Pulmonary Vein Cardiomyocytes Research Articles

Enhanced Late INa Induces Intracellular Ion Disturbances and Automatic Activity in the Guinea Pig Pulmonary Vein Cardiomyocytes.

The effects of enhanced late INa, 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 INa, caused increases in the basal Na+ and Ca2+ concentrations, increases in the number of Ca2+ sparks and Ca2+ waves, and the generation of repetitive Ca2+ transients. These phenomena were inhibited by eleclazine, a blocker of the late INa; SEA0400, an inhibitor of the Na+/Ca2+ exchanger (NCX); H89, a protein kinase A (PKA) inhibitor; and KN-93, a Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor. These results suggest that enhancement of late INa in the pulmonary vein cardiomyocytes causes disturbance of the intracellular ion environment through activation of the NCX and Ca2+-dependent enzymes. Such mechanisms are probably involved in the ectopic electrical activity of the pulmonary vein myocardium.

Circadian rhythm in pro-arrhythmic activity of pulmonary vein cardiomyocytes

Abstract Introduction Cardiomyocytes in the pulmonary vein (PV) sleeves are a major source of ectopic activity driving atrial fibrillation (AF) and episodes of AF in patients are more prevalent at night. While it is known that circadian clocks within the heart regionally control 24-hour variation in pacemaking and ventricular repolarisation, the mechanisms underlying the nighttime preponderance in AF are unknown. Purpose This study addresses the hypothesis that circadian rhythms exist in the pro-arrhythmic activity of PV cardiomyocytes. Methods Male Wistar rats maintained in a 24-hour cycle of 12-hr light/dark (lights-on at Zeitgeber time, ZT=0; lights-off, ZT12) were subject to terminal general anaesthesia (140 mg/kg Na pentobarbital i.p.) at ZT=0, 6, 12 or 18 hr and the hearts removed. RNA was extracted from left atrial (LA) appendage (LAA) and proximal PV at the LA/PV junction (3 rats per ZT) and RNA sequencing conducted. Reads were mapped to the rat genome, counts normalised and models in which ZT was or was not included as a factor compared (Likelihood Ratio Test, adjusted-P<0.01). Whole-cell current clamp recordings were made from cardiomyocytes isolated from the proximal PV (n=308) and the LAA (n=264) (N=74 rats). The effects of noradrenaline (NA, 1 µM) and acetylcholine (ACh, 1 µM) were examined. Data were plotted against the ZT of the time of recording and fitted to a sine wave to establish circadian rhythmicity (P<0.05, extra-sum-of-squares F-test). The effect of ≥24 hr constant dark in the period immediately before experiment was examined. Data are reported as mean ± standard error. Results The expression of 1368 genes varied significantly with ZT, including circadian clock components (e.g. Bmal1, Per1). PV cells were larger than LAA cells (73±1.6 pF vs 53±1.3 pF, P<0.0001) and had more depolarised resting membrane potential (-69±0.2 mV vs -72±0.1 mV, P<0.0001). Both cell types showed circadian variation in action potential duration at 90% repolarisation (APD90) and frequency of pro-arrhythmic activity. Pro-arrhythmic activity was greatest in PV cells and the frequency was greater during the rest phase (ZT0-12) in both cell types. In contrast, the circadian rhythm in APD90 differed between cell type, with the longest APD­­90 recorded during the active phase in PV cells (ZT12-24) but during the rest phase in LAA cells. Pro-arrhythmic activity was increased by NA and decreased by ACh in both cell types with maximal effect during the rest phase. Conclusion Circadian variation in APD90 and proarrhythmic-activity has been demonstrated in isolated proximal PV and LAA cardiomyocytes, with differences in rhythm between the two cell types. RNA sequencing suggests the presence of peripheral clocks in LAA and PV cardiomyocytes. Understanding these mechanisms is important in development of future therapeutic options for AF.

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.

