RESUMO
The E-twenty-six variant 1 (ETV1)-dependent transcriptome plays an important role in atrial electrical and structural remodelling and the occurrence of atrial fibrillation (AF), but the underlying mechanism of ETV1 in AF is unclear. In this study, cardiomyocyte-specific ETV1 knockout (ETV1f/fMyHCCre/+, ETV1-CKO) mice were constructed to observe the susceptibility to AF and the underlying mechanism in AF associated with ETV1-CKO mice. AF susceptibility was examined by intraesophageal burst pacing, induction of AF was increased obviously in ETV1-CKO mice than WT mice. Electrophysiology experiments indicated shortened APD50 and APD90, increased incidence of DADs, decreased density of ICa,L in ETV1-CKO mice. There was no difference in VINACT,1/2 and VACT,1/2, but a significantly longer duration of the recovery time after inactivation in the ETV1-CKO mice. The recording of intracellular Ca2+ showed that there was significantly increased in the frequency of calcium spark, Ca2+ transient amplitude, and proportion of SCaEs in ETV1-CKO mice. Reduction of Cav1.2 rather than NCX1 and SERCA2a, increase RyR2, p-RyR2 and CaMKII was reflected in ETV1-CKO group. This study demonstrates that the increase in calcium spark and SCaEs corresponding to Ca2+ transient amplitude may trigger DAD in membrane potential in ETV1-CKO mice, thereby increasing the risk of AF.
Assuntos
Fibrilação Atrial , Cálcio , Átrios do Coração , Camundongos Knockout , Miócitos Cardíacos , Fatores de Transcrição , Animais , Miócitos Cardíacos/metabolismo , Camundongos , Fibrilação Atrial/metabolismo , Fibrilação Atrial/genética , Cálcio/metabolismo , Átrios do Coração/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Sinalização do Cálcio , Potenciais de Ação , Potenciais da Membrana , MasculinoRESUMO
Increased adenosine A2A receptor (A2AR) expression and activation underlies a higher incidence of spontaneous calcium release in atrial fibrillation (AF). Adenosine A3 receptors (A3R) could counteract excessive A2AR activation, but their functional role in the atrium remains elusive, and we therefore aimed to address the impact of A3Rs on intracellular calcium homeostasis. For this purpose, we analyzed right atrial samples or myocytes from 53 patients without AF, using quantitative PCR, patch-clamp technique, immunofluorescent labeling or confocal calcium imaging. A3R mRNA accounted for 9% and A2AR mRNA for 32%. At baseline, A3R inhibition increased the transient inward current (ITI) frequency from 0.28 to 0.81 events/min (p < 0.05). Simultaneous stimulation of A2ARs and A3Rs increased the calcium spark frequency seven-fold (p < 0.001) and the ITI frequency from 0.14 to 0.64 events/min (p < 0.05). Subsequent A3R inhibition caused a strong additional increase in the ITI frequency (to 2.04 events/min; p < 0.01) and increased phosphorylation at s2808 1.7-fold (p < 0.001). These pharmacological treatments had no significant effects on L-type calcium current density or sarcoplasmic reticulum calcium load. In conclusion, A3Rs are expressed and blunt spontaneous calcium release at baseline and upon A2AR-stimulation in human atrial myocytes, pointing to A3R activation as a means to attenuate physiological and pathological elevations of spontaneous calcium release events.
Assuntos
Fibrilação Atrial , Receptores Purinérgicos P1 , Humanos , Adenosina/metabolismo , Fibrilação Atrial/metabolismo , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Homeostase , Miócitos Cardíacos/metabolismo , Receptores Purinérgicos P1/metabolismo , RNA Mensageiro/metabolismo , Retículo Sarcoplasmático/metabolismoRESUMO
Intracellular Ca2+-calmodulin (CaM) signaling plays an important role in Ca2+-CaM-dependent kinase (CaMKII) and calcineurin (CaN)-mediated cardiac biology. While neurogranin (Ng) is known as a major Ca2+-CaM modulator in the brain, its pathophysiological role in cardiac hypertrophy has never been studied before. In the present study, we report that Ng is expressed in the heart and depletion of Ng dysregulates Ca2+ homeostasis and promotes cardiac failure in mice. 10-month-old Ng null mice demonstrate significantly increased heart-to-body weight ratios compared to wild-type. Using histological approaches, we identified that depletion of Ng increases cardiac hypertrophy, fibrosis, and collagen deposition near perivascular areas in the heart tissue of Ng null mice. Ca2+ spark experiments revealed that cardiac myocytes isolated from Ng null mice have decreased spark frequency and width, while the duration of sparks is significantly increased. We also identified that a lack of Ng increases CaMKIIδ signaling and periostin protein expression in these mouse hearts. Overall, we are the first study to explore how Ng expression in the heart plays an important role in Ca2+ homeostasis in cardiac myocytes as well as the pathophysiology of cardiac hypertrophy and fibrosis.
