RESUMO
Early embryonic development represents a sensitive time-window during which the foetus might be vulnerable to the exposure of environmental contaminants, potentially leading to heart diseases also later in life. Bisphenol A (BPA), a synthetic chemical widely used in plastics manufacturing, has been associated with heart developmental defects, even in low concentrations. This study aims to investigate the effects of environmentally relevant doses of BPA on developing cardiomyocytes using a human induced pluripotent stem cell (hiPSC)-derived model. Firstly, a 2D in vitro differentiation system to obtain cardiomyocytes from hiPSCs (hiPSC-CMs) have been established and characterised to provide a suitable model for the early stages of cardiac development. Then, the effects of a repeated BPA exposure, starting from the undifferentiated stage throughout the differentiation process, were evaluated. The chemical significantly decreased the beat rate of hiPSC-CMs, extending the contraction and relaxation time in a dose-dependent manner. Quantitative proteomics analysis revealed a high abundance of basement membrane (BM) components (e.g., COL4A1, COL4A2, LAMC1, NID2) and a significant increase in TNNC1 and SERBP1 proteins in hiPSC-CMs treated with BPA. Network analysis of proteomics data supported altered extracellular matrix remodelling and provided a disease-gene association with well-known pathological conditions of the heart. Furthermore, upon hypoxia-reoxygenation challenge, hiPSC-CMs treated with BPA showed higher rate of apoptotic events. Taken together, our results revealed that a long-term treatment, even with low doses of BPA, interferes with hiPSC-CMs functionality and alters the surrounding cellular environment, providing new insights about diseases that might arise upon the toxin exposure. Our study contributes to the current understanding of BPA effects on developing human foetal cardiomyocytes, in correlation with human clinical observations and animal studies, and it provides a suitable model for New Approach Methodologies (NAMs) for environmental chemical hazard and risk assessment.
Assuntos
Células-Tronco Pluripotentes Induzidas , Miócitos Cardíacos , Animais , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Diferenciação CelularRESUMO
TRPV3 (transient receptor potential vanilloid 3) is a pro-inflammatory ion channel mostly expressed by keratinocytes of the human skin. Previous studies have shown that the expression of TRPV3 is markedly upregulated in the lesional epidermis of atopic dermatitis (AD) patients suggesting a potential pathogenetic role of the ion channel in the disease. In the current study, we aimed at defining the molecular and functional expression of TRPV3 in non-lesional skin of AD patients as previous studies implicated that healthy-appearing skin in AD is markedly distinct from normal skin with respect to terminal differentiation and certain immune function abnormalities. By using multiple, complementary immunolabelling and RT-qPCR technologies on full-thickness and epidermal shave biopsy samples from AD patients (lesional, non-lesional) and healthy volunteers, we provide the first evidence that the expression of TRPV3 is markedly upregulated in non-lesional human AD epidermis, similar to lesional AD samples. Of further importance, by using the patch-clamp method on cultured healthy and non-lesional AD keratinocytes, we also show that this upregulation is functional as determined by the significantly augmented TRPV3-specific ion current (induced by agonists) on cultured non-lesional AD keratinocytes when compared to healthy ones.
Assuntos
Dermatite Atópica , Dermatite Atópica/metabolismo , Epiderme/metabolismo , Humanos , Queratinócitos/metabolismo , Pele/metabolismo , Regulação para CimaRESUMO
Enhancement of the late Na+ current (INaL) increases arrhythmia propensity in the heart, while suppression of the current is antiarrhythmic. GS967 is an agent considered as a selective blocker of INaL. In the present study, effects of GS967 on INaL and action potential (AP) morphology were studied in canine ventricular myocytes by using conventional voltage clamp, action potential voltage clamp and sharp microelectrode techniques. The effects of GS967 (1 µM) were compared to those of the class I/B antiarrhythmic compound mexiletine (40 µM). Under conventional voltage clamp conditions, INaL was significantly suppressed by GS967 and mexiletine, causing 80.4 ± 2.2% and 59.1 ± 1.8% reduction of the densities of INaL measured at 50 ms of depolarization, and 79.0 ± 3.1% and 63.3 ± 2.7% reduction of the corresponding current integrals, respectively. Both drugs shifted the voltage dependence of the steady-state inactivation curve of INaL towards negative potentials. GS967 and mexiletine dissected inward INaL profiles under AP voltage clamp conditions having densities, measured at 50% of AP duration (APD), of -0.37 ± 0.07 and -0.28 ± 0.03 A/F, and current integrals of -56.7 ± 9.1 and -46.6 ± 5.5 mC/F, respectively. Drug effects on peak Na+ current (INaP) were assessed by recording the maximum velocity of AP upstroke (V+max) in multicellular preparations. The offset time constant was threefold faster for GS967 than mexiletine (110 ms versus 289 ms), while the onset of the rate-dependent block was slower in the case of GS967. Effects on beat-to-beat variability of APD was studied in isolated myocytes. Beat-to-beat variability was significantly decreased by both GS967 and mexiletine (reduction of 42.1 ± 6.5% and 24.6 ± 12.8%, respectively) while their shortening effect on APD was comparable. It is concluded that the electrophysiological effects of GS967 are similar to those of mexiletine, but with somewhat faster offset kinetics of V+max block. However, since GS967 depressed V+max and INaL at the same concentration, the current view that GS967 represents a new class of drugs that selectively block INaL has to be questioned and it is suggested that GS967 should be classified as a class I/B antiarrhythmic agent.
