Selective Inhibition of Pulmonary Vein Excitability by Constitutively Active GIRK Channels Blockade in Rats.

Pulmonary veins (PV) are the main source of ectopy, triggering atrial fibrillation. This study investigated the roles of G protein-coupled inwardly rectifying potassium (GIRK) channels in the PV and the left atrium (LA) of the rat. Simultaneous intracellular microelectrode recording from the LA and the PV of the rat found that in the presence or absence of acetylcholine, the GIRK channel blocker tertiapin-Q induced AP duration elongation in the LA and the loss of over-shooting AP in the PV, suggesting the presence of constitutively active GIRK channels in these tissues. Patch-clamp recordings from isolated myocytes showed that tertiapin-Q inhibited a basal inwardly rectified background current in PV cells with little effect in LA cells. Experiments with ROMK1 and KCa1.1 channel blockers ruled out the possibility of an off-target effect. Western blot showed that GIRK4 subunit expression was greater in PV cardiomyocytes, which may explain the differences observed between PV and LA in response to tertiapin-Q. In conclusion, GIRK channels blockade abolishes AP only in the PV, providing a molecular target to induce electrical disconnection of the PV from the LA.

Spiky: An ImageJ Plugin for Data Analysis of Functional Cardiac and Cardiomyocyte Studies.

INTRODUCTION AND OBJECTIVE: Nowadays, investigations of heart physiology and pathophysiology rely more and more upon image analysis, whether for the detection and characterization of events in single cells or for the mapping of events and their characteristics across an entire tissue. These investigations require extensive skills in image analysis and/or expensive software, and their reproducibility may be a concern. Our objective was to build a robust, reliable and open-source software tool to quantify excitation-contraction related experimental data at multiple scales, from single isolated cells to the whole heart. METHODS AND RESULTS: A free and open-source ImageJ plugin, Spiky, was developed to detect and analyze peaks in experimental data streams. It allows rapid and easy analysis of action potentials, intracellular calcium transient and contraction data from cardiac research experiments. As shown in the provided examples, both classical bi-dimensional data (XT signals) and video data obtained from confocal microscopy and optical mapping experiments (XYT signals) can be analyzed. Spiky was written in ImageJ Macro Language and JAVA, and works under Windows, Mac and Linux operating systems. CONCLUSION: Spiky provides a complete working interface to process and analyze cardiac physiology research data.

Automatic Activity Arising in Cardiac Muscle Sleeves of the Pulmonary Vein.

Ectopic activity in the pulmonary vein cardiac muscle sleeves can both induce and maintain human atrial fibrillation. A central issue in any study of the pulmonary veins is their difference from the left atrial cardiac muscle. Here, we attempt to summarize the physiological phenomena underlying the occurrence of ectopic electrical activity in animal pulmonary veins. We emphasize that the activation of multiple signaling pathways influencing not only myocyte electrophysiology but also the means of excitation-contraction coupling may be required for the initiation of triggered or automatic activity. We also gather information regarding not only the large-scale structure of cardiac muscle sleeves but also recent studies suggesting that cellular heterogeneity may contribute to the generation of arrythmogenic phenomena and to the distinction between pulmonary vein and left atrial heart muscle.

Selective inhibition of electrical conduction within the pulmonary veins by α1-adrenergic receptors activation in the Rat.