Assuntos
Cálcio , Neurogranina , Animais , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Calmodulina/metabolismo , Cardiomegalia/metabolismo , Fibrose , Camundongos , Camundongos Knockout , Miócitos Cardíacos/metabolismo , Neurogranina/genética , Neurogranina/metabolismoRESUMO
KEY POINTS: Increasing sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) pump activity enhances sarcoplasmic reticulum calcium (Ca) load, which increases both ryanodine receptor opening and driving force of Ca release flux. Both of these effects promote Ca spark formation and wave propagation. However, increasing SERCA activity also accelerates local cytosolic Ca decay as the wave front travels to the next cluster, which limits wave propagation. As a result, increasing SERCA pump activity has a biphasic effect on the propensity of arrhythmogenic Ca waves, but a monotonic effect to increase Ca spark frequency and amplitude. ABSTRACT: Waves of sarcoplasmic reticulum (SR) calcium (Ca) release can cause arrhythmogenic afterdepolarizations in cardiac myocytes. Ca waves propagate when Ca sparks at one Ca release unit (CRU) recruit new Ca sparks in neighbouring CRUs. Under normal conditions, Ca sparks are too small to recruit neighbouring Ca sparks where Ca sensitivity is also low. However, under pathological conditions such as a Ca overload or ryanodine receptor (RyR) sensitization, Ca sparks can be larger and propagate more readily as macro-sparks or full Ca waves. Increasing SERCA pump activity promotes SR Ca load, which promotes RyR opening and increases driving force of the Ca release flux from SR to cytosol, promoting Ca waves. However, high sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) activity can also decrease local cytosolic [Ca] as it approaches the next CRU, thereby reducing wave appearance and propagation. In this study, we use a physiologically detailed model of subcellular Ca cycling and experiments in phospholamban-knockout mice, to show how Ca waves are initiated and propagate and how different conditions contribute to the generation and propagation of Ca waves. We show that reducing diffusive coupling between Ca sparks by increasing SERCA activity prevents Ca waves by reducing [Ca] at the next CRU, as do Ca buffers, low intra-SR Ca diffusion and distance between CRUs. Increasing SR Ca uptake rate has a biphasic effect on Ca wave propagation; initially it enhances Ca spark probability and amplitude and CRU coupling, thereby promoting arrhythmogenic Ca wave propagation, but at higher levels SR Ca uptake can abort those arrhythmogenic Ca waves.
Assuntos
Sinalização do Cálcio , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Animais , Cálcio/metabolismo , Camundongos , Miócitos Cardíacos/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismoRESUMO
KEY POINTS: In cardiac myocytes, subcellular local calcium release signals, calcium sparks, are recruited to form each cellular calcium transient and activate the contractile machinery. Abnormal timing of recovery of sparks after their termination may contribute to arrhythmias. We developed a method to interrogate recovery of calcium spark trigger probabilities and their amplitude over time using two-photon photolysis of a new ultra-effective caged calcium compound. The findings confirm the utility of the technique to define an elevated sensitivity of the calcium release mechanism in situ and to follow hastened recovery of spark trigger probabilities in a mouse model of an inherited cardiac arrhythmia, which was used for validation. Analogous methods are likely to be applicable to investigate other microscopic subcellular signalling systems in a variety of cell types. ABSTRACT: In cardiac myocytes Ca2+ -induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) through ryanodine receptors (RyRs) governs activation of contraction. Ca2+ release occurs via subcellular Ca2+ signalling events, Ca2+ sparks. Local recovery of Ca2+ release depends on both SR refilling and restoration of Ca2+ sensitivity of the RyRs. We used two-photon (2P) photolysis of the ultra-effective caged Ca2+ compound BIST-2EGTA and laser-scanning confocal Ca2+ imaging to probe refractoriness of local Ca2+ release in control conditions and in the presence of cAMP or low-dose caffeine (to stimulate CICR) or cyclopiazonic acid (CPA; to slow SR refilling). Permeabilized cardiomyocytes were loaded with BIST-2EGTA and rhod-2. Pairs of short 2P photolytic pulses (1 ms, 810 nm) were applied with different intervals to test Ca2+ release amplitude recovery and trigger probability for the second spark in a pair. Photolytic and biological events were distinguished by classification with a self-learning support vector machine (SVM) algorithm. In permeabilized myocytes data recorded in the presence of CPA showed a lower probability of triggering a second spark compared to control or cAMP conditions. Cardiomyocytes from a mouse model harbouring the arrhythmogenic RyRR420Q mutation were used for further validation and revealed a higher Ca2+ sensitivity of CICR. This new 2P approach provides composite information of Ca2+ release amplitude and trigger probability recovery reflecting both SR refilling and restoration of CICR and RyR Ca2+ sensitivity. It can be used to measure the kinetics of local CICR recovery, alterations of which may be related to premature heart beats and arrhythmias.
