and attempts to substitute either Cd⁺⁺, Co⁺⁺, Mg⁺⁺, Ba⁺⁺, or Sr⁺⁺ for Ca⁺⁺ were unsuccessful. However, K⁺ appeared to compete with Ca⁺⁺ for these sites, and the evoked tension could be reduced by prestimulation of the muscle fibers with high K⁺ salines. The results of studies on the influx of ⁴⁵Ca during potassium contractures were compatible with the view of muscle activation by the entry of extracellular calcium.     

Role of inositol 1,4,5-trisphosphate in excitation-contraction coupling in skeletal muscle

The sarcoplasmic reticulum (SR) of skeletal muscle is an intracellular membranous network that controls the myoplasmic Ca2+ concentration and the contraction-relaxation cycle. Ca2+ release from the terminal cisternae (TC) region of the SR evokes contraction. How electrical depolarization of the transverse tubule is linked to Ca2+ release from the junctionally associated TC is still largely unknown. Independent evidence has been recently obtained indicating that either inositol trisphosphate (IP3) or (and) Ca2+ is (are) the chemical transmitter(s) of excitation-contraction coupling. Here we outline the experimental data in support of each transmitter and discuss possible interactive roles of Ca2+ and IP3.     

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.     

Excitation-contraction coupling in crab muscle fibers with swollen T tubules

Single crab (Callinectes danae) fibers were equilibrated with isotonic, high KCl solutions and were subsequently returned to the control saline. This caused marked swelling of the T tubules. Fibers treated with 100 mM KCl had a 2.5-mV residual depolarization, a 50% decrease in effective membrane resistance (Reff) and a 75% reduction in membrane time constant (tau m). These fibers exhibited large increases in membrane conductance upon depolarization and were inexcitable; membrane depolarization with current pulses elicited no contraction. The effects of the KCl treatment on membrane properties were not reproduced by treatment with high potassium gluconate solutions, which did not cause tubular swelling. Tetrabutylammonium (10 mM) or Ba ions (10-20 mM), but not tetraethylammonium (40-100 mM), Sr ions (15-70 mM), or procaine (1-8 mM) reversed the effects of the KCl treatment on Reff, tau m, membrane excitability, and excitation-contraction coupling. The time course of the Ba effects was consistent with the suggestion that the KCl treatment increases the K conductance of the tubular membranes, which in turn prevents the activation of voltage-dependent Ca channels located in the membranes of the T system. This results in inhibition of the Ca-dependent electrogenesis and consequently, the absence of contraction upon depolarization of the plasma membrane.     

Mitochondrial calcium uptake regulates rapid calcium transients in skeletal muscle during excitation-contraction (E-C) coupling

Defective coupling between sarcoplasmic reticulum and mitochondria during control of intracellular Ca(2+) signaling has been implicated in the progression of neuromuscular diseases. Our previous study showed that skeletal muscles derived from an amyotrophic lateral sclerosis (ALS) mouse model displayed segmental loss of mitochondrial function that was coupled with elevated and uncontrolled sarcoplasmic reticulum Ca(2+) release activity. The localized mitochondrial defect in the ALS muscle allows for examination of the mitochondrial contribution to Ca(2+) removal during excitation-contraction coupling by comparing Ca(2+) transients in regions with normal and defective mitochondria in the same muscle fiber. Here we show that Ca(2+) transients elicited by membrane depolarization in fiber segments with defective mitochondria display an ~10% increased amplitude. These regional differences in Ca(2+) transients were abolished by the application of 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, a fast Ca(2+) chelator that reduces mitochondrial Ca(2+) uptake. Using a mitochondria-targeted Ca(2+) biosensor (mt11-YC3.6) expressed in ALS muscle fibers, we monitored the dynamic change of mitochondrial Ca(2+) levels during voltage-induced Ca(2+) release and detected a reduced Ca(2+) uptake by mitochondria in the fiber segment with defective mitochondria, which mirrored the elevated Ca(2+) transients in the cytosol. Our study constitutes a direct demonstration of the importance of mitochondria in shaping the cytosolic Ca(2+) signaling in skeletal muscle during excitation-contraction coupling and establishes that malfunction of this mechanism may contribute to neuromuscular degeneration in ALS.     

Theory of excitation-contraction coupling in cardiac muscle.

