RESUMEN
Dynamic systems such as cells or tissues generate, either spontaneously or in response to stimuli, transient signals that carry information about the system. Characterization of recorded transients is often hampered by a low signal-to-noise ratio (SNR). Reduction of the noise by filtering has limited use due to partial signal distortion. Occasionally, transients can be approximated by a mathematical function, but such a function may not hold correctly if recording conditions change. We introduce here the model-independent approximation method for general noisy transient signals based on the Gaussian process regression. The method was implemented in the software TransientAnalyzer, which detects transients in a record, finds their best approximation by the Gaussian process, constructs a surrogate spline function, and estimates specified signal parameters. The method and software were tested on a cellular model of the calcium concentration transient corrupted by various SNR levels and recorded at a low sampling frequency. Statistical analysis of the model data sets provided the error of estimation <7.5% and the coefficient of variation of estimates <17% for peak SNR = 5. The performance of Gaussian process regression on signals of diverse experimental origin was even better than fitting by a function. The software and its description are available on GitHub.
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Algoritmos , Programas Informáticos , Relación Señal-RuidoRESUMEN
[Figure: see text].
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Señalización del Calcio , Proteínas de la Membrana/metabolismo , Miocitos Cardíacos/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/genética , Taquicardia Ventricular/genética , Potenciales de Acción , Animales , Sitios de Unión , Células Cultivadas , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Mutación , Miocitos Cardíacos/fisiología , Miocitos Cardíacos/ultraestructura , Unión Proteica , Canal Liberador de Calcio Receptor de Rianodina/química , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Taquicardia Ventricular/metabolismo , Taquicardia Ventricular/patologíaRESUMEN
Developing cardiac myocytes undergo substantial structural and functional changes transforming the mechanism of excitation-contraction coupling from the embryonic form, based on calcium influx through sarcolemmal DHPR calcium channels, to the adult form, relying on local calcium release through RYR calcium channels of sarcoplasmic reticulum stimulated by calcium influx. We characterized day-by-day the postnatal development of the structure of sarcolemma, using techniques of confocal fluorescence microscopy, and the development of the calcium current, measured by the whole-cell patch-clamp in isolated rat ventricular myocytes. We characterized the appearance and expansion of the t-tubule system and compared it with the appearance and progress of the calcium current inactivation induced by the release of calcium ions from sarcoplasmic reticulum as structural and functional measures of direct DHPR-RYR interaction. The release-dependent inactivation of calcium current preceded the development of the t-tubular system by several days, indicating formation of the first DHPR-RYR couplons at the surface sarcolemma and their later spreading close to contractile myofibrils with the growing t-tubules. Large variability of both of the measured parameters among individual myocytes indicates uneven maturation of myocytes within the growing myocardium.
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Calcio/metabolismo , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Animales , Canales de Calcio/metabolismo , Proliferación Celular , Fenómenos Electrofisiológicos , Femenino , Masculino , RatasRESUMEN
Human ryanodine receptor 2 (hRyR2) mediates calcium release from the sarcoplasmic reticulum, enabling cardiomyocyte contraction. The N-terminal region of hRyR2 (amino acids 1-606) is the target of >30 arrhythmogenic mutations and contains a binding site for phosphoprotein phosphatase 1. Here, the solution and crystal structures determined under near-physiological conditions, as well as a homology model of the hRyR2 N-terminal region, are presented. The N-terminus is held together by a unique network of interactions among its three domains, A, B and C, in which the central helix (amino acids 410-437) plays a prominent stabilizing role. Importantly, the anion-binding site reported for the mouse RyR2 N-terminal region is notably absent from the human RyR2. The structure concurs with the differential stability of arrhythmogenic mutations in the central helix (R420W, I419F and I419F/R420W) which are owing to disparities in the propensity of mutated residues to form energetically favourable or unfavourable contacts. In solution, the N-terminus adopts a globular shape with a prominent tail that is likely to involve residues 545-606, which are unresolved in the crystal structure. Docking the N-terminal domains into cryo-electron microscopy maps of the closed and open RyR1 conformations reveals C(α) atom movements of up to 8â Å upon channel gating, and predicts the location of the leucine-isoleucine zipper segment and the interaction site for spinophilin and phosphoprotein phosphatase 1 on the RyR surface.