Assuntos
Cálcio , Retículo Sarcoplasmático , Animais , Cálcio/metabolismo , Sinalização do Cálcio , Camundongos , Miócitos Cardíacos/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismoRESUMO
Intracellular Ca signals represent a universal mechanism of cell function. Messages carried by Ca are local, rapid, and powerful enough to be delivered over the thermal noise. A higher signal-to-noise ratio is achieved by a cooperative action of Ca release channels such as IP3 receptors or ryanodine receptors arranged in clusters (release units) containing a few to several hundred release channels. The channels synchronize their openings via Ca-induced Ca release, generating high-amplitude local Ca signals known as puffs in neurons and sparks in muscle cells. Despite the positive feedback nature of the activation, Ca signals are strictly confined in time and space by an unexplained termination mechanism. Here we show that the collective transition of release channels from an open to a closed state is identical to the phase transition associated with the reversal of magnetic field in an Ising ferromagnet. Our simple quantitative criterion closely predicts the Ca store depletion level required for spark termination for each cluster size. We further formulate exact requirements that a cluster of release channels should satisfy in any cell type for our mapping to the Ising model and the associated formula to remain valid. Thus, we describe deterministically the behavior of a system on a coarser scale (release unit) that is random on a finer scale (release channels), bridging the gap between scales. Our results provide exact mapping of a nanoscale biological signaling model to an interacting particle system in statistical physics, making the extensive mathematical apparatus available to quantitative biology.
Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Animais , Citoplasma/metabolismo , Coração/fisiologia , Temperatura Alta , Bicamadas Lipídicas , Campos Magnéticos , Modelos Biológicos , Modelos Estatísticos , Retículo Sarcoplasmático/metabolismo , Razão Sinal-RuídoRESUMO
Previous studies have shown that ventricular myocytes from female rats have smaller contractions and Ca(2+) transients than males. As cardiac contraction is regulated by the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway, we hypothesized that sex differences in cAMP contribute to differences in Ca(2+) handling. Ca(2+) transients (fura-2) and ionic currents were measured simultaneously (37°C, 2Hz) in ventricular myocytes from adult male and female C57BL/6 mice. Under basal conditions, diastolic Ca(2+), sarcoplasmic reticulum (SR) Ca(2+) stores, and L-type Ca(2+) current did not differ between the sexes. However, female myocytes had smaller Ca(2+) transients (26% smaller), Ca(2+) sparks (6% smaller), and excitation-contraction coupling gain in comparison to males (23% smaller). Interestingly, basal levels of intracellular cAMP were lower in female myocytes (0.7±0.1 vs. 1.7±0.2fmol/µg protein; p<0.001). Importantly, PKA inhibition (2µM H-89) eliminated male-female differences in Ca(2+) transients and gain, as well as Ca(2+) spark amplitude. Western blots showed that PKA inhibition also reduced the ratio of phospho:total RyR2 in male hearts, but not in female hearts. Stimulation of cAMP production with 10µM forskolin abolished sex differences in cAMP levels, as well as differences in Ca(2+) transients, sparks, and gain. To determine if the breakdown of cAMP differed between the sexes, phosphodiesterase (PDE) mRNA levels were measured. PDE3 expression was similar in males and females, but PDE4B expression was higher in female ventricles. The inhibition of cAMP breakdown by PDE4 (10µM rolipram) abolished differences in Ca(2+) transients and gain. These findings suggest that female myocytes have lower levels of basal cAMP due, in part, to higher expression of PDE4B. Lower cAMP levels in females may attenuate PKA phosphorylation of Ca(2+) handling proteins in females, and may limit positive inotropic responses to stimulation of the cAMP/PKA pathway in female hearts.