Assuntos
Potenciais de Ação/efeitos dos fármacos , Antiarrítmicos/farmacologia , Coração/efeitos dos fármacos , Mexiletina/farmacologia , Piridinas/farmacologia , Triazóis/farmacologia , Animais , Cães , Feminino , Frequência Cardíaca/efeitos dos fármacos , Masculino , Miocárdio , Miócitos Cardíacos/efeitos dos fármacosRESUMO
The cardiac late sodium current (INa,late) is the small sustained component of the sodium current active during the plateau phase of the action potential. Several studies demonstrated that augmentation of the current can lead to cardiac arrhythmias; therefore, INa,late is considered as a promising antiarrhythmic target. Fundamentally, enlarged INa,late increases Na+ influx into the cell, which, in turn, is converted to elevated intracellular Ca2+ concentration through the Na+/Ca2+ exchanger. The excessive Ca2+ load is known to be proarrhythmic. This review describes the behavior of the voltage-gated Na+ channels generating INa,late in health and disease and aims to discuss the physiology and pathophysiology of Na+ and Ca2+ homeostasis in context with the enhanced INa,late demonstrating also the currently accessible antiarrhythmic choices.
Assuntos
Sódio , Cálcio , Canal de Sódio Disparado por Voltagem NAV1.5 , Trocador de Sódio e CálcioRESUMO
Based on recent findings, an increased late sodium current (INa,late) plays an important pathophysiological role in cardiac diseases, including rhythm disorders. The article first describes what is INa,late and how it functions under physiological circumstances. Next, it shows the wide range of cellular mechanisms that can contribute to an increased INa,late in heart diseases, and also discusses how the upregulated INa,late can play a role in the generation of cardiac arrhythmias. The last part of the article is about INa,late inhibiting drugs as potential antiarrhythmic agents, based on experimental and preclinical data as well as in the light of clinical trials.
RESUMO
Calcium ions (Ca2+) play a major role in the cardiac excitation-contraction coupling. Intracellular Ca2+ concentration increases during systole and falls in diastole thereby determining cardiac contraction and relaxation. Normal cardiac function also requires perfect organization of the ion currents at the cellular level to drive action potentials and to maintain action potential propagation and electrical homogeneity at the tissue level. Any imbalance in Ca2+ homeostasis of a cardiac myocyte can lead to electrical disturbances. This review aims to discuss cardiac physiology and pathophysiology from the elementary membrane processes that can cause the electrical instability of the ventricular myocytes through intracellular Ca2+ handling maladies to inherited and acquired arrhythmias. Finally, the paper will discuss the current therapeutic approaches targeting cardiac arrhythmias.
RESUMO
Although late sodium current (INa-late) has long been known to contribute to plateau formation of mammalian cardiac action potentials, lately it was considered as possible target for antiarrhythmic drugs. However, many aspects of this current are still poorly understood. The present work was designed to study the true profile of INa-late in canine and guinea pig ventricular cells and compare them to INa-late recorded in undiseased human hearts. INa-late was defined as a tetrodotoxin-sensitive current, recorded under action potential voltage clamp conditions using either canonic- or self-action potentials as command signals. Under action potential voltage clamp conditions the amplitude of canine and human INa-late monotonically decreased during the plateau (decrescendo-profile), in contrast to guinea pig, where its amplitude increased during the plateau (crescendo profile). The decrescendo-profile of canine INa-late could not be converted to a crescendo-morphology by application of ramp-like command voltages or command action potentials recorded from guinea pig cells. Conventional voltage clamp experiments revealed that the crescendo INa-late profile in guinea pig was due to the slower decay of INa-late in this species. When action potentials were recorded from multicellular ventricular preparations with sharp microelectrode, action potentials were shortened by tetrodotoxin, which effect was the largest in human, while smaller in canine, and the smallest in guinea pig preparations. It is concluded that important interspecies differences exist in the behavior of INa-late. At present canine myocytes seem to represent the best model of human ventricular cells regarding the properties of INa-late. These results should be taken into account when pharmacological studies with INa-late are interpreted and extrapolated to human. Accordingly, canine ventricular tissues or myocytes are suggested for pharmacological studies with INa-late inhibitors or modifiers. Incorporation of present data to human action potential models may yield a better understanding of the role of INa-late in action potential morphology, arrhythmogenesis, and intracellular calcium dynamics.