Pulmonary veins (PV) are involved in the pathophysiology of paroxysmal atrial fibrillation. In the rat, left atrium (LA) and PV cardiomyocytes have different reactions to α1-adrenergic receptor activation. In freely beating atria-PV preparations, we found that electrical field potential (EFP) originated from the sino-atrial node propagated through the LA and the PV. The α1-adrenergic receptor agonist cirazoline induced a progressive loss of EFP conduction in the PV whereas it was maintained in the LA. This could be reproduced in preparations electrically paced at 5 Hz in LA. During pacing at 10 Hz in the PV where high firing rate ectopic foci can occur, cirazoline stopped EFP conduction from the PV to the LA, which allowed the sino-atrial node to resume its pace-making function. Loss of conduction in the PV was associated with depolarization of the diastolic membrane potential of PV cardiomyocytes. Adenosine, which reversed the cirazoline-induced depolarization of the diastolic membrane potential of PV cardiomyocytes, restored full over-shooting action potentials and EFP conduction in the PV. In conclusion, selective activation of α1-adrenergic receptors results in the abolition of electrical conduction within the PV. These results highlight a potentially novel pharmacological approach to treat paroxysmal atrial fibrillation by targeting directly the PV myocardium.

A microsensing system for the in vivo real-time detection of local drug kinetics.

Real-time recording of the kinetics of systemically administered drugs in in vivo microenvironments may accelerate the development of effective medical therapies. However, conventional methods require considerable analyte quantities, have low sampling rates and do not address how drug kinetics correlate with target function over time. Here, we describe the development and application of a drug-sensing system consisting of a glass microelectrode and a microsensor composed of boron-doped diamond with a tip of around 40 µm in diameter. We show that, in the guinea pig cochlea, the system can measure-simultaneously and in real time-changes in the concentration of bumetanide (a diuretic that is ototoxic but applicable to epilepsy treatment) and the endocochlear potential underlying hearing. In the rat brain, we tracked the kinetics of the drug and the local field potentials representing neuronal activity. We also show that the actions of the antiepileptic drug lamotrigine and the anticancer reagent doxorubicin can be monitored in vivo. Our microsensing system offers the potential to detect pharmacological and physiological responses that might otherwise remain undetected.

A TTX-sensitive resting Na+ permeability contributes to the catecholaminergic automatic activity in rat pulmonary vein.

INTRODUCTION: Ectopic activity arising from pulmonary veins (PV) plays a prominent role in the onset of atrial fibrillation in humans. Rat PV cardiac muscle cells have a lower resting membrane potential (RMP) than the left atria (LA) and presents in the presence of norepinephrine an automatic activity, which occurs in bursts. This study investigated the role of Na channels upon the RMP and the catecholaminergic automatic activity (CAA) in PV cardiac muscle. METHODS AND RESULTS: RMP and CAA experiments were performed in male Wistar rat PV. Whole-cell INa was recorded in isolated PV and LA cardiomyocytes. PV has a higher tetrodotoxin (TTX)-sensitive basal Na(+) permeability than the LA, due to a ∼ 5 mV more negative Na window current in the former tissue. TTX, quinidine, and ranolazine (1 to 10 µM each) decreased CAA incidence and arrhythmias by increasing burst intervals because of a reduction of the slope of slow depolarization between bursts. TTX and ranolazine also reduced burst duration. At 1 Hz, 10 µM quinidine, ranolazine, and TTX inhibited peak INa by 33%, 28%, and 98%, respectively. Each reduced the Na window current. There was no evidence for a TTX- or ranolazine-sensitive late Na current. CONCLUSION: Na channels confer a TTX-sensitive basal Na(+) permeability to rat PV cardiac muscle cells and contribute to the slope of slow depolarization between bursts of CAA. Na channel blockers act mostly via reduction of the Na window current. Ranolazine also has an anti-α1 adrenergic effect, which contributed to its antiarrhythmic effect.

ANO1 contributes to angiotensin-II-activated Ca2+-dependent Cl- current in human atrial fibroblasts.

