Péter Szentesi

ATP utilization for calcium uptake and force production in different types of human skeletal muscle fibres

1 The contractile properties and ATPase activity of skinned human skeletal muscle fibres from vastus lateralis were examined. Fibre types were resolved from single fibre segments by SDS‐polyacrylamide gel electrophoresis. ATPase activity was determined by enzymatic coupling of ATP resynthesis to the oxidation of NADH. 2 The partitioning of ATPase activity into (a) calcium‐activated activity due to actomyosin (AM) interaction, (b) calcium‐activated activity of the sarcoplasmic reticular (SR) calcium pump, and (c) basal (calcium independent) activity was investigated by comparing ATP utilization before and after exposure of the preparations for 30 min to a solution containing 0.5 % Triton X‐100, which effectively abolished the SR ATPase activity. 3 Partitioning of ATPase activity was also determined by measuring ATP utilization and force at different concentrations of butanedione monoxime (BDM), which inhibits AM interaction. 4 The results obtained with Triton X‐100 and BDM were similar. At saturating Ca2+ concentrations and 20 °C, the AM, SR and basal ATPase activities per litre cell volume (±s.e.m.) amounted to 46 ± 4, 51 ± 4 and 19 ± 2 μm s−1 in type I fibres (n = 21), 139 ± 14, 69 ± 8 and 30 ± 3 μm s−1 in type IIA fibres (n = 25), 137 ± 22, 175 ± 28 and 26 ± 8 μm s−1 in type IIA/B fibres (n = 4) and 108 ± 13, 169 ± 42 and 32 ± 8 μm s−1 in type IIB fibres (n = 2). 5 These results indicate that ATP utilization for SR Ca2+ pumping in fast fibres is considerably larger than in slow fibres. The SR ATPase activity in human muscle represents a considerable fraction of the total (AM + SR + basal) ATPase activity.

Action potentials and potassium currents in rat ventricular muscle during experimental diabetes

Time course of the surface electrical activity was studied in left ventricular trabeculae of Wistar rats made diabetic using streptozotocin. The action potentials were recorded in Tyrodes solution at 32 degrees C, their duration considerably increased in diabetes. By the 8th week, the prolongation was 64% at 25% of repolarization; 112% at 50% and 118% at 75%. Insulin treatment reduced the prolongation of the action potentials although a complete restoration was not achieved. 0.1 mM La3+ moderately shortened the electrical activity both in control and in diabetic trabeculae. Three mM 4-aminopyridine made the time course of control action potentials very similar to the diabetic ones while the action potentials from the diabetic animals were prolonged further to a smaller extent. Whole-cell clamp experiments in isolated ventricular myocytes (20-23 degrees C) showed a considerable decrease and a somewhat accelerated inactivation of the transient outward current (Ito) in diabetes. The steady-state inactivation and the rate of recovery from inactivation of Ito did not change. No alterations in the magnitude and voltage dependence of inward rectifier (IK1) were found around the resting membrane potential. The diabetes-related suppression of Ito explains the decreased repolarization rate of action potentials.

Sarcoplasmic reticulum Ca2+ refilling controls recovery from Ca2+-induced Ca2+ release refractoriness in heart muscle

In cardiac muscle Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) is initiated by Ca2+ influx via L-type Ca2+ channels. At present, the mechanisms underlying termination of SR Ca2+ release, which are required to ensure stable excitation-contraction coupling cycles, are not precisely known. However, the same mechanism leading to refractoriness of SR Ca2+ release could also be responsible for the termination of CICR. To examine the refractoriness of SR Ca2+ release, we analyzed Na+-Ca2+ exchange currents reflecting cytosolic Ca2+ signals induced by UV-laser flash-photolysis of caged Ca2+. Pairs of UV flashes were applied at various intervals to examine the time course of recovery from CICR refractoriness. In cardiomyocytes isolated from guinea-pigs and mice, &bgr;-adrenergic stimulation with isoproterenol-accelerated recovery from refractoriness by ≈2-fold. Application of cyclopiazonic acid at moderate concentrations (<10 &mgr;mol/L) slowed down recovery from refractoriness in a dose-dependent manner. Compared with cells from wild-type littermates, those from phospholamban knockout (PLB-KO) mice exhibited almost 5-fold accelerated recovery from refractoriness. Our results suggest that SR Ca2+ refilling mediated by the SR Ca2+-pump corresponds to the rate-limiting step for recovery from CICR refractoriness. Thus, the Ca2+ sensitivity of CICR appears to be regulated by SR Ca2+ content, possibly resulting from a change in the steady-state Ca2+ sensitivity and in the gating kinetics of the SR Ca2+ release channels (ryanodine receptors). During Ca2+ release, the concomitant reduction in Ca2+ sensitivity of the ryanodine receptors might also underlie Ca2+ spark termination by deactivation.

