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The adult isoform of voltage-gated L-type calcium channel (CaV1.1a) opens slowly at strong depolarizations, its contribution to the calcium influx during an action potential is negligible in skeletal muscle. In contrast, calcium influx through the embryonic splice variant (CaV1.1e) substantially contributes to depolarization-induced calcium transients as this isoform displays an altered voltage-dependence and gating kinetics as compared to CaV1.1a. Utilizing a genetically modified mouse model (CaV1.1αE29), which exclusively expresses the embryonic CaV1.1e variant in adult muscle, spontaneous or depolarization-evoked calcium release events were recorded in enzymatically isolated, intact adult skeletal muscle fibers from the m. flexor digitorum brevis, using the fluorescent calcium probe Fluo-8 and the Zeiss Live fast confocal microscope equipped with an Axon Multiclamp 700b. The events occurred at preferred locations as the distribution of spark number in the given position was different from the theoretical Poisson distribution which represents random event occurrence. Calcium events with both short (spark-like, duration = 46.0±1.5 ms, n = 125) and long (ember-like, duratio n= 473±14 ms, n = 218) duration were present on the same cell. In addition, events with complex kinetics, including late openings within an event also appeared. Voltage-activated (500 ms long pulses to the threshold of SR calcium release) with similar properties were also observed.

Mitochondria play key roles in cellular energy generation and lifespan of most eukaryotes. To understand the functions of four nuclear-encoded genes predicted to be related to the maintenance of mitochondrial morphology and function in Aspergillus nidulans, systematic characterization was carried out. The deletion and overexpression mutants of aodA, dnmA, mnSOD and pimA encoding alternative oxidase, dynamin related protein, manganese superoxide dismutase and Lon protease, respectively, were generated and examined for their growth, stress tolerances, respiration, autolysis, cell death, sterigmatocystin production, hyphal morphology and size and mitochondrial superoxide production as well as development. Overall, genetic manipulation of these genes had less effect on cellular physiology and ageing in A. nidulans than that of their homologs in another fungus Podospora anserina with a well-characterized senescence. The observed interspecial phenotypic differences can be explained by the dissimilar intrinsic stabilities of the mitochondrial genomes in A. nidulans and P. anserina. Furthermore, the marginally altered phenotypes observed in A. nidulans mutants indicate the presence of effective compensatory mechanisms for the complex networks of mitochondrial defense and quality control. Importantly, these findings can be useful for developing novel platforms for heterologous protein production, or on new biocontrol and bioremediation technologies based on Aspergillus species.

Intracellular calcium transients have long been known to play an important role in platelet activation. The main processes involved are the IP3 mediated release of calcium from the endoplasmic reticulum and the consequent activation of the store-operated calcium entry (SOCE) mechanism, consisting of the STIM-Orai system and TRPC channels. Traditionally the following methods have been used to measure the calcium level in platelets: flow cytometry, spectrofluorimetry and fluorescent microscopy, with Fura-2-AM used as a calcium indicator. These methods, however, are either incapable of visualising the time course of the calcium transient for the same cell during the activation process or show only the composite calcium level for all the cells.Combining co-loading with Fluo-4-AM and Fura-Red-AM and high-speed confocal imaging (Zeiss LSM Live) we have been able to visualize the calcium transients in case of platelet activation. We investigated the effects of thrombin receptor activating peptide (TRAP), acting on the receptor involved in the physiological platelet activation.Our results indicate that cellular calcium response to TRAP was highly heterogeneous, with only 22.2% (95% CI: 15.1-30.8%) of the platelets exhibiting a calcium transient after activation by TRAP. The non-responding cells still showed an increase of intracellular calcium level when calcium ionophore A23187 was applied. By first using a calcium-free buffer and re-adding calcium after TRAP activation we were also able to distinctly measure SOCE on these cells. Also, by directly activating IP3 receptors with thimerosal, we have been able to measure the effects of inhibitors of the phospholipase C pathway on SOCE.We believe that the proposed imaging method is useful for studying the calcium homeostasis of platelets and possibly other blood cells. The observed heterogeneity of platelet responses may have important clinical and pharmacological consequences.

Disruption of epigenetic regulation and characteristic metabolic alterations (known as the Warburg-effect) are well-known hallmarks of cancer. In our study we investigated the expression levels of microRNAs and histone deacetylase enzymes via RT-qPCR in bone marrow specimens of adult patients suffering from hematological malignancies (total cohort n = 40), especially acute myeloid leukemia (n = 27). The levels of the three examined Warburg-effect related microRNAs (miR-378*, miR-23b, miR-26a) positively correlated with each other and the oncogenic miR-155 and miR-125b, while negatively with the level of the tumorsuppressor miR-124. Significant relationships have been confirmed between the levels of SIRT6, HDAC4 and the microRNAs listed above. In NPM1-mutated AML (n = 6), the level of miR-125b was significantly lower than in the group of AML patients not carrying this mutation (n = 13) (p

Intracellular calcium concentration ([Ca(2+)](i)) is a key player in a wide range of cellular functions from long-term effects that determine the fate of the cell to immediate responses as secretion and motility. To initiate contraction, calcium ions in skeletal muscle are released into the myoplasm through the calcium channels, the ryanodine receptors, of the sarcoplasmic reticulum. The opening of these channels give rise to localised increases in [Ca(2+)](i), originally termed calcium sparks, that fuse and generate the global calcium transient. Whereas calcium sparks in amphibians are abundant and stereotyped, events in mammalian skeletal muscle are scarce and morphologically diverse. This review compares the different forms of calcium release events, occurring spontaneously or evoked by a depolarising pulse, observed in the different classes of vertebrates. It then addresses the questions whether or not these events can be considered as elementary and how the global calcium transient can be reconstructed from them.

It is now widely recognised that changes of the intracellular calcium concentration have deep impact on the differentiation of various non-excitable cells including the elements of the vertebrate skeleton. It has become evident that purinergic signalling is one of the most ancient cellular mechanisms that can cause such alterations in the intracellular Ca(2+)-homeostasis, which are precisely set either spatially or temporally. Purinergic signalling is believed to regulate intracellular Ca(2+)-concentration of developing cartilage and skeletal muscle cells and suggested to play roles in the modulation of various cellular functions. This idea is supported by the fact that pluripotent mesenchymal cells, chondroprogenitors or muscle precursors, as well as mature chondrocytes all are capable of releasing ectonucleotides, and express various types of purinoreceptors and ectonucleotidases. The presence of the basic components of purinergic signalling proves that cells of the chondrogenic lineage can utilise this mechanism for modulating their intracellular Ca(2+) concentration independently from the surrounding skeletal muscle and bone tissues, which are well known to release ectopurines during development and mechanical stress. In this review, we summarize accumulating experimental evidence supporting the importance of purinergic signalling in the regulation of chondrogenesis and during skeletal muscle formation.

Human herpesvirus 6 (HHV-6) frequently reactivates in human immunodeficiency virus 1 (HIV-1) infected patients, and is thought to be a cofactor in AIDS progression. Macrophages are targets and reservoirs of HIV-1 and HHV-6; hence, they have an important role in dissemination and pathogenesis of these viruses. The present study examined the effects of HHV-6 A variant on replication of R5 variants of HIV-1 in macrophages. For this purpose, HIV-1 replication was investigated in macrophages infected with HIV-1 alone or along with HHV-6A. Our results demonstrated that HHV-6A significantly suppressed HIV-1 replication in coinfected cultures. HHV-6A infection resulted in increased secretion of RANTES and IL-8. Experiments with exogenous RANTES and IL-8 revealed that these chemokines also significantly suppressed HIV-1 replication in infected macrophages. RANTES is able to induce desensitization and internalization of CCR5, the chemokine coreceptor of R5 variants. In addition, IL-8 receptor activation results in cross-desensitization and cross-internalization of CCR5. We found that CCR5 sensitivity and expression level is diminished in HHV-6A-infected macrophage cultures compared with uninfected cells. Taken together, our results indicate that HHV-6A infection decreases the susceptibility of macrophages to R5 variants of HIV-1 in which the HHV-6A induced RANTES and IL-8 may have importance.

