Julianna Cseri

A purinergic signal transduction pathway in mammalian skeletal muscle cells in culture

Abstract. The effects of adenosine 5′-triphosphate (ATP) on human and mouse skeletal muscle fibres in primary culture were investigated. ATP-evoked changes in intracellular calcium concentration ([Ca2+]i) were measured and compared with those induced by agonists of the nicotinic acetylcholine (Ach)- and P2X purinoreceptors. While ATP was effective on both myoblasts and multi-nucleated myotubes in the micromolar range, Ach failed to induce any change in [Ca2+]i at early stages of development. In contrast, myofibres with peripheral nuclei showed little response to ATP but responded to Ach with a large change in [Ca2+]i. The responsiveness of the myotubes to Ach paralleled that to potassium. The removal of external calcium abolished the response to ATP. P2X receptor agonists mimicked the response to ATP with the order of potency being ATP>2′,3′-O-(4-benzoyl)-benzoyl-ATP>β,γ-methylene-ATP>α,β-methylene-ATP. Under voltage-clamp conditions ATP induced an inward current that showed little inactivation. These results are consistent with the existence of P2X receptor-mediated signal transduction pathway in cultured mammalian skeletal 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 micro M 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 micro M. 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 micro M 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.

Contribution from P2X and P2Y purinoreceptors to ATP-evoked changes in intracellular calcium concentration on cultured myotubes.

Although the alteration of purinoreceptor pattern on skeletal muscle is known to accompany physiological muscle differentiation and the pathogenesis of muscle dystrophy, the exact identity of and the relative contribution from the individual receptor subtypes to the purinergic signal have been controversial. To identify these subtypes in cultured myotubes of 5–10 nuclei, changes in intracellular calcium concentration and surface membrane ionic currents were detected and calcium fluxes calculated after the application of the subtype-specific agonists 2′3′-O-(benzoyl-4-benzoyl)-ATP (BzATP), 2-methyltio-ADP and UTP. The effectiveness of these agonists together with positive immunocytochemical staining revealed the presence of P2X4, P2X5, P2X7, P2Y1 and P2Y4 receptors. siRNA-reduced protein expression of P2X5, P2X7 and P2Y1 receptors was accompanied by reduction in the ATP-evoked calcium transients. Furthermore, anti-P2X7 siRNA caused a significant drop in the early peak and delayed steady component of the calculated calcium flux. The use of its antagonist, oxidized ATP, similarly to transfection with anti-P2X7 siRNA caused significant reduction in the agonist-elicited ionic currents IATP and IBzATP, with a greater drop in the latter. Our results demonstrate that the activation of ionotropic P2X4, P2X5 and P2X7 and metabotropic P2Y1 and P2Y4 purinoreceptors participates in forming the calcium transients of multinucleated myotubes.

Differential effects of maurocalcine on Ca2+ release events and depolarization-induced Ca2+ release in rat skeletal muscle.

Maurocalcine (MCa), a 33 amino acid toxin obtained from scorpion venom, has been shown to interact with the isolated skeletal‐type ryanodine receptor (RyR1) and to strongly modify its calcium channel gating. In this study, we explored the effects of MCa on RyR1 in situ to establish whether the functional interaction of RyR1 with the voltage‐sensing dihydropyridine receptor (DHPR) would modify the ability of MCa to interact with RyR1. In developing skeletal muscle cells the addition of MCa into the external medium induced a calcium transient resulting from RyR1 activation and strongly inhibited the effect of the RyR1 agonist chloro‐m‐cresol. In contrast, MCa failed to affect the depolarization‐induced Ca2+ release. In intact adult fibres MCa did not induce any change in the cytosolic Ca2+ concentration. However, when the surface membrane was permeabilized and calcium release events were readily observable, MCa had a time‐dependent dual effect: it first increased event frequency, from 0.060 ± 0.002 to 0.150 ± 0.007 sarcomere−1 s−1, and reduced the amplitude of individual events without modifying their spatial distribution. Later on it induced the appearance of long‐lasting events resembling the embers observed in control conditions but having a substantially longer duration. We propose that the functional coupling of DHPRs and RyR1s within a Ca2+ release unit prevents MCa from either reaching its binding site or from being able to modify the gating not only of the RyR1s physically coupled to DHPRs but all RyR1s within the Ca2+ release unit.

CONCENTRATION-DEPENDENT EFFECTS OF TETRACAINE ON EXCITATION-CONTRACTION COUPLING IN FROG SKELETAL MUSCLE FIBRES

SummaryThe effects of low (10–100 μm) concentrations of tetracaine on intramembrane charge movement and on the rate of calcium release (Rrel) from the sarcoplasmic reticulum (SR) were studied in cut skeletal muscle fibres of the frog using the voltage clamp technique. The fibres were mounted in a single or double vaseline gap chamber to study the events near the contraction threshold or in a wide membrane potential range. Although the ‘hump’ component of charge movement (Qγ) was suppressed to some extent, the voltage dependence and the parameters of the Boltzmann distribution were not modified significantly at tetracaine concentrations below 50 μm. At 50 and 100 μm of tetracaine the midpoint voltage of the Boltzmann distribution was shifted to higher membrane potentials and the steepness was decreased. The total available charge remained the same at all concentrations tested. Using fura-2 to measure calcium transients at 100 μm tetracaine the threshold for calcium release was found to be significantly shifted to more positive membrane potentials. Tetracaine reversibly suppressed both the early inactivating peak and the steady-level of Rrel but the concentration dependence of these effects was markedly different. The inactivating component of calcium release was decreased with a Hill coefficient of approximately 1 and half effective concentration of 11.8 μm while the steady-level was decreased with a Hill coefficient of greater than 2 and a half effective concentration of 47.0 μm. These results favour two sites of action where tetracaine would suppress the calcium release from the SR.

HMGB1 expression and muscle regeneration in idiopathic inflammatory myopathies and degenerative joint diseases

The High-Mobility Group Box 1 protein (HMGB1) is a known nuclear protein which may be released from the nucleus into the cytoplasm and the extracellular space. It is believed that the mobilized HMGB1 plays role in the autoimmune processes as an alarmin, stimulating the immune response. In addition, muscle regeneration and differentiation may also be altered in the inflammatory surroundings. Biopsy specimens derived from patients with idiopathic inflammatory myopathies (IIM) such as polymyositis or dermatomyositis were compared to muscle samples from patients undergoing surgical interventions for coxarthrosis. The biopsy and surgery specimens were used for Western blot analysis, for immunohistochemical detection of HMGB1 in histological preparations and for cell culturing to examine cell proliferation and differentiation. Our data show lower HMGB1 expression, impaired proliferation and slightly altered fusion capacity in the primary cell cultures started from IIM specimens than in cultures of coxarthrotic muscles. The ratio of regenerating muscle fibres with centralised nuclei (myotubes) is lower in the IIM samples than in the coxarthrotic ones but corticosteroid treatment shifts the ratio towards the coxarthrotic value. Our data suggest that the impaired regeneration capacity should also be considered to be behind the muscle weakness in IIM patients. The role of HMGB1 as a pathogenic signal requires further investigation.