Cardiac fibroblasts are an integral part of the myocardial tissue and contribute to its remodelling. This study characterises for the first time the calcium-dependent chloride channels (CaCC) in the plasma membrane of primary human atrial cardiac fibroblasts by means of the iodide efflux and the patch clamp methods. The calcium ionophore A23187 and Angiotensin II (Ang II) activate a chloride conductance in cardiac fibroblasts that shares pharmacological similarities with calcium-dependent chloride channels. This chloride conductance is depressed by RNAi-mediated selective Anoctamine 1 (ANO1) but not by Anoctamine 2 (ANO2) which has been revealed as CaCC and is inhibited by the selective ANO1 inhibitor, T16inh-A01. The effect of Ang II on anion efflux is mediated through AT1 receptors (with an EC50 = 13.8 ± 1.3 nM). The decrease of anion efflux by calphostin C and bisindolylmaleimide I (BIM I) suggests that chloride conductance activation is dependent on PKC. We conclude that ANO1 contributes to CaCC current in human cardiac fibroblasts and that this is regulated by Ang II acting via the AT1 receptor pathway.

The ß1-subunit of Na(v)1.5 cardiac sodium channel is required for a dominant negative effect through α-α interaction.

Brugada syndrome (BrS) is an inherited autosomal dominant cardiac channelopathy. Several mutations on the cardiac sodium channel Na(v)1.5 which are responsible for BrS lead to misfolded proteins that do not traffic properly to the plasma membrane. In order to mimic patient heterozygosity, a trafficking defective mutant, R1432G was co-expressed with Wild Type (WT) Na(v)1.5 channels in HEK293T cells. This mutant significantly decreased the membrane Na current density when it was co-transfected with the WT channel. This dominant negative effect did not result in altered biophysical properties of Na(v)1.5 channels. Luminometric experiments revealed that the expression of mutant proteins induced a significant reduction in membrane expression of WT channels. Interestingly, we have found that the auxiliary Na channel ß(1)-subunit was essential for this dominant negative effect. Indeed, the absence of the ß(1)-subunit prevented the decrease in WT sodium current density and surface proteins associated with the dominant negative effect. Co-immunoprecipitation experiments demonstrated a physical interaction between Na channel α-subunits. This interaction occurred only when the ß(1)-subunit was present. Our findings reveal a new role for ß(1)-subunits in cardiac voltage-gated sodium channels by promoting α-α subunit interaction which can lead to a dominant negative effect when one of the α-subunits shows a trafficking defective mutation.

Stringent programming of DNA methylation in humans.

We describe a PCR-based method called Amplified Methylation Polymorphism (AMP) for scanning genomes for DNA methylation changes. AMP detects tissue-specific DNA methylation signatures often representing junctions between methylated and unmethylated DNA close to intronexon junctions and/or associated with CpG islands. Identical AMP profiles are detected for healthy, young, monozygotic twins.

Inwardly rectifying potassium channels: their structure, function, and physiological roles.

Inwardly rectifying K(+) (Kir) channels allow K(+) to move more easily into rather than out of the cell. They have diverse physiological functions depending on their type and their location. There are seven Kir channel subfamilies that can be classified into four functional groups: classical Kir channels (Kir2.x) are constitutively active, G protein-gated Kir channels (Kir3.x) are regulated by G protein-coupled receptors, ATP-sensitive K(+) channels (Kir6.x) are tightly linked to cellular metabolism, and K(+) transport channels (Kir1.x, Kir4.x, Kir5.x, and Kir7.x). Inward rectification results from pore block by intracellular substances such as Mg(2+) and polyamines. Kir channel activity can be modulated by ions, phospholipids, and binding proteins. The basic building block of a Kir channel is made up of two transmembrane helices with cytoplasmic NH(2) and COOH termini and an extracellular loop which folds back to form the pore-lining ion selectivity filter. In vivo, functional Kir channels are composed of four such subunits which are either homo- or heterotetramers. Gene targeting and genetic analysis have linked Kir channel dysfunction to diverse pathologies. The crystal structure of different Kir channels is opening the way to understanding the structure-function relationships of this simple but diverse ion channel family.

Catecholaminergic automatic activity in the rat pulmonary vein: electrophysiological differences between cardiac muscle in the left atrium and pulmonary vein.