Assuntos
Ventrículos do Coração/metabolismo , Ativação do Canal Iônico , Miocárdio/metabolismo , Canais de Sódio/metabolismo , Potenciais de Ação/efeitos dos fármacos , Animais , Venenos de Cnidários/toxicidade , Cães , Cobaias , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Masculino , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Tetrodotoxina/farmacologiaRESUMO
It has long been known that the electrophysiological effects of many cardioactive drugs strongly depend on the rate dependent frequency. This was recognized first for class I antiarrhythmic agents: their Vmax suppressive effect was attenuated at long cycle lengths. Later many Ca2+ channel blockers were also found to follow such kinetics. The explanation was provided by the modulated and the guarded receptor theories. Regarding the duration of cardiac action potentials (APD) an opposite frequency-dependence was observed, i.e. the drug-induced changes in APD were proportional with the cycle length of stimulation, therefore it was referred as "reverse rate-dependency". The beat-to-beat, or short term variability of APD (SV) has been recognized as an important proarrhythmic mechanism, its magnitude can be used as an arrhythmia predictor. SV is modulated by several cardioactive agents, however, these drugs modify also APD itself. In order to clear the drug-specific effects on SV from the concomitant unspecific APD-change related ones, the term of "relative variability" was introduced. Relative variability is increased by ion channel blockers that decrease the negative feedback control of APD (i.e. blockers of ICa, IKr and IKs) and also by elevation of cytosolic Ca2+. Cardiac arrhythmias are also often categorized according to the characteristic heart rate (tachy- and bradyarrhythmias). Tachycardia is proarrhythmic primarily due to the concomitant Ca2+ overload causing delayed afterdepolarizations. Early afterdepolarizations (EADs) are complications of the bradycardic heart. What is common in the reverse rate-dependent nature of drug action on APD, increased SV and EAD incidence associated with bradycardia.
Assuntos
Potenciais de Ação/fisiologia , Antiarrítmicos/farmacologia , Arritmias Cardíacas/tratamento farmacológico , Frequência Cardíaca/efeitos dos fármacos , Ventrículos do Coração/efeitos dos fármacos , Bloqueadores dos Canais de Potássio/farmacologia , Animais , Bradicardia/fisiopatologia , Cães , Avaliação Pré-Clínica de Medicamentos , Eletrofisiologia , Cobaias , Humanos , Íons , Cinética , Masculino , Camundongos , Miócitos Cardíacos/fisiologia , Preparações Farmacêuticas , Coelhos , Taquicardia/fisiopatologiaRESUMO
The role of transient receptor potential melastatin 4 (TRPM4) channels has been frequently tested using their inhibitor 9-phenanthrol in various cardiac preparations; however, the selectivity of the compound is uncertain. Therefore, in the present study, the concentration-dependent effects of 9-phenanthrol on major ionic currents were studied in canine isolated ventricular cells using whole-cell configuration of the patch-clamp technique and 10 mM BAPTA-containing pipette solution to prevent the Ca2+-dependent activation of TRPM4 channels. Transient outward (Ito1), rapid delayed rectifier (IKr), and inward rectifier (IK1) K+ currents were suppressed by 10 and 30 µM 9-phenanthrol with the blocking potency for IK1 < IKr < Ito1 and partial reversibility. L-type Ca2+ current was not affected up to the concentration of 30 µM. In addition, a steady outward current was detected at voltages positive to -40 mV in 9-phenanthrol, which was larger at more positive voltages and larger 9-phenanthrol concentrations. Action potentials were recorded using microelectrodes. Maximal rate of depolarization, phase-1 repolarization, and terminal repolarization were decreased and the plateau potential was depressed by 9-phenanthrol (3-30 µM), congruently with the observed alterations of ionic currents. Significant action potential prolongation was observed by 9-phenanthrol in the majority of the studied cells, but only at 30 µM concentration. In conclusion, 9-phenanthrol is not selective to TRPM4 channels in canine ventricular myocardium; therefore, its application as a TRPM4 blocker can be appropriate only in expression systems but not in native cardiac cells.