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Arritmias Cardíacas/genética , Mutación , Canal Liberador de Calcio Receptor de Rianodina/química , Secuencia de Aminoácidos , Animales , Arritmias Cardíacas/metabolismo , Sitios de Unión , Cloruros/metabolismo , Cristalografía por Rayos X , Humanos , Ratones , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Conformación Proteica , Estructura Terciaria de Proteína , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Alineación de SecuenciaRESUMEN
BACKGROUND: Phosphoinositide 3-kinase γ (PI3Kγ) signaling engaged by ß-adrenergic receptors is pivotal in the regulation of myocardial contractility and remodeling. However, the role of PI3Kγ in catecholamine-induced arrhythmia is currently unknown. METHODS AND RESULTS: Mice lacking PI3Kγ (PI3Kγ(-/-)) showed runs of premature ventricular contractions on adrenergic stimulation that could be rescued by a selective ß(2)-adrenergic receptor blocker and developed sustained ventricular tachycardia after transverse aortic constriction. Consistently, fluorescence resonance energy transfer probes revealed abnormal cAMP accumulation after ß(2)-adrenergic receptor activation in PI3Kγ(-/-) cardiomyocytes that depended on the loss of the scaffold but not of the catalytic activity of PI3Kγ. Downstream from ß-adrenergic receptors, PI3Kγ was found to participate in multiprotein complexes linking protein kinase A to the activation of phosphodiesterase (PDE) 3A, PDE4A, and PDE4B but not of PDE4D. These PI3Kγ-regulated PDEs lowered cAMP and limited protein kinase A-mediated phosphorylation of L-type calcium channel (Ca(v)1.2) and phospholamban. In PI3Kγ(-/-) cardiomyocytes, Ca(v)1.2 and phospholamban were hyperphosphorylated, leading to increased Ca(2+) spark occurrence and amplitude on adrenergic stimulation. Furthermore, PI3Kγ(-/-) cardiomyocytes showed spontaneous Ca(2+) release events and developed arrhythmic calcium transients. CONCLUSIONS: PI3Kγ coordinates the coincident signaling of the major cardiac PDE3 and PDE4 isoforms, thus orchestrating a feedback loop that prevents calcium-dependent ventricular arrhythmia.
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Catecolaminas/toxicidad , Fosfatidilinositol 3-Quinasa Clase Ib/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Taquicardia Ventricular/enzimología , Taquicardia Ventricular/prevención & control , Animales , Animales Recién Nacidos , Biorretroalimentación Psicológica/fisiología , Señalización del Calcio/genética , Fosfatidilinositol 3-Quinasa Clase Ib/deficiencia , Fosfatidilinositol 3-Quinasa Clase Ib/genética , Técnicas de Sustitución del Gen , Isoenzimas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Miocitos Cardíacos/enzimologíaRESUMEN
In mammalian cardiac myocytes, the elementary calcium releases triggered by step voltage stimuli manifest either as solitary or as twin spikes that vary widely in kinetics and amplitude for unknown reasons. Here we examined the variability of calcium spikes measured using line-scanning confocal microscopy in patch-clamped rat ventricular myocytes. Amplitude distributions of the single and of the first of twin spikes were broader than those of the second spikes. All could be best approximated by a sum of a few elementary Gaussian probability distribution functions. The latency distributions of the single and the first spikes were identical, much shorter and less variable than those of the second spikes. The multimodal distribution of spike amplitudes and the probability of occurrence of twin spikes were stochastically congruent with activation of only a few of the many RyR2 channels present in the release site cluster. The occurrence of twin release events was rare due to refractoriness of release, induced with a probability proportional to the number of RyR2s activated in the primary release event. We conclude that the variability of the elementary calcium release events supports a calcium signalling mechanism that arises from stochastics of RyR2 gating and from inactivation of local origin.