Inhibitory Effect of a Late Sodium Current Blocker, NCC-3902, on the Automaticity of the Guinea Pig Pulmonary Vein Myocardium.

The effect of blocking the persistent component of the sodium channel current (late INa) on the automatic activity of the isolated guinea pig pulmonary vein myocardium was examined. NCC-3902 blocked late INa, but did not affect other major ion channel currents stably expressed in cell lines. In isolated pulmonary vein cardiomyocytes, NCC-3902 blocked the late INa induced by a ramp depolarizing voltage clamp pulse similar to that of the pacemaker depolarization observed in the pulmonary vein myocardium. In isolated pulmonary vein tissue, NCC-3902 decreased the frequency of automatic firing of the myocardium through a reduction of the pacemaker depolarization slope. In isolated pulmonary vein cardiomyocytes, NCC-3902 significantly reduced the firing frequency of Ca2+ transients, but had no effect on Ca2+ sparks. NCC-3902 affected neither the spontaneous beating rate of the right atrium nor the contractile force of the ventricular myocardium. Selective blockers of late INa like NCC-3902, which inhibit the automatic activity of the pulmonary vein myocardium, appear to be promising as drugs for the pharmacological treatment of atrial fibrillation.

Effect of macrophage migration inhibitory factor on pulmonary vein arrhythmogenesis through late sodium current.

Macrophage migration inhibitory factor (MIF), a pleiotropic inflammatory cytokine, is highly expressed in patients with atrial fibrillation (AF). Inflammation increases the risk of AF and is primarily triggered by pulmonary vein (PV) arrhythmogenesis. This study investigated whether MIF can modulate the electrical activity of the PV and examined the underlying mechanisms of MIF. A conventional microelectrode, a whole-cell patch clamp, western blotting, and immunofluorescent confocal microscopy were used to investigate electrical activity, calcium (Ca2+) regulation, protein expression, ionic currents, and cytosolic reactive oxygen species (ROS) in rabbit PV tissue and isolated single cardiomyocytes with and without MIF incubation (100 ng/mL, treated for 6 h). The MIF (100 ng/mL)-treated PV tissue (n = 8) demonstrated a faster beating rate (1.8 ± 0.2 vs. 2.6 ± 0.1 Hz, P < 0.05), higher incidence of triggered activity (12.5 vs. 100%, P < 0.05), and premature atrial beat (0 vs. 100%, P < 0.05) than the control PV tissue (n = 8). Compared with the control PV cardiomyocytes, MIF-treated single PV cardiomyocytes had larger Ca2+ transients (0.6 ± 0.1 vs. 1.0 ± 0.1, ΔF/F0, P < 0.05), sarcoplasmic reticulum Ca2+ content (0.9 ± 0.20 vs. 1.7 ± 0.3 mM of cytosol, P < 0.05), and cytosolic ROS (146.8 ± 5.3 vs. 163.7 ± 3.8, ΔF/F0, P < 0.05). Moreover, MIF-treated PV cardiomyocytes exhibited larger late sodium currents (INa-Late), L-type Ca2+ currents, and Na+/Ca2+ exchanger currents than the control PV cardiomyocytes. KN93 [a selective calcium/calmodulin-dependent protein kinase II (CaMKII) blocker, 1 μM], ranolazine (an INa-Late inhibitor, 10 μM), and N-(mercaptopropionyl) glycine (ROS inhibitor, 10 mM) reduced the beating rates and the incidence of triggered activity and premature captures in the MIF-treated PV tissue. Macrophage migration inhibitory factor increased PV arrhythmogenesis through Na+ and Ca2+ dysregulation through the ROS activation of CaMKII signalling, which may contribute to the genesis of AF during inflammation. Anti-CaMKII treatment may reverse PV arrhythmogenesis. Our results clearly reveal a key link between MIF and AF and offer a viable therapeutic target for AF treatment.

Decoding the PITX2-controlled genetic network in atrial fibrillation.