Assuntos
Ventrículos do Coração/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Fenantrenos/farmacologia , Potássio/metabolismo , Canais de Cátion TRPM/antagonistas & inibidores , Potenciais de Ação/efeitos dos fármacos , Animais , Cálcio/metabolismo , Cães , Feminino , Masculino , Miócitos Cardíacos/citologiaRESUMO
Transient receptor potential (TRP) ion channels were first characterized on neurons, where they are classically implicated in sensory functions; however, research in recent decades has shown that many of these channels are also expressed on nonneuronal cell types. Emerging findings have highlighted the role of TRP channels in the skin, where they have been shown to be important in numerous cutaneous functions. Of particular interest is TRPV3, which was first described on keratinocytes. Its functional importance was supported when its gain-of-function mutation was linked to Olmsted syndrome, which is characterized by palmoplantar keratoderma, periorifacial hyperkeratosis, diffuse hypotrichosis and alopecia, and itch. Despite these exciting results, we have no information about the role and functionality of TRPV3 on keratinocytes at the cellular level. In this study, we identified TRPV3 expression both on human skin and cultured epidermal keratinocytes. TRPV3 stimulation was found to function as a Ca2+-permeable ion channel that suppresses proliferation of epidermal keratinocytes and induces cell death. Stimulation of the channel also triggers a strong proinflammatory response via the NF-κB pathway. Collectively, our data show that TRPV3 is functionally expressed on human epidermal keratinocytes and that it plays a role in cutaneous inflammatory processes.
Assuntos
Apoptose/imunologia , Dermatite/imunologia , Epiderme/imunologia , Queratinócitos/imunologia , Canais de Cátion TRPV/imunologia , Cálcio/metabolismo , Cátions Bivalentes/metabolismo , Proliferação de Células , Dermatite/patologia , Epiderme/metabolismo , Células HEK293 , Voluntários Saudáveis , Humanos , Queratinócitos/metabolismo , Canais de Cátion TRPV/metabolismoRESUMO
The role of Ca2+-activated Cl- current (ICl(Ca)) in cardiac arrhythmias is still controversial. It can generate delayed afterdepolarizations in Ca2+-overloaded cells while in other studies incidence of early afterdepolarization (EAD) was reduced by ICl(Ca). Therefore our goal was to examine the role of ICl(Ca) in spatial and temporal heterogeneity of cardiac repolarization and EAD formation. Experiments were performed on isolated canine cardiomyocytes originating from various regions of the left ventricle; subepicardial, midmyocardial and subendocardial cells, as well as apical and basal cells of the midmyocardium. ICl(Ca) was blocked by 0.5mmol/L 9-anthracene carboxylic acid (9-AC). Action potential (AP) changes were tested with sharp microelectrode recording. Whole-cell 9-AC-sensitive current was measured with either square pulse voltage-clamp or AP voltage-clamp (APVC). Protein expression of TMEM16A and Bestrophin-3, ion channel proteins mediating ICl(Ca), was detected by Western blot. 9-AC reduced phase-1 repolarization in every tested cell. 9-AC also increased AP duration in a reverse rate-dependent manner in all cell types except for subepicardial cells. Neither ICl(Ca) density recorded with square pulses nor the normalized expressions of TMEM16A and Bestrophin-3 proteins differed significantly among the examined groups of cells. The early outward component of ICl(Ca) was significantly larger in subepicardial than in subendocardial cells in APVC setting. Applying a typical subepicardial AP as a command pulse resulted in a significantly larger early outward component in both subepicardial and subendocardial cells, compared to experiments when a typical subendocardial AP was applied. Inhibiting ICl(Ca) by 9-AC generated EADs at low stimulation rates and their incidence increased upon beta-adrenergic stimulation. 9-AC increased the short-term variability of repolarization also. We suggest a protective role for ICl(Ca) against risk of arrhythmias by reducing spatial and temporal heterogeneity of cardiac repolarization and EAD formation.
