Biomechanics of the sarcolemma and costameres in single skeletal muscle fibers from normal and dystrophin-null mice.

We studied the biomechanical properties of the sarcolemma and its links through costameres to the contractile apparatus in single mammalian myofibers of Extensor digitorum longus muscles isolated from wild (WT) and dystrophin-null (mdx) mice. Suction pressures (P) applied through a pipette to the sarcolemma generated a bleb, the height of which increased with increasing P. Larger increases in P broke the connections between the sarcolemma and myofibrils and eventually caused the sarcolemma to burst. We used the values of P at which these changes occurred to estimate the tensions and stiffness of the system and its individual elements. Tensions of the whole system and the sarcolemma, as well as the maximal tension sustained by the costameres, were all significantly lower (1.8-3.3 fold) in muscles of mdx mice compared to WT. Values of P at which separation and bursting occurred, as well as the stiffness of the whole system and of the isolated sarcolemma, were ~2-fold lower in mdx than in WT. Our results indicate that the absence of dystrophin reduces muscle stiffness, increases sarcolemmal deformability, and compromises the mechanical stability of costameres and their connections to nearby myofibrils.

A novel thienylhydrazone, (2-thienylidene)3,4-methylenedioxybenzoylhydrazine, increases inotropism and decreases fatigue of skeletal muscle.

This study was designed to investigate the effects on single skeletal muscle fibers of a novel thienylhydrazone, referred to as LASSBio-294, which is a bioisoster of pyridazinone compounds that inhibit the cyclic AMP-specific phosphodiesterase (PDE) 4. Twitch and fatigue were analyzed in single skeletal muscle fibers isolated from either the semitendinous or the tibialis anterior muscles dissected from the frog Rana pipiens. LASSBio-294 (12.5-100 microM) increased twitch tension, accelerated the maximal rate of tension decay during relaxation, and had very little effect in the maximal rate of tension development of muscle fibers directly stimulated at < or =30 Hz. The positive inotropic effect of LASSBio-294 developed slowly, reaching its maximum at 40 min and was inversely proportional to the frequency of stimulation, becoming negligible at 60 and 90 Hz. The concentration-response relationship for LASSBio-294-induced potentiation of twitch tension was bell-shaped, with maximal effect occurring at 25 microM. In addition, LASSBio-294 reduced development of fatigue induced by tetanic stimulation of the muscle fibers and reduced the time needed for 80% prefatigue tension recovery after fatigue had developed to 50% of the maximal pretetanic force. These effects of LASSBio-294 can be fully explained by stimulation of the sarcoplasmic reticulum Ca2+ pump and could be ascribed to an increase in cellular levels of cyclic AMP due to PDE inhibition. The novel thienylhydrazone LASSBio-294 may be useful for treatment of patients suffering from conditions in which muscle fatigue is a debilitating symptom (e.g., chronic heart failure).

Plasmalemmal transport of magnesium in excitable cells.

In excitable cells, the concentration of intracellular free Mg2+ ([Mg2+]i) is several hundred times lower than expected if Mg2+ ions were at electrochemical equilibrium. Since Mg2+ is a permeant ion across the plasmalemma, it must be constantly extruded. An ATP-dependent Na/Mg exchanger has been proposed as the sole mechanism responsible for Mg2+ extrusion. However, this hypothesis fails to explain numerous observations including the fact that K+ and Cl- appear to be involved in Mg2+ transport. Until now three main limitations have hampered the studies of plasmalemmal Mg2+ transport: i) 28Mg, the only useful radioactive isotope of Mg2+, has a short half-life and is difficult to obtain; ii) squid giant axons, the ideal preparation to carry out transport studies under "zero-trans" conditions, are only available during the summer months; and iii) the ionic fluxes mediated by the Mg2+ transporter are very small and difficult to measure. The purpose of this manuscript is to review how these limitations have been recently overcame and to propose a novel hypothesis for the plasmalemmal Mg2+ transporter in squid axons and barnacle muscle cells. Overcoming the limitations for studying the plasmalemmal Mg2+ transporter has been possible as a result of the following findings: i) the Mg2+ exchanger can operate in "reverse", thus extracellular Mg2+-dependent ionic fluxes (e.g., Na+ efflux) can be utilized to measure its activity; ii) internally perfused, voltage-clamped barnacle muscle cells which are available all year long can be used in addition to squid axons; and iii) phosphoinositides (e.g., PIP2) produce an 8-fold increase in the ionic fluxes mediated by the Mg2+ exchanger. The hypothesis that we postulate is that, in squid giant axons and barnacle muscle cells, a 2Na+2K+2Cl:1Mg exchanger is responsible for transporting Mg2+ across the plasmalemma and for maintaining [Mg2+]i under steady-state conditions.