Ectopic activity in cardiac muscle within pulmonary veins (PVs) is associated with the onset and the maintenance of atrial fibrillation in humans. The mechanism underlying this ectopic activity is unknown. Here we investigate automatic activity generated by catecholaminergic stimulation in the rat PV. Intracellular microelectrodes were used to record electrical activity in isolated strips of rat PV and left atrium (LA). The resting cardiac muscle membrane potential was lower in PV [-70 +/- 1 (SE) mV, n = 8] than in LA (-85 +/- 1 mV, n = 8). No spontaneous activity was recorded in PV or LA under basal conditions. Norepinephrine (10(-5) M) induced first a hyperpolarization (-8 +/- 1 mV in PV, -3 +/- 1 mV in LA, n = 8 for both) then a slowly developing depolarization (+21 +/- 2 mV after 15 min in PV, +1 +/- 2 mV in LA) of the resting membrane potential. Automatic activity occurred only in PV; it was triggered at approximately -50 mV, and it occurred as repetitive bursts of slow action potentials. The diastolic membrane potential increased during a burst and slowly depolarized between bursts. Automatic activity in the PV was blocked by either atenolol or prazosine, and it could be generated with a mixture of cirazoline and isoprenaline. In both tissues, cirazoline (10(-6) M) induced a depolarization (+37 +/- 2 mV in PV, n = 5; +5 +/- 1 mV in LA, n = 5), and isoprenaline (10(-7) M) evoked a hyperpolarization (-11 +/- 3 mV in PV, n = 7; -3 +/- 1 mV in LA, n = 6). The differences in membrane potential and reaction to adrenergic stimulation lead to automatic electrical activity occurring specifically in cardiac muscle in the PV.

Microstructure-based Monte Carlo simulation of Ca2+ dynamics evoking cardiac calcium channel inactivation.

Ca(2+) dynamics underlying cardiac excitation-contraction coupling are essential for heart functions. In this study, we constructed microstructure-based models of Ca(2+) dynamics to simulate Ca(2+) influx through individual L-type Calcium channels (LCCs), an effective Ca(2+) diffusion within the cytoplasmic space and in the dyadic space, and the experimentally observed calcium-dependent inactivation (CDI) of the LCCs induced by local and global Ca(2+) sensing. The models consisted of LCCs with distal and proximal Ca(2+) (Calmodulin-Ca(2+) complex) binding sites. In one model, the intra-cellular space was organelle-free cytoplasmic space, and the other was with a dyadic space including sarcoplasmic reticulum membrane. The Ca(2+) dynamics and CDI of the LCCs in the model with and without the dyadic space were then simulated using the Monte Carlo method. We first showed that an appropriate set of parameter values of the models with effectively extra-slow Ca(2+) diffusion enabled the models to reproduce major features of the CDI process induced by the local and global sensing of Ca(2+) near LCCs as measured with single and two spatially separated LCCs by Imredy and Yue (Neuron. 1992;9:197-207). The effective slow Ca(2+) diffusion might be due to association and dissociation of Ca(2+) and Calmodulin (CaM). We then examined how the local and global CDIs were affected by the presence of the dyadic space. The results suggested that in microstructure modeling of Ca(2+) dynamics in cardiac myocytes, the effective Ca(2+) diffusion under CaM-Ca(2+) interaction, the nanodomain structure of LCCs for detailed CDI, and the geometry of subcellular space for modeling dyadic space should be considered.

In silico risk assessment for drug-induction of cardiac arrhythmia.