Assuntos
Potenciais de Ação/efeitos dos fármacos , Anoctamina-1/biossíntese , Antracenos/farmacologia , Arritmias Cardíacas/metabolismo , Bestrofinas/biossíntese , Miócitos Cardíacos/metabolismo , Animais , Arritmias Cardíacas/induzido quimicamente , Arritmias Cardíacas/patologia , Cães , Miócitos Cardíacos/patologiaRESUMO
Cardiac contractility is regulated by changes in intracellular Ca concentration ([Ca2+]i). Normal function requires that [Ca2+]i be sufficiently high in systole and low in diastole. Much of the Ca needed for contraction comes from the sarcoplasmic reticulum and is released by the process of calcium-induced calcium release. The factors that regulate and fine-tune the initiation and termination of release are reviewed. The precise control of intracellular Ca cycling depends on the relationships between the various channels and pumps that are involved. We consider 2 aspects: (1) structural coupling: the transporters are organized within the dyad, linking the transverse tubule and sarcoplasmic reticulum and ensuring close proximity of Ca entry to sites of release. (2) Functional coupling: where the fluxes across all membranes must be balanced such that, in the steady state, Ca influx equals Ca efflux on every beat. The remainder of the review considers specific aspects of Ca signaling, including the role of Ca buffers, mitochondria, Ca leak, and regulation of diastolic [Ca2+]i.
Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/fisiologia , Acoplamento Excitação-Contração/fisiologia , Mitocôndrias Cardíacas/fisiologia , Miócitos Cardíacos/fisiologia , Animais , Humanos , Líquido Intracelular/fisiologia , Retículo Sarcoplasmático/fisiologiaRESUMO
KEY POINTS: For the heart to function as a pump, intracellular calcium concentration ([Ca2+ ]i ) must increase during systole to activate contraction and then fall, during diastole, to allow the myofilaments to relax and the heart to refill with blood. The present study investigates the control of diastolic [Ca2+ ]i in rat ventricular myocytes. We show that diastolic [Ca2+ ]i is increased by manoeuvres that decrease sarcoplasmic reticulum function. This is accompanied by a decrease of systolic [Ca2+ ]i such that the time-averaged [Ca2+ ]i remains constant. We report that diastolic [Ca2+ ]i is controlled by the balance between Ca2+ entry and Ca2+ efflux during systole. The results of the present study identify a novel mechanism by which changes of the amplitude of the systolic Ca transient control diastolic [Ca2+ ]i . ABSTRACT: The intracellular Ca concentration ([Ca2+ ]i ) must be sufficently low in diastole so that the ventricle is relaxed and can refill with blood. Interference with this will impair relaxation. The factors responsible for regulation of diastolic [Ca2+ ]i , in particular the relative roles of the sarcoplasmic reticulum (SR) and surface membrane, are unclear. We investigated the effects on diastolic [Ca2+ ]i that result from the changes of Ca cycling known to occur in heart failure. Experiments were performed using Fluo-3 in voltage clamped rat ventricular myocytes. Increasing stimulation frequency increased diastolic [Ca2+ ]i . This increase of [Ca2+ ]i was larger when SR function was impaired either by making the ryanodine receptor leaky (with caffeine or ryanodine) or by decreasing sarco/endoplasmic reticulum Ca-ATPase activity with thapsigargin. The increase of diastolic [Ca2+ ]i produced by interfering with the SR was accompanied by a decrease of the amplitude of the systolic Ca transient, such that there was no change of time-averaged [Ca2+ ]i . Time-averaged [Ca2+ ]i was increased by ß-adrenergic stimulation with isoprenaline and increased in a saturating manner with increased stimulation frequency; average [Ca2+ ]i was a linear function of Ca entry per unit time. Diastolic and time-averaged [Ca2+ ]i were decreased by decreasing the L-type Ca current (with 50 µm cadmium chloride). We conclude that diastolic [Ca2+ ]i is controlled by the balance between Ca entry and efflux during systole. Furthermore, manoeuvres that decrease the amplitude of the Ca transient (without decreasing Ca influx) will therefore increase diastolic [Ca2+ ]i . This identifies a novel mechanism by which changes of the amplitude of the systolic Ca transient control diastolic [Ca2+ ]i .