Interaction of D-600 with the transmembrane domain of the sarcoplasmic reticulum Ca(2+)-ATPase.

Experiments were performed to determine whether the organic Ca(2+) channel blocker D-600 (gallopamil), which penetrates into muscle cells, affects sarcoplasmic reticulum (SR) Ca(2+) uptake by directly inhibiting the light SR Ca(2+)-ATPase. We have previously shown that at 10 microM, D-600 inhibits LSR ATP-dependent Ca(2+) uptake by 50% but has no effect on ATPase activity (21). These data suggest that the SR Ca(2+)-ATPase might be a potential target for D-600. The ATPase activity of the enzyme is associated with its hydrophilic cytoplasmic domain, whereas Ca(2+) binding and translocation are associated with the transmembrane domain (18). In the present experiments, we determined which of the two domains of the ATPase is affected by D-600. Thermal inactivation experiments using the SR Ca(2+)-ATPase demonstrated that D-600 decreased the thermal stability of Ca(2+) transport but had no effect on the stability of ATPase activity. In addition, D-600 at a concentration of 160 microM did not have any leaking effect of Ca(2+) on the Ca(2+)-loaded SR. Thermal denaturation profiles of SR membranes revealed that D-600 interacts directly with the transmembrane domain of the Ca(2+)-ATPase. No evidence for interaction with the nucleotide domain was obtained. We conclude that the Ca(2+) blocker D-600 inhibits the SR Ca(2+) pump specifically by interacting with the transmembrane Ca(2+)-binding domain of the Ca(2+)-ATPase.

Alterations in heart failure of cyclic AMP-dependent inotropic and lusitropic properties of cardiac and skeletal muscle.

A central working hypothesis in our laboratory is that deficient cellular cyclic AMP concentrations may be responsible, at least in part, for striated muscle dysfunction, both cardiac and skeletal, in heart failure. These results suggest that therapy aimed at restoring cyclic AMP to normal levels may be effective with regard to improving systolic and diastolic function in the heart and may decrease the development of fatigue in skeletal muscle of patients with failure. The use of cyclic AMP-dependent drugs in clinical practice has been limited by side effects associated with raising total cellular content of this cyclic nucleotide. However, evidence suggesting that separate pools of cyclic AMP may exist within the cell raises the possibility that those pools associated with excitation/contraction coupling could serve as more specific therapeutic targets.

Effect of the organic Ca2+ channel blocker D-600 on sarcoplasmic reticulum Ca2+ uptake in skeletal muscle.

Experiments were undertaken to study the possibility that the calcium channel blocker D-600 (gallopamil), which penetrates into muscle cells (20), facilitates excitation-contraction coupling in skeletal muscle (7) by a direct effect on the sarcoplasmic reticulum (SR). The effects of D-600 were studied on single phasic muscle fibers, either intact or split open. D-600 potentiated twitches in intact fibers at concentrations lower than those reported in whole muscles. In split fibers, the force produced by caffeine-induced Ca2+ release from the SR was reversibly inhibited by 5 microM D-600, when added to the Ca2+ loading solution. This inhibitory effect was inversely related to temperature, and it was dose dependent. When D-600 was added after Ca2+ loading and before caffeine exposure, or during the caffeine exposure itself, it did not inhibit Ca2+ release, but rather increased the development of force. We conclude that, apart from the blocking effect that D-600 may have on the voltage sensor, the drug penetrates into the myoplasm and affects excitation-contraction coupling by inhibiting the SR Ca2+ pump. This may be the consequence of a conformational change in the transmembrane Ca2+ binding domain of the ATPase.

Deficient cellular cyclic AMP may cause both cardiac and skeletal muscle dysfunction in heart failure.

Deficient myocardial cyclic AMP concentrations contribute to abnormal Ca2+ handling and systolic and diastolic dysfunction in chronic heart failure (CHF). We tested the hypothesis that decreased cyclic AMP in skeletal muscle of animals with failure may contribute to the weakness and easy fatiguability also common in patients with CHF. We compared intracellular Ca2+ signaling and contractility in skeletal muscle preparations from rats 6 weeks after myocardial infarction-induced CHF versus sham-operated controls. Bundles of 100 to 200 cells were dissected from the extensor digitorum longus (EDL) muscle of control and CHF rats. Muscles from CHF rats exhibited depressed tension development compared with control muscles during twitches. Treatment with 2mM dibutyryl cyclic AMP returned tension and Ca2+ towards normal levels. There was no evidence of cellular atrophy in the CHF rats. In conclusion, EDL skeletal muscle from rats with CHF had intrinsic abnormalities in excitation-contraction coupling that could be reversed with cyclic AMP supplementation as previously reported for the heart. This suggests that deficient cyclic AMP levels may contribute to both cardiac and skeletal muscle dysfunction in CHF.