Atrial fibrillation (AF), the most common sustained cardiac arrhythmia and a major risk factor for stroke, often arises through ectopic electrical impulses derived from the pulmonary veins (PVs). Sequence variants in enhancers controlling expression of the transcription factor PITX2, which is expressed in the cardiomyocytes (CMs) of the PV and left atrium (LA), have been implicated in AF predisposition. Single nuclei multiomic profiling of RNA and analysis of chromatin accessibility combined with spectral clustering uncovered distinct PV- and LA-enriched CM cell states. Pitx2-mutant PV and LA CMs exhibited gene expression changes consistent with cardiac dysfunction through cell type–distinct, PITX2-directed, cis-regulatory grammars controlling target gene expression. The perturbed network targets in each CM were enriched in distinct human AF predisposition genes, suggesting combinatorial risk for AF genesis. Our data further reveal that PV and LA Pitx2-mutant CMs signal to endothelial and endocardial cells through BMP10 signaling with pathogenic potential. This work provides a multiomic framework for interrogating the basis of AF predisposition in the PVs of humans.

Characterization of constitutively active GIRK ion channels in rat pulmonary vein

In human, pulmonary vein (PV) cardiomyocytes (CM) generate abnormal electrical activity, which is responsible for the triggering of atrial fibrillation. In rat PV, activation of α1-adrenergic receptors results in the depolarization of PV CM that lead to the inhibition of electrical conduction from PV toward left atria (LA). The mechanism underlying the depolarization remain unknown but might involve G-protein activated K+ channels (GIRK) which is responsible for the IkAch current. The aim of this study was to characterize and compare IkAch in PV and LA CM. All experiments were carried out in rat. Patch clamp technique in whole cell configuration was used to measure background and IkAch currents in CM. Membrane potential of PV and LA CM was recorded in tissues using sharp microelectrodes. The expression of Kir3.1 and Kir3.4 subunits of GIRK channels was quantified by western blot experiments. In patch clamp experiments, carbachol (Cch) (1 μM) induced the activation of an inward rectifying current which was fully blocked by tertiapin-Q (TQ, 300 nM), a selective blocker of IkAch in both PV and LA CM. In the absence of Cch, TQ blocked an inward rectifier current, which inverted at equilibrium potential of K+ in the PV but not in the LA CM. This suggests the existence of a constitutively active IKAch only in the PV CM. In accordance with these results, the western blot experiments indicate that the expression level of Kir3.4 subunits which contribute to the constitutive activity of GIRK channels was about 1.73 times more abundant in PV CM than in LA ones ( P < 0.001). Finally, sharp microelectrodes experiments showed that TQ (150 nM) lead to membrane depolarization of PV CM (16.7 ± 1 mV, P < 0.001) but not in LA CM (2.1 ± 0.6 mV, P = 0.173). However, this depolarization was less than that induced by the alpha adrenergic agonist cirazoline (steady state diastolic membrane potential in presence of cirazoline = − 44.1 ± 1.7 mV vs −57.2 ± 0.9 mV in presence of TQ ( P < 0.001)) suggesting that blockade of IkAch is not the only mechanism involved in the PV depolarization. These results suggest the presence of constitutively active GIRK channels only in PV CM. Their blockade could be a promising pharmacological target to block the electrical conduction between PV and LA, and thus containing abnormal electrical activity in PV.

Preferential Expression of Ca2+-Stimulable Adenylyl Cyclase III in the Supraventricular Area, including Arrhythmogenic Pulmonary Vein of the Rat Heart.