Triadin Deletion Induces Impaired Skeletal Muscle Function

Triadin is a multiple proteins family, some isoforms being involved in muscle excitation-contraction coupling, and some having still unknown functions. To obtain clues on triadin functions, we engineered a triadin knock-out mouse line and characterized the physiological effect of triadin ablation on skeletal muscle function. These mice presented a reduced muscle strength, which seemed not to alter their survival and has been characterized in the present work. We first checked in these mice the expression level of the different proteins involved in calcium homeostasis and observed in fast muscles an increase in expression of dihydropyridine receptor, with a large reduction in calsequestrin expression. Electron microscopy analysis of KO muscles morphology demonstrated the presence of triads in abnormal orientation and a reduction in the sarcoplasmic reticulum terminal cisternae volume. Using calcium imaging on cultured myotubes, we observed a reduction in the total amount of calcium stored in the sarcoplasmic reticulum. Physiological studies have been performed to evaluate the influence of triadin deletion on skeletal muscle function. Muscle strength has been measured both on the whole animal model, using hang test or electrical stimulation combined with NMR analysis and strength measurement, or on isolated muscle using electrical stimulation. All the results obtained demonstrate an important reduction in muscle strength, indicating that triadin plays an essential role in skeletal muscle function and in skeletal muscle structure. These results indicate that triadin alteration leads to the development of a myopathy, which could be studied using this new animal model.

Intramembrane charge movement and sarcoplasmic calcium release in enzymatically isolated mammalian skeletal muscle fibres.

1 Single muscle fibres were dissociated enzymatically from the extensor digitorum longus and communis muscles of rats and guinea‐pigs. The fibres were mounted into a double Vaseline gap experimental chamber and the events in excitation–contraction coupling were studied under voltage clamp conditions. 2 The voltage dependence of intramembrane charge movement followed a two‐state Boltzmann distribution with maximal available charge of 26.1 ± 1.5 and 26.1 ± 1.3 nC μF−1, mid‐point voltage of –35.1 ± 5.0 and –42.2 ± 1.2 mV and steepness of 16.7 ± 2.2 and 17.0 ± 1.9 mV (means ±s.e.m., n= 7 and 4) in rats and guinea‐pigs, respectively. 3 Intracellular calcium concentration ([Ca2+]i) was monitored using the calcium‐sensitive dyes antipyrylazo III, fura‐2 and mag‐fura‐5. Resting [Ca2+]i was similar in rats and guinea‐pigs with 125 ± 18 and 115 ± 8 nm (n= 10 and 9), respectively, while the maximal increase for a 100 ms depolarization to 0 mV was larger in rats (6.3 ± 1.0 μm; n= 7), than in guinea‐pigs (2.8 ± 0.3; n= 4). 4 The rate of calcium release (Rrel) from the sarcoplasmic reticulum (SR) displayed an early peak followed by a fast and a slow decline to a quasi maintained steady level. After normalizing Rrel to the estimated SR calcium content (1.2 ± 0.1 and 0.9 ± 0.1 mm in rats and guinea‐pigs, respectively) and correcting for depletion of calcium in the SR the peak and steady levels at 0 mV, respectively, were found to be 2.50 ± 0.08 and 0.81 ± 0.06% ms−1 in rats and 2.43 ± 0.25 and 0.88 ± 0.01 % ms−1 in guinea‐pigs. The voltage dependence was essentially the same in both species, but different from that in amphibians. 5 These experiments show that enzymatic isolation yields functionally intact mammalian skeletal muscle fibres for Vaseline gap experiments. The data also suggest a close connection in the regulation of the different kinetic components of SR calcium release in mammalian skeletal muscle.

MEASUREMENTS OF INTRACELLULAR MG2+ CONCENTRATION IN MOUSE SKELETAL MUSCLE FIBERS WITH THE FLUORESCENT INDICATOR MAG-INDO-1

Measurements of intracellular free magnesium concentration ([Mg2+]i) were performed on enzymatically isolated skeletal muscle fibers from mice, using the fluorescent ratiometric indicator mag-indo-1. An original procedure was developed to calibrate the dye response within the fibers: fibers were first permeabilized with saponin in the presence of a given extracellular magnesium concentration and were then embedded in silicone grease. The dye was then pressure microinjected into the saponin-permeabilized silicone-embedded fibers, and fluorescence was measured. The results show that for all tested [Mg2+], the value of the measured fluorescence ratio was higher than that found in aqueous solutions. Furthermore, the apparent binding curve that could be fit to the in vivo ratio data was shifted toward higher [Mg2+] by a factor of approximately 2. Using the in vivo calibration parameters, the mean resting [Mg2+]i was found to be 1.53 +/- 0.16 mM (n = 7). In an attempt to gain insight into the myoplasmic magnesium buffering capacity, we measured, together with mag-indo-1 fluorescence, the current elicited by the application of carbamylcholine (CCh) to the endplate of isolated fibers, in the presence of a high extracellular magnesium concentration. The results show that, under these conditions, a change in [Mg2+]i displaying a time course and amplitude qualitatively consistent with the CCh-induced inward current can be measured.