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Señalización del Calcio/fisiología , Calcio/fisiología , Canal Liberador de Calcio Receptor de Rianodina/fisiología , Animales , Técnicas In Vitro , Activación del Canal Iónico , Masculino , Miocitos Cardíacos/fisiología , Ratas , Ratas WistarRESUMEN
Ryanodine receptor channels at calcium release sites of cardiac myocytes operate on the principle of calcium-induced calcium release. In vitro experiments revealed competition of Ca2+ and Mg2+ in the activation of ryanodine receptors (RyRs) as well as inhibition of RyRs by Mg2+. The impact of RyR modulation by Mg2+ on calcium release is not well understood due to the technical limitations of in situ experiments. We turned instead to an in silico model of a calcium release site (CRS), based on a homotetrameric model of RyR gating with kinetic parameters determined from in vitro measurements. We inspected changes in the activity of the CRS model in response to a random opening of one of 20 realistically distributed RyRs, arising from Ca2+/Mg2+ interactions at RyR channels. Calcium release events (CREs) were simulated at a range of Mg2+-binding parameters at near-physiological Mg2+ and ATP concentrations. Facilitation of Mg2+ binding to the RyR activation site inhibited the formation of sparks and slowed down their activation. Impeding Mg-binding to the RyR activation site enhanced spark formation and speeded up their activation. Varying Mg2+ binding to the RyR inhibition site also dramatically affected calcium release events. Facilitation of Mg2+ binding to the RyR inhibition site reduced the amplitude, relative occurrence, and the time-to-end of sparks, and vice versa. The characteristics of CREs correlated dose-dependently with the effective coupling strength between RyRs, defined as a function of RyR vicinity, single-channel calcium current, and Mg-binding parameters of the RyR channels. These findings postulate the role of Mg2+ in calcium release as a negative modulator of the coupling strength among RyRs in a CRS, translating to damping of the positive feedback of the calcium-induced calcium-release mechanism.
RESUMEN
The dyads of cardiac myocytes contain ryanodine receptors (RYRs) that generate calcium sparks upon activation. To test how geometric factors of RYR distribution contribute to the formation of calcium sparks, which cannot be addressed experimentally, we performed in silico simulations on a large set of models of calcium release sites (CRSs). Our models covered the observed range of RYR number, density, and spatial arrangement. The calcium release function of CRSs was modeled by RYR openings, with an open probability dependent on concentrations of free Ca2+ and Mg2+ ions, in a rapidly buffered system, with a constant open RYR calcium current. We found that simulations of spontaneous sparks by repeatedly opening one of the RYRs in a CRS produced three different types of calcium release events (CREs) in any of the models. Transformation of simulated CREs into fluorescence signals yielded calcium sparks with characteristics close to the observed ones. CRE occurrence varied broadly with the spatial distribution of RYRs in the CRS but did not consistently correlate with RYR number, surface density, or calcium current. However, it correlated with RYR coupling strength, defined as the weighted product of RYR vicinity and calcium current, so that CRE characteristics of all models followed the same state-response function. This finding revealed the synergy between structure and function of CRSs in shaping dyad function. Lastly, rearrangements of RYRs simulating hypothetical experiments on splitting and compaction of a dyad revealed an increased propensity to generate spontaneous sparks and an overall increase in calcium release in smaller and more compact dyads, thus underlying the importance and physiological role of RYR arrangement in cardiac myocytes.
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Calcio , Miocitos Cardíacos , Calcio/metabolismo , Señalización del Calcio , Simulación por Computador , Miocitos Cardíacos/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismoRESUMEN
We report the domain analysis of the N-terminal region (residues 1-759) of the human cardiac ryanodine receptor (RyR2) that encompasses one of the discrete RyR2 mutation clusters associated with catecholaminergic polymorphic ventricular tachycardia (CPVT1) and arrhythmogenic right ventricular dysplasia (ARVD2). Our strategy utilizes a bioinformatics approach complemented by protein expression, solubility analysis and limited proteolytic digestion. Based on the bioinformatics analysis, we designed a series of specific RyR2 N-terminal fragments for cloning and overexpression in Escherichia coli. High yields of soluble proteins were achieved for fragments RyR2(1-606)xHis(6), RyR2(391-606)xHis(6), RyR2(409-606)xHis(6), Trx.RyR2(384-606)xHis(6), TrxxRyR2(391-606)xHis(6) and Trx.RyR2(409-606)xHis(6). The folding of RyR2(1-606)xHis(6) was analyzed by circular dichroism spectroscopy resulting in alpha-helix and beta-sheet content of approximately 23% and approximately 29%, respectively, at temperatures up to 35 degrees C, which is in agreement with sequence based secondary structure predictions. Tryptic digestion of the largest recombinant protein, RyR2(1-606)xHis(6), resulted in the appearance of two specific subfragments of approximately 40 and 25 kDa. The 25 kDa fragment exhibited greater stability. Hybridization with anti-His(6).Tag antibody indicated that RyR2(1-606)xHis(6) is cleaved from the N-terminus and amino acid sequencing of the proteolytic fragments revealed that digestion occurred after residues 259 and 384, respectively.