Ectopic excitability in pulmonary veins (PVs) is the major cause of atrial fibrillation. We previously reported that the inositol trisphosphate receptor in rat PV cardiomyocytes cooperates with the Na+-Ca2+ exchanger to provoke ectopic automaticity in response to norepinephrine. Here, we focused on adenylyl cyclase (AC) as another effector of norepinephrine stimulation. RT-PCR, immunohistochemistry, and Western blotting revealed that the abundant expression of Ca2+-stimulable AC3 was restricted to the supraventricular area, including the PVs. All the other AC isotypes hardly displayed any region-specific expressions. Immunostaining of isolated cardiomyocytes showed an enriched expression of AC3 along the t-tubules in PV myocytes. The cAMP-dependent response of L-type Ca2+ currents in the PV and LA cells is strengthened by the 0.1 mM intracellular Ca2+ condition, unlike in the ventricular cells. The norepinephrine-induced automaticity of PV cardiomyocytes was reversibly suppressed by 100 µM SQ22536, an adenine-like AC inhibitor. These findings suggest that the specific expression of AC3 along t-tubules may contribute to arrhythmogenic automaticity in rat PV cardiomyocytes.

Free Feeding of CpG-Oligodeoxynucleotide Particles Prophylactically Attenuates Allergic Airway Inflammation and Hyperresponsiveness in Mice.

CpG-oligodeoxynucleotides (CpG-ODNs) constitute an attractive alternative for asthma treatment. However, very little evidence is available from studies on the oral administration of CpG-ODNs in animals. Previously, we developed acid-resistant particles (named ODNcap) as an oral delivery device for ODNs. Here, we showed that free feeding of an ODNcap-containing feed prophylactically attenuates allergic airway inflammation, hyperresponsiveness, and goblet cell hyperplasia in an ovalbumin-induced asthma model. Using transcriptomics-driven approaches, we demonstrated that injury of pulmonary vein cardiomyocytes accompanies allergen inhalation challenge, but is inhibited by ODNcap feeding. We also showed the participation of an airway antimicrobial peptide (Reg3γ) and fecal microbiota in the ODNcap-mediated effects. Collectively, our findings suggest that daily oral ingestion of ODNcap may provide preventive effects on allergic bronchopulmonary insults via regulation of mechanisms involved in the gut-lung connection.

Ceramide modulates electrophysiological characteristics and oxidative stress of pulmonary vein cardiomyocytes.

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. 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 H2 O2 (2mM), MitoQ, wortmannin or 740 YP. A whole-cell patch clamp and fluorescence imaging were used to record the ionic currents, calcium (Ca2+ ) transients, and intracellular reactive oxygen species (ROS) and sodium (Na+ ) in isolated single PV cardiomyocytes before and after ceramide (1μM) treatment. Ceramide (0.1, 0.3, 1 and 3μM) reduced the beating rate of PV tissues. Furthermore, ceramide (1μM) suppressed the 2mM H2 O2 -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 H2 O2 -induced faster PV beating rate could be counteracted by MitoQ or wortmannin with no additive effect from the ceramide. Ceramide inhibited pPI3K. Ceramide reduced Ca2+ transients, sarcoplasmic reticulum Ca2+ contents, L-type Ca2+ currents, Na+ currents, late Na+ currents, Na+ -hydrogen exchange currents, and intracellular ROS and Na+ in PV cardiomyocytes, but did not change Na+ -Ca2+ exchange currents. 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.

Vascular endothelial growth factor modulates pulmonary vein arrhythmogenesis via vascular endothelial growth factor receptor 1/NOS pathway

Atrial fibrillation (AF) is a common form of arrhythmia with serious public health impacts, but its underlying mechanisms are not yet fully understood. Vascular endothelial growth factor (VEGF) is highly expressed in the atrium of patients with AF, but whether VEGF affects AF pathogenesis remains unclear. Pulmonary veins (PVs) are important sources for the genesis of atrial tachycardia or AF. Therefore, this study assessed the effects of VEGF on PV electrophysiological properties and evaluated its underlying mechanisms. Conventional microelectrodes and whole-cell patch clamps were performed using isolated rabbit PV preparations or single isolated PV cardiomyocytes before and after VEGF or VEGF receptor (VEGFR), Akt, NOS inhibitor administration. We found that VEGF (0.1, 1, and 10 ng/mL) reduced the PV beating rate in a dose-dependent manner. Furthermore, VEGF (10 ng/mL) reduced late diastolic depolarization and diastolic tension. Isoproterenol increased PV beating and burst firing, which was attenuated by VEGF (1 ng/mL). In the presence of VEGFR-1 inhibition (ZM306416 at 10 μM) and L-NAME (100 μM), VEGF (1 ng/mL) did not alter PV spontaneous activity. In isolated PV cardiomyocytes, VEGF (1 ng/mL) decreased L-type calcium, sodium/calcium exchanger, and late sodium currents. In conclusion, we found that VEGF reduces PV arrhythmogenesis by modulating sodium/calcium homeostasis through VEGFR-1/NOS signaling pathway.

