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The effects of enhanced late I Na , a persistent component of the Na + channel current, on the intracellular ion dynamics and the automaticity of the pulmonary vein cardiomyocytes were studied with fluorescent microscopy. Anemonia viridis toxin II (ATX- II), an enhancer of late I Na , caused increases in the basal Na + and Ca 2+ concentrations, increases in the number of Ca 2+ sparks and Ca 2+ waves, and the generation of repetitive Ca 2+ transients. These phenomena were inhibited by eleclazine, a blocker of the late I Na ; SEA0400, an inhibitor of the Na + /Ca 2+ exchanger (NCX); H89, a protein kinase A (PKA) inhibitor; and KN-93, a Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) inhibitor. These results suggest that enhancement of late I Na in the pulmonary vein cardiomyocytes causes disturbance of the intracellular ion environment through activation of the NCX and Ca 2+ -dependent enzymes. Such mechanisms are probably involved in the ectopic electrical activity of the pulmonary vein myocardium.

Background: Ceramide is involved in regulating metabolism and energy expenditure, and its abnormal myocardial accumulation may contribute to heart injury or lipotoxic cardiomyopathy. Whether ceramide can modulate the electrophysiology of pulmonary veins (PVs) remains unknown., Materials and Methods: We used conventional microelectrodes to measure the electrical activity of isolated rabbit PV tissue preparations before and after treatment with various concentrations of ceramide with or without H 2 O 2 (2 mM), MitoQ, wortmannin or 740 YP. A whole-cell patch clamp and fluorescence imaging were used to record the ionic currents, calcium (Ca 2+ ) transients, and intracellular reactive oxygen species (ROS) and sodium (Na + ) in isolated single PV cardiomyocytes before and after ceramide (1 μM) treatment., Results: Ceramide (0.1, 0.3, 1 and 3 μM) reduced the beating rate of PV tissues. Furthermore, ceramide (1 μM) suppressed the 2 mM H 2 O 2 -induced faster PV beating rate, triggered activities and burst firings, which were further reduced by MitoQ. In the presence of wortmannin, ceramide did not change the PV beating rate. The H 2 O 2 -induced faster PV beating rate could be counteracted by MitoQ or wortmannin with no additive effect from the ceramide. Ceramide inhibited pPI3K. Ceramide reduced Ca 2+ transients, sarcoplasmic reticulum Ca 2+ contents, L-type Ca 2+ currents, Na + currents, late Na + currents, Na + -hydrogen exchange currents, and intracellular ROS and Na + in PV cardiomyocytes, but did not change Na + -Ca 2+ exchange currents., Conclusion: C2 ceramide may exert the distinctive electrophysiological effect of modulating PV activities, which may be affected by PI3K pathway-mediated oxidative stress, and might play a role in the pathogenesis of PV arrhythmogenesis., (© 2021 Stichting European Society for Clinical Investigation Journal Foundation. Published by John Wiley & Sons Ltd.)

Delayed rectifier K + current (I Ks ) is a key contributor to repolarization of action potentials. This study investigated the mechanisms underlying the adrenoceptor-induced potentiation of I Ks in pulmonary vein cardiomyocytes (PVC). PVC were isolated from guinea pig pulmonary vein. The action potentials and I Ks current were recorded using perforated and conventional whole-cell patch-clamp techniques. The expression of I Ks was examined using immunocytochemistry and Western blotting. KCNQ1, a I Ks pore-forming protein was detected as a signal band approximately 100 kDa in size, and its immunofluorescence signal was found to be mainly localized on the cell membrane. The I Ks current in PVC was markedly enhanced by both β 1 - and β 2 -adrenoceptor stimulation with a negative voltage shift in the current activation, although the potentiation was more effectively induced by β 2 -adrenoceptor stimulation than β 1 -adrenoceptor stimulation. Both β-adrenoceptor-mediated increases in I Ks were attenuated by treatment with the adenylyl cyclase (AC) inhibitor or protein kinase A (PKA) inhibitor. Furthermore, the I Ks current was increased by α 1 -adrenoceptor agonist but attenuated by the protein kinase C (PKC) inhibitor. PVC exhibited action potentials in normal Tyrode solution which was slightly reduced by HMR-1556 a selective I Ks blocker. However, HMR-1556 markedly reduced the β-adrenoceptor-potentiated firing rate. The stimulatory effects of β- and α 1 -adrenoceptor on I Ks in PVC are mediated via the PKA and PKC signal pathways. HMR-1556 effectively reduced the firing rate under β-adrenoceptor activation, suggesting that the functional role of I Ks might increase during sympathetic excitation under in vivo conditions., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Introduction: Phosphodiesterase (PDE) isoform inhibitors have mechanical and electrical effects on the heart. Inhibition of PDE-1 enzymes is a novel strategy for treating heart failure. However, the electrophysiological effects of PDE-1 inhibition on the heart remain unclear. This study explored the effects of PDE-1 inhibition using ITI-214 on electrical activity in the pulmonary vein (PV), the most common trigger of atrial fibrillation, and investigated the underlying ionic mechanisms., Methods: Conventional microelectrodes or whole-cell patch clamps were employed to study the effects of ITI-214 (0.1-10 μM) on PV electrical activity, mechanical responses and ionic currents in isolated rabbit PV tissue specimens and isolated single PV cardiomyocytes., Results: ITI-214 at 1 μM and 10 μM (but not 0.1 μM) significantly reduced PV spontaneous beating rate (10 ± 2% and 10 ± 3%, respectively) and PV diastolic tension (11 ± 3% and 17 ± 3%, respectively). ITI-24 (1 μM) significantly reduced late sodium current (I Na-Late ), L-type calcium current (I Ca-L ) and the reverse mode of the sodium-calcium exchanger (NCX), but it did not affect peak sodium currents., Conclusions: ITI-214 reduces PV spontaneous activity and PV diastolic tension by reducing I Na-Late , I Ca-L and NCX current. Considering its therapeutic potential in heart failure, targeting PDE-1 inhibition may provide a novel strategy for managing atrial arrhythmogenesis., (© 2021 Stichting European Society for Clinical Investigation Journal Foundation. Published by John Wiley & Sons Ltd.)