Membrane healing and restoration of contractility after mechanical injury in isolated skeletal muscle fibers of the frog.

In single isolated skeletal muscle fibers of the frog, we studied (i) the recovery from large sarcolemmal mechanical injuries of the response to electric stimulation and (ii) the integrity of the sarcolemma under the light microscope. In Ringer's solution, the damaged cells stopped contracting and deteriorated completely within 1 hr. In the presence of phosphatidylcholine (0.025 g/ml in Ringer's solution), the injured cells initially responded with local twitches. Within 0.5 hr, contractility and membrane integrity started to recover and both were back to control levels within 3 hr. When these cells were placed back in normal Ringer's solution, they remained viable and active for several hours. Our results suggest that phosphatidylcholine can protect muscle fibers from the effects of sarcolemmal injury.

Extracellular Mg(2+)-dependent Na+, K+, and Cl- efflux in squid giant axons.

An extracellular Na+ (Nao)-dependent Mg2+ efflux process that requires intracellular ATP has been proposed as the sole mechanism responsible for Mg2+ extrusion in internally dialyzed squid axons (12). We have shown that this exchanger can also "reverse" and mediate an extracellular Mg2+ (Mgo)-dependent Na+ efflux (16). We have extended these studies and found that, in the presence of ouabain, bumetanide, tetrodotoxin, and K+ channel blockers and in the absence of extracellular Na+, K+, and bicarbonate, intracellular K+ and Cl- are also involved in the Mgo-dependent Na+ efflux process. Two main observations support this view: 1) operation of the Mgo-dependent Na+ efflux requires the presence of intracellular K+ and Cl-, and 2) Mgo removal produces a reversible and nearly identical reduction in the magnitude of the simultaneous efflux of the ionic pairs K(+)-Na+ and Cl(-)-Na+. These results suggest that the putative bumetanide-insensitive Na-Mg exchanger also transports K+ and Cl-.

Non-homogeneous Ca release in isolated frog skeletal muscle fibres.

We have examined the spatial distribution of [Ca2+]i during tetanic stimulation in frog skeletal muscle cells using a fluorescence imaging method. We have found a completely unexpected pattern of Ca release: Ca is released forming gradients composed of spots of very significant and slow fluctuations of calcium release. Our findings could be explained if the calcium release process in skeletal muscle is influenced significantly by [Ca2+]i, such as in cardiac muscle, and suggests that the SR/Ca release control can include the established voltage-dependent plus a cardiac-like process of calcium-induced Ca release and a Ca release inhibition by Ca.

Alterations in contractility and intracellular Ca2+ transients in isolated bundles of skeletal muscle fibers from rats with chronic heart failure.

To determine if chronic heart failure (CHF) leads to functional or structural alterations of skeletal muscle, we compared intracellular Ca2+ signaling, contractility, and the rate of fatigue development, together with electron microscopy (EM), in skeletal muscle preparations from rats with myocardial infarction-induced CHF versus sham-operated control rats. Bundles of 100 to 200 cells were dissected from the extensor digitorum longus (EDL) muscle of control (n = 13) and CHF (n = 19) rats and were either loaded with aequorin or fixed for EM. Muscles from CHF rats exhibited depressed tension development compared with control muscles during twitches (1.4 +/- 0.2 versus 2.8 +/- 0.7 g/mm2, P < .05) and maximal tetani (5.3 +/- 1.4 versus 10.7 +/- 2.4 g/mm2, P < .05). Depressed tension in CHF was accompanied by reduced quantitative [Ca2+]i release during twitches (0.7 +/- 0.1 versus 0.4 +/- 0.1 microM, P < .05) and during maximal tetani (1.8 +/- 0.3 versus 0.9 +/- 0.2 microM, P < .05). Skeletal muscle from CHF rats also demonstrated prolonged intracellular Ca2+ transients during twitches and tetani and accelerated fatigue development. EM revealed a lack of cellular atrophy in the CHF rats. In conclusion, EDL skeletal muscle from rats with CHF had intrinsic abnormalities in excitation-contraction coupling unrelated to cellular atrophy. These findings indicate that CHF is a condition accompanied by EDL skeletal muscle dysfunction.

A chemical method for intracellular loading of the calcium indicator aequorin in mammalian skeletal muscle.