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Biología Computacional/métodos , Proteínas Recombinantes/biosíntesis , Canal Liberador de Calcio Receptor de Rianodina/química , Secuencia de Aminoácidos , Dicroismo Circular , Histidina/metabolismo , Humanos , Datos de Secuencia Molecular , Oligopéptidos/metabolismo , Fragmentos de Péptidos/química , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Análisis de Secuencia de Proteína , SolubilidadRESUMEN
The function of ion channels to mediate the flux of ions through membranes of living cells depends on their number, conductance, and open probability. The open probability, PO, characterizes gating of channels that is sensitive to experimental conditions and that can be determined in single-channel experiments. Individual experimental records and even whole series of single-channel activity measurements represent random samples of the stochastic gating continuous in time. The aim of this study was to understand the relationship between the accuracy (trueness and precision) of PO determination and the method of single-channel activity data collection. We used simulated single-channel experiments with variable settings of data collection for a range of open probability values. We found that at low PO, the trueness of PO determination depends on the average number of channel openings per record, while the precision of PO determination depends on the total number of channel openings in the whole dataset and on the distribution of open and closed times. We derived relationships that allow planning of single-channel experiments for the required accuracy of PO determination over a large span of open probabilities.
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Electrofisiología/métodos , Activación del Canal Iónico , Canales Iónicos/fisiología , Animales , Humanos , Cinética , Potenciales de la Membrana , Miocitos Cardíacos/citología , Técnicas de Placa-Clamp , Probabilidad , Reproducibilidad de los ResultadosRESUMEN
Cardiac excitation-contraction coupling relies on dyads, the intracellular calcium synapses of cardiac myocytes, where the plasma membrane contacts sarcoplasmic reticulum and where electrical excitation triggers calcium release. The morphology of dyads and dynamics of local calcium release vary substantially. To better understand the correspondence between the structure and the functionality of dyads, we estimated incidences of structurally different dyads and of kinetically different calcium release sites and tested their responsiveness to experimental myocardial injury in left ventricular myocytes of rats. According to the structure of dyads estimated in random electron microscopic images of myocardial tissue, the dyads were sorted into 'compact' or 'loose' types. The calcium release fluxes, triggered at local calcium release sites in patch-clamped ventricular myocytes and recorded by laser scanning confocal fluorescence microscopy, were decomposed into 'early' and 'late' components. ANOVA tests revealed very high correlation between the relative amplitudes of early and late calcium release flux components and the relative occurrences of compact and loose dyads in the control and in the injured myocardium. This finding ascertained the relationship between the structure of dyads and the functionality of calcium release sites and the responsiveness of calcium release sites to physical load in cardiac myocytes.
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Canales de Calcio/metabolismo , Señalización del Calcio , Calcio/metabolismo , Ventrículos Cardíacos/fisiopatología , Contracción Miocárdica , Miocitos Cardíacos/fisiología , Animales , Acoplamiento Excitación-Contracción , Masculino , Miocitos Cardíacos/citología , Ratas , Ratas Wistar , Retículo Sarcoplasmático/metabolismoRESUMEN
We established that Endosidin2 (ES2) affected the trafficking routes of both newly synthesized and endocytic pools of PIN-FORMED2 (PIN2) in Arabidopsis root epidermal cells. PIN2 populations accumulated in separated patches, which gradually merged into large and compact ES2 aggregates (ES2As). FM4-64 endocytic tracer labeled ES2As as well. Both PIN2 pools also appeared in vacuoles. Accelerated endocytosis of PIN2, its aggregation in the cytoplasm, and redirection of PIN2 flows to vacuoles led to a substantial reduction of the abundance of this protein in the plasma membrane. Whereas PIN-FORMED3 and PIN-FORMED4 also aggregated in the cytoplasm, SYT1 was not sensitive to ES2 treatment and did not appear either in the cytoplasmic aggregates or vacuoles. Ultrastructural analysis revealed that ES2 affects the Golgi apparatus so that stacks acquired cup-shape and even circular shape surrounded by several vesicles. Abnormally shaped Golgi stacks, stack remnants, multi-lamellar structures, separated Golgi cisterna rings, tubular structures, and vesicles formed discrete clusters.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Endocitosis/efectos de los fármacos , Limoninas/farmacología , Proteínas de Transporte de Membrana/metabolismo , Pared Celular/metabolismo , Citoplasma/metabolismo , Aparato de Golgi/metabolismo , Raíces de Plantas/citología , Raíces de Plantas/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Transporte de Proteínas/efectos de los fármacos , Sinaptotagmina I/metabolismoRESUMEN