Pulmonary vein (PV) cardiomyocytes have the potential to generate spontaneous activity, in contrast to working myocytes of atria. Different electrophysiological properties underlie the potential automaticity of PV cardiomyocytes, one being the hyperpolarization-activated inward current (I h ), which facilitates the slow diastolic depolarization. In the present study, we examined pharmacological characteristics of the I h of PV cardiomyocytes in rat, guinea pig and rabbit. The results showed that guinea pig and rat PV cardiomyocytes possessed sizeable amplitudes of the I h , and the I h of guinea pig was suppressed by Cs + , a blocker of the hyperpolarization-activated cation current. However, the I h of rat was not suppressed by Cs + , but by Cd 2+ , a blocker of the Cl - current. The current density of the I h of rabbit PV cardiomyocytes was significantly smaller than those of other species. This suggests that the ion channels that carry the I h of PV cardiomyocytes differ among the animal species.

Pulmonary veins (PVs) are the major origin of atrial fibrillation. Recently, we recorded hyperpolarization-activated Cl - current ( I Cl, h ) in rat PV cardiomyocytes. Unlike the well-known chloride channel protein 2 (CLCN2) current, the activation curve of I Cl, h was hyperpolarized as the Cl - ion concentration ([Cl - ] i ) increased. This current could account for spontaneous activity in PV cardiomyocytes linked to atrial fibrillation. In this study, we aimed to identify the channel underlying I Cl, h Using RT-PCR amplification specific for Clcn2 or its homologs, a chloride channel was cloned from rat PV and detected in rat PV cardiomyocytes using immunocytochemistry. The gene sequence and electrophysiological functions of the protein were identical to those previously reported for Clcn2 , with protein activity observed as a hyperpolarization-activated current by the patch-clamp method. However, the [Cl - ] i dependence of activation was entirely different from the observed I Cl, h of PV cardiomyocytes; the activation curve of the Clcn2 -transfected cells shifted toward positive potential with increased [Cl - ] i , whereas the I Cl, h of PV and left ventricular cardiomyocytes showed a leftward shift. Therefore, we used MS to explore the possibility of additional proteins interacting with CLCN2 and identified an individual 71-kDa protein, HSPA8, that was strongly expressed in rat PV cardiomyocytes. With co-expression of HSPA8 in HEK293 and PC12 cells, the CLCN2 current showed voltage-dependent activation and shifted to negative potential with increasing [Cl - ] i Molecular docking simulations further support an interaction between CLCN2 and HSPA8. These findings suggest that CLCN2 in rat heart contains HSPA8 as a unique accessory protein., (© 2019 Okamoto et al.)

The role of the Na + current in the automaticity of the pulmonary vein myocardium was examined in isolated guinea pig pulmonary vein cardiomyocytes and tissue preparations. Tetrodotoxin inhibited the automaticity of pulmonary vein tissue preparations by suppressing the diastolic depolarization of the action potential. ATX-II, which increased the density of persistent component of the Na + current (late I Na ), induced a depolarization of the resting membrane potential followed by spontaneous firing of action potentials. GS-458967, which inhibited the late I Na , suppressed the diastolic depolarization and the firing of action potentials. Pilsicainide, which inhibited only the transient component of Na + current (peak I Na ), had no effect on the firing frequency. GS-458967 had no effect on the contractile force of the working myocardium. In conclusion, late I Na is involved in the diastolic depolarization and automaticity of the pulmonary vein myocardium. Late I Na inhibitors appear to be effective therapeutic agents for atrial fibrillation with minimum adverse effects on the working myocardium., (Copyright © 2019 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

Cardiomyocytes and myocardial sleeves dissociated from pulmonary veins (PVs) potentially generate ectopic automaticity in response to noradrenaline (NA), and thereby trigger atrial fibrillation. We developed a mathematical model of rat PV cardiomyocytes (PVC) based on experimental data that incorporates the microscopic framework of the local control theory of Ca 2+ release from the sarcoplasmic reticulum (SR), which can generate rhythmic Ca 2+ release (limit cycle revealed by the bifurcation analysis) when total Ca 2+ within the cell increased. Ca 2+ overload in SR increased resting Ca 2+ efflux through the type II inositol 1,4,5-trisphosphate (IP 3 ) receptors (InsP 3 R) as well as ryanodine receptors (RyRs), which finally triggered massive Ca 2+ release through activation of RyRs via local Ca 2+ accumulation in the vicinity of RyRs. The new PVC model exhibited a resting potential of -68 mV. Under NA effects, repetitive Ca 2+ release from SR triggered spontaneous action potentials (APs) by evoking transient depolarizations (TDs) through Na + /Ca 2+ exchanger (AP TD s). Marked and variable latencies initiating AP TD s could be explained by the time courses of the α1- and β1-adrenergic influence on the regulation of intracellular Ca 2+ content and random occurrences of spontaneous TD activating the first AP TD . Positive and negative feedback relations were clarified under AP TD generation.

Mechanisms underlying ectopic activity in the pulmonary vein (PV) which triggers paroxysmal atrial fibrillation are unknown. Although several studies have suggested that calcium signalling might be involved in these arrhythmias, little is known about calcium cycling in PV cardiomyocytes (CM). We found that individual PV CM showed a wide range of transverse tubular incidence and organization, going from their virtual absence, as described in atrial CM, to well transversally organised tubular systems, like in ventricular CM. These different types of CM were found in groups scattered throughout the tissue. The variability of the tubular system was associated with cell to cell heterogeneity of calcium channel (Ca v 1.2) localisation and, thereby, of Ca v 1.2-Ryanodine receptor coupling. This was responsible for multiple forms of PV CM calcium transient. Spontaneous calcium sparks and waves were not only more abundant in PV CM than in LA CM but also associated with a higher depolarising current. In conclusion, compared with either the atrium or the ventricle, PV myocardium presents marked structural and functional heterogeneity.