The main components of repolarization reserve for the ventricular action potential (AP) are the rapid (I(Kr)) and slow (I(Ks)) delayed outward K(+) currents. While many drugs block I(Kr) and cause life-threatening arrhythmias including torsades de pointes, the frequency of arrhythmias varies between different I(Kr)-blockers. Different types of block of I(Kr) cause distinct phenotypes of prolongation of action potential duration (APD), increase in transmural dispersion of repolarization (TDR) and, accordingly, occurrence of torsades de pointes. Therefore the assessment of a drug's proarrhythmic risk requires a method that provides quantitative and comprehensive comparison of the effects of different forms of I(Kr)-blockade upon APDs and TDR. However, most currently available methods are not adapted to such an extensive comparison. Here, we introduce I(Kr)-I(Ks) two-dimensional maps of APD and TDR as a novel risk-assessment method. Taking the kinetics of I(Kr)-blockade into account, APDs can be calculated upon a ventricular AP model which systematically alters the magnitudes of I(Kr) and I(Ks). The calculated APDs are then plotted on a map where the x axis represents the conductance of I(Kr) while the y axis represents that of I(Ks). TDR is simulated with models corresponding to APs in epicardial, midcardial and endocardial myocardium. These two-dimensional maps of APD and TDR successfully account for differences in the risk resulting from three distinct types of I(Kr)-blockade which correspond to the effects of dofetilide, quinidine and vesnarinone. This method may be of use to assess the arrhythmogenic risk of various I(Kr)-blockers.

Physiological modulation of voltage-dependent inactivation in the cardiac muscle L-type calcium channel: a modelling study.

The inactivation of the L-type Ca2+ current is composed of voltage-dependent and calcium-dependent mechanisms. The relative contribution of these processes is still under dispute and the idea that the voltage-dependent inactivation could be subject to further modulation by other physiological processes had been ignored. This study sought to model physiological modulation of inactivation of the current in cardiac ventricular myocytes, based upon the recent detailed experimental data that separated total and voltage-dependent inactivation (VDI) by replacing extracellular Ca2+ with Mg2+ and monitoring L-type Ca2+ channel behaviour by outward K+ current flowing through the channel in the absence of inward current flow. Calcium-dependent inactivation (CDI) was based upon Ca2+ influx and formulated from data that was recorded during beta-adrenergic stimulation of the myocytes. Ca2+ influx and its competition with non-selective monovalent cation permeation were also incorporated into channel permeation in the model. The constructed model could closely reproduce the experimental Ba2+ and Ca2+ current results under basal condition where no beta-stimulation was added after a slight reduction of the development of fast voltage-dependent inactivation with depolarization. The model also predicted that under beta-adrenergic stimulation voltage-dependent inactivation is lost and calcium-dependent inactivation largely compensates it. The developed model thus will be useful to estimate the respective roles of VDI and CDI of L-type Ca2+ channels in various physiological and pathological conditions of the heart which would otherwise be difficult to show experimentally.

Contractile and relaxant properties of rat-isolated pulmonary veins related to localization and histology.

The aim of this study was to investigate the in vitro vasomotor properties of rat extra-and intralobar pulmonary veins (PVs) related to their localization and to assess the modulatory role of endothelium on these properties. Segments from PVs were mounted in small vessel myograph and stretched at various diameters (D(10), D(20), D(30)) corresponding to intraluminal pressures of 10, 20 or 30 mmHg. At D(10) or D(20), contractile responses to phenylephrine, U46619 and angiotensin II of distal intralobar part of PVs were smaller compared with those of proximal extralobar part, but no longer different when distal part was stretched at D(30). When submitted to an NO donor, sodium nitroprusside, distal part of PV relaxed more strongly when stretched at D(30) compared with D(10). Acetylcholine and bradykinin were devoid of relaxing effect on distal parts stretched at D(10), but in contrast to acetylcholine, bradykinin slightly relaxed preparations stretched at D(30). Isoprenaline strongly relaxed PVs ( approximately 80% of initial precontraction), with the distal part exhibiting a higher sensitivity to the agonist compared with the proximal part. This relaxation was also observed with salbutamol and suppressed with ICI 118551, which is in favour of the involvement of beta(2)-adrenoceptors in this effect. Preincubation of the preparations with N(G)-nitro-l-arginine methyl ester (10(-4) m) and indomethacin (10(-5) m) did not modify the contractile responses to U46619, nor the relaxing response to isoprenaline, which support that endothelium does not appear to play a significant modulatory role in these responses. Histological and electron microscopical examinations of proximal and distal sections of the same vein show that the layers of smooth muscle cells and cardiomyocytes were thicker in the proximal compared with the distal part. This study shows that, because of morphological heterogeneity of the PVs, the site of dissection and the initial condition of tension can play a significant role upon the sensitivity and the magnitude of the responses to both contractile and relaxing agonists.