The bioluminescent calcium indicator aequorin was loaded into bundles of skeletal muscle fibers from the rat extensor digitorum longus by macroinjection, a technique previously applied only to cardiac muscle. After loading, the amplitude and time course of the twitch returned to control values, indicating lack of damage to the fibers. Individual light signals (i.e., calcium transients) were recorded during each twitch or tetanus without the need for signal averaging. The calcium transients obtained were qualitatively and quantitatively similar to those reported previously with microinjection of aequorin. Our data suggest that macroinjection may be the method of choice for loading aequorin into mammalian skeletal muscle.

Differential activation of myofibrils during fatigue in phasic skeletal muscle cells.

In fatigued muscles the T-system is swollen; thus the action potential may fail to travel along the T-system or the T-tubule terminal cisternae signal may fail to bring about TC Ca2+ release. This would lead to a decrease in the number of myofibrils activated and in force development, but if fatigue is the result of a generalized process, all the myofibrils would be affected equally leading to a lower activation of all of them. We have investigated this possibility in isolated twitch muscle fibres by giving them repetitive tetanic stimulations until fatigue developed. The behaviour of myofibrils was followed with cinemicrophotography. Before fatigue, no lack of shortening of myofibrils could be found. During fatigue groups of myofibrils became wavy. When exposed to caffeine, the wavy myofibrils disappeared and tension similar to the control developed. The tension-caffeine concentration relationship was shifted to the left after development of fatigue. In low Na+ solution fatigue developed faster and after reintroducing normal Ringer, tension recovered substantially. K-contractures were smaller during fatigue. These results indicate that in this type of fatigue, a step in the EC coupling chain of events is involved in its development.

Extracellular magnesium-dependent sodium efflux in squid giant axons.

Experiments were designed to determine whether the putative Na(+)-Mg2+ exchanger previously demonstrated to mediate Mg2+ efflux (R. DiPolo and L. Beagué. Biochim. Biophys. Acta 946: 424-428, 1988) could also mediate the efflux of Na+ (presumably a Na+ efflux-Mg2+ influx exchange) in squid giant axons. The effects of external Mg2+ (Mg(o)) on 22Na efflux were measured in internally dialyzed, ATP-fueled axons in which the contribution to Na+ efflux by other pathways was inhibited. To facilitate measurement of Mg(o)-dependent Na+ efflux, the intracellular concentration of Na+ was increased. To prevent Na(+)-Na+ exchange, external Na+ was replaced by tris(hydroxymethyl)aminomethane. To assess the effect of Mg(o) on Na+ efflux without altering the total divalent cation concentrations, Mg(o) was replaced mole-for-mole by external Ba2+ (Ba(o)). This manipulation produced reversible reductions in Na+ efflux. These reductions were neither due to membrane hyperpolarization nor to a direct effect of Bao but were due instead to the reduction in Mg(o). The Mg(o)-dependent Na+ efflux was inhibited by external amiloride but was spared by bumetanide. In the absence of external Na+, the Mgo-dependent Na+ efflux increased as a function of external Mg2+ with Michaelis-Menten kinetics. These results indicate that the Na(+)-Mg2+ exchange can mediate the efflux of Na+ (operate in Na+ efflux-Mg2+ influx mode of exchange).

Intracellular free magnesium in frog skeletal muscle fibres measured with ion-selective micro-electrodes.

Intracellular free Mg2+ concentration [( Mg2+]i) was measured in frog skeletal muscle fibres, using Mg2+-selective micro-electrodes based on the neutral ligand ETH-1117. In calibration solutions the electrodes showed significant interference from K+, and to a lesser extent from Na+, at concentrations found intracellularly. Therefore, in order to calibrate the electrodes properly, it was necessary first to obtain an accurate value for intracellular free Na+ and K+ concentrations ([Na+]i and [K+]i), using the appropriate liquid ion exchanger micro-electrodes. In fibres from muscles maintained in Ringer solution, the mean value for [Na+]i was 6.2 +/- 0.4 mM (S.E. of mean; n = 20 fibres in five muscles), while [K+]i was 104 +/- 1.7 mM (range 83-122 mM; n = 25 fibres in eight muscles). Due to the substantial variability found for [K+]i, not only between fibres from different muscles, but also between fibres belonging to the same muscle, it was necessary to measure [Mg2+]i and [K+]i simultaneously in the same fibre to determine as accurately as possible the degree of K+ interference on Mg2+-selective micro-electrode response. In nineteen fibres from six muscles maintained in Ringer solution, the mean [K+]i was 91.7 +/- 2.7 mM (range 71-110 mM), while the mean [Mg2+]i was 0.80 +/- 0.07 mM (range 0.2-1.2 mM). The mean resting potential was -79.3 +/- 0.4 mV (S.E. of mean). In fifteen fibres from four muscles equilibrated in Ringer solution containing 0.5 mM-Mg2+, the mean [K+]i was 115.5 +/- 0.1 mM (range 97-129 mM) and the mean [Mg2+]i measured simultaneously in the same fibres was 1.69 +/- 0.21 mM (range 0.2-2.7 mM). The mean resting potential was -83 +/- 0.7 mV. The mean [K+]i and [Mg2+]i found in these fibres was significantly higher (P less than 0.0001) than those measured in fibres from muscles maintained in standard Ringer solution (i.e. without external Mg2+). Possible explanations for this finding are discussed. Whether in the presence (0.5 mM) or in the absence of external Mg2+, our values for [Mg2+]i are distinctly lower than those previously reported by others, using the same type of Mg2+-selective micro-electrodes but calibrated simply from assumptions about the actual level of K+ and Na+ interference on Mg2+-selective micro-electrode response.(ABSTRACT TRUNCATED AT 400 WORDS)