The collar of the pulmonary vein (PV) is the focal point for the initiation of atrial arrhythmias, but the mechanisms underlying how PV cells differ from neighboring left atrial tissue are unclear. We examined the biophysical and molecular properties of I Na in cells isolated from the canine pulmonary sleeve and compared the properties to left atrial tissue. PV and left atrial myocytes were isolated and patch clamp techniques were used to record I Na . Action potential recordings from either tissue type were made using high-resistance electrodes. mRNA was determined using quantitative RT-PCR and proteins were determined by Western blot. Analysis of the action potential characteristics showed that PV tissue had a lower Vmax compared with left atrial tissue. Fast I Na showed that current density was slightly lower in PV cells compared with LA cells (-96 ± 18.7 pA/pF vs. -120 ± 6.7 pA/pF, respectively, p < 0.05). The recovery from inactivation of I Na in PV cells was slightly slower but no marked difference in steady-state inactivation was noted. Analysis of late I Na during a 225-ms pulse showed that late I Na was significantly smaller in PV cells compared to LA cells at all measured time points into the pulse. These results suggest PV cells have lower density of both peak and late I Na . Molecular analysis of Nav1.5 and the four beta subunits showed lower levels of Nav1.5 as well as Navβ1 subunits, confirming the biophysical findings. These data show that a lower density of I Na may lead to depression of excitability and predispose the PV collar to re-entrant circuits under pathophysiological conditions.

The electrophysiological properties underlying the automaticity of the guinea pig pulmonary vein myocardium were studied. About 30% of the isolated pulmonary vein tissue preparations showed spontaneous electrical activity, as shown by glass microelectrode recordings from their myocardial layer. The remaining quiescent preparations had a resting membrane potential less negative than that in the left atria. Blockade of the acetylcholine activated potassium current (I K-ACh ) by tertiapin induced a depolarizing shift of the resting membrane potential and automatic electrical activity in the pulmonary vein, but not in the atria. The tertiapin-induced electrical activity, as well as the spontaneous activity, was inhibited by the application of carbachol or by chelation of intracellular Ca 2+ by BAPTA. The isolated pulmonary vein cardiomyocytes had an I K-ACh density similar to that of the atrial cardiomyocytes, but a lower density of the inwardly-rectifying potassium current (I K1 ). Spontaneous Ca 2+ transients were observed in about 30% of the isolated pulmonary vein cardiomyocytes, but not in atrial cardiomyocytes. The Ca 2+ transients in the pulmonary vein cardiomyocytes were induced by tertiapin and inhibited by carbachol. These results indicate that the pulmonary vein cardiomyocytes have a reduced density of the inwardly-rectifying potassium current, which plays a permissive role in their intracellular Ca 2+ -dependent automaticity., (Copyright © 2017 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

Fibroblast growth factor 23 (FGF23), elevated in chronic renal failure, increases atrial arrhythmogenesis and dysregulates calcium homeostasis. Late sodium currents (INa-Late) critically induces ectopic activity of pulmoanry vein (the most important atrial fibrillation trigger). This study was to investigate whether FGF23 activates the INa-Late leading to calcium dysregulation and increases PV arrhythmogenesis. Patch clamp, western blot, and confocal microscopy were used to evaluate the electrical activities, calcium homeostasis, and mitochondrial reactive oxygen species (ROS) in PV cardiomyocytes with or without FGF23 (0.1 or 1 ng/mL) incubation for 4~6 h. Compared to the control, FGF23 (1 ng/mL, but not 0.1 ng/mL)-treated PV cardiomyocytes had a faster beating rate. FGF23 (1 ng/mL)-treated PV cardiomyocytes had larger INa-Late, calcium transients, and mitochondrial ROS than controls. However, ranolazine (an inhibitor of INa-Late) attenuated FGF23 (1 ng/mL)-increased beating rates, calcium transients and mitochondrial ROS. FGF23 (1 ng/mL)-treated PV cardiomyocytes exhibited larger phosphorylation of calcium/calmodulin-dependent protein kinase II (CaMKII). Chelerythrine chloride (an inhibitor of protein kinase C) decreased INa-Late in FGF23 (1 ng/mL)-treated PV cardiomyocytes. However, KN93 (a selective CaMKII blocker) decreased INa-Late in control and FGF23 (1 ng/mL)-treated PV cardiomyocytes to a similar extent. In conclusion, FGF23 increased PV arrhythmogenesis through sodium and calcium dysregulation by acting protein kinase C signaling.

The pulmonary vein has a unique electrophysiological property showing an autonomic electrical activity, and this phenomenon has been further focused on as a source of triggers of atrial fibrillation. The pulmonary vein cardiomyocytes have shorter action potential duration, less negative resting membrane potential, and smaller maximum upstroke velocity than those in the left atrium, whose underlying cellular mechanisms may generate arrhythmogenic substrates such as abnormal automaticity and triggered activity. In diseased conditions including sustained atrial tachycardia or chronic volume overload, its arrhythmogenic profile can be further modified through abbreviation of action potential duration of the pulmonary vein myocardium, which may become a cause of reentry. Recently, antiarrhythmic effects of various drugs have been extensively investigated in isolated pulmonary vein preparations. The present review article highlights the recent advances in our understanding of electrophysiological and pharmacological profiles of the pulmonary vein.