International audience; In skeletal muscle, sarcoplasmic reticulum (SR) calcium release is controlled by the plasma membrane voltage through interactions between the voltage-sensing dihydropyridine receptor (DHPr) and the ryanodine receptor (RYr) calcium release channel. Maurocalcine (MCa), a scorpion toxin peptide presenting some homology with a segment of a cytoplasmic loop of the DHPr, has been previously shown to strongly affect the activity of the isolated RYr. We injected MCa into mouse skeletal muscle fibers and measured intracellular calcium under voltage-clamp conditions. Voltage-activated calcium transients exhibited similar properties in control and in MCa-injected fibers during the depolarizing pulses, and the voltage dependence of calcium release was similar under the two conditions. However, MCa was responsible for a pronounced sustained phase of Ca(2+) elevation that proceeded for seconds following membrane repolarization, with no concurrent alteration of the membrane current. The magnitude of the underlying uncontrolled extra phase of Ca(2+) release correlated well with the peak calcium release during the pulse. Results suggest that MCa binds to RYr that open on membrane depolarization and that this interaction specifically alters the process of repolarization-induced closure of the channels.

The antifungal protein AFP is a small, cystein-rich protein secreted by the imperfect ascomycete Aspergillus giganteus. The protein efficiently inhibits the growth of filamentous fungi, including a variety of serious human and plant pathogens mainly of the genera Aspergillus and Fusarium, whereas AFP does not affect the growth of yeast and bacteria. This restricted susceptibility range makes it very attractive for medical or biotechnological use to combat fungal infection and contamination. We, therefore, analyzed whether AFP affects the growth or function of a number of mammalian cells. Here we show that the protein neither provokes any cytotoxic effects on human endothelial cells isolated from the umbilical vein nor activates the immune system. Moreover, potassium currents of neurons and astrocytes do not change in the presence of AFP and neither excitatory processes nor the intracellular calcium homeostasis of cultured skeletal muscle myotubes are affected by AFP. Our data, therefore, suggest that AFP is indeed a promising candidate for the therapeutic or biotechnological use as a potential antifungal agent.

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.

ATP acts as a neuro-modulator through purinoceptors in many different tissues. Many subtypes of these receptors have been identified in the inner ear, but so far only two types have been shown to be present in the membrane of the isolated outer hair cells (OHCs). The aim of this study was to detect and visualize the existence and distribution of purinoceptor subtypes as well as to study the [Ca(2+)](i) response of these cells in response to stimulation with ATP. Four P2X and three P2Y receptor subtypes were identified with different expression pattern in the membrane of guinea pig outer hair cells. Whereas intense labeling was observed for P2X1, P2X2, P2X4, P2Y1, P2Y2, and P2Y4, the labeling for the subtype P2X7 was weak. There was a marked difference in the distribution of the receptors along the surface of the cells with a homogenous distribution in cases of P2X1, P2X4, and P2Y1. In contrast, P2X2 and P2Y2 receptor density was high mainly at the apical, while P2X7 and P2Y4 at the basal pole of the cells. Similarly a heterogeneity was observed in the ATP-induced transient elevation in [Ca(2+)](i), which had either fast kinetics without desensitization or slow rise with desensitization.

Cytosolic [Ca 2+ ] transients elicited by voltage clamp depolarization were examined by confocal line scanning of rat skeletal muscle fibres. Ca 2+ sparks were observed in the fibres’ membranepermeabilized ends, but not in responses to voltage in the membrane-intact area. Elementary events of the depolarization-evoked response could be separated either at low voltages (near −50 mV) or at −20mV in partially inactivated cells. These were of lower amplitude, narrower and of much longer duration than sparks, similar to ‘lone embers’ observed in the permeabilized segments. Their average amplitude was 0.19 and spatial half-width 1.3 µm. Other parameters depended on voltage. At −50 mV average duration was 111 ms and latency 185 ms. At −20 mV duration was 203 ms and latency 24 ms. Ca 2+ release current, calculated on an average of events, was nearly steady at 0.5‐0.6 pA. Accordingly, simulations of the fluorescence event elicited by a subresolution source of 0.5 pA open for 100 ms had morphology similar to the experimental average. Because 0.5 pA is approximately the current measured for single RyR channels in physiological conditions, the elementary fluorescence events in rat muscle probably reflect opening of a single RyR channel. A reconstruction of cell-averaged release flux at −20 mV based on the observed distribution of latencies and calculated elementary release had qualitatively correct but slower kinetics than the release flux in prior whole-cell measurements. The qualitative agreement indicates that global Ca 2+ release flux results from summation of these discrete events. The quantitative discrepancies suggest that the partial inactivation strategy may lead to events of greater duration than those occurring physiologically in fully polarized cells.

Changes in intracellular calcium concentration ([Ca2+]i) evoked by prolonged depolarisation (120 mM KCl) or by the application of 15 mM caffeine were measured on skeletal muscle cells in primary culture. The extrusion rate (PVmax) of calcium from the myoplasm was determined, which in turn enabled the calculation of the calcium flux (Fl) underlying the measured calcium transients. PVmax was found to increase during differentiation, from 107 +/- 10 microM/s at the early myotube stage to 596 +/- 36 microM/s in secondary myotubes. This was paralleled by a decrease in resting [Ca2+]i from 99 +/- 4 to 51 +/- 2 nM. The depolarisation-evoked Fl rose to peak and then ceased despite the continuous presence of KCl. In contrast, the caffeine-induced Fl showed a peak and a clear steady-level with a peak-to-steady ratio of 5.6 +/- 1.2. Removal of external calcium suppressed the depolarisation--induced flux by 88 +/- 5% indicating that both an influx and a release from the SR underlie the K(+)-evoked calcium transients. Subsequent applications of caffeine resulted in essentially identical fluxes indicating an efficient refilling of the internal stores. Moreover, if a depolarisation-induced calcium transient preceded the second caffeine-evoked release, the latter was significantly larger than the first suggesting that much of the calcium that entered was stored in the SR rather than extruded.

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.

Aim: The aim of this study was to compare the action potential configuration, contractility, intracellular Ca 2+ and H + concentrations in mammalian cardiac tissues bathed with Krebs and Tyrode solutions at 37 °C. Results: In Langendorff-perfused guinea-pig hearts, loaded with the fluorescent Ca 2+ -indicator Fura-2, or H + -sensitive dye carboxy-SNARF, shifts from Krebs to Tyrode solution caused intra-cellular acidification, increased diastolic pressure and [Ca 2+ ] i , decreased systolic pressure and [Ca 2+ ] i , leading to a reduction in the amplitude of [Ca 2+ ] i transients and pulse pressure. Contractility was also depressed in canine ventricular trabeculae when transferred from Krebs to Tyrode solution. Shifts from Krebs to Tyrode solution increased the duration of action potentials in multicellular cardiac preparations excised from canine and rabbit hearts but not in isolated cardiomyocytes. All these changes in action potential morphology, contractility, [Ca 2+ ] i and [H + ] i were readily reversible by addition of 26 mmol L -1 bicarbonate to Tyrode solution. Effects of dofetilide and CsCl, both blockers of the delayed rectifier K current, on action potential duration were compared in Krebs and Tyrode solutions. Dofetilide lengthened rabbit ventricular action potentials in a significantly greater extent in Tyrode than in Krebs solution. Exposure of canine Purkinje fibres to CsCl evoked early after depolarizations within 40 min in all preparations incubated with Tyrode solution, but not in those bathed with Krebs solution. Conclusion: It is concluded that the marked differences in action potential morphology, [Ca 2+ ] i , [H + ] i and contractility observed between preparations bathed with Krebs and Tyrode solutions are more likely attributable to differences in the intracellular buffering capacities of the two media.

Dystrophin-deficient muscle fibers from mdx mice are believed to suffer from increased calcium entry and elevated submembranous calcium level, the actual source and functional consequences of which remain obscure. Here we compare the properties of the dihydropyridine receptor as voltage sensor and calcium channel in control and mdx muscle fibers, using the silicone-voltage clamp technique. In control fibers charge movement followed a two-state Boltzmann distribution with values for maximal charge, midpoint voltage, and steepness of 23 +/- 2 nC/ micro F, -37 +/- 3 mV, and 13 +/- 1 mV (n = 7). Essentially identical values were obtained in mdx fibers and the time course of charge recovery from inactivation was also similar in the two populations (tau approximately 6 s). In control fibers the voltage dependence of the slow calcium current elicited by 100-ms-long pulses gave values for maximal conductance, apparent reversal potential, half-activation potential, and steepness factor of 156 +/- 15 S/F, 65.5 +/- 2.9 mV, -0.76 +/- 1.2 mV, and 6.2 +/- 0.5 mV (n = 17). In mdx fibers, the half-activation potential of the calcium current was slightly more negative (-6.2 +/- 1.2 mV, n = 16). Also, when using longer pulses, the time constant of calcium current decay was found to be significantly larger (by a factor of 1.5-2) in mdx than in control fibers. These changes in calcium current properties are unlikely to be primarily responsible for a dramatic alteration of intracellular calcium homeostasis. They may be speculated to result, at least in part, from remodeling of the submembranous cytoskeleton network due to the absence of dystrophin.