Assuntos
Cálcio/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Ventrículos do Coração/citologia , Miócitos Cardíacos/metabolismo , Retículo Sarcoplasmático/metabolismo , Caracteres Sexuais , Transdução de Sinais , Adenilil Ciclases/metabolismo , Animais , Sinalização do Cálcio , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Diástole , Ativação Enzimática , Acoplamento Excitação-Contração , Feminino , Espaço Intracelular/metabolismo , Ativação do Canal Iônico , Masculino , Camundongos Endogâmicos C57BL , Fosforilação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismoRESUMO
A hallmark of atrial fibrillation is an excess of spontaneous calcium release events, which can be mimicked by ß1- or ß2-adrenergic stimulation. Because ß1-adrenergic receptor blockers (ß1-blockers) are primarily used in clinical practice, we here examined the impact of ß2-adrenergic stimulation on spontaneous calcium release and assessed whether the R- and S-enantiomers of the non-selective ß- blocker carvedilol could reverse these effects. For this purpose, human atrial myocytes were isolated from patients undergoing cardiovascular surgery and subjected to confocal calcium imaging or immunofluorescent labeling of the ryanodine receptor (RyR2). Interestingly, the ß2-adrenergic agonist fenoterol increased the incidence of calcium sparks and waves to levels observed with the non-specific ß-adrenergic agonist isoproterenol. Moreover, fenoterol increased both the amplitude and duration of the sparks, facilitating their fusion into calcium waves. Subsequent application of the non ß-blocking R-Carvedilol enantiomer reversed these effects of fenoterol in a dose-dependent manner. R-Carvedilol also reversed the fenoterol-induced phosphorylation of the RyR2 at Ser-2808 dose-dependently, and 1 µM of either R- or S-Carvedilol fully reversed the effect of fenoterol. Together, these findings demonstrate that ß2-adrenergic stimulation alone stimulates RyR2 phosphorylation at Ser-2808 and spontaneous calcium release maximally, and points to carvedilol as a tool to attenuate the pathological activation of ß2-receptors.
RESUMO
AIMS: It is unknown how ß-adrenergic stimulation affects calcium dynamics in individual RyR2 clusters and leads to the induction of spontaneous calcium waves. To address this, we analysed spontaneous calcium release events in green fluorescent protein (GFP)-tagged RyR2 clusters. METHODS: Cardiomyocytes from mice with GFP-tagged RyR2 or human right atrial tissue were subjected to immunofluorescent labelling or confocal calcium imaging. RESULTS: Spontaneous calcium release from single RyR2 clusters induced 91.4% ± 2.0% of all calcium sparks while 8.0% ± 1.6% were caused by release from two neighbouring clusters. Sparks with two RyR2 clusters had 40% bigger amplitude, were 26% wider, and lasted 35% longer at half maximum. Consequently, the spark mass was larger in two- than one-cluster sparks with a median and interquartile range for the cumulative distribution of 15.7 ± 20.1 vs 7.6 ± 5.7 a.u. (P < .01). ß2-adrenergic stimulation increased RyR2 phosphorylation at s2809 and s2815, tripled the fraction of two- and three-cluster sparks, and significantly increased the spark mass. Interestingly, the amplitude and mass of the calcium released from a RyR2 cluster were proportional to the SR calcium load, but the firing rate was not. The spark mass was also higher in 33 patients with atrial fibrillation than in 36 without (22.9 ± 23.4 a.u. vs 10.7 ± 10.9; P = .015). CONCLUSIONS: Most sparks are caused by activation of a single RyR2 cluster at baseline while ß-adrenergic stimulation doubles the mass and the number of clusters per spark. This mimics the shift in the cumulative spark mass distribution observed in myocytes from patients with atrial fibrillation.
Assuntos
Fibrilação Atrial , Canal de Liberação de Cálcio do Receptor de Rianodina , Adrenérgicos , Animais , Fibrilação Atrial/metabolismo , Cálcio/metabolismo , Sinalização do Cálcio , Humanos , Camundongos , Miócitos Cardíacos/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismoRESUMO
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.