Pulmonary veins (PVs) are believed to be a crucial origin of atrial fibrillation. We recently reported that rat PV cardiomyocytes exhibit arrhythmogenic automaticity in response to norepinephrine. Herein, we further characterized the electrophysiological properties underlying the potential arrhythmogenicity of PV cardiomyocytes. Patch clamping studies revealed a time dependent hyperpolarization-activated inward current in rat PV cardiomyocytes, but not in left atrial (LA) myocytes. The current was Cs(+) resistant, and was not affected by removal of external Na(+) or K(+). The current was inhibited with Cd(2+), and the reversal potential was sensitive to changes in [Cl(-)] on either side of the membrane in a manner consistent with a Cl(-) selective channel. Cl(-) channel blockers attenuated the current, and slowed or completely inhibited the norepinephrine-induced automaticity. The biophysical properties of the hyperpolarization-activated Cl(-) current in rat PVs were different from those of ClC-2 currents previously reported: (i) the voltage-dependent activation of the Cl(-) current in rat PVs was shifted to negative potentials as [Cl(-)]i increased, (ii) the Cl(-) current was enhanced by extracellular acidification, and (iii) extracellular hyper-osmotic stress increased the current, whereas hypo-osmotic cell swelling suppressed the current. qPCR analysis revealed negligible ClC-2 mRNA expression in the rat PV. These findings suggest that rat PV cardiomyocytes possess a peculiar voltage-dependent Cl(-) channel, and that the channel may play a functional role in norepinephrine-induced automaticity., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Cardiomyocyte-like cells have been reported in thoracic veins of rodents and other mammals, but their differentiation state and relationship to the muscle mass in the heart remain to be characterized. Here we investigated the distribution, ultrastructure, expression and developmental regulation of myofilament proteins of mouse and rat pulmonary and azygos venous cardiomyocytes. Tracing cardiomyocytes in transgenic mouse tissues using a lacZ reporter gene driven by a cloned rat cardiac troponin T promoter demonstrated scattered distribution of cardiomyocytes discontinuous from the atrial sleeves. The longitudinal axis of venous cardiomyocytes is perpendicular to that of the vessel. These cells contain typical sarcomere structures and intercalated discs as shown in electron microscopic images, and express cardiac isoforms of troponin T, troponin I and myosin. The expression of troponin I isoform genes and the alternative splicing of cardiac troponin T in thoracic venous cardiomyocytes are regulated during postnatal development in precise synchrony with that in the heart. However, the patterns of cardiac troponin T splicing in adult rat thoracic venous cardiomyocytes are slightly but clearly distinct from those in the atrial and ventricular muscles. The data indicate that mouse and rat thoracic venous cardiomyocytes residing in extra-cardiac tissue possess a physiologically differentiated state and an intrinsically pre-set developmental clock, which are apparently independent of the very different hemodynamic environments and functional features of the vessels and heart., (© 2012 The Authors Journal compilation © 2012 FEBS.)

Background: Amyloid peptides modulate cardiac calcium homoeostasis and play an important role in the pathophysiology of atrial fibrillation. Pulmonary veins (PVs) are critical in the genesis of atrial fibrillation and contain abundant amyloid peptides. Therefore, the purpose of this study is to investigate whether amyloid peptides may change the PV electrical activity through regulating calcium homoeostasis., Methods and Results: The channel and calcium-handling protein expressions, intracellular calcium and ionic currents were studied in isolated rabbit PV cardiomyocytes in the presence and absence (control) of beta-amyloid (Aβ(25-35) ) for 4-6 h, using Western blot analysis, indo-1 fluorimetric ratio and whole-cell patch clamp techniques. Aβ(25-35) decreased the expressions of Ca(V) 1.2, total or Ser16-phosphorylated phospholamban (p-PLB), p-PLB/PLB ratio, sodium/calcium exchanger, but did not change ryanodine receptor, sarcoplasmic reticulum (SR) ATPase and K(+) channel proteins (Kir2.1, Kir2.3, Kv1.4, Kv1.5 and Kv4.2). Aβ(25-35) -treated cardiomyocytes had smaller calcium transient, SR calcium store, L-type calcium current and sodium/calcium exchanger current than control cardiomyocytes. Moreover, Aβ(25-35) -treated cardiomyocytes (n = 20) had shorter 90% of the action potential duration (82 ± 3 vs. 93 ± 5 ms, P < 0·05) than control cardiomyocytes (n = 16)., Conclusion: Aβ(25-35) has direct electrophysiological effects on PV cardiomyocytes., (© 2011 The Authors. European Journal of Clinical Investigation © 2011 Stichting European Society for Clinical Investigation Journal Foundation.)

Atrial fibrillation, the most common sustained arrhythmia, is believed to be triggered by ectopic electrical activity originating in the myocardial sleeves surrounding the pulmonary veins (PVs). It has been reported that myocardial sleeves have the potential to generate automaticity in response to norepinephrine. This study investigated the cellular mechanisms underlying norepinephrine-induced automaticity in PV cardiomyocytes isolated from rats. Application of 10 μM norepinephrine to PV cardiomyocytes induced repetitive and transient increases in intracellular Ca(2+) concentrations. The Ca(2+) transient was accompanied by depolarization, and induced automatic rhythmic action potentials at approximately 4Hz in perforated patch clamp preparations in 27% of myocytes were observed. When the recording mode was switched from current-clamp to voltage-clamp mode during the continuous presence of automaticity, an oscillatory current was observed. The oscillatory current was always inward, irrespective of the membrane potential, indicating that the current was derived mainly from the Na(+)-Ca(2+) exchanger (NCX). The norepinephrine-induced automaticity was suppressed by blocking either the β(1)- or α(1)-adrenoceptor. Additionally, this automaticity was blocked by inhibitors of phospholipase C and the inositol 1,4,5-triphosphate receptor (IP(3)R) but not by a protein kinase C inhibitor. We observed that the transverse-tubule system was enriched in cardiomyocytes in the PV, in contrast to those of the atrium, and that the NCX and IP(3)R were co-localized along transverse tubules. These findings suggest that a functional coupling between the NCX and IP(3)R causes arrhythmic excitability of the PV during the presence of combined β(1)- and α(1)-adrenoceptor stimulation., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Ectopic foci originating from the pulmonary veins (PVs) have been suggested as the underlying cause for generating atrial arrhythmias that include atrial fibrillation (AF). Recent experimental findings indicate two types of PV cells: pacemaking and non-pacemaking. In this study, we have developed two mathematical models for human PV cardiomyocytes with and without pacemaking activities. The models were reconstructed by modifying an existing model of the human right atrium to incorporate extant experimental data on the electrical differences between the two cell types. Differences in their action potential (AP) profiles and automaticity were reproduced by the models, which can be attributed to the observed differences in the current densities of I(NCX), I(to), I(Na) and I(Ca-L), as well as the difference in the channel kinetics of I(Ca-L) and inclusion of the I(f) and I(Ca-T) currents in the pacemaking cells. The developed models provide a useful tool suitable for studying the substrates for generating AF.