Computer assisted image acquisition techniques, including confocal microscopy, require efficient tools for an automatic sorting of vast amount of generated image data. The complexity of the classification process, absence of adequate tools, and insufficient amount of reference data has made the automated processing of images challenging. Mastering of this issue would allow implementation of statistical analysis in research areas such as in research on formation of t-tubules in cardiac myocytes. We developed a system aimed at automatic assessment of cardiomyocyte development stages (SAACS). The system classifies confocal images of cardiomyocytes with fluorescent dye stained sarcolemma. We based SAACS on a densely connected convolutional network (DenseNet) topology. We created a set of labelled source images, proposed an appropriate data augmentation technique and designed a class probability graph. We showed that the DenseNet topology, in combination with the augmentation technique is suitable for the given task, and that high-resolution images are instrumental for image categorization. SAACS, in combination with the automatic high-throughput confocal imaging, will allow application of statistical analysis in the research of the tubular system development or remodelling and loss.
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Procesamiento de Imagen Asistido por Computador/métodos , Miocitos Cardíacos/citología , Animales , Inteligencia Artificial , Diferenciación Celular , Colorantes Fluorescentes , Aprendizaje Automático , Microscopía Confocal/métodos , Modelos Cardiovasculares , Miocitos Cardíacos/ultraestructura , Redes Neurales de la Computación , Ratas , Sarcolema/ultraestructuraRESUMEN
Wolframin (Wfs1) is a membrane protein of the sarco/endoplasmic reticulum. Wfs1 mutations are responsible for the Wolfram syndrome, characterized by diabetic and neurological symptoms. Although Wfs1 is expressed in cardiac muscle, its role in this tissue is not clear. We have characterized the effect of invalidation of Wfs1 on calcium signaling-related processes in isolated ventricular myocytes of exon5-Wfs1 deficient rats (Wfs1-e5/-e5) before the onset of overt disease. Calcium transients and contraction were measured in field-stimulated isolated myocytes using confocal microscopy with calcium indicator fluo-3 AM and sarcomere length detection. Calcium currents and their calcium release-dependent inactivation were characterized in whole-cell patch-clamp experiments. At 4 months, Wfs1-e5/-e5 animals were euglycemic, and echocardiographic examination revealed fully compensated cardiac function. In field-stimulated isolated ventricular myocytes, both the amplitude and the duration of contraction of Wfs1-e5/-e5 animals were elevated relative to control Wfs1+/+ littermates. Increased contractility of myocytes resulted largely from prolonged cytosolic calcium transients. Neither the amplitude of calcium currents nor their voltage dependence of activation differed between the two groups. Calcium currents in Wfs1-e5/-e5 myocytes showed a larger extent of inactivation by short voltage prepulses applied to selectively induce calcium release-dependent inactivation of calcium current. Neither the calcium content of the sarcoplasmic reticulum, measured by application of 20 mmol/l caffeine, nor the expression of SERCA2, determined from Western blots, differed significantly in myocytes of Wfs1-e5/-e5 animals compared to control ones. These experiments point to increased duration of calcium release in ventricular myocytes of Wfs1-e5/-e5 animals. We speculate that the lack of functional wolframin might cause changes leading to upregulation of RyR2 channels resulting in prolongation of channel openings and/or a delay in termination of calcium release.
RESUMEN
The plasminogen system is harnessed in a wide variety of physiological processes, such as fibrinolysis, cell migration, or efferocytosis; and accordingly, it is essential upon inflammation, tissue remodeling, wound healing, and for homeostatic maintenance in general. Previously, we identified a plasminogen receptor in the mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R, CD222). Here, we demonstrate by means of genetic knockdown, knockout, and rescue approaches combined with functional studies that M6P/IGF2R is up-regulated on the surface of macrophages, recognizes plasminogen exposed on the surface of apoptotic cells, and mediates plasminogen-induced efferocytosis. The level of uptake of plasminogen-coated apoptotic cells inversely correlates with the TNF-α production by phagocytes indicating tissue clearance without inflammation by this mechanism. Our results reveal an up-to-now undetermined function of M6P/IGF2R in clearance of apoptotic cells, which is crucial for tissue homeostasis.
