Na(+)-Ca2+ exchange induces low Na+ contracture in frog skeletal muscle fibers after partial inhibition of sarcoplasmic reticulum Ca(2+)-ATPase.

International audienceContractile responses due to reduction in external sodium concentration ([Na+]o) were investigated in twitch skeletal muscle fibers of frog semitendinosus. Experiments were conducted after partial inhibition of sarcoplasmic reticulum Ca(2+)-ATPase by cyclopiazonic acid (CPA). In the absence of CPA, Na+ withdrawal failed to produce any change in resting tension. In the presence of CPA (2-10 microM), [Na+]o reduction induced a transient contracture without a significant change in the resting membrane potential. The amplitude of the contracture displayed a step dependence on [Na+]o, was increased by K(+)-free medium and was prevented in Ca(2+)-free medium. This contracture was inhibited by various blockers of the Na(+)-Ca2+ exchange but was little affected by inhibitors of sarcolemmal Ca(2+)-ATPase or mitochondria. When sarcoplasmic reticulum function was impaired, low-Na+ solutions caused no contracture. These results provide evidence that skeletal muscle fibers possess a functional Na(+)-Ca2+ exchange which can mediate sufficient Ca2+ entry to activate contraction by triggering Ca2+ release from sarcoplasmic reticulum when the sodium electrochemical gradient is reduced, and sarcoplasmic reticulum Ca(2+)-ATPase is partially inhibited. This indicates that when the sarcoplasmic reticulum Ca(2+)-ATPase is working (no CPA), Ca2+ fluxes produced by the exchanger are buffered by the sarcoplasmic reticulum. Thus the Na(+)-Ca2+ exchange may be one of the factors determining sarcoplasmic reticulum Ca2+ content and thence the magnitude of the release of Ca2+ from the sarcoplasmic reticulum

Changes in voltage activation of contraction in frog skeletal muscle fibres as a result of sarcoplasmic reticulum Ca2+-ATPase activity

International audienceThe effects of cyclopiazonic acid, a specific sarcoplasmic reticulum Ca2+-ATPase inhibitor, on isometric tension were studied in response to prolonged steady-state depolarization induced by a rapid change in extracellular potassium concentration (potassium contractures) in frog semitendinosus muscle fibres. Cyclopiazonic acid (1-10 microM) enhanced the amplitude and time-course of relaxation of 146 mM potassium contracture. In the presence of cyclopiazonic acid 0.5 microM, the relationship between the amplitude of potassium contractures and the membrane potential shifted to more negative potentials, whereas the steady-state inactivation curve was unchanged. These observations suggest that cyclopiazonic acid has no effect on voltage sensors. The difference between potassium contractures in the absence and presence of cyclopiazonic acid in skeletal muscle fibres implies that the amplitude and slow relaxation of tension during prolonged steady-state depolarization may be expected to depend not only on inactivation of the process regulating calcium release from the sarcoplasmic reticulum but also on the ability of the sarcoplasmic reticulum to pump calcium

Cyclopiazonic acid and thapsigargin reduce Ca2+ influx in frog skeletal muscle fibres as a result of Ca2+ store depletion

International audienceWe have investigated the influence of the sarcoplasmic reticulum (SR) Ca2+ content on the retrograde control of skeletal muscle L-type Ca2+ channels activity by ryanodine receptors (RyR). The effects of cyclopiazonic acid (CPA) and thapsigargin (TG), two structurally unrelated inhibitors of SR Ca(2+)-adenosine triphosphatase (ATPase), were examined on the SR Ca2+ content, the calcium current and contraction in single frog semitendinosus fibres using the double mannitol-gap technique. At moderate concentrations that only partially inhibited Ca2+ sequestration by the SR, CPA (2-4 microM) induces a concentration dependent depression of contraction and Ca2+ current amplitudes. When Ba2+ is the charge carrier, the inward current is not changed by CPA suggesting that this Ca(2+)-pump inhibitor does not directly affect dihydropyridine Ca2+ channels. Similar effects were obtained with TG (1-5 microM). Changes in Ca2+ currents and contraction were accompanied by a reduced Ca2+ loading of the SR. We attribute the modulation of the Ca2+ current to the selective inhibition of the SR Ca2+ ATPase, resulting in a decreased Ca2+ release and thereby a reduced activation of calcium inward currents. This is therefore taken to represent a calcium release-dependent modulation of skeletal muscle L-type Ca2+ channels

Measurement of sarcomere length during fast contraction of muscle fibers by digital image analysis.

International audienceA low-cost, high-resolution (spatial and temporal) image analysis system was developed to measure sarcomere length (Sl) during fast twitch of isolated striated muscle fibers at different temperatures. Fiber images were examined during twitch with an imaging rate of 220 Hz. To increase temporal resolution beyond 220 Hz, consecutive temporally shifted image sequences (N sequences) were acquired. Individual or average Sl was directly measured from a horizontal profile without spatial-frequency assessment. Measurement precision (E) was determined and expressed as: E(%) = 100xPs/(IsxSl), where Ps is the pixel size and Is the involved sarcomere number. At 18 degrees C during isometric twitch, Sls were measured with 220 Hz temporal and 0.2% spatial resolutions. Sl shortened in the central region (0.21+/-0.12 microm) as tension developed, reaching a maximal shortening of 8.09 + 2.05% (at rest, Sl = 2.59+/-0.05 microm, n = 4) in 32.5+/-1.96 ms. At 30 degrees C, Sl variations were examined with 880 Hz temporal resolution, in which case maximal S1 shortening was reached in 15.74+/-1.99 ms, and then decreased to 5.19+/-1.97% (at rest, S1 = 2.6+/-0.06 microm). The twitch tension developed by the whole fiber was recorded and compared with sarcomere length behavior. Sarcomere length variations in the central region were representative of overall developed tensions at 18 and 30 degrees C

Voltage clamp with double sucrose gap technique. External series resistance compensation.

In this paper we deal with the double sucrose-gap voltage clamp technique. To perform a reliable clamp or to analyze the intracellular potential distribution, any external series resistance in the artificial node must be taken into account for it induces an instability in the external potential as soon as a current develops. A circuit was designed to compensate for this error, it has been found effective on an analog model and on experimental uni- or multicellular preparations. The attenuation in series resistance frequently causes ringing in the step response. This behavior was studied theoretically and also simulated with analog models where a selective bridged-T network was found to represent the electrical characteristics of the preparation when associated with the chamber and control electronics. A residual series resistance was found and is considered to be a part of the preparation. Characteristics necessary to obtain best results are proposed, for a preparation to be studied in experiments utilizing the double sucrose gap technique with external series resistance compensation