Assuntos
Cálcio/fisiologia , Diástole/fisiologia , Miócitos Cardíacos/fisiologia , Sístole/fisiologia , Animais , Ventrículos do Coração/citologia , Masculino , Ratos Wistar , Rianodina/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina/fisiologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/antagonistas & inibidores , Tapsigargina/farmacologiaRESUMO
The Frank-Starling mechanism allows the amount of blood entering the heart from the veins to be precisely matched with the amount pumped out to the arterial circulation. As the heart fills with blood during diastole, the myocardium is stretched and oxidants are produced. Here we show that protein kinase G Iα (PKGIα) is oxidant-activated during stretch and this form of the kinase selectively phosphorylates cardiac phospholamban Ser16-a site important for diastolic relaxation. We find that hearts of Cys42Ser PKGIα knock-in (KI) mice, which are resistant to PKGIα oxidation, have diastolic dysfunction and a diminished ability to couple ventricular filling with cardiac output on a beat-to-beat basis. Intracellular calcium dynamics of ventricular myocytes isolated from KI hearts are altered in a manner consistent with impaired relaxation and contractile function. We conclude that oxidation of PKGIα during myocardial stretch is crucial for diastolic relaxation and fine-tunes the Frank-Starling response.
Assuntos
Proteína Quinase Dependente de GMP Cíclico Tipo I/genética , Diástole/fisiologia , Ventrículos do Coração/enzimologia , Miocárdio/enzimologia , Miócitos Cardíacos/enzimologia , Animais , Fenômenos Biomecânicos , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Débito Cardíaco/fisiologia , Proteína Quinase Dependente de GMP Cíclico Tipo I/metabolismo , Dissulfetos/química , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Técnicas de Introdução de Genes , Ventrículos do Coração/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Contração Miocárdica/fisiologia , Miocárdio/citologia , Miócitos Cardíacos/citologia , Técnicas de Cultura de Órgãos , Oxirredução , Estresse Oxidativo , Fosforilação , Cultura Primária de Células , Serina/metabolismo , Especificidade por SubstratoRESUMO
Ca(2+)-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. We aimed to study the profile of ICl(Ca) systematically under physiological conditions (normal Ca(2+) cycling and AP voltage-clamp) as well as in conditions designed to change [Ca(2+)]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. The profile of ICl(Ca), identified as a 9-anthracene carboxylic acid-sensitive current under AP voltage-clamp conditions, contained an early fast outward and a late inward component, overlapping early and terminal repolarizations, respectively. Both components were moderately reduced by ryanodine, while fully abolished by BAPTA, but not EGTA. [Ca(2+)]i was monitored using Fura-2-AM. Setting [Ca(2+)]i to the systolic level measured in the bulk cytoplasm (1.1µM) decreased ICl(Ca), while application of Bay K8644, isoproterenol, and faster stimulation rates increased the amplitude of ICl(Ca). Ca(2+)-entry through L-type Ca(2+) channels was essential for activation of ICl(Ca). TMEM16A and Bestrophin-3 showed strong co-localization with one another and also with Cav1.2 channels, when assessed using immunolabeling and confocal microscopy in both canine myocytes and human ventricular myocardium. Activation of ICl(Ca) in canine ventricular cells requires Ca(2+)-entry through neighboring L-type Ca(2+) channels and is only augmented by SR Ca(2+)-release. Substantial activation of ICl(Ca) requires high Ca(2+) concentration in the dyadic clefts which can be effectively buffered by BAPTA, but not EGTA.
Assuntos
Potenciais de Ação , Canais de Cálcio Tipo L/metabolismo , Canais de Cloreto/metabolismo , Ventrículos do Coração/metabolismo , Miócitos Cardíacos/metabolismo , Retículo Sarcoplasmático/metabolismo , Potenciais de Ação/efeitos dos fármacos , Animais , Biomarcadores , Bloqueadores dos Canais de Cálcio/farmacologia , Cães , Fenômenos Eletrofisiológicos , Humanos , Miócitos Cardíacos/efeitos dos fármacos , Técnicas de Patch-ClampRESUMO
Beat-to-beat variability of action potential duration (short-term variability, SV) is an intrinsic property of mammalian myocardium. Since the majority of agents and interventions affecting SV may modify also action potential duration (APD), we propose here the concept of relative SV (RSV), where changes in SV are normalized to changes in APD and these data are compared to the control SV-APD relationship obtained by lengthening or shortening of action potentials by inward and outward current injections. Based on this concept the influence of the several experimental conditions like stimulation frequency, temperature, pH, redox-state and osmolarity were examined on RSV in canine ventricular myocytes using sharp microelectrodes. RSV was increased by high stimulation frequency (cycle lengths <0.7 s), high temperature (above 37ºC), oxidative agents (H2O2), while it was decreased by reductive environment. RSV was not affected by changes in pH (within the range of 6.4-8.4) and osmolarity of the solution (between 250-350 mOsm). The results indicate that changes in beat-to-beat variability of APD must be evaluated exclusively in terms of RSV; furthermore, some experimental conditions, including the stimulation frequency, redox-state and temperature have to be controlled strictly when analyzing alterations in the short-term variability of APD.