Effects of tetracaine on sarcoplasmic calcium release in mammalian skeletal muscle fibres

1 Single muscle fibres were dissociated enzymatically from the extensor digitorum communis muscle of rats. The fibres were mounted into a double Vaseline gap experimental chamber and the events in excitation‐contraction coupling were studied under voltage clamp conditions in the presence and absence of the local anaesthetic tetracaine. 2 Changes in intracellular calcium concentration ([Ca2+]i) were monitored using the calcium sensitive dyes antipyrylazo III and fura‐2 and the rate of calcium release (Rrel) from the sarcoplasmic reticulum (SR) was calculated. Tetracaine decreased the maximal attained [Ca2+]i and suppressed, in a dose‐dependent manner, both the early peak and the steady level of Rrel in the voltage range examined. 3 The concentration dependence of the effects on the two kinetic components of Rrel were almost identical with a half‐effective concentration (K50) of 70 and 71 μm and a Hill coefficient (nH) of 2.7 and 2.3 for the peak and the steady level, respectively. Furthermore, the drug did not alter the peak to steady level ratio up to a concentration (50 μm) that caused a 35 ± 5% reduction in calcium release. Higher concentrations did suppress the ratio but the degree of suppression was voltage independent. 4 Tetracaine (50 μm) neither influenced the total available intramembrane charge nor altered its membrane potential dependence. It shifted the transfer function, the normalized SR permeability versus normalized charge to the right, indicating that similar charge transfer caused a smaller increase in SR permeability. 5 To explore the site of action of tetracaine further the ryanodine receptor (RyR) calcium release channel of the SR was purified and reconstituted into planar lipid bilayers. The reconstituted channel had a conductance of 511 ± 14 pS (n= 8) in symmetric 250 mm KCl that was not affected by tetracaine. Tetracaine decreased the open probability of the channel in a concentration‐dependent manner with K50= 68 μm and nH= 1.5. 6 These experiments show that tetracaine suppresses SR calcium release in enzymatic isolated mammalian skeletal muscle fibres. This effect is due, presumably, to the decreased open probability of the RyR in the presence of the drug. Since both the inactivating peak and the steady level of Rrel were equally affected by tetracaine, our observations suggest that there is a tight coupling between these kinetic components of SR calcium release in mammalian skeletal muscle.

Effects of cardiac glycosides on excitation-contraction coupling in frog skeletal muscle fibres.

1. The effects of digoxin and ouabain on the calcium release flux from the sarcoplasmic reticulum (SR), isometric tension and intramembrane charge movement were studied in voltage clamped skeletal muscle fibres of the frog. 2. Both cardiac glycosides increased both calcium transients and simultaneously recorded tension at all membrane potentials, showing different effects on the peak and on the steady components of the calcium release flux. These effects were attained at an extracellular digoxin concentration of 5 nM and an estimated intracellular ouabain concentration of 1‐2 nM. Digoxin and ouabain thus exerted their effects at the same concentration on calcium release in skeletal muscle as previously observed in isolated cardiac‐type ryanodine receptor (RyR) calcium release channels. 3. The peak of SR calcium release increased at all voltages, with the largest potentiation at intermediate membrane potentials. This increase in calcium release flux was attained despite an unchanged SR calcium content. The attenuated release rate therefore reflected an increased number of open RyR channels rather than increased SR loading. 4. These effects could be attributed to an increase in calcium release activation and not a decrease in the rate of inactivation. Rather, the rate of inactivation was enhanced at all voltages as expected from the increased calcium concentration in the triadic junction. 5. In contrast, CMA (17 alpha‐acetoxy‐6‐chloro‐4, 6‐pregnadiene‐3,20‐dione; 5 microM), a Na(+)‐K(+)‐ATPase inhibitor with no positive inotropic effects on the heart, neither influenced SR calcium release nor antagonized the effects of ouabain. 6. Both digoxin and ouabain preserved total intramembrane charge apart from a small negative shift in the mid‐point voltage and increase in slope factor. 7. Both digoxin and ouabain induced calcium release from heavy SR vesicles at rates comparable to that induced by ryanodine or caffeine. 8. It is concluded that at least part of the inactivating component of SR calcium release involves distinct RyR calcium release channels that resemble the cardiac RyR isoform in its specific sensitivity to cardiac glycosides.

Melanoma cells exhibit strong intracellular TASK-3-specific immunopositivity in both tissue sections and cell culture

Abstract.Amplification of the kcnk9 gene and overexpression of the encoded channel protein (TASK-3) seems to be involved in carcinogenesis. In the present work, TASK-3 expression of melanoma cells has been studied. For the investigation of TASK-3-specific immunolabelling, a monoclonal antibody has been developed and applied along with two, commercially available polyclonal antibodies targeting different epitopes of the channel protein. Both primary and metastatic melanoma cells proved to be TASK-3 positive, showing prominent intracellular TASK-3-specific labelling; mostly concentrating around or in the proximity of the nuclei. The immunoreaction was associated with the nuclear envelope, and with the processes of the cells and it was also present in the cell surface membrane. Specificity of the immunolabelling was confirmed by Western blot and transfection experiments. As TASK-3 immunopositivity of benign melanocytes could also be demonstrated, the presence or absence of TASK-3 channels cannot differentiate between malignant and non-malignant melanocytic tumours.

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.