Myalgia and muscle weakness may appreciably contribute to the poor adherence to statin therapy. Although the pathomechanism of statin-induced myopathy is not completely understood, changes in calcium homeostasis and reduced coenzyme Q10 levels are hypothesized to play important roles. In our experiments, fluvastatin and/or coenzyme Q10 was administered chronically to normocholesterolaemic or hypercholaestherolaemic rats, and the modifications of the calcium homeostasis and the strength of their muscles were investigated. While hypercholesterolaemia did not change the frequency of sparks, fluvastatin increased it on muscles both from normocholesterolaemic and from hypercholesterolaemic rats. This effect, however, was not mediated by a chronic modification of the ryanodine receptor as shown by the unchanged ryanodine binding in the latter group. While coenzyme Q10 supplementation significantly reduced the frequency of the spontaneous calcium release events, it did not affect their amplitude and spatial spread in muscles from fluvastatin-treated rats. This indicates that coenzyme Q10 supplementation prevented the spark frequency increasing effect of fluvastatin without having a major effect on the amount of calcium released during individual sparks. In conclusion, we have found that fluvastatin, independently of the cholesterol level in the blood, consistently and specifically increased the frequency of calcium sparks in skeletal muscle cells, an effect which could be prevented by the addition of coenzyme Q10 to the diet. These results support theories favouring the role of calcium handling in the pathophysiology of statin-induced myopathy and provide a possible pathway for the protective effect of coenzyme Q10 in statin treated patients symptomatic of this condition.

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.

Transient receptor potential canonical‐6 (TRPC6) ion channels, expressed at high levels in podocytes of the filtration barrier, are recently implicated in the pathogenesis of various forms of proteinuric kidney diseases. Indeed, inherited or acquired up‐regulation of TRPC6 activities are suggested to play a role in podocytopathies. Yet, we possess limited information about the regulation of TRPC6 in human podocytes. Therefore, in this study, we aimed at defining how the protein kinase C (PKC) system, one of the key intracellular signalling pathways, regulates TRPC6 function and expression. On human differentiated podocytes, we identified the molecular expressions of both TRPC6 and several PKC isoforms. We also showed that TRPC6 channels are functional since the TRPC6 activator 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG) induced Ca2+‐influx to the cells. By assessing the regulatory roles of the PKCs, we found that inhibitors of the endogenous activities of classical and novel PKC isoforms markedly augmented TRPC6 activities. In contrast, activation of the PKC system by phorbol 12‐myristate 13‐acetate (PMA) exerted inhibitory actions on TRPC6 and suppressed its expression. Importantly, PMA treatment markedly down‐regulated the expression levels of PKCα, PKCβ, and PKCη reflecting their activation. Taken together, these results indicate that the PKC system exhibits a ‘tonic’ inhibition on TRPC6 activity in human podocytes suggesting that pathological conditions altering the expression and/or activation patterns of podocyte‐expressed PKCs may influence TRPC6 activity and hence podocyte functions. Therefore, it is proposed that targeted manipulation of certain PKC isoforms might be beneficial in certain proteinuric kidney diseases with altered TRPC6 functions.

The embryonic splice variant of the voltage-gated L-type calcium channel (CaV1.1e) displays an altered voltage-dependence and gating kinetics as compared to that expressed in adult skeletal muscle. Because the adult CaV1.1a only opens slowly at strong depolarizations, its contribution as a source of calcium influx during action potentials is negligible. In contrast, calcium influx through the embryonic CaV1.1e substantially contributes to depolarization-induced calcium transients in fetal muscles and in cultured myotubes. In a genetically modified mouse (CaV1.1αE29), which exclusively expresses the embryonic CaV1.1e variant also in adult muscle the calcium influx component is maintained throughout life. Utilizing this mouse model, calcium release events - calcium sparklets - were recorded in enzymatically isolated, intact adult skeletal muscle fibers from the m. flexor digitorum longus using the fluorescent calcium probe fluo-8 and the fast confocal scanner (ZeissLive) in the x-y mode. While control animals did not display such events, CaV1.1αE29 mice spontaneously generated sparklets with a frequency of 9.2∗10-4±6∗10-4 Hz/μm2 (19 fibers; mean±SEM). The role of external calcium as the trigger was tested by either removing calcium from the external solution or by the application of 5μM nizoldipine to block the calcium current through CaV1.1e. Both interventions resulted in a complete loss of the events. Identified sparklets (n=311) were characterized by an average amplitude (αF/F0) of 0.287±0.005, a full-width at half-maximum of 3.05±0.05 µm, and duration of 235±4 ms, clearly different from the properties of calcium sparks on saponin-permeabilized adult mammalian skeletal muscle fibers. These findings indicate that the sustained expression of the CaV1.1e splice variant gives rise to spontaneous calcium entry events (sparklets) in adult muscle fibers and that their properties are distinct from calcium sparks arising from ryanodine receptors. Support: OTKA-NN107765, FWF P23479, W1101, TAMOP-4.2.4.A./2-11-1-2012-0001.

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 ([Ca(2+)](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 [Ca(2+)](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 [Ca(2+)](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 ATP2',3'- O-(4-benzoyl)-benzoyl-ATPbeta,gamma-methylene-ATPalpha,beta-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.

Capacitive calcium influx is associated with the release of calcium from internal stores and participates in intracellular calcium homeostasis. In keratinocytes, its activation is linked to the stimulation of the phospho-inositide (PI) pathway and seems to be altered in psoriasis. An overnight treatment of isolated HaCaT keratinocytes with phorbol 12-myristate 13-acetate (PMA) selectively downregulated the classical, calcium-dependent protein kinase C (PKC) isoenzyme PKC alpha in preconfluent cells. This was parallelled by an increased capacitative calcium influx with no effects on the PI pathway. These observations were strengthened in measurements using cyclopiazonic acid which revealed a 47% increase in PMA pretreated as compared with control cells in the calcium influx rate through store-operated calcium channels (SOC-s) following the emptying of the intracellular calcium stores. In confluent as compared with preconfluent cultures PKC epsilon was markedly increased, while other isoenzymes were not affected. In parallel, the kinetics of capacitative calcium influx were altered, showing clear inactivation. PMA pretreatment in these cells had little effect on PKC alpha but downregulated both PKC beta and PKC epsilon, and did not increase the influx through SOC-s. These observations support the differential regulation of SOC-s by PKC and suggest the involvement of several PKC isoenzymes in human keratinocytes.

The sarcoplasmic/endoplasmic reticulum Ca2+ATPases (SERCAs) are the main Ca2+ pumps which decrease the intracellular Ca2+ level by reaccumulating Ca2+ into the sarcoplasmic reticulum. The neonatal SERCA1b is the major Ca2+ pump in myotubes and young muscle fibers. To understand its role during skeletal muscle differentiation its synthesis has been interfered with specific shRNA sequence. Stably transfected clones showing significantly decreased SERCA1b expression (cloneC1) were selected for experiments. The expression of the regulatory proteins of skeletal muscle differentiation was examined either by Western-blot at the protein level for MyoD, STIM1, calsequestrin (CSQ), and calcineurin (CaN) or by RT-PCR for myostatin and MCIP1.4. Quantitative analysis revealed significant alterations in CSQ, STIM1, and CaN expression in cloneC1 as compared to control cells. To examine the functional consequences of the decreased expression of SERCA1b, repeated Ca2+-transients were evoked by applications of 120 mM KCl. The significantly higher [Ca2+]i measured at the 20th and 40th seconds after the beginning of KCl application (112±3 and 110±3 nM vs. 150±7 and 135±5 nM, in control and in cloneC1 cells, respectively) indicated a decreased Ca2+-uptake capability which was quantified by extracting the maximal pump rate (454±41 μM/s vs. 144±24 μM/s, in control and in cloneC1 cells). Furthermore, the rate of calcium release from the SR (610±60 vs. 377±64 μM/s) and the amount of calcium released (843±75 μM vs. 576±80 μM) were also significantly suppressed. These changes were also accompanied by a reduced activity of CaN in cells with decreased SERCA1b. In parallel, cloneC1 cells showed inhibited cell proliferation and decreased myotube nuclear numbers. Moreover, while cyclosporineA treatment suppressed the proliferation of parental cultures it had no effect on cloneC1 cells. SERCA1b is thus considered to play an essential role in the regulation of [Ca2+]i and its ab ovo gene silencing results in decreased skeletal muscle differentiation.