RESUMO
Ca2+ sparks are instrumental to understand physiological and pathological Ca2+ signaling in the heart. High-speed two spatially dimensional (2D) confocal imaging (>120â¯Hz) enables acquisition of sparks with high-content information, however, owing to a wide variety of different acquisition modalities the question arises: how much they reflect the "true" Ca2+ spark properties. To address this issue, we compared a fast point and a 2D-array scanner equipped with a range of different detectors. As a quasi-standard biological sample, we employed Ca2+ sparks in permeabilized and intact mouse ventricular myocytes and utilized an unbiased, automatic Ca2+ spark analysis tool, iSpark. Data from the point scanner suffered from low pixel photon fluxes (PPF) concomitant with high Poissonian noise. Images from the 2D-array scanner displayed substantially increased PPF, lower Poissonian noise and almost 3-fold increased sign-to-noise ratios. Noteworthy, data from the 2D scanner suffered from considerable inter-pinhole crosstalk evident for the permeabilized cells. Spark properties, such as frequency, amplitude, decay time and spatial spread were distinctly different for any scanner/detector combination. Our study reveals that the apparent Ca2+ spark properties differ dependent on the particular recording modality and set-up employed, quantitatively.
Assuntos
Sinalização do Cálcio , Imageamento Tridimensional , Microscopia Confocal , Animais , Ventrículos do Coração/citologia , Camundongos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , SemicondutoresRESUMO
In amphibian skeletal muscle calcium (Ca2+) sparks occur both as voltage-dependent and voltage-independent ligand-activated release events. However, whether their properties and their origin show similarities are still in debate. Elevated K+, constant Cl- content solutions were used to initiate small depolarizations of the resting membrane potential to activate dihydropyridine receptors (DHPR) and caffeine to open ryanodine receptors (RyR) on intact fibers. The properties of Ca2+ sparks observed under control conditions were compared to those measured on depolarized cells and those after caffeine treatment. Calcium sparks were recorded on intact frog skeletal muscle fibers using high time resolution confocal microscopy (x-y scan: 30 Hz). Sparks were elicited by 1 mmol/l caffeine or subthreshold depolarization to different membrane potentials. Both treatments increased the frequency of sparks and altered their morphology. Images were analyzed by custom-made computer programs. Both the amplitude (in ΔF/F0; 0.259 ± 0.001 vs. 0.164 ± 0.001; n = 24942 and 43326, respectively; mean ± SE, p < 0.001) and the full width at half maximum (FWHM, in µm; parallel with fiber axis: 2.34 ± 0.01 vs. 1.92 ± 0.01, p < 0.001; perpendicular to fiber axis: 2.08 ± 0.01 vs. 1.68 ± 0.01, p < 0.001) of sparks was significantly greater after caffeine treatment than on depolarized cells. 9.8% of the sparks detected on depolarized fibers and about one third of the caffeine activated sparks (29.7%) overlapped with another one on the previous frame on x-y scans. Centre of overlapping sparks travelled significantly longer distances between consecutive frames after caffeine treatment then after depolarization (in µm; 1.66 ± 0.01 vs. 0.95 ± 0.01, p < 0.001). Our results suggest that the two types of ryanodine receptors, the junctional RyRs controlled by DHPRs and the parajunctional RyRs are activated independently, using alternate ways, with the possibility of cooperation between neighboring release channels.
RESUMO
Analysis of calcium sparks in cardiomyocytes can provide valuable information about functional changes of calcium handling in health and disease. As a part of the calcium sparks analysis, sparks detection and characterization is necessary. Here, we describe a new open-source platform for automatic calcium sparks detection from line scan confocal images. The developed software is tailored for detecting only calcium sparks, allowing us to design a graphical user interface specifically for this task. The software enables detecting sparks automatically as well as adding, removing, or adjusting regions of interest marking each spark. The results of the analysis are stored in an SQL database, allowing simple integration with statistical tools. We have analyzed the performance of the algorithm using a large set of synthetic images with varying spark sizes and noise levels and also compared the analysis results with results obtained by software established in the field. The use of our software is illustrated by an analysis of the effect of isoprenaline (ISO) on spark frequency, amplitude, and spatial and temporal characteristics. For that, cardiomyocytes from C57BL/6 mice were used. We demonstrated an increase in spark frequency, tendency of having larger spark amplitudes, sparks with a longer duration, and occurrence of multiple sparks from the same site in the presence of ISO. We also show that the duration and the width of sparks with the same amplitude were similar in the absence and presence of ISO. The software was released as an open source repository and is available for free use and collaborative development.