Cardiomyocytes consist of single- and bi-nucleus myocytes. However, the electrophysiological characteristics of mononucleated and binucleated myocytes have never been elucidated. Left atrium (LA) and pulmonary veins (PVs) are important substrate and initiators of atrial fibrillation. The purposes of this study were to evaluate the electrical properties and calcium homeostasis in mononucleated and binucleated cardiomyocytes in the LA and PVs. A whole-cell clamp, fluo-4 fluorescence, and immunocytostaining were used to investigated mononucleated and binucleated cardiomyocytes in the LA and PVs. Both mononucleated PV and LA cardiomyocytes had more positive resting membrane potential than respective PV and LA binucleated cardiomyocytes. Additionally, mononucleated PV cardiomyocytes (n = 36) had faster beating rates (2.1 ± 0.2 Hz versus 1.0 ± 0.2 Hz, P < 0.05) than binucleated (n = 10) PV cardiomyocytes. The PV (n = 18) and LA (n = 15) mononucleated cardiomyocytes had larger [Ca²⁺](i) transients (F/F₀ 1.64 ± 0.09 versus 1.20 ± 0.03 IU, P < 0.05; 1.52 ± 0.06 versus 1.19 ± 0.05 IU, P < 0.05) than the binucleated PV (n = 10) and LA (n = 10) cardiomyocytes. The immunostaining showed that mononucleated cardiomyocytes had lower Kir 2.3 and higher ryanodine receptor densities than did binucleated cardiomyocytes both in the PV and LA. In conclusions, mononucleated PV and LA cardiomyocytes contain distinctive electrophysiological characteristics with a higher arrhythmogenic activity, which indicates that cell nucleus number may potentially determine the electrical activity and calcium handling in cardiomyocytes., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Pulmonary veins (PVs) contain cardiomyocytes with a complex cellular morphology and high arrhythmogenesis. Ca(2+) regulation and Ca(2+) sparks play a pivotal role in the electrical activity of cardiomyocytes. The purpose of this study was to investigate whether the cell morphology can determine the PV electrical activity and Ca(2+) homeostasis. Through confocal microscopy with fluo-3 Ca(2+) fluorescence, Ca(2+) sparks and Ca(2+) transients were evaluated in isolated single rabbit left atria (LA) and PV cardiomyocytes according to the cell morphology (rod, rod-spindle and spindle/bifurcated). Twenty-two (20%) rod, 49 (43%) rod-spindle and 41 (37%) spindle/bifurcated cardiomyocytes were identified in the LA (n = 29) and PV (n = 83) cardiomyocytes. The PV cardiomyocytes with pacemaker activity had a higher incidence of spindle/bifurcated morphology than LA and PV cardiomyocytes without pacemaker activity. As compared to those in the rod or rod-spindle cardiomyocytes, spindle/bifurcated cardiomyocytes had a larger Ca(2+) transient amplitude and higher frequency of the Ca(2+) sparks with larger amplitude and longer duration. In contrast, rod-spindle and rod cardiomyocytes had similar Ca(2+) transients and Ca(2+) sparks. The cell length correlated well with the amplitude of the Ca(2+) transient and Ca(2+) spark duration with a linear regression. In conclusion, cell morphology and cell length play a potential role in the Ca(2+) homeostasis and Ca(2+) spark. The large Ca(2+) transients and high frequency of Ca(2+) sparks in spindle/bifurcated cardiomyocytes may cause a high arrhythmogenesis in the PV cardiomyocytes with pacemaker activity.

Background: Pulmonary veins (PVs) are the most important sources of ectopic beats with the initiation of paroxysmal atrial fibrillation, or the foci of ectopic atrial tachycardia and focal atrial fibrillation. Elimination of nitric oxide (NO) enhances cardiac triggered activity, and NO can decrease PV arrhythmogenesis through mechano-electrical feedback. However, it is not clear whether NO may have direct electrophysiological effects on PV cardiomyocytes. This study is aimed to study the effects of nitroprusside (NO donor), on the ionic currents and arrhythmogenic activity of single cardiomyocytes from the PVs., Methods: Single PV cardiomyocytes were isolated from the canine PVs. The action potential and ionic currents were investigated in isolated single canine PV cardiomyocytes before and after sodium nitroprusside (80 muM,) using the whole-cell patch clamp technique., Results: Nitroprusside decreased PV cardiomyocytes spontaneous beating rates from 1.7 +/- 0.3 Hz to 0.5 +/- 0.4 Hz in 9 cells (P < 0.05); suppressed delayed after depolarization in 4 (80%) of 5 PV cardiomyocytes. Nitroprusside inhibited L-type calcium currents, transient outward currents and transient inward current, but increased delayed rectified potassium currents., Conclusion: Nitroprusside regulates the electrical activity of PV cardiomyocytes, which suggests that NO may play a role in PV arrhythmogenesis.