Patterns of sarcomere activation, temperature dependence, and effect of ryanodine in chemically skinned cardiac fibers.

Functionally skinned and electrochemically shunted myocytes were prepared by perfusing rat hearts with collagenase in order to obtain a technically improved measurement of sarcomere dynamics and to evaluate the role of sarcoplasmic reticulum in situ with respect to contractile activation. In the presence of micromolar calcium, the myocytes exhibited phasic and propagated contraction waves beginning at one end and proceeding along the myocyte. Beating rates, the propagation velocity of the activation wave, and single sarcomere shortening and relaxation velocities were obtained by manual or automated analysis of 16-mm film recorded at 170 frames/s from a camera attached to a microscope that was equipped with a temperature-controlled stage. In parallel experiments, calcium accumulation by the sarcoplasmic reticulum of the myocytes in situ was measured by direct isotopic tracer methods. The frequency (10-38 min-1) of spontaneous contractions, the velocity (1.9-7.4 microns . s-1) of sarcomere shortening, and the velocity (1.7-6.8 microns . s-1) of sarcomere relaxation displayed identical temperature dependences (Q10 = 2.2), which are similar to that of the calcium pump of sarcoplasmic reticulum and are consistent with a rate limit imposed by enzyme-catalyzed mechanisms on all these parameters. On the other hand, the velocity (77-159 microns . s-1) of sequential sarcomere activation displayed a lower temperature dependence (Q10 = 1.5), which is consistent with a diffusion-limited and self-propagating release of calcium from one sarcomere to the other. The phasic contractile activity of the dissociated myocytes was inhibited by 10(-8)-10(6) M ryanodine (and not by myolemmal calcium blockers) under conditions in which calcium accumulation by sarcoplasmic reticulum in situ was demonstrated to proceed optimally. The effect of ryanodine is attributed to an interaction of this drug with sarcotubular structures, producing inhibition of calcium release from the sarcoplasmic reticulum. The consequent lack of sarcomere activation underlines the role of sarcoplasmic reticulum uptake and release in the phasic contractile activation of the electrochemically shunted myocytes.

Electron probe X-ray microanalysis of post-tetanic Ca2+ and Mg2+ movements across the sarcoplasmic reticulum in situ.

Ca2+ and Mg2+ movements across the sarcoplasmic reticulum (SR) of frog skeletal muscle fibers were measured in situ by electron probe microanalysis of muscles rapidly frozen following a tetanus. At 400 ms following a 1.2-s tetanus at room temperature, the force had relaxed to base-line, and 0.3 mmol of Ca2+/liter of cytoplasmic H2O had been pumped by the SR, indicating that the in situ pumping of the SR Ca-ATPase is sufficiently high to account for the removal of Ca2+ from the Ca2+-specific sites of troponin (0.18 mmol of Ca2+-specific sites/liter of cytoplasmic H2O) and for the rate of relaxation from a tetanus at room temperature. The half-time of the return of the total 1.0 mmol of Ca2+/liter of cytoplasmic H2O released during a tetanus was 1.1 s, comparable to the slow Koff rate of Ca2+ from (carp) parvalbumin (1.0 s-1) and consistent with the hypothesis that the return of this Ca2+ to the terminal cisternae is rate-limited by the Ca2+ off-rate from parvalbumin. The return of the Mg2+ taken up by the terminal cisternae during a tetanus to resting levels was significantly slower than the time course of the Ca2+ movements, suggesting that the Mg2+ permeability of the SR in situ is low and may be transiently increased during tetanic stimulation.