Physiological modulation of inactivation in L-type Ca2+ channels: one switch.

The relative contributions of voltage- and Ca(2+)-dependent mechanisms of inactivation to the decay of L-type Ca(2+) channel currents (I(CaL)) is an old story to which recent results have given an unexpected twist. In cardiac myocytes voltage-dependent inactivation (VDI) was thought to be slow and Ca(2+)-dependent inactivation (CDI) resulting from Ca(2+) influx and Ca(2+)-induced Ca(2+)-release (CICR) from the sarcoplasmic reticulum provided an automatic negative feedback mechanism to limit Ca(2+) entry and the contribution of I(CaL) to the cardiac action potential. Physiological modulation of I(CaL) by Beta-adrenergic and muscarinic agonists then involved essentially more or less of the same by enhancing or reducing Ca(2+) channel activity, Ca(2+) influx, sarcoplasmic reticulum load and thus CDI. Recent results on the other hand place VDI at the centre of the regulation of I(CaL). Under basal conditions it has been found that depolarization increases the probability that an ion channel will show rapid VDI. This is prevented by Beta-adrenergic stimulation. Evidence also suggests that a channel which shows rapid VDI inactivates before CDI can become effective. Therefore the contributions of VDI and CDI to the decay of I(CaL) are determined by the turning on, by depolarization, and the turning off, by phosphorylation, of the mechanism of rapid VDI. The physiological implications of these ideas are that under basal conditions the contribution of I(CaL) to the action potential will be determined largely by voltage and by Ca(2+) following Beta-adrenergic stimulation.

High efficiency activation of L-type Ca2+ current by 5-HT in human atrial myocytes.

In human atrial myocytes, serotonin rather than sympathetic, stimulation is more frequently associated with atrial fibrillation. So does the arrhythmogenic effect of serotonin result from the mechanism of action of the receptor or the context of its action upon cardiac myocytes? The capacity of agonists to produce cAMP followed the sequence 5-HT < Iso < Forskolin to increase ICaL with 5-HT = Iso = Forskolin. The simultaneous application of threshold concentrations of 5-HT and Iso maximally increased ICaL. We will show that the effect of 5-HT upon human atrial myocytes is an imbalance between low production of cAMP and maximal activation of ICaL.

Low-voltage triggering of Ca2+ release from the sarcoplasmic reticulum in cardiac muscle cells.

This study investigated the interaction between L-type Ca2+ current (ICaL) and Ca2+ release from the sarcoplasmic reticulum (SRCR) in whole cell voltage-clamped guinea pig ventricular myocytes. Quasiphysiological cation solutions (Nao+:KI+) were used for most experiments. In control conditions, there was no obvious interaction between ICaL and SRCR. In isoproterenol, activation of ICaL from voltages between -70 and -50 mV reduced the amplitude and accelerated the decay of the current. Short (50 ms), small-amplitude voltage steps applied 60 or 510 ms before stimulating ICaL inhibited and facilitated the current, respectively. These changes were blocked by ryanodine. Low-voltage activated currents such as T-type Ca2+ current, TTX-sensitive ICa (ICaTTX), or "slip mode" Ca2+ conductance via INa+ were not responsible for low-voltage SRCR. However, L-type Ca2+ currents could be distinguished at voltages as negative as -45 mV. It is concluded that in the presence of isoproterenol, Ca2+ release from the SR at negative potentials is due to activation of L-type Ca2+ channels.