Background: Several lines of evidence point to a particularly important role of the left atrium (LA) in initiating and maintaining atrial fibrillation (AF). This role may be related to the location of pulmonary veins (PVs) in the LA. The aim of the present study was to investigate the action potential (AP) and ionic currents in LA-PV cardiomyocytes isolated from Bio14.6 myopathic Syrian hamsters (36-57 week-old) versus age-matched F1B healthy control hamsters., Methods and Results: Whole-cell patch-clamp techniques were used to record AP in current-clamp mode and ionic currents in voltage-clamp mode. The results obtained show that in both healthy and myopathic LA-PV tissue spontaneously discharging cardiomyocytes can be found, but they are more numerous in myopathic (9/29) than in healthy hamsters (4/42, p < 0.05 by chi2 analysis). Myopathic myocytes have shorter AP duration (APD) with smaller ICa,L and INCX than the healthy control. The currents ITO, IK, IK1 and ICa,T are not significantly different in myopathic versus healthy cells., Conclusions: Our results indicate that in myopathic Syrian hamsters LA-PV cardiomyocytes are more prone to automatic rhythms. Also, they show altered electrophysiologic properties, which may be due to abnormal Ca2+ channels and may account for contractile dysfunction.

Objective: To investigate the effects of ShenSongYangXin on action potential and some current channels in isolated rabbit pulmonary vein cardiomyocytes and to elucidate its possible mechanism of curing atrial fibrillation., Methods: Action potentials as well as L-type calcium channel current (I(Ca-L)), transient outward potassium current (I(K1)) and inward rectifier potassium current (I(To)) were recorded using whole cell patch-clamp technique., Results: ShenSongYangXin prolonged the action potential duration (APD) significantly, APD90 was enlarged from (187 +/- 49) ms to (286 +/- 76) ms at the concentration of 5 mg/ml and to (312 +/- 82) ms at the concentration of 10 mg/dl respectively (P < 0.05). It also depressed the current of I(Ca-L), I(K1) and Iro in a concentration-dependent manner., Conclusion: ShenSongYangXin can be used to treat atrial fibrillation through its actions on multiple current channels.

Pulmonary veins (PVs) contain cardiomyocytes with spontaneous activity that may be responsible for PV arrhythmia. Abnormal Ca(2+) regulation is known to contribute to PV arrhythmogenesis. The purpose of this study was to investigate whether PV cardiomyocytes with spontaneous activity have different intracellular Ca(2+) ([Ca(2+)](i)) transients, Ca(2+) sparks and responses to isoproterenol and ryanodine receptor modulators (magnesium and FK506) than do PV cardiomyocytes without spontaneous activity and left atrial (LA) cardiomyocytes. Through fluorescence and confocal microscopy, we evaluated the [Ca(2+)](i) transients and Ca(2+) sparks in isolated rabbit PV and LA cardiomyocytes. PV cardiomyocytes with spontaneous activity had larger [Ca(2+)](i) transients and sarcoplasmic reticulum (SR) Ca(2+) stores than PV cardiomyocytes without spontaneous activity or LA cardiomyocytes. PV cardiomyocytes with spontaneous activity also had a higher incidence and frequency of Ca(2+) sparks, and had Ca(2+) sparks with larger amplitudes than other cardiomyocytes. Magnesium (5.4 mM) reduced the [Ca(2+)](i) transient amplitude and beating rate in PV cardiomyocytes with spontaneous activity. However, in contrast with other cardiomyocytes, low doses (1.8 mM) of magnesium did not reduce the [Ca(2+)](i) transients amplitude in PV cardiomyocytes with spontaneous activity. FK506 (1 microM) diminished the SR Ca(2+) stores in PV cardiomyocytes with spontaneous activity to a lesser extent than that in other cardiomyocytes. Isoproterenol (10 nM) increased the [Ca(2+)](i) transient amplitude to a lesser extent in LA cardiomyocytes than in PV cardiomyocytes with or without spontaneous activity. In conclusion, our results suggest that enhanced [Ca(2+)](i) transients, increased Ca(2+) sparks and SR Ca(2+) stores may contribute to the spontaneous activity of PV cardiomyocytes.

Evidence is growing of a relationship between atrial dilation and atrial fibrillation (AF), the most prevalent type of arrhythmia. Pulmonary veins, which are important ectopic foci for provoking AF, are of increasing interest in relation to the early development of AF. Here, using single cardiomyocytes isolated from rabbit pulmonary veins, we characterised the stretch-activated currents induced by swelling and axial mechanical stretching. Swelling induced both a stretch-activated nonselective cationic current (NSC) and a Cl(-) current. The swelling-induced Cl(-) current (I Cl,swell) was inhibited by DIDS, whereas the swelling-induced NSC (I NSC,swell) was inhibited by Gd3+. The cationic selectivity of the I NSC,swell was K+ >Cs+ >Na+ >Li+, whilst the PK/PNa, PCs/PNa, and PLi/PNa permeability ratios were 2.84, 1.86, and 0.85, respectively. Activation of the I NSC,swell was faster than that of the I Cl,swell. Given a high K+ concentration in the bath solution, the I NSC,swell showed limited amplitude (<-70 mV). Mechanical stretching induced an immediate Gd3+- and streptomycin-sensitive NSC (I NSC,stretch) that was permeable to Na+, K+, Cs+ and NMDG. Persistent stretching activated a DIDS-sensitive current (I Cl,stretch). The I NSC,stretch, but not the I NSC,swell, was completely blocked by 400 microM streptomycin; therefore, the two currents may not be associated with the same channel. In addition, the type of current induced may depend on the type of stretching. Thus, stretch-induced anionic and cationic currents are functionally present in the cardiomyocytes of the main pulmonary veins of rabbits, and they may have pathophysiological roles in the development of AF under stretched conditions.