The endogenous lipid agent N-arachidonoylethanolamine (anandamide), among other effects, has been shown to be involved in nociceptive processing both in the central and peripheral nervous systems. Anandamide is thought to be synthesised by several enzymatic pathways both in a Ca(2+)-sensitive and Ca(2+)-insensitive manner, and rat primary sensory neurons produce anandamide. Here, we show for the first time, that cultured rat primary sensory neurons express at least four of the five known Ca(2+)-insensitive enzymes implicated in the synthesis of anandamide, and that application of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-arachidonoyl, the common substrate of the anandamide-synthesising pathways, results in anandamide production which is not changed by the removal of extracellular Ca(2+). We also show that anandamide, which has been synthesised in primary sensory neurons following the application of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-arachidonoyl induces a transient receptor potential vanilloid type 1 ion channel-mediated excitatory effect that is not inhibited by concomitant activation of the cannabinoid type 1 receptor. Finally, we show that sub-populations of transient receptor potential vanilloid type 1 ion channel-expressing primary sensory neurons also express some of the putative Ca(2+)-insensitive anandamide-synthesising enzymes. Together, these findings indicate that anandamide synthesised by primary sensory neuron via a Ca(2+)-insensitive manner has an excitatory rather than an inhibitory role in primary sensory neurons and that excitation is mediated predominantly through autocrine signalling. Regulation of the activity of the Ca(2+)-insensitive anandamide-synthesising enzymes in these neurons may be capable of regulating the activity of these cells, with potential relevance to controlling nociceptive processing.

Iron is an essential element for all microorganisms. Bacteria and fungi produce versatile siderophores for binding and storing this essential transition metal when its availability is limited in the environment. The aim of the study was to optimize the fermentation medium ofAspergillus fumigatusfor siderophore production. Triacetyl-fusarinine C and ferricrocin yields were dependent on glucose and glycine supplementations as well as the initial pH of the culture media. The optimal fermentation medium for triacetylfusarinine C production contained 8% glucose, 0.4% glycine and the initial pH was set to 5.9. Meanwhile, maximal ferricrocin yields were recorded in the presence of 10% glucose, 0.5% glycine and at an initial pH of 7.4. Under optimized fermentation conditions, the yields for triacetylfusarinine C and ferricrocin increased up to 2.9 g/l culture medium and 18.9 mg/g mycelium, respectively.

Skeletal muscle excitation–contraction (E–C) coupling is altered in several models of phosphatidylinositol phosphate (PtdInsP) phosphatase deficiency and ryanodine receptor activity measured in vitro was reported to be affected by certain PtdInsPs, thus prompting investigation of the physiological role of PtdInsPs in E–C coupling. We measured intracellular Ca2+ transients in voltage-clamped mouse muscle fibres microinjected with a solution containing a PtdInsP substrate (PtdIns(3,5)P 2 or PtdIns(3)P) or product (PtdIns(5)P or PtdIns) of the myotubularin phosphatase MTM1. No significant change was observed in the presence of either PtdIns(5)P or PtdIns but peak SR Ca2+ release was depressed by ~30% and 50% in fibres injected with PtdIns(3,5)P 2 and PtdIns(3)P, respectively, with no concurrent alteration in the membrane current signals associated with the DHPR function as well as in the voltage dependence of Ca2+ release inactivation. In permeabilized muscle fibres, the frequency of spontaneous Ca2+ release events was depressed in the presence of the three tested phosphorylated forms of PtdInsP with PtdIns(3,5)P 2 being the most effective, leading to an almost complete disappearance of Ca2+ release events. Results support the possibility that pathological accumulation of MTM1 substrates may acutely depress ryanodine receptor-mediated Ca2+ release. Overexpression of a mCherry-tagged form of MTM1 in muscle fibres revealed a striated pattern consistent with the triadic area. Ca2+ release remained although unaffected by MTM1 overexpression and was also unaffected by the PtdIns-3-kinase inhibitor LY2940002, suggesting that the 3-phosphorylated PtdIns lipids active on voltage-activated Ca2+ release are inherently maintained at a low level, inefficient on Ca2+ release in normal conditions. This work was supported by grants from Centre National de la Recherche Scientifique (CNRS), Université Lyon 1, Association Française contre les Myopathies (AFM), Hubert Curien Partnership Balaton (TeT_10-1-2011-0723) and OTKA K107765. E.G.R. was a recipient of a fellowship from the Spanish Ministry of Education and Science (MEC, José Castillejo Program).

Ca2+ sparks represent synchronous opening of the ryanodine receptor (RyR) Ca2+ release channels located at the sarcoplasmic reticulum (SR) membrane. Whereas a quantal nature of Ca2+ sparks has been defined in cardiac muscle, the regulation of Ca2+ sparks in skeletal muscle has not been well-studied. Osmotic-stress applied to an intact skeletal muscle fiber can produce brief Ca2+ sparks and prolonged Ca2+ burst events. Here, we show that termination of Ca2+ bursts occurs in a step wise and quantal manner. Ca2+ burst events display kinetic features that are consistent with the involvement of both stochastic attrition and coordinated closure of RyR channels in the termination of SR Ca2+ release. Elemental unitary transition steps could be defined with a mean deltaF/F0 of approximately 0.28. corresponding to the gating of 1-2 RyR channels. Moreover, the amplitude of the elemental transition steps declines at the later stage of the burst event. In tandem Ca2+ burst events where two Ca2+ bursts occur at the same position within a fiber in rapid succession, the trailing event is consistently of lower amplitude than the initial event. These two complementary results suggest that SR Ca2+ release may be associated with local depletion of SR Ca2+ stores in mammalian skeletal muscle.

1 Enzymatically dissociated fibres from the extensor digitorum communis muscle of rats were mounted into a double Vaseline gap chamber. The rate of calcium release (Rrel) from the sarcoplasmic reticulum (SR) and changes in SR permeability to Ca2+ (PSR) were calculated from measured changes in intracellular calcium concentration. 2 Calcium release during a prepulse attenuated the inactivating component of PSR of the subsequent test pulse. The suppression was graded, larger release causing greater suppression, as expected from a calcium-dependent inactivation process. However, if the dissociation constant of the putative inhibitory calcium binding site (Kd) was estimated using different test pulses different affinities were obtained: a smaller test pulse yielded a smaller Kd. 3 Comparing the suppression of the inactivatable component of PSR during the test pulse (suppression) with the inactivatable component during the prepulse (pre-inactivation) revealed a linear relationship with a regression coefficient close to unity. 4 Lowering intracellular magnesium by decreasing its concentration to 25 μm in the internal solution altered the time course of PSR. The maximal peak-to-steady-level ratio was increased to 6·3 ± 0·4 (n= 10, mean ± s.e.m.) from a control value of 3·0 ± 0·2 (n= 19). Despite the apparent change in steady-state inactivation, suppression remained equal to that pre-inactivation. 5 Our results support the view that a depolarizing pulse always recruits the same set of calcium release channels and a portion of these channels undergoes a deterministic inactivation process.

Using isolated cells from a spontaneously immortalized human keratinocyte cell line (HaCaT) loaded with Fura-2 the regulation of intracellular Ca 2+ concentration ([Ca 2+ ] i ) was studied. The continuous presence of ATP induced a biphasic response in high external Ca 2+ . The first component reflected the release of calcium from intracellular stores since it was present after the removal of external calcium with ethylene-glycol-bis-N,N,N',N'-tetraacetic acid (EGTA). The second phase of [Ca 2+ ] i increase was not detectable in the absence of external calcium and raising the extracellular [Ca 2+ ] increased the rate of rise in [Ca 2+ ] i suggesting that it was influenced by the external environment. Furthermore, after adenosine triphosphate (ATP) had emptied the intracellular store in a calcium-free milieu, the elevation of external Ca 2+ induced a secondary increase in [Ca 2+ ] i that did not require the presence of ATP Depleting the intracellular calcium store with a Ca-ATP-ase inhibitor (cyclopiasonic acid, 10 μM) also induced calcium entry. The depletion induced calcium entry was inhibited by econazole (100 μM). These findings indicate the presence of a calcium release activated calcium influx pathway in HaCaT keratinocytes.