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Fagocitosis/efectos de los fármacos , Plasminógeno/farmacología , Receptor IGF Tipo 2/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Técnicas de Inactivación de Genes , Humanos , Células Jurkat , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Células THP-1 , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The principal role of calcium current in the triggering of calcium release in cardiac myocytes is well recognized. The mechanism of how calcium current (I(Ca)) controls the intensity of calcium release is not clear because of the stochastic nature of voltage-dependent gating of calcium channels (DHPRs) and of calcium-dependent gating of ryanodine receptors (RyRs). To disclose the relation between DHPR openings and the probability of calcium release, local calcium release activation by I(Ca) was investigated in rat ventricular myocytes using patch-clamp and confocal microscopy. Calcium spikes were activated by temporally synchronized DHPR calcium current triggers, generated by instantaneous 'tail' I(Ca) and modulated by prepulse duration, by tail potential, and by the DHPR agonist BayK 8644. The DHPR-RyR coupling fidelity was determined from the temporal distribution of calcium spike latencies using a model based on exponentially distributed DHPR open times. The analysis provided a DHPR mean open time of approximately 0.5 ms, RyR activation time constant of approximately 0.6 ms, and RyR activation kinetics of the 4th order. The coupling fidelity was low due to the inherent prevalence of very short DHPR openings but was increased when DHPR openings were prolonged by BayK 8644. The probability of calcium release activation was high, despite low coupling fidelity, due to the activation of many DHPRs at individual release sites. We conclude that the control of calcium release intensity by physiological stimuli can be achieved by modulating the number and duration of DHPR openings at low coupling fidelity, thus avoiding the danger of inadvertently triggering calcium release events.
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Potenciales de Acción/fisiología , Canales de Calcio Tipo L/fisiología , Señalización del Calcio/fisiología , Calcio/metabolismo , Activación del Canal Iónico/fisiología , Potenciales de la Membrana/fisiología , Miocitos Cardíacos/fisiología , Animales , Células Cultivadas , Masculino , Ratas , Ratas WistarRESUMEN
We have constructed a three-dimensional reaction-diffusion model of the mammalian cardiac calcium release unit. We analyzed effects of diffusion coefficients, single channel current amplitude, density of RyR channels, and reaction kinetics of ATP(2-) with Ca(2+) and Mg(2+) ions on spatiotemporal concentration profiles of Ca(2+), Mg(2+), and ATP(2-) in the dyadic cleft during Ca(2+) release. The model revealed that Ca(2+) concentration gradients persist near RyRs in the steady state. Even with low number of open RyRs, peak [Ca(2+)] in the dyadic space reached values similar to estimates of luminal [Ca(2+)] in approximately 1 ms, suggesting that during calcium release the Ca(2+) gradient moves from the cisternal membrane towards the boundary of the dyadic space with the cytosol. The released Ca(2+) bound to ATP(2-), and thus substantially decreased ATP(2-) concentration in the dyadic space. The released Ca(2+) could also replace Mg(2+) in its complex with ATP(2-) during first milliseconds of release if dissociation of MgATP was fast. The results suggest that concentration changes of Ca(2+), Mg(2+), and ATP(2-) might be large and fast enough to reduce dyadic RyR activity. Thus, under physiological conditions, termination of calcium release may be facilitated by the synergic effect of the construction and chemistry of mammalian cardiac dyads.