The consequences of cardiac excitation-contraction coupling by calcium-induced calcium release were studied theoretically, using a series of idealized models solved by analytic and numerical methods. "Common-pool" models, those in which the trigger calcium and released calcium pass through a common cytosolic pool, gave nearly all-or-none regenerative calcium releases (in disagreement with experiment), unless their loop gain was made sufficiently low that it provided little amplification of the calcium entering through the sarcolemma. In the linear (small trigger) limit, it was proven rigorously that no common-pool model can give graded high amplification unless it is operated on the verge of spontaneous oscillation. To circumvent this problem, we considered two types of "local-control" models. In the first type, the local calcium from a sarcolemmal L-type calcium channel directly stimulates a single, immediately opposed SR calcium release channel. This permits high amplification without regeneration, but requires high conductance of the SR channel. This problem is avoided in the second type of local control model, in which one L-type channel triggers a regenerative cluster of several SR channels. Statistical recruitment of clusters results in graded response with high amplification. In either type of local-control model, the voltage dependence of SR calcium release is not exactly the same as that of the macroscopic sarcolemmal calcium current, even though calcium is the only trigger for SR release. This results from the existence of correlations between the stochastic openings of individual sarcolemmal and SR channels. Propagation of regenerative calcium-release waves (under conditions of calcium overload) was analyzed using analytically soluble models in which SR calcium release was treated phenomenalogically. The range of wave velocities observed experimentally is easily explained; however, the observed degree of refractoriness to wave propagation requires either a strong dependence of SR calcium release on the rate of rise of cytosolic calcium or localization of SR release sites to one point in the sarcomere. We conclude that the macroscopic behavior of calcium-induced calcium release depends critically on the spatial relationships among sarcolemmal and SR calcium channels, as well as on their kinetics.     

Pharmacological studies of excitation-contraction coupling in skeletal muscle

The use of drugs in the study of excitation-contraction (E-C) coupling in skeletal muscle during the 25-30 years and the role of these studies in the development of the "trigger-calcium" hypothesis was reviewed. In early studies, caffeine was used as a tool to test the function of the intracellular contraction apparatus when the twitch or depolarization contracture was eliminated by a procedure that was thought to block the coupling part of the E-C coupling process. Later it was shown that caffeine produced contractures by releasing Ca2+ ions from intracellular binding sites and then that caffeine produced this effect by sensitizing the sarcoplasmic reticulum to Ca2+-induced Ca2+ release. More recently, organic calcium channel blocking drugs (verapamil, D-600, and nitrendipine) were used to confirm earlier results showing that depolarization contractures but not twitches require the entrance into the cells via the slow Ca2+ channels of extracellular calcium ions for E-C coupling. Most recently, we have investigated the effects of TMB-8 (8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate) on E-C coupling in frog skeletal muscle. This compound was shown by other workers to act in several tissues by stabilizing Ca2+ bound at intracellular sites. It was found that at the appropriate concentration TMB-8 blocked twitches but neither high K+ nor caffeine induced contractures. These results suggest that TMB-8 blocks twitches by preventing the release of Ca2+ ions bound to the intracellular surface of the t-tubular membrane, which is often called the store of "trigger-calcium" ions.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of calcium in excitation--contraction coupling in crustacean muscle fibers

The evidence that calcium (Ca) plays an important role in electrical activity and an essential role in excitation--contraction (E--C) coupling in crustacean muscles is reviewed. These muscles produce graded electrical and mechanical responses to applied depolarizations. Removal of Ca from the bath solution eliminates both responses. Addition of Ba2+ or Sr2+ to Ca-free saline restores membrane electrogenesis, and all-or-none action potentials can be induced. With Sr2+ vigorous contractions are produced, whereas Ba action potentials evoke minimal or no tension, showing that rapid depolarization of the membrane potential is not sufficient per se for E--C coupling in crab and barnacle muscle. Several inorganic (e.g., multivalent cations) and organic (e.g., aminoglycoside antibiotics) which block membrane Ca channels block electrogenesis and contraction. However, the "Ca antagonists" verapamil and D600 also block Ca uptake at intracellular storage sites, resulting in spontaneous contractions and the delayed relaxation of small contractions associated with residual Ca currents. The evidence that the Ca which enters the fibres needs to release Ca from intracellular storage sites to produce contractions is detailed and discussed. Finally, a model for E--C coupling is discussed. This model includes the sites and mechanisms of action for several chemicals which modify E--C coupling in crustacean muscle fibres.     

Inositol trisphosphate and excitation-contraction coupling in skeletal muscle

The role of inositol trisphosphate as a chemical messenger in excitation-contraction coupling is discussed, both in terms of positive and negative results. The evidence presented includes experiments on the effect of inositol trisphosphate in intact and skinned fibers, in calcium release from isolated sarcoplasmic reticulum vesicles, in activation of single calcium release channels incorporated in planar bilayers, and biochemical experiments that have established the presence of all the intermediate steps involved in the metabolism of phosphoinositides, both in intact muscle and in isolated membranes. From these results, it is clear that a role for inositol triphosphate in skeletal muscle function is highly likely; whether this molecule is the physiological messenger in excitation-contraction coupling remains to be established.