Concentration-dependent effects of bimoclomol, the novel heat shock protein coinducer, on intracellular calcium transients and contractility were studied in Langendorff-perfused guinea-pig hearts loaded with the fluorescent calcium indicator dye Fura-2. Bimoclomol had a biphasic effect on contractility: both peak left ventricular pressure and the rate of force development significantly increased at a concentration of 10 nM or higher. The maximal effect was observed between 0.1 and 1 μM, and the positive inotropic action disappeared by further increasing the concentration of bimoclomol. The drug increased systolic calcium concentration with a similar concentration-dependence. In contrast, diastolic calcium concentration increased monotonically in the presence of bimoclomol. Thus low concentrations of the drug (10–100 nM) increased, whereas high concentrations (10 μM) decreased the amplitude of intracellular calcium transients. Effects of bimoclomol on action potential configuration was studied in isolated canine ventricular myocytes. Action potential duration was increased at low (10 nM), unaffected at intermediate (0.1–1 μM) and decreased at high (10–100 μM) concentrations of the drug. In single canine sarcoplasmic calcium release channels (ryanodine receptor), incorporated into artificial lipid bilayer, bimoclomol significantly increased the open probability of the channel in the concentration range of 1–10 μM. The increased open probability was associated with increased mean open time. The effect of bimoclomol was again biphasic: the open probability decreased below the control level in the presence of 1 mM bimoclomol. Bimoclomol (10 μM–1 mM) had no significant effect on the rate of calcium uptake into sarcoplasmic reticulum vesicles of the dog, indicating that in vivo calcium reuptake might not substantially be affected by the drug. In conclusion, the positive inotropic action of bimoclomol is likely due to the activation of the sarcoplasmic reticulum calcium release channel in mammalian ventricular myocardium. Keywords: Heat shock protein, heart, ion currents, action potential duration, intracellular calcium transient, sarcoplasmic reticulum, ryanodine receptor, calcium release, calcium uptake Introduction Bimoclomol, the recently developed cytoprotective hydroxylamine derivative (N-2-hydroxy-3-(1-piperidinyl) propoxy]-3-pyridine-carboximidoyl chloride maleate, BRLP-42; see in Figure 1), was shown to have a wide variety of effects in healthy and diseased animals. Chronic treatment with the compound was effective against several types of diabetic dysfunction, including neuropathy (Biro et al., 1994), retinopathy (Hegedus et al., 1994) and abnormal vascular reactivity (Jednakovits et al., 1994). Bimoclomol exerted beneficial effects in rat and canine hearts exposed to coronary artery occlusion and vasospasm (Jaszlits et al., 1993), effects associated probably with enhanced expression of the heat shock protein HSP70 (Vigh et al., 1997). In addition, the drug showed strong dose-dependent acute antiarrhythmic action in isolated rat heart and also in anaesthetized animals. Recent electrophysiological studies indicate that bimoclomol may activate calciumdependent outward currents in mammalian ventricular cardiomyocytes (Magyar et al., 1999). Currently, the compound is in human phase 2 clinical trial. The present study was aimed to characterize the effects of the drug on calcium handling (i.e. on calcium release, calcium uptake and [Ca2+]i transients) in mammalian ventricular myocardium. Figure 1 Chemical structure of bimoclomol, applied as maleate salt. Methods Recording of contractility and [Ca2+]i transients in Langendorff-perfused guinea-pig heart Male guinea-pigs (weighing 300–500 g) were i.v. heparinized and anaesthetized with pentobarbitone-Na (150 mg kg−1). After opening the chest the heart was rapidly removed and fixed to the cannula of a Langendorff-perfusion device. The heart was perfused with modified Krebs solution containing (mM): NaCl 118, KCl 4.7, CaCl2 2.5, MgSO4 1.2, NaHCO3 25, Na2EDTA 0.5, KH2PO4 0.23 and glucose 5.5. The pH of this perfusate was set to 7.4 when gassed with a mixture of 5% CO2 and 95% O2 at 37°C. The coronary flow rate, controlled by a peristaltic pump, was adjusted to 10 ml min−1 g−1. Left ventricular pressure (LVP) was continuously monitored using a Braun 2021-02 arterial pressure transducer that was connected to the left ventricular cavity (Edes & Kranias, 1990). Heart rate was maintained 200 beats min−1 by left atrial pacing. Experiments were performed in a cumulative concentration-dependent manner. After taking control records, bimoclomol was applied at 1 nM, 10 nM, 100 nM, 1 μM, 3 μM and 10 μM concentration, each for 10 min. This protocol was performed in six guinea-pig hearts. To record [Ca2+]i transients, the heart was loaded with the acetoxymethylester of the fluorescent dye, Fura-2 (5 mM), added to the superfusate together with probenecid (0.6 mM), Synperonic (1.25 g l−1) and albumin (50 g l−1). Probenecid was used to inhibit the unspecific anion-exchanger of the cell membrane in order to avoid the extrusion of Fura-2 from the cells. Synperonic and albumin enhanced loading with Fura-2-AM. Under these conditions, stable calcium signals were obtained for periods as long as 120 min, allowing concentration-dependent studies (Kristof et al., 1998) to be performed. Fluorescence was excited at both 340 and 380 nm wavelengths. The emitted light was collected at 510 nm using a trifurcated quartz fibre optic bundle connected to a Deltascan device (Photon Technology International, New Brunswick, NJ, U.S.A.). [Ca2+]i was measured as a fluorescent ratio (F340/F380). The analogue signals of fluorescence and LVP were sampled at 1 kHz. In each case ten subsequent beats were averaged and stored for analysis. The following parameters were analysed: peak left ventricular pressure, maximum and minimum values of the first time derivative of pressure, systolic and diastolic [Ca2+]i (expressed as fluorescent ratios), and amplitude of the [Ca2+]i transient (defined as the difference between systolic and diastolic values of [Ca2+]i).

Following prolonged exercise, muscle force production is often impaired. One possible cause of this force deficit is impaired intracellular activation. We have used single skeletal muscle fibers from the lumbrical muscle of Xenopus laevis to study the effects of fatigue on excitation-contraction coupling. Fatigue was induced in 13 intact fibers. Five fibers recovered in normal Ringer only (fatigued-only fibers). The remaining eight fibers were subjected to a brief hypotonic treatment (F-H fibers) that is known to prolong the effects of fatigue. Intramembrane charge movement, changes in intracellular calcium concentration ([Ca2+]i) and force transients were measured in a single Vaseline gap chamber under voltage clamp. In F-H fibers, membrane capacitance was reduced. Confocal microscopy showed that this was not due to closure of the transverse tubules. The amount of normalized intramembrane charge was reduced from 21.0 +/- 2.8 nC/microF (n = 10) in rested fibers to 12.2 +/- 1.1 nC/microF in F-H fibers. However, the voltage dependence of intramembrane charge movement was unchanged. In F-H fibers, force production was virtually abolished. This was the consequence of the greatly reduced [Ca2+]i accompanying a depolarizing pulse. In recovering fatigued-only fibers, while the maximal available charge was not significantly smaller (18.3 +/- 1.1 nC/ microF), both calcium and force were reduced, albeit to a lesser extent than in F-H fibers. The data are consistent with a model where fatigue reduces the number of voltage sensors in the t-tubules and, in addition, alters the coupling between the remaining functional voltage sensors and the calcium channels of the sarcoplasmic reticulum.

The regulation by calcium of the ryanodine receptor/SR calcium release channel (RyR) from rat skeletal muscle was studied under isolated conditions and in situ. RyRs were either solubilized and incorporated into lipid bilayers or single fibres were mounted into a Vaseline gap voltage clamp. Single channel data were compared to parameters determined from the calculated calcium release flux. With K+ (250 mM) being the charge carrier the single channel conductance was 529 pS at 50 μM Ca2+ cis and trans, and decreased with increasing cis [Ca2+]. Open probability showed a bell shaped calcium dependence revealing an activatory and an inhibitory Ca2+ binding site (Hill coefficients of 1.18 and 1.28, respectively) with half activatory and inhibitory concentrations of 9.4 and 298 μM. The parameters of the inhibitory site agreed with the calcium dependence of channel inactivation deduced from the decline in SR calcium release in isolated fibres. Mean open time showed slight [Ca2+] dependence following a single exponential at every Ca2+ concentration tested. Closed time histograms, at high [Ca2+], were fitted with three exponentials, from which the longest was calcium independent, and resembled the recovery time constant of SR inactivation (115 ± 15 ms) obtained in isolated fibres. The data are in agreement with a model where calcium binding to the inhibitory site on RyR would be responsible for the calcium dependent inactivation in situ.