RESUMO
The objective of the study is to investigate the propagation of Ca(2+) waves in full-width cardiac myocytes and carry out sensitivity analysis to study the effects of various physiological parameters on global Ca(2+) waves. Based on the anomalous subdiffusion of Ca(2+) sparks, a mathematical model was proposed to characterize the Ca(2+) waves. The computed results were in agreement with the experimental measurements using confocal microscopy. This model includes variables of current through the Ca(2+) release unit (CRU; ICRU), duration of current flow through CRU (Topen), Ca(2+) sensitivity parameter (K), the longitudinal and transverse spatial separation of CRUs (lx and ly, where x denotes longitudinal direction (x-axis) and y denotes transverse direction (y-axis)) and Ca(2+) diffusion coefficients (Dx, Dy). The spatio-temporal mechanism of the anomalous Ca(2+) sparks led to results that were different from those based on Fick's law. The major findings were reported as: ICRU affected the dynamic properties of Ca(2+) waves more significantly than Topen; the effect of K on the properties of Ca(2+) waves was negligible; ly affected the amplitude significantly, but lx affected the longitudinal velocity significantly; in turn, the limitation and significance of the study are discussed.
Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Miócitos Cardíacos/citologia , Animais , Anisotropia , Cálcio/química , Difusão , Microscopia Confocal , Modelos Teóricos , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Fatores de TempoRESUMO
Increased oxidative stress contributes to heart dysfunction via impaired Ca(2+) homeostasis in diabetes. Abnormal RyR2 function related with altered cellular redox state is an important factor in the pathogenesis of diabetic cardiomyopathy, while its underlying mechanisms remain poorly understood. In the present study, we used a streptozotocin-induced rat model of diabetic cardiomyopathy and tested a hypothesis that diabetes-related alteration in RyR2 function is related with ROS-induced posttranslational modifications. For this, we used heart preparations from either a diabetic rat or a sodium selenate (NaSe)-treated (0.3 mg/kg for 4 weeks) diabetic rat as well as either NaSe- (100 nmol/L) or thioredoxin (Trx; 5 µmol/L)-incubated (30 min) diabetic cardiomyocytes. Experimental approaches included imaging of intracellular free-Ca(2+) ([Ca(2+)]i) under both electrically stimulated and resting Fluo-3-loaded cardiomyocytes. RyR2-mediated SR-Ca(2+) leak was significantly enhanced in diabetic cardiomyocytes, resulting in reduced amplitude and prolonged time courses of [Ca(2+)]i transients compared to those of controls. Both SR-Ca(2+) leak and [Ca(2+)]i transients were normalized by treating diabetic rats with NaSe or by incubating diabetic myocytes with NaSe or Trx. Moreover, exposure of diabetic cardiomyocytes to antioxidants significantly improved [Ca(2+)]i handling factors such as phosphorylation/protein levels of RyR2, amount of RyR2-bound FKBP12.6 and activities of both protein kinase A and CaMKII. NaSe treatment also normalized the oxidative stress/antioxidant defense biomarkers in plasma as well as Trx activity and nuclear factor-κB phosphorylation in the diabetic rat heart. Collectively, these findings suggest that redox modification through Trx-system besides the glutathione system contributes to abnormal function of RyR2s in hyperglycemic cardiomyocytes, presenting a potential therapeutic target for treating diabetics to preserve cardiac function.
Assuntos
Cardiotônicos/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Ácido Selênico/farmacologia , Tiorredoxinas/farmacologia , Animais , Cálcio/metabolismo , Cardiomiopatias/tratamento farmacológico , Diabetes Mellitus Experimental , Glutationa/sangue , Peroxidação de Lipídeos , Masculino , Miócitos Cardíacos/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Oxirredução , Estresse Oxidativo , Fosforilação , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Proteínas de Ligação a Tacrolimo/genética , Proteínas de Ligação a Tacrolimo/metabolismoRESUMO
Calcium as a ubiquitous messenger participates in the pathophysiology progresses of various cells. Synchronized calcium signaling transduction is related to normal cardiac myocyte excitation-contraction coupling and beating rhythm. Calcium homeostasis abnormality in atrial myocyte is an important influencing mechanism of atrial fibrillation (AF), and it is mostly resulted from calcium leak caused by destabilization of sarcoplasmic reticulum calcium release channel ryanodine receptor 2 (RyR2). Calcium leaks, including calcium sparks and calcium waves, can trigger extra depolarization and even action potential, and cause AF on the basis of atrial pathology altering matrices. In this review, we summarized the manifestations and influencing factors of calcium leak, and its potential role in the development and progress of AF.