Assuntos
Potenciais de Ação/fisiologia , Adaptação Fisiológica/fisiologia , Estimulação Elétrica/métodos , Frequência Cardíaca/fisiologia , Miócitos Cardíacos/química , Miócitos Cardíacos/fisiologia , Animais , Relógios Biológicos/fisiologia , Células Cultivadas , Cães , Concentração de Íons de Hidrogênio , Miócitos Cardíacos/citologia , TemperaturaRESUMO
PURPOSE: To compare the androgen receptor (AR) status of the appendix testis (AT) in congenital undescended and retractile testes. MATERIALS AND METHODS: Twenty-four appendix testes (AT) were removed from 21 boys to detect AR expression by immunohistochemistry and immunofluorescence staining. Group 1 (n = 3) includes ATs from three patients with unilateral and group 2 (n = 6) with bilateral congenital undescended testis. All patients with bilateral form had been previously treated with human chorionic gonadotropin (hCG) without an acceptable effect. Group 3 (n = 12) includes ATs collected from 12 boys with acquired undescended testis or retractile testicle. Group 4 (n = 3) includes ATs from three young adults who received hCG treatment for undescended testis in their childhood and underwent open testicular biopsy to investigate infertility. Further seven ATs were collected to detect AR mRNA using RT-PCR analysis. RESULTS: Both immunohistochemistry and immunofluorescence staining of ATs showed AR expression in 100 % of the cases in groups 3 and 4 (12/12 and 3/3), but there was no visible AR expression in groups 1 and 2 (0/3 and 0/6); however, RT-PCR analysis revealed mRNA expression of AR both in congenital undescended and in retractile testicles. CONCLUSIONS: The presence of AR in the epithelial cells of AT in patients with retractile testicle and its absence in patients with congenital undescended testis can be a possible cause of the effectiveness of hormonal treatment in retractile testis and its inefficacy in patients with congenital undescended testis.
Assuntos
Gonadotropina Coriônica/uso terapêutico , Criptorquidismo/metabolismo , RNA Mensageiro/genética , Receptores Androgênicos/genética , Biópsia , Pré-Escolar , Criptorquidismo/tratamento farmacológico , Criptorquidismo/genética , Humanos , Imuno-Histoquímica , Lactente , Masculino , Microscopia Confocal , Receptores Androgênicos/biossíntese , Substâncias para o Controle da Reprodução/uso terapêutico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Testículo/metabolismo , Testículo/patologiaRESUMO
Profound changes in tissue redox potential occur in the heart under conditions of oxidative stress frequently associated with cardiac arrhythmias. Since beat-to-beat variability (short term variability, SV) of action potential duration (APD) is a good indicator of arrhythmia incidence, the aim of this work was to study the influence of redox changes on SV in isolated canine ventricular cardiomyocytes using a conventional microelectrode technique. The redox potential was shifted toward a reduced state using a reductive cocktail (containing dithiothreitol, glutathione, and ascorbic acid) while oxidative changes were initiated by superfusion with H2O2. Redox effects were evaluated as changes in "relative SV" determined by comparing SV changes with the concomitant APD changes. Exposure of myocytes to the reductive cocktail decreased SV significantly without any detectable effect on APD. Application of H2O2 increased both SV and APD, but the enhancement of SV was the greater, so relative SV increased. Longer exposure to H2O2 resulted in the development of early afterdepolarizations accompanied by tremendously increased SV. Pretreatment with the reductive cocktail prevented both elevation in relative SV and the development of afterdepolarizations. The results suggest that the increased beat-to-beat variability during an oxidative stress contributes to the generation of cardiac arrhythmias.