1. Enzymatically dissociated single muscle fibres of the rat were studied under voltage clamp conditions in a double Vaseline gap experimental chamber. Intramembrane charge movement and changes in intracellular calcium concentration ([Ca2+]i) were measured and the rate of calcium release (Rrel) from the sarcoplasmic reticulum (SR) was calculated. This enabled the determination of SR permeability and thus the estimation of the transfer function between intramembrane charge movement and SR permeability. 2. Perchlorate (3 mM) shifted the membrane potential dependence of intramembrane charge movement to more negative voltages without any effect on the steepness or on the maximal available charge. The drug increased SR permeability at every membrane potential but did not alter the peak-to-steady level ratio. It also increased the slope of the transfer function, indicating a more efficient coupling between the voltage sensors and the ryanodine receptors. 3. Caffeine (1 mM), on the other hand, increased SR permeability without altering the voltage dependence of intramembrane charge movement. It neither prolonged the depolarization-induced increase in [Ca2+]i at short pulse durations nor altered the time to peak of Rrel. The augmentation of SR permeability by the drug was more pronounced during the peak caffeine response than during its steady level. This was manifested in a leftward shift of the transfer function rather than an increase in its slope. 4. These observations indicate that perchlorate and caffeine alter the coupling between the voltage sensors and SR calcium release channels in mammalian skeletal muscle. They do not, however, share a common mechanism for enhancing the depolarization-induced release of calcium from the SR.

Myostatin, a member of the transforming growth factor β family, is a potent negative regulator of skeletal muscle growth, as myostatin-deficient mice show a great increase in muscle mass. Yet the physical performance of these animals is reduced. As an explanation for this, alterations in the steps in excitation-contraction coupling were hypothesized and tested for in mice with the 12 bp deletion in the propeptide region of the myostatin precursor (Mstn(Cmpt-dl1Abc) or Cmpt). In voluntary wheel running, control C57BL/6 mice performed better than the mutant animals in both maximal speed and total distance covered. Despite the previously described lower specific force of Cmpt animals, the pCa-force relationship, determined on chemically permeabilized fibre segments, did not show any significant difference between the two mouse strains. While resting intracellular Ca(2+) concentration ([Ca(2+)]i) measured on single intact flexor digitorum brevis (FDB) muscle fibres using Fura-2 AM was similar to control (72.0 ± 1.7 vs. 78.1 ± 2.9 nM, n = 38 and 45), the amplitude of KCl-evoked calcium transients was smaller (360 ± 49 vs. 222 ± 45 nM, n = 22) in the mutant strain. Similar results were obtained using tetanic stimulation and Rhod-2 AM, which gave calcium transients that were smaller (2.42 ± 0.11 vs. 2.06 ± 0.10 ΔF/F0, n = 14 and 13, respectively) on Cmpt mice. Sarcoplasmic reticulum (SR) calcium release flux calculated from these transients showed a reduced peak (23.7 ± 3.0 vs. 15.8 ± 2.1 mM s(-1)) and steady level (5.7 ± 0.7 vs. 3.7 ± 0.5 mM s(-1)) with no change in the peak-to-steady ratio. The amplitude and spatial spread of calcium release events detected on permeabilized FDB fibres were also significantly smaller in mutant mice. These results suggest that reduced SR calcium release underlies the reduced muscle force in Cmpt animals.

High-speed confocal microscopy is a method now commonly used to investigate subcellular calcium release events, but whose potential is rarely used fully without an effective standard analysis method.Here a detailed evaluation of a standalone analysis package for line-scan and X-Y images is presented. The algorithms used are based on a one-dimensional stationary wavelet transform. Computer generated images mimicking the properties of real recordings were used to assess the accuracy of the algorithms.On line-scan images the minimum usable detection amplitude for stereotypical calcium sparks at a sensitivity of ca. 80% was found to be 0.085. Sparks of amplitude (ΔF/F0) 0.15 and above are detected with a sensitivity of at least 98%. Due to the large number of points in a calcium spark, specificity exceeds 95% in all cases, enabling automatic analysis without manual correction. Furthermore, the temporal characteristics of sparks can be determined with a very high accuracy.On series of X-Y images the cell orientation and its sarcomeric structure is determined. Here, the minimal detection amplitude for generated sparks with 2 μm FWHM is 0.08 at a sensitivity level of 94% with a specificity level exceeding 98%. Using optimal detection parameters, sparks of 0.2 amplitude are found with a sensitivity of 100%. A separation distance of 3.6 μm is required between the centers of two sparks. The high temporal resolution makes it possible to study the spatiotemporal evolution of a calcium release event in 2D since events found on subsequent images at identical locations are automatically linked to each other.As the levels of detection parameters show, the assessed toolset, available upon request, is highly effective in the analysis of muscle calcium release events and may be useful for the analysis of other fluorescent calcium images.

Transient receptor potential (TRP) cation channels are emerging in vascular biology. In particular, the expression of the capsaicin receptor (TRPV1) was reported in vascular smooth muscle cells. This study characterized the arteriolar TRPV1 function and expression in the rat. TRPV1 mRNA was expressed in various vascular beds. Six commercially available antibodies were tested for TRPV1 specificity. Two of them were specific (immunostaining was abolished by blocking peptides) for neuronal TRPV1 and one recognized vascular TRPV1. TRPV1 was expressed in blood vessels in the skeletal muscle, mesenteric and skin tissues, as well as in the aorta and carotid arteries. TRPV1 expression was found to be regulated at the level of individual blood vessels, where some vessels expressed, while others did not express TRPV1 in the same tissue sections. Capsaicin (a TRPV1 agonist) evoked constrictions in skeletal muscle arteries and in the carotid artery, but had no effect on the femoral and mesenteric arteries or the aorta. In blood vessels, TRPV1 expression was detected in most of the large arteries, but there were striking differences at level of the small arteries. TRPV1 activity was suppressed in some isolated arteries. This tightly regulated expression and function suggests a physiological role for vascular TRPV1.

Presence of the pituitary adenylate cyclase-activating polypeptide (PACAP) signalling has been proved in various peripheral tissues. PACAP can activate protein kinase A (PKA) signalling via binding to pituitary adenylate cyclase-activating polypeptide type I receptor (PAC1), vasoactive intestinal polypeptide receptor (VPAC) 1 or VPAC2 receptor. Since little is known about the role of this regulatory mechanism in bone formation, we aimed to investigate the effect of PACAP on osteogenesis of UMR-106 cells. PACAP 1-38 as an agonist and PACAP 6-38 as an antagonist of PAC1 were added to the culture medium. Surprisingly, both substances enhanced protein expressions of collagen type I, osterix and alkaline phosphatase, along with higher cell proliferation rate and an augmented mineralisation. Although expression of PKA was elevated, no alterations were detected in the expression, phosphorylation and nuclear presence of CREB, but increased nuclear appearance of Runx2, the key transcription factor of osteoblast differentiation, was shown. Both PACAPs increased the expressions of bone morphogenetic proteins (BMPs) 2, 4, 6, 7 and Smad1 proteins, as well as that of Sonic hedgehog, PATCH1 and Gli1. Data of our experiments indicate that activation of PACAP pathway enhances bone formation of UMR-106 cells and PKA, BMP and Hedgehog signalling pathways became activated. We also found that PACAP 6-38 did not act as an antagonist of PACAP signalling in UMR-106 cells.