Selective activation of adrenoceptors potentiates IKs current in pulmonary vein cardiomyocytes through the protein kinase A and C signaling pathways

Delayed rectifier K+ current (IKs) is a key contributor to repolarization of action potentials. This study investigated the mechanisms underlying the adrenoceptor-induced potentiation of IKs in pulmonary vein cardiomyocytes (PVC). PVC were isolated from guinea pig pulmonary vein. The action potentials and IKs current were recorded using perforated and conventional whole-cell patch-clamp techniques. The expression of IKs was examined using immunocytochemistry and Western blotting. KCNQ1, a IKs 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 IKs current in PVC was markedly enhanced by both β1- and β2-adrenoceptor stimulation with a negative voltage shift in the current activation, although the potentiation was more effectively induced by β2-adrenoceptor stimulation than β1-adrenoceptor stimulation. Both β-adrenoceptor-mediated increases in IKs were attenuated by treatment with the adenylyl cyclase (AC) inhibitor or protein kinase A (PKA) inhibitor. Furthermore, the IKs current was increased by α1-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 IKs blocker. However, HMR-1556 markedly reduced the β-adrenoceptor-potentiated firing rate. The stimulatory effects of β- and α1-adrenoceptor on IKs 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 IKs might increase during sympathetic excitation under in vivo conditions.

Neonatal hyperoxia inhibits proliferation and survival of atrial cardiomyocytes by suppressing fatty acid synthesis.

Preterm birth increases the risk for pulmonary hypertension and heart failure in adulthood. Oxygen therapy can damage the immature cardiopulmonary system and may be partially responsible for the cardiovascular disease in adults born preterm. We previously showed that exposing newborn mice to hyperoxia causes pulmonary hypertension by 1 year of age that is preceded by a poorly understood loss of pulmonary vein cardiomyocyte proliferation. We now show that hyperoxia also reduces cardiomyocyte proliferation and survival in the left atrium and causes diastolic heart failure by disrupting its filling of the left ventricle. Transcriptomic profiling showed that neonatal hyperoxia permanently suppressed fatty acid synthase (Fasn), stearoyl-CoA desaturase 1 (Scd1), and other fatty acid synthesis genes in the atria of mice, the HL-1 line of mouse atrial cardiomyocytes, and left atrial tissue explanted from human infants. Suppressing Fasn or Scd1 reduced HL-1 cell proliferation and increased cell death, while overexpressing these genes maintained their expansion in hyperoxia, suggesting that oxygen directly inhibits atrial cardiomyocyte proliferation and survival by repressing Fasn and Scd1. Pharmacologic interventions that restore Fasn, Scd1, and other fatty acid synthesis genes in atrial cardiomyocytes may, thus, provide a way of ameliorating the adverse effects of supplemental oxygen on preterm infants.