Atrial fibrillation is the most prevalent arrhythmia, but the mechanisms by which it develops are not clear. Recently, over 90% of paroxysmal atrial fibrillation was found to be located inside the main pulmonary veins (PVs). We found that single cardiac myocytes isolated from the main PVs of rabbits generate spontaneous action potentials (SAP). We therefore assayed the electrical characteristics of these cardiomyocytes. Among the diverse ionic currents identified were INa, ICa,L, IK1, IKr, IKs, Ito, IKsus, Incx, Ipump, IKH and ICl,Ca. In contrast, IK1 was minimal, IKs could be detected only in the presence of 10 microM forskolin, and we were unable to detect If and ICa,T, the most important currents for pacemaking activity in sinoatrial node cells. To identify the main cause of SAP, we developed a model that can explain the electrical properties of these cardiomyocytes. After reconstructing the ionic currents based on experimental observations, we were able to use our model to successfully reconstruct the characteristics of the SAP of PV cardiomyocytes. The simulation showed that the major currents contributing to pacemaking depolarization were ICaL, IKr, a background current and Na+-K+ pump current. Deactivation kinetics of IKr was one of the major determinants of the rate of pacemaking depolarization. The steady state inactivation of Ito was shifted to the negative voltage and the activity of Ito was minimal in the range of the SAP. The major currents for the repolarization were IKr and Ipump. The amplitude of most currents in these cardiac myocytes was small and no currents did not exceed 30 pA during the SAP, indicating that slight activation of other inward or outward currents will have profound effects on the SAP. To our knowledge, this report is the first to show the simulation of SAP of PV cardiomyocytes. This model may help to study on the electrophysiological basis of paroxysmal atrial fibrillation originating from PVs.

Cardiac tissue in the pulmonary vein sleeves plays an important role in clinical atrial fibrillation. Mechanisms leading to pulmonary vein activity in atrial fibrillation remain unclear. Indirect experimental evidence points to pulmonary vein Ca(2+) handling as a potential culprit, but there are no direct studies of pulmonary vein cardiomyocyte Ca(2+) handling in the literature. We used the Ca(2+)-sensitive dye indo-1 AM to study Ca(2+) handling in isolated canine pulmonary vein and left atrial myocytes. Results were obtained at 35 degrees C and room temperature in cells from control dogs and in cardiomyocytes from dogs subjected to 7-day rapid atrial pacing. We found that basic Ca(2+)-transient properties (amplitude: 186 +/- 28 vs. 216 +/- 25 nM; stimulus to half-decay time: 192 +/- 9 vs. 192 +/- 9 ms; atria vs. pulmonary vein, respectively, at 1 Hz), beat-to-beat regularity, propensity to alternans, beta-adrenergic response (amplitude increase at 0.4 Hz: 96 +/- 52 vs. 129 +/- 61%), number of spontaneous Ca(2+)-transient events after Ca(2+) loading (in normal Tyrode: 0.9 +/- 0.2 vs. 1.3 +/- 0.2; with 1 microM isoproterenol: 7.6 +/- 0.3 vs. 5.1 +/- 1.8 events/min), and caffeine-induced Ca(2+)-transient amplitudes were not significantly different between atrial and pulmonary vein cardiomyocytes. In an arrhythmia-promoting model (dogs subjected to 7-day atrial tachypacing), Ca(2+)-transient amplitude and kinetics were the same in cells from both pulmonary veins and atrium. In conclusion, the similar Ca(2+)-handling properties of canine pulmonary vein and left atrial cardiomyocytes that we observed do not support the hypothesis that intrinsic Ca(2+)-handling differences account for the role of pulmonary veins in atrial fibrillation.

In recent studies, we recorded transiently activated outward currents by the application of three-step voltage pulses to induce a reverse mode of Na+-Ca2+ exchange (NCX). We found that these currents were mediated by a Ca2+-activated Cl- current. Based on the recent reports describing the atrial Ca2+ transients, the Ca2+ transient at the subsarcolemmal space was initiated and then diffused into the cytosolic space. Because the myocardium in the pulmonary vein is an extension of the atrium, the Ca2+-activated Cl- current may reflect the subsarcolemmal Ca2+ dynamics. We tried to predict the subsarcolemmal Ca2+ dynamics by simulating these current traces. According to recent reports on the geometry of atrial myocytes, we assumed that there were three compartments of sarcoplasmic reticulum (SR): a network SR, a junctional SR and a central SR. Based on these structures, we also divided the cytosolic space into three compartments: the junctional, subsarcolemmal and cytosolic spaces. Geometry information and cellular capacitance suggested that there were essentially no T-tubules in these cells. The basic physical data, such as the compartmental volumes, the diffusion coefficients and the stability coefficients of the Ca2+ buffers, were obtained from the literature. In the simulation, we incorporated the NCX, the L-type Ca2+ channel, the rapid activating outward rectifier K+ channel, the Na+-K+ pump, the SR Ca2+-pump, the ryanodine receptor, the Ca2+-activated Cl- channel and the dynamics of Na+, K+, Ca2+ and Cl-. In these conditions, we could successfully reconstruct the Ca2+-activated Cl- currents. The simulation allowed estimation of the Ca2+ dynamics of each compartment and the distribution of the Ca2+-activated Cl- channel and the NCX in the sarcolemma on the junctional or subsarcolemmal space.

To investigate the characteristics of action potentials and their ionic mechanism in cardiomyocytes from rabbit pulmonary vein sleeves (PVC), and to compare them with those in left atrial cardiomyocytes (LAC), the technique of whole-cell patch clamp was applied. We used current-clamp technique to record action potentials, and voltage-clamp technique to record ionic currents. PVC had longer action potential duration (APD) than LAC, and therefore a second plateau response could be induced easily, suggesting a strong tendency of early afterdepolarization (EAD) genesis in PVC. Non-selective cation current (I(NSCC)) was first recorded in both LAC and PVC. This I(NSCC)was permeable to K(+), Na(+) and Cs(+), sensitive to GdCl3 but not sensitive to 4-AP. The current densities of inward rectifier potassium current (I(K1)), transient outward potassium current (I(To)) and I(NSCC) were all significantly less in PVC than those in LAC. These differences in repolarizing ionic currents between PVC and LAC form a basis of the differences in their action potential configurations and might be an important ionic mechanism of the arrhythmogenic characteristics of pulmonary vein muscle sleeves.