In our mouse model, a naturally occuring 12-bp deletion in the myostatin gene is considered responsible for the compact phenotype (Mstn(Cmpt-dl1Abc)) labeled by a tremendous increase in body weight along with signs of muscle weakness and easier fatigability.Western blot screenings showed significantly reduced endogenous STIM1 and Orai1 protein levels in the compact mouse muscle samples. As a consequence, we hypothesized that SOCE may be consequently altered. Enzymatically isolated fluo-8 AM loaded FDB fibers from wild type and Mstn(Cmpt-dl1Abc) mice were used. To elicit a massive SR Ca2+-release a RyR1 agonist (4-chloro-meta-cresol) was applied in a Ca2+ free medium and in the presence of the SR Ca2+ pump inhibitor (thapsigargin). The above cocktail triggered a deep transient store depletion which in turn switched on SOCE in a wild type cell. The peak of the Ca2+ transient (triggered by the entry of the external Ca2+ into the cytoplasm following the internal store depletion) was normalized to the peak of the SR Ca2+ release transient (1.06±0.11, n=17 vs 0.8±0.08, n=9). On isolated FDB fibers under voltage clamp, we found that the voltage dependence of the normalized fluorescence of the calcium transients in response to membrane depolarizations ranging between −60 and +30 mV, with a 10 mV increments were well fitted with a Boltzmann distribution (V0.5: −23.22±1.35 mV vs −24.15±0.77 mV with respective k values of 6.14±1.15 vs. 6.93±0.65). Fatigue inducing protocols are being currently tested. Collectively, these data point toward altered Ca2+ homeostasis in the compact mice. Understanding the mechanisms behind them may help developing new strategies in muscle atrophy, ageing and wasting diseases. To our knowledge, we are the first to employ Mstn(Cmpt-dl1Abc)mice and perform a comparative study to characterize their Ca2+ homeostasis in light of SOCE.

Evaluation of voice quality parameters of esophageal speech in different neoglottis forms after total laryngectomy.Presentation of voice analysis of 20 patients, who underwent total laryngectomy. The success of acquiring this technique was estimated by means of a voice analyzing program (pitch, sound-holding, loudness, spectrogram),and by the intelligibility via the telephone. Shape of the different types of neoglottis that developed and its functioning during vocalization and continuous speech were observed by nasal endoscopy. Data obtained from the voice analysis were compared among the observed three different neoglottis forms.The average dysphonia index of the 20 patients was 1.67 ± 0.38 (mean ± SD). Nasal fiberoscopic examination revealed three different types of neoglottis forms – a small mucosal button, two mucosal battens, and a mucosal lip. Voice quality of the esophageal speech of the patients with the mucosal button was found to be the closest to normal by subjective and objective acoustical evaluation.These findings emphasize the importance of the proper wound closure technique which can facilitate the development of a special button shape neoglottis form and help to acquire esophageal speech with the best quality parameters shortly after total laryngectomy.

Excitation-contraction coupling events leading to the onset of contraction were studied in single skeletal frog muscle fibers. This entailed the simultaneous measurement of the changes in intracellular calcium concentration using antipyrylazo III and fura-2, isometric force, and clamp voltage in a modified single vaseline gap chamber for the first time. The calcium transients were incorporated into an analysis of calcium binding to regulatory sites of troponin C (TnC) that permitted both a linear and a cooperative interaction. The analysis assumed that the onset of mechanical activation corresponds with a particular TnC saturation with calcium setting constraints for the calcium binding parameters of the regulatory sites. Using a simple model that successfully reproduced both the time course and the relative amplitudes of the measured isometric force transients over a wide membrane potential range, k(off) of TnC was calculated to be 78 s(-1) for the cooperative model at 10 degrees C. Together with the above constraints this gave a dissociation constant of 8.8 +/- 2.5 microM and a relative TnC saturation at the threshold (Sth) that would cause just detectable movement of 0.17 +/- 0.03 (n = 13; mean +/- SE). The predictions were found to be independent of the history of calcium binding to the regulatory sites. The observed delay between reaching Sth and the onset of fiber movement (8.7 +/- 1.0 ms; mean +/- SE, n = 37; from seven fibers) was independent of the membrane potential giving an upper estimate for the delay in myofilament activation. We thus emerge with quantitative values for the calcium binding to the regulatory sites on TnC under maintained structural conditions close to those in vivo.

TASK-3 (KCNK9 or K2P9.1) channels are thought to promote proliferation and/or survival of malignantly transformed cells, most likely by increasing their hypoxia tolerance. Based on our previous results that suggested mitochondrial expression of TASK-3 channels, we hypothesized that TASK-3 channels have roles in maintaining mitochondrial activity. In the present work we studied the effect of reduced TASK-3 expression on the mitochondrial function and survival of WM35 and A2058 melanoma cells. TASK-3 knockdown cells had depolarized mitochondrial membrane potential and contained a reduced amount of mitochondrial DNA. Compared to their scrambled shRNA-transfected counterparts, they demonstrated diminished responsiveness to the application of the mitochondrial uncoupler [(3-chlorophenyl)hydrazono]malononitrile (CCCP). These observations indicate impaired mitochondrial function. Further, TASK-3 knockdown cells presented reduced viability, decreased total DNA content, altered cell morphology, and reduced surface area. In contrast to non- and scrambled shRNA-transfected melanoma cell lines, which did not present noteworthy apoptotic activity, almost 50 % of the TASK-3 knockdown cells exhibited strong Annexin-V-specific immunofluorescence signal. Sequestration of cytochrome c from the mitochondria to the cytosol, increased caspase 3 activity, and translocation of the apoptosis-inducing factor from mitochondria to cell nuclei were also demonstrated in TASK-3 knockdown cells. Interference with TASK-3 channel expression, therefore, induces caspase-dependent and -independent apoptosis of melanoma cells, most likely via causing mitochondrial depolarization. Consequently, TASK-3 channels may be legitimate targets of future melanoma therapies.

The effects of low (10-100 microM) concentrations of tetracaine on intermembrane 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 (Q gamma) was suppressed to some extent, the voltage dependence and the parameters of the Boltzmann distribution were not modified significantly at tetracaine concentrations below 50 microM. At 50 and 100 microM 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 microM 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 the effects was markedly different. The inactivation component of calcium release was decreased with a Hill coefficient of approximately 1 and half effective concentration of 11.8 microM while the steady-level was decreased with a Hill coefficient of greater than 2 and a half effective concentration of 47.0 microM. These results favour two sites of action where tetracaine would suppress the calcium release from the SR.

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.

Background: Calcium activated Cl− current (ICl(Ca)) mediated by TMEM16A and/or Bestrophin-3 may contribute to cardiac arrhythmias. The true profile of ICl(Ca) during an actual ventricular action potential (AP), however, is poorly understood. Our goal was to study the current profile under normal calcium cycling and AP voltage-clamp condition as well as in case of altered intracellular calcium concentration ([Ca2+]i). The expression of TMEM16A and/or Bestrophin-3 in canine and human left ventricular myocytes was examined. The possible spatial distribution of these proteins and their co-localization with Cav1.2 was also studied.Methods: Whole-cell configuration of the patch-clamp technique and action potential voltage-clamp were used to monitor ICl(Ca), detected as 9-anthracene carboxylic acid (9-AC)-sensitive current. FURA-2-AM dye was used to measure [Ca2+]i. Expression and cellular localization of Cav1.2, Bestrophin-3 and TMEM16A was analyzed with immunocytochemistry and confocal microscopy.Results: Under AP voltage-clamp conditions the profile of ICl(Ca) contained an early fast outward (1.62±0.06 A/F) and a late inward component (−0.16±0.02 A/F), overlapping early and terminal repolarizations, respectively. Both components were reduced by ryanodine (1.05±0.03 A/F; −0.07±0.03 A/F), while fully abolished by BAPTA (0.13±0.10 A/F; −0.08±0.02 A/F), but not EGTA (1.17±0.09 A/F; −0.13±0.02 A/F). Setting [Ca2+]i to the systolic level (1.1 µM) decreased ICl(Ca), while application of Bay K8644, isoproterenol, and faster stimulation rates increased the amplitude of ICl(Ca). Both L-type Ca2+ current and ICl(Ca) were eliminated by nisoldipine. TMEM16A and Bestrophin-3 showed strong co-localization with one another and also with Cav1.2 channels both canine myocytes and human ventricular myocardium.Conclusions: Activation of ICl(Ca) in canine ventricular cells requires calcium entry through neighboring L-type Ca2+ channels and is only augmented by SR Ca2+-release. Substantial activation of ICl(Ca) requires high Ca2+ in the dyadic clefts which can be effectively buffered by BAPTA, but not EGTA.