Assuntos
Potenciais de Ação/fisiologia , Arritmias Cardíacas/etiologia , Frequência Cardíaca/fisiologia , Miocárdio/metabolismo , Miócitos Cardíacos/fisiologia , Estresse Oxidativo/fisiologia , Potenciais de Ação/efeitos dos fármacos , Animais , Antioxidantes/farmacologia , Arritmias Cardíacas/metabolismo , Células Cultivadas , Cães , Feminino , Frequência Cardíaca/efeitos dos fármacos , Masculino , Miócitos Cardíacos/efeitos dos fármacos , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Fatores de TempoRESUMO
This study was designed to investigate the influence of cytosolic Ca(2+) levels ([Ca(2+)]i) on action potential duration (APD) and on the incidence of early afterdepolarizations (EADs) in canine ventricular cardiomyocytes. Action potentials (AP) of isolated cells were recorded using conventional sharp microelectrodes, and the concomitant [Ca(2+)]i was monitored with the fluorescent dye Fura-2. EADs were evoked at a 0.2 Hz pacing rate by inhibiting the rapid delayed rectifier K(+) current with dofetilide, by activating the late sodium current with veratridine, or by activating the L-type calcium current with BAY K8644. These interventions progressively prolonged the AP and resulted in initiation of EADs. Reducing [Ca(2+)]i by application of the cell-permeant Ca(2+) chelator BAPTA-AM lengthened the AP at 1.0 Hz if it was applied alone, in the presence of veratridine, or in the presence of BAY K8644. However, BAPTA-AM shortened the AP if the cells were pretreated with dofetilide. The incidence of the evoked EADs was strongly reduced by BAPTA-AM in dofetilide, moderately reduced in veratridine, whereas EAD incidence was increased by BAPTA-AM in the presence of BAY K8644. Based on these experimental data, changes in [Ca(2+)]i have marked effects on APD as well as on the incidence of EADs; however, the underlying mechanisms may be different, depending on the mechanism of EAD generation. As a consequence, reduction of [Ca(2+)]i may eliminate EADs under some, but not all, experimental conditions.
Assuntos
Potenciais de Ação/fisiologia , Arritmias Cardíacas/metabolismo , Cálcio/metabolismo , Citosol/metabolismo , Ventrículos do Coração/metabolismo , Miócitos Cardíacos/metabolismo , Éster Metílico do Ácido 3-Piridinacarboxílico, 1,4-Di-Hidro-2,6-Dimetil-5-Nitro-4-(2-(Trifluormetil)fenil)/farmacologia , Potenciais de Ação/efeitos dos fármacos , Animais , Arritmias Cardíacas/fisiopatologia , Agonistas dos Canais de Cálcio/farmacologia , Quelantes de Cálcio/farmacologia , Células Cultivadas , Citosol/efeitos dos fármacos , Cães , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Feminino , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/fisiopatologia , Masculino , Miócitos Cardíacos/efeitos dos fármacos , Fenetilaminas/farmacologia , Sulfonamidas/farmacologia , Fatores de Tempo , Veratridina/farmacologiaRESUMO
Understanding the role of ionic currents in shaping the cardiac action potential (AP) has great importance as channel malfunctions can lead to sudden cardiac death by inducing arrhythmias. Therefore, researchers frequently use inhibitors to selectively block a certain ion channel like 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and 9-anthracene carboxylic acid (9-AC) for calcium-activated chloride current (ICl(Ca)). This study aims to explore which blocker is preferable to study ICl(Ca). Whole-cell voltage-clamp technique was used to record ICa,L, IKs, IKr and IK1, while action potentials were measured using sharp microelectrodes. DIDS- (0.2 mM) and 9-AC-sensitive (0.5 mM) currents were identical in voltage-clamp conditions, regardless of intracellular Ca(2+) buffering. DIDS-sensitive current amplitude was larger with the increase of stimulation rate and correlated well with the rate-induced increase of calcium transients. Both drugs increased action potential duration (APD) to the same extent, but the elevation of the plateau potential was more pronounced with 9-AC at fast stimulation rates. On the contrary, 9-AC did not influence either the AP amplitude or the maximal rate of depolarization (V max), but DIDS caused marked reduction of V max. Both inhibitors reduced the magnitude of phase-1, but, at slow stimulation rates, this effect of DIDS was larger. All of these actions on APs were reversible upon washout of the drugs. Increasing concentrations of 9-AC between 0.1 and 0.5 mM in a cumulative manner gradually reduced phase-1 and increased APD. 9-AC at 1 mM had no additional actions upon perfusion after 0.5 mM. The half-effective concentration of 9-AC was approximately 160 µM with a Hill coefficient of 2. The amplitudes of ICa,L, IKs, IKr and IK1 were not changed by 0.5 mM 9-AC. These results suggest that DIDS is equally useful to study ICl(Ca) during voltage-clamp but 9-AC is superior in AP measurements for studying the physiological role of ICl(Ca) due to the lack of sodium channel inhibition. 9-AC has also no action on other ion currents (ICa,L, IKr, IKs, IK1); however, ICa,L tracings can be contaminated with ICl(Ca) when measured in voltage-clamp condition.