Study of the Characteristics of the Pulmonary Vein and Superior Vena Cava of Rabbits

Background: This study aims to (1) investigate the characteristics of the action potential and triggering activity of cardiomyocytes in the pulmonary vein (PV) and superior vena cava (SVC) of rabbits and (2) study the features of cation currents in cardiomyocytes in rabbit PV and SVC-inward rectifier potassium current (IK1), transient outward potassium current (Ito), and non-selective cation currents (INSCC). Methods: The standard glass microelectrode and whole-cell patch-clamp techniques were used to record the action potential and various currents in the above cells. Results: (1) Cardiomyocytes in either PV or SVC had longer action potential durations than in the adjacent atrium, and spontaneous early after depolarization (EAD) could occur in both PV and SVC under normal physiological conditions. (2) The action potential in PV cardiomyocytes had a relative refractory period but did not have an absolute refractory period, and this characteristic enabled a premature beat that triggered a second plateau response, which led to EAD. (3) INSCC was found for the first time in the PV, SVC, and atria. (4) The current intensity of IK1, Ito, and INSCC was significantly lower in the PV and SVC than in the left and right atria, and the difference in the current intensity in INSCC could influence the action potential. Conclusions: PV and SVC can both initiate and maintain AF, but PV is the primary ectopic foci in initiating AF. The present study found that the second plateau response was easily induced in cardiomyocytes in PA shortly after depolarization. This was a specific characteristic of the action potential of PV. In addition, we preliminarily analyzed the differences in the main outward currents and noted a voltage-dependent INSCC in both PV and SVC rabbits’ cardiomyocytes. Furthermore, the current intensities of IK1, Ito, and INSCC were significantly lower in the PV and SVC than in the left and right atria, and the difference in the current intensity of INSCC influenced the action potential. The different permeability of INSCC for cations at different phases may play a role in inducing EAD.

Co-existence of a fast tetrodotoxin-sensitive low voltage-activated non selective Na/Ca/K channel and of T-type Ca channels in rat isolated pulmonary vein cardiomyocytes

Ectopic foci in pulmonary veins (PV) are involved in the onset of atrial fibrillation. Norepinephrine (NE) induces an automatic activity occurring in bursts in rat PV cardiomyocytes (CM) which depends on Ca (upstroke) and Na (inter-burst) channels. To characterize low voltage-activated (LVA) Ca currents in rat PVCM susceptible to trigger ectopic foci. Whole-cell ICa (5 mM Ca) was recorded from -100 mV with classical Na- and K-free solutions at room temperature in enzymatically isolated PVCM from male Wistar rats. A fast LVA ICa (FLVA-ICa; −1.5 ± 0.2 pA/pF, n = 45; 26% of peak ICa, max recorded at + 20 mV) was activated between −55 and −20 mV in ∼56% of PVCM and blocked by 10 μM tetrodotoxin (TTX). In that, it was similar to the reported TTX-sensitive Ca current. However, it was markedly increased by addition of NaCl (1 or 3 mM) or KCl (5 or 10 mM). Permeability ratios P’Ca/PNa and P’Ca/PK calculated for bi-ionic conditions were respectively 2.25 ± 0.51 (n = 6) and 1.88 ± 0.25 (n = 14), and not different from a value of 2. NE (10 μM, n = 13) increased FLVA-ICa and negatively shifted its activation threshold, an effect partially reversed by 5 μM Atenolol. It was partially blocked by 5 μM Nifedipine (n = 5) and increased by 300 nM BayK8644 (n = 6), but not blocked by Ranolazine (10 μM, n = 6). NiCl2 (40 μM, n = 9) and TTA-A2 (10 or 100 nM, n = 11) increased FLVA-ICa. Neither Ba nor Sr alone could permeate the FLVA channel or block Ca influx but revealed a large slower activating and inactivating LVA Ca current (SLVA-ICa), present in 10 out of 80 PVCM and absent in LACM. SLVA-ICa was partially blocked by 100 nM TTA-A2. The ionic channel underlying FLVA-ICa is likely a fast voltage-gated non-selective channel permeable to Na/Ca/K with a dihydropyridine binding site. SLVA-ICa might correspond to Ca influx through Cav3.1 channels. The respective role of these channels to triggering ectopic foci in the PV myocardial sleeve remained to be determined.