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Adenosina Trifosfato/metabolismo , Señalización del Calcio , Magnesio/metabolismo , Modelos Cardiovasculares , Miocitos Cardíacos/metabolismo , Retículo Sarcoplasmático/metabolismo , Animales , Simulación por Computador , Citosol/metabolismo , Difusión , Humanos , Cinética , Proyectos de Investigación , Canal Liberador de Calcio Receptor de Rianodina/metabolismoRESUMEN
Antidepressants inhibit many membrane receptors and ionic channels, including the L-type calcium channel. Here, we investigated the inhibition of calcium current (I(Ca)) by antidepressants in enzymatically isolated rat ventricular myocytes using whole-cell patch clamp. The molecular mechanism of inhibition was studied by comparing the voltage and state dependence of antidepressant inhibition of I(Ca) to the respective properties of calcium antagonists, and by studying the effect of (+/-)-1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-[trifluoromethyl]phenyl)-3-pyridine carboxylic acid methyl ester (Bay K8644) or diltiazem on the inhibitory potency of the antidepressants. All selected antidepressants inhibited calcium currents reversibly and concentration-dependently. At a stimulation frequency of 0.33 Hz, the antidepressants imipramine, clomipramine, desipramine, amitriptyline, maprotiline, citalopram, and dibenzepin blocked I(Ca), with IC(50) values of 8.3, 11.6, 11.7, 23.2, 31.0, 64.5, and 364 muM. The antidepressant drugs shifted steady-state inactivation curves of I(Ca) to negative voltages. The extent of the shift was similar to that induced by diltiazem or verapamil, but it was significantly smaller than that induced by felodipine. The use-dependent component of the antidepressant-induced block was similar to that of diltiazem, and it was significantly more and less, respectively, than those of felodipine and verapamil. In the presence of Bay K8644, antidepressants were more effective in inhibiting I(Ca). However, the inhibitory effect of antidepressants was also augmented by diltiazem, suggesting that these drugs do not compete with diltiazem for a single binding site. These data suggest that antidepressants exert their inhibitory action on cardiac L-type calcium channels by a specific interaction at a receptor site similar to, but distinct from, the benzothiazepine site.
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Antidepresivos/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo L/fisiología , Miocitos Cardíacos/fisiología , Animales , Relación Dosis-Respuesta a Droga , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Miocitos Cardíacos/efectos de los fármacos , Ratas , Ratas WistarRESUMEN
In underground hibernacula temperate northern hemisphere bats are exposed to Pseudogymnoascus destructans, the fungal agent of white-nose syndrome. While pathological and epidemiological data suggest that Palearctic bats tolerate this infection, we lack knowledge about bat health under pathogen pressure. Here we report blood profiles, along with body mass index (BMI), infection intensity and hibernation temperature, in greater mouse-eared bats (Myotis myotis). We sampled three European hibernacula that differ in geomorphology and microclimatic conditions. Skin lesion counts differed between contralateral wings of a bat, suggesting variable exposure to the fungus. Analysis of blood parameters suggests a threshold of ca. 300 skin lesions on both wings, combined with poor hibernation conditions, may distinguish healthy bats from those with homeostatic disruption. Physiological effects manifested as mild metabolic acidosis, decreased glucose and peripheral blood eosinophilia which were strongly locality-dependent. Hibernating bats displaying blood homeostasis disruption had 2 °C lower body surface temperatures. A shallow BMI loss slope with increasing pathogen load suggested a high degree of infection tolerance. European greater mouse-eared bats generally survive P. destructans invasion, despite some health deterioration at higher infection intensities (dependant on hibernation conditions). Conservation measures should minimise additional stressors to conserve constrained body reserves of bats during hibernation.
Asunto(s)
Ascomicetos/fisiología , Quirópteros/microbiología , Hibernación , Interacciones Huésped-Patógeno , Micosis/veterinaria , Enfermedades de la Piel/veterinaria , Animales , Índice de Masa Corporal , Quirópteros/sangre , Quirópteros/fisiología , Micosis/sangre , Micosis/microbiología , Micosis/patología , Enfermedades de la Piel/sangre , Enfermedades de la Piel/microbiología , Enfermedades de la Piel/patologíaRESUMEN
White-nose syndrome (WNS) is a fungal disease caused by Pseudogymnoascus destructans that is devastating to Nearctic bat populations but tolerated by Palearctic bats. Temperature is a factor known to be important for fungal growth and bat choice of hibernation. Here we investigated the effect of temperature on the pathogenic fungal growth in the wild across the Palearctic. We modelled body surface temperature of bats with respect to fungal infection intensity and disease severity and were able to relate this to the mean annual surface temperature at the site. Bats that hibernated at lower temperatures had less fungal growth and fewer skin lesions on their wings. Contrary to expectation derived from laboratory P. destructans culture experiments, natural infection intensity peaked between 5 and 6°C and decreased at warmer hibernating temperature. We made predictive maps based on bat species distributions, temperature and infection intensity and disease severity data to determine not only where P. destructans will be found but also where the infection will be invasive to bats across the Palearctic. Together these data highlight the mechanistic model of the interplay between environmental and biological factors, which determine progression in a wildlife disease.