Background: Myocardial cells in the pulmonary veins (PVs) are thought to play a major role in the initiation and maintenance of atrial arrhythmias, including atrial fibrillation. However, systematic single-cell microelectrode recordings from different regions in intact PV-atrial tissues are lacking., Objectives: The purpose of this study was to determine the transmembrane action potential properties of myocardial cells in different regions of the PV and the left atrium (LA) and assess their arrhythmogenic potential during perfusion with isoproterenol (ISO) and rapid atrial pacing., Methods: Glass microelectrode recordings of action potentials were made from the left PV and the LA in Langendorff-perfused young (3-4 month) male rats (Fisher344) (n = 9)., Results: Action potential duration (APD) of atrial and PV cells had similar duration at a pacing cycle length (CL) of 200 ms. However, shortening of the pacing CL to 100 ms led to heterogeneous repolarization of PV cells. Mid-PV cells had a significantly higher maximum slope of APD restitution than atrial or other PV sites. Intra-PV conduction block developed at rates when LA and proximal PV cells manifested 1:1 capture. Perfusion of ISO and rapid atrial pacing promoted the emergence of early afterdepolarization (EAD) and triggered beats in two out of nine tissues, causing premature atrial activation. No difference in resting potential or AP amplitude could be detected among the PV and LA cells., Conclusions: PV myocardial cells develop marked heterogeneity in repolarization, and there is a slight ease of developing EAD and triggered activity in response to rapid pacing and ISO infusion.

Background: Cardiomyocytes in pulmonary vein (PV) sleeves are important in atrial fibrillation (AF), but underlying mechanisms are poorly understood. Pulmonary veins have different ionic current properties compared to left atrium, with pulmonary vein inward-rectifier currents being smaller and delayed-rectifier currents larger than in left atrium., Methods: Expression and distribution of the inward-rectifier subunits Kir2.1 and Kir2.3, the rapid delayed-rectifier alpha-subunit ERG, the slow delayed-rectifier alpha-subunit KvLQT1, the beta-subunit minK, the L-type Ca(2+)-subunit Ca(v)1.2, and the Na(+),Ca(2+)-exchanger were quantified by Western blot on isolated cardiomyocytes and localized by immunohistochemistry in tissue sections obtained from canine hearts., Results: Western blotting indicated significantly greater expression of ERG (by 28%, P<0.05) and KvLQT1 (by 34%, P<0.05) in pulmonary vein versus left atrial (LA) cardiomyocytes, but smaller Kir2.3 and similar Kir2.1, Ca(v)1.2 and Na(+),Ca(2+)-exchanger expression in PV. Kir2.1 exhibited weak transverse tubular distribution in both regions. Kir2.3 localized to intercalated disks in both regions, and to transverse tubules in left atrium but not pulmonary vein. ERG staining was more intense in pulmonary vein than left atrium, localizing to transverse tubules in both regions and intercalated disks in pulmonary veins. KvLQT1 was more intensely expressed in pulmonary veins, with a transverse tubular and intercalated disk localization, versus a more diffuse signal in left atrium. The Na(+),Ca(2+)-exchanger localized to transverse tubules, plasma membranes and intercalated disks with similar intensity in each region., Conclusions: Greater ERG and KvLQT1 abundance in pulmonary vein cardiomyocytes, lower abundance of Kir2.3 in pulmonary veins and differential pulmonary vein subcellular distribution of Kir2.3, ERG and KvLQT1 subunits may contribute to ionic current differences between pulmonary vein and left atrial cardiomyocytes.

Ethanol consumption has been considered to contribute to the occurrences of paroxysmal atrial fibrillation. Pulmonary veins are known to initiate atrial fibrillation. This study investigated whether ethanol may induce atrial fibrillation through increasing arrhythmogenic activity of pulmonary vein cardiomyocytes. Using the whole-cell clamp technique, the action potential and ionic currents were investigated in rabbit single pulmonary vein beating cardiomyocytes with and without (control) incubation of ethanol. Compared with control cardiomyocytes, pulmonary vein cardiomyocytes receiving 0.3 mg/ml or 1 mg/ml ethanol had shorter action potential duration, but had similar beating rates (2.6+/-1.3, 2.7+/-1.2, 2.7+/-1.2 Hz) and incidences (45%, 41%, 32%) of delayed after depolarization. Pulmonary vein cardiomyocytes receiving ethanol had smaller L-type Ca(2+) currents and larger transient outward currents, but had similar transient inward, delayed rectified outward, inward rectified and pacemaker currents. These results suggest that ethanol has no direct effect on the arrhythmogenic potential of pulmonary vein cardiomyocytes.

Pulmonary vein (PV) cardiomyocytes play an important role in atrial fibrillation; however, little is known about their specific cellular electrophysiological properties. We applied standard microelectrode recording and whole-cell patch-clamp to evaluate action potentials and ionic currents in canine PVs and left atrium (LA) free wall. Resting membrane potential (RMP) averaged -66 +/- 1 mV in PVs and -74 +/- 1 mV in LA (P < 0.0001) and action potential amplitude averaged 76 +/- 2 mV in PVs vs. 95 +/- 2 mV in LA (P < 0.0001). PVs had smaller maximum phase 0 upstroke velocity (Vmax: 98 +/- 9 vs. 259 +/- 16 V s(-1), P < 0.0001) and action potential duration (APD): e.g. at 2 Hz, APD to 90% repolarization in PVs was 84 % of LA (P < 0.05). Na+ current density under voltage-clamp conditions was similar in PV and LA, suggesting that smaller Vmax in PVs was due to reduced RMP. Inward rectifier current density in the PV cardiomyocytes was approximately 58% that in the LA, potentially accounting for the less negative RMP in PVs. Slow and rapid delayed rectifier currents were greater in the PV (by approximately 60 and approximately 50 %, respectively), whereas transient outward K+ current and L-type Ca2+ current were significantly smaller (by approximately 25 and approximately 30%, respectively). Na(+)-Ca(2+)-exchange (NCX) current and T-type Ca2+ current were not significantly different. In conclusion, PV cardiomyocytes have a discrete distribution of transmembrane ion currents associated with specific action potential properties, with potential implications for understanding PV electrical activity in cardiac arrhythmias.