Role of LVA Ca channels in norepinephrine-induced automatic activity in rat pulmonary vein myocardial sleeve

Ectopic foci in pulmonary veins (PV) are involved in the onset of atrial fibrillation. Norepinephrine (NE) induces in rat PV cardiomyocytes (CM) an automatic activity in saw-tooth pattern (catecholaminergic automatic activity or CAA), consisting of bursts of slow action potentials which depends on Ca (upstroke) and Na (inter burst) channels. We have previously shown the co-existence in rat PVCM, of a fast LVA (FLVA) non selective TTX-sensitive cationic channel and of T-type Ca channels. To evaluate in male Wistar rat PV myocardial sleeve the contribution of these channels to triggering NE-induced ectopic foci. Whole-cell currents were studied at room temperature in enzymatically isolated PVCM. Mechanical activity of PV rings was recorded in an organ bath at 37 °C and CAA was induced by application of 10 μM NE. The FLVA Ca current was increased by the Cav3.x blockers TTA-A2 (10-100 nM, n = 11) and NiCl2 (40 μM, n = 9) whereas a large LVA Ca current, present in 10 out of 80 PVCM, was partially reduced by 100 nM TTA-A2. Study of K currents revealed the presence of an inward rectifying current sensitive to TRAM-34 (1 μM, n = 6), a blocker of the Ca-activated K channel SK4. In PV rings, TTA-A2 (1-5 μM) decreased CAA incidence. Total arrhythmia duration, measured over the last 10 min of a 30 min long drug application, was reduced as well as burst duration and the number of spontaneous twitches within a burst. On the other hand, 40 μM NiCl2 or 1-2 μM TRAM-34 increased total arrhythmia duration by decreasing the interval between bursts (no significant effect on the burst characteristics). Only NiCl2 had a significant negative inotropic effect (∼80% inhibition of L-type calcium current). Our results suggest that Ca influx through Cav3.1 channels contribute to triggering bursts of automatic activity and that Cav3.2 Ca channels, through activation of SK4 channels, are involved in the regulation of the interval between bursts.

Klotho modulates electrical activity and calcium homeostasis in pulmonary vein cardiomyocytes via PI3K/Akt signalling.

Klotho, a potential antiageing protein has remarkable cardiovascular effects, which is lower in the patients with chronic kidney disease (CKD). Chronic kidney disease increases the risk of atrial fibrillation, majorly triggered by pulmonary vein (PV) arrhythmogenesis. This study investigated whether klotho protein can modulate PV electrical activity and the underlying potential mechanisms. A conventional microelectrode and whole-cell patch clamp were used to investigate the action potentials and ionic currents in isolated rabbit PV tissue preparations and single cardiomyocytes before and after klotho administration. Phosphoinositide 3-kinase (PI3K)/Akt signalling was studied using western blotting. Klotho significantly reduced PV spontaneous beating rates in PV tissue preparations at 1.0 and 3.0 ng/mL (but not at 0.1 and 0.3 ng/mL). In the presence of the Akt inhibitor (10 µM), klotho (1.0 and 3.0 ng/mL) did not change PV electrical activities. Klotho (1.0 ng/mL) significantly decreased the late sodium current (INa-Late) and L-type calcium current (ICa-L), similar to the Akt inhibitor (10 µM). Western blots demonstrated that klotho (1.0 ng/mL)-treated PV cardiomyocytes had less phosphorylation of Akt (Ser473) compared with klotho-untreated cardiomyocytes. Compared with control PVs, klotho at relatively lower concentrations (0.1 and 0.3 ng/mL) significantly reduced beating rates and decreased the amplitudes of delay afterdepolarizations in CKD PVs. Klotho modulated PV electrical activity by inhibiting PI3K/Akt signalling, which may provide a novel insight into CKD-induced arrhythmogenesis.

Low voltage-activated channels in rat pulmonary vein cardiomyocytes: coexistence of a non-selective cationic channel and of T-type Ca channels.