In aging decreased physical activity and reduced muscle mass (sarcopenia) leads to impaired muscle force and increased fatigability accompanied by a decline in sarcoplasmic reticulum (SR) calcium release. As an essential trace element selenium plays a significant role in muscle functions, as in selenium deficiency skeletal muscle disorders manifesting in muscle pain, fatigue, proximal weakness, and serum creatine kinase elevation could develop. Here in vivo physical activity of control and myostatin deficient (Cmpt) mice during 22 months were examined. Their performance in grip and voluntary wheel tests reached its maximum 3 month after birth and declined after 10 month. This decrease was faster in Cmpt mice as the average daily running distance decreased to 37% in control and to 25% in Cmpt mice in old age. In vitro force was measured on soleus and extensor digitorum longus (EDL) muscles from 20-month-old control, Cmpt, and selenium supplemented mice. Albeit the absolute force was higher in Cmpt than in control mice, after normalization to cross section EDL was significantly stronger in the latter (4.70±0.88 vs 7.61±0.96 mN/mm2, respectively). Selenium supplementation significantly increased the maximal force of EDL (10.89±0.60 mN/mm2). Changes in intracellular calcium concentration were measured on enzymatically isolated intact flexor digitorum brevis muscle fibers using Fura-2. The rate of KCl depolarization-evoked SR calcium release was greater in selenium supplemented than in control animals (691±100 vs 481±33 µM/s, respectively). Western-blot analysis revealed no change in the expression of the dihydropyridine receptor in the three animal groups while that of the ryanodine receptor declined with aging which was reversed by long-term training. Our results support the positive effects of selenium and training on SR calcium release in old age associated muscle weakness. On the other hand, the increased muscle mass of Cmpt mice during their lifespan doesn’t improve their physical performance in old age. Supported by: NKFIH-K-115461

Chondrogenesis is known to be regulated by calcium-dependent signalling pathways in which temporal aspects of calcium homeostasis are of key importance. We aimed to better characterise calcium influx and release functions with respect to rapid calcium oscillations in cells of chondrifying chicken high density cultures. We found that differentiating chondrocytes express the α1 subunit of voltage-operated calcium channels (VOCCs) at both mRNA and protein levels, and that these ion channels play important roles in generating Ca(2+) influx for oscillations as nifedipine interfered with repetitive calcium transients. Furthermore, VOCC blockade abrogated chondrogenesis and almost completely blocked cell proliferation. The contribution of internal Ca(2+) stores via store-operated Ca(2+) entry (SOCE) seems to be indispensable to both Ca(2+) oscillations and chondrogenesis. Moreover, this is the first study to show the functional expression of STIM1/STIM2 and Orai1, molecules that orchestrate SOCE, in chondrogenic cells. Inhibition of SOCE combined with ER calcium store depletion abolished differentiation and severely diminished proliferation, suggesting the important role of internal pools in calcium homeostasis of differentiating chondrocytes. Finally, we present an integrated model for the regulation of calcium oscillations of differentiating chondrocytes that may have important implications for studies of chondrogenesis induced in various stem cell populations.

High-speed confocal microscopy is a relatively new method used to investigate localized calcium release events, most notably calcium sparks. Line scan images can reliably be recorded at speeds up to 60000 lines/s while for X-Y images 120 frame/s rate is achievable. The increased amount of recorded data raises the necessity of efficient automatic analysis methods. A set of images recorded on frog skeletal muscle cells after caffeine treatment was used to test the described methods.We present a collection of methods useful for the automatic analysis of high speed line scan and X-Y recordings, mostly based on stationary wavelet transform (SWT). Our one-dimensional SWT based ember-detection method was adapted to detect sparks on high-speed line scan recordings. The centerline of detected sparks is automatically marked on each scan line to assess the spatial and temporal characteristics of the spark. 3.66% of all sparks (n=23704) in the test image set had complex spatial properties revealed by high-speed imaging. Amplitude and full width at half maximum (FWHM) is determined for each scan line to calculate the amount of calcium released (signal mass).On X-Y image series two-dimensional SWT was used to denoise the images and to detect sparks on each frame. The orientation of the skeletal muscle cell and the position of sarcomeres are determined using fast Fourier-transform on the stationary components of the image series. The position of the center for each spark is determined relatively to the sarcomeres. FWHM is calculated in the axes perpendicular (FWHMX) and parallel (FWHMY) to the sarcomeres. For the test dataset (n=22426), FWHMX/FWHMY is 1.22.The above-described methods enable us to automatically analyze high-speed confocal images and to reveal some of their properties which were hidden on conventional confocal images.

Myostatin, a member of the transforming growth factor β family was shown to be a potent negative regulator of skeletal muscle growth. It is strongly expressed in skeletal muscle and myostatin deficient mice have a great increase in muscle mass. Yet, the physical performance of these animals is not improved or even suppressed as compared to control mice. To understand the possible underlying mechanisms we investigated the calcium homeostasis of skeletal muscle fibers from mice with the compact myostatin mutation (MstnCmpt-dlAbc).Resting membrane potentials were essentially identical on control and MstnCmpt-dlAbc mice (−79.2±0.3 vs. −78.0±0.3 mV). Action potentials (AP), evoked by field stimulation through a pair of platinum electrodes on single intact flexor digitorum brevis (FDB) muscle fibers, on the other hand, were significantly shorter on MstnCmpt-dlAbc animals. The average time between the peak and 90% repolarization of the AP corresponded to 0.92±0.03 vs. 0.78±0.02 ms on control and on MstnCmpt-dlAbc fibers, respectively. The other parameters of the APs were essentially identical.The resting and the depolarization-evoked changes in intracellular Ca2+ concentration ([Ca2+]i) were measured on single FDB fibers with the fluorescent dye Fura-2. While resting [Ca2+]i was higher (57.6±1.4 vs. 65.4±2.8 nM), the amplitude of the KCl-evoked calcium transients was smaller (360±49 vs. 215±44 nM) in MstnCmpt-dlAbc mice. Similar experiments were conducted on Fluo-3 loaded fibers using a confocal microscope and repeated (at 100 Hz for 200 ms) field stimulation. AP-evoked calcium transients were again smaller (2.75±0.15 vs. 2.20±0.14) on MstnCmpt-dlAbc fibers. Elementary calcium release events were detected using Fluo-3 on permeabilized FDB fibers. No significant difference in the parameters of sparks was observed between control and MstnCmpt-dlAbc mice.These results indicate that an alteration in calcium signaling might underlie the reduced muscle performance in myostatin deficient mice.

The antifungal protein of Penicillium chrysogenum (PAF) inhibits the growth of important pathogenic filamentous fungi, including members of the Aspergillus family and some dermatophytes. Furthermore, PAF was proven to have no toxic effects on mammalian cells in vitro. To prove that PAF could be safely used in therapy, experiments were carried out to investigate its in vivo effects. Adult mice were inoculated with PAF intranasally in different concentrations, up to 2700 μg·kg− 1 daily, for 2 weeks. Even at the highest concentration – a concentration highly toxic in vitro for all affected molds – used, animals neither died due to the treatment nor were any side effects observed. Histological examinations did not find pathological reactions in the liver, in the kidney, and in the lungs. Mass spectrometry confirmed that a measurable amount of PAF was accumulated in the lungs after the treatment. Lung tissue extracts from PAF treated mice exerted significant antifungal activity. Small-animal positron emission tomography revealed that neither the application of physiological saline nor that of PAF induced any inflammation while the positive control lipopolysaccharide did. The effect of the drug on the skin was examined in an irritative dermatitis model where the change in the thickness of the ears following PAF application was found to be the same as in control and significantly less than when treated with phorbol-12-myristate-13-acetate used as positive control. Since no toxic effects of PAF were found in intranasal application, our result is the first step for introducing PAF as potential antifungal drug in therapy.

The expression pattern of cardiac ion channels displays marked changes during ontogeny. This study was designed to follow the developmental changes in the expression of major ventricular and atrial ion channel proteins (including both pore forming and regulatory subunits) in canine cardiac tissues at the mRNA level using competitive reverse transcription polymerase chain reaction. Therefore, the corresponding mRNA levels were compared in myocardial tissues excised from embryonic (25-60 days of gestation) and adult (2-3 years old) canine hearts. Expression level of Kv4.3, Kv1.4, KChIP2, KvLQT1, and Cav3.2 mRNAs were higher in the adult than in the embryonic hearts, while expression of Nav1.5 and minK mRNAs were higher in the embryonic than in the adult myocardium. No change in Kir2.1, HERG, Kv1.5, and Cav1.2 mRNA was observed during ontogeny. Direction of the developmental change in the mRNA level, determined for any specific channel protein, was identical in the atrial and ventricular samples. The age-dependent increase observed in the expression of Kv4.3, Kv1.4, KChIP2, and KvLQT1 is congruent with the greater repolarization reserve of the adult myocardium, associated with higher densities of Ito and IKs. The results indicate that age-dependent changes in the expression pattern of many ion channels are similar in canine and healthy human myocardium, therefore, canine cardiac muscle can be considered as a good model of studying developmental changes in the human heart.