In rat pulmonary vein (PV) cardiomyocytes (CM), norepinephrine (NE) induces an automatic activity consisting of bursts of slow action potentials which depend on Ca2+ (upstroke) and Na+ (inter-burst) channels. Our objective was to characterize low voltage-activated (LVA) currents in rat PVCM susceptible to trigger this activity. Whole-cell ICa (5 mM Ca2+) was recorded from - 100 mV with classical Na+- and K+-free solutions. A fast LVA ICa (FLVA-ICa), present in ≈ 56% of PVCM between ~ - 50 to - 20 mV, was blocked by 10 μM TTX and markedly increased by addition of NaCl (1 or 3 mM) or KCl (5 or 10 mM). Permeability ratios P'Ca/PNa and P'Ca/PK calculated for bi-ionic conditions were respectively 2.25 ± 0.51 and 1.88 ± 0.25, and not different from a value of 2. FLVA-ICa was increased by 10 μM NE and 300 nM BayK8644, decreased by 5 μM nifedipine but not blocked by ranolazine (10 μM). NiCl2 (40 μM) and TTA-A2 (10 or 100 nM) increased FLVA-ICa. Similar results were obtained in left atrial (LA) CM. Neither Ba2+ nor Sr2+ alone could permeate the FLVA channel or block Ca2+ influx but revealed a large slower activating and inactivating LVA Ca2+ current (SLVA-ICa), present in 10 out of 80 PVCM, absent in LACM, and partially inhibited by 100 nM TTA-A2. Therefore, the ionic channel underlying FLVA-ICa is likely a fast voltage-gated non-selective channel with a dihydropyridine binding site. SLVA-ICa might correspond to Ca2+ influx through Cav3.x channels and contribute to triggering NE-induced automatic activity in the PV myocardial sleeve.

Ion currents, action potentials, and noradrenergic responses in rat pulmonary vein and left atrial cardiomyocytes.

The electrophysiological properties of pulmonary vein (PV)‐cardiomyocytes, and their responses to the sympathetic neurotransmitter, noradrenaline (NA), are thought to differ from those of the left atrium (LA) and contribute to atrial ectopy. The aim of this study was to examine rat PV cardiomyocyte electrophysiology and responses to NA in comparison with LA cells. LA and PV cardiomyocytes were isolated from adult male Wistar rat hearts, and membrane potentials and ion currents recorded at 36°C using whole‐cell patch‐clamp techniques. PV and LA cardiomyocytes did not differ in size. In control, there were no differences between the two cell‐types in zero‐current potential or action potential duration (APD) at 1 Hz, although the incidence of early afterdepolarizations (EADs) was greater in PV than LA cardiomyocytes. The L‐type Ca2+ current (I CaL) was ~×1.5 smaller (p = .0029, Student's t test) and the steady‐state K+ current (I Kss) was ~×1.4 larger (p = .0028, Student's t test) in PV than in LA cardiomyocytes. PV cardiomyocyte inward‐rectifier current (I K1) was slightly smaller than LA cardiomyocyte I K1. In LA cardiomyocytes, NA significantly prolonged APD30. In PV cells, APD30 responses to 1 μM NA were heterogeneous: while the mean percentage change in APD30 was not different from 0 (16.5 ± 9.7%, n cells/N animals = 12/10, p = .1177, one‐sample t test), three cells showed shortening (‐18.8 ± 6.0%) whereas nine showed prolongation (28.3 ± 10.1%, p = .008, Student's t test). NA had no effect on I K1 in either cell‐type but inhibited PV I Kss by 41.9 ± 4.1% (n/N = 23/11 p < .0001), similar to LA cells. NA increased I CaL in most PV cardiomyocytes (median × 2.2‐increase, p < .0001, n/N = 32/14, Wilcoxon‐signed‐rank test), although in 7/32 PV cells I CaL was decreased following NA. PV cardiomyocytes differ from LA cells and respond heterogeneously to NA.

Selective inhibition of electrical conduction within the pulmonary veins by \u03b11-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.