Rat Atrial Cells Research Articles

Non-Selective Cation Currents Mediated by Cx43 Hemichannel-P2X4 Receptor Signaling Pathway in Rat Atrial Myocytes under Shear Stress

Cardiac myocytes are exposed to shear stress under physiological conditions and hemodynamic disturbances. Shear stress has been thought to trigger two distinct global Ca2+ waves in atrial myocytes through autocrine activation of P2X4 receptor (P2X4R) and P2Y1-receptor (P2Y1R) via connexin 43 (Cx43)-mediated ATP release. In this study, we examined whether such shear stress-triggered Cx43-ATP-P2 receptor signaling generates depolarizing currents in atrial myocytes. Whole-cell patch clamp and dye-flux assays were used with genetic and pharmacological interventions in rat atrial myocytes and HL-1 cells. We found that shear stress activated a non-selective inward cation (Cs+) current (IS,Cs) at resting potential which was eliminated by Cx43 blockade using siRNA, antibodies, carbenoxolone or La3+. IS,Cs was enhanced by removal of external Ca2+, whereas internal Ca2+ buffering suppressed IS,Cs. Hemichannel-mediated dye (calcein red-orange) flux was induced by shear stress. This response was augmented by removing external Ca2+ but was not affected by pannexin blocker (probenecid). The shear-activated current was significantly reduced when Cs+ was replaced with NMDG+, and this was enhanced by quinine and suppressed by La3+. P2X4R antagonists (5-BDBD and PSB-12054), P2XR-blocker (iso-PPADS) and anti-P2X4R antibodies each blocked IS,Cs by approximately 50%. About 25% of IS,Cs was suppressed by P2Y1R inhibitor (MRS2179) and/or transient receptor potential melastatin 4 blocker (9-phenanthrol). P2X4R, but not P2X5R, was co-localized with Cx43 at the cell ends. P2X4R-dependent IS,Cs was augmented in atrial myocytes from hypertrophied rat hearts by short-term aortic constriction. Our data suggest that shear stress triggers Cx43 hemichannels to gate P2X4Rs in their vicinity. This shear stress signaling may play an important role in membrane depolarization at negative potentials and in atrial remodeling under increased afterload.

Distinct shear-induced Ca2+ signaling in the left and right atrial myocytes: Role of P2 receptor context

Atrial myocytes are continuously exposed to shear stress during cardiac cycles. Previous reports have shown that shear stress induces two different types of global Ca2+ signaling in atrial myocytes–longitudinal Ca2+ waves (L-waves) and action potential-involved transverse waves (T-waves), and suggested an underlying role of the autocrine activation of P2 receptors. We explored the correlations between ATP release and Ca2+ wave generation in atrial myocytes and investigated why the cells develop two Ca2+-wave types during the same shear force. We examined whether ATP release correlates with different shear-stress (~16 dyn/cm2)-mediated Ca2+ signaling by simultaneous measurement of local Ca2+ and ATP release in individual atrial myocytes using two-dimensional confocal imaging and sniffer patch techniques, respectively. Functional P2X7-receptor-expressing HEK293 cells were established as sniffer cells, which generated currents in real time in response to ATP released from a closely positioned atrial myocyte. Both shear-stress-induced L- and T-waves were preceded by sniffer currents with no difference in the current magnitude. Left atrial (LA) myocytes had two- to three-fold larger sniffer currents than right atrial (RA) cells, as was confirmed by ATP chemiluminescence assay. Shear-stress-induced ATP release was eliminated by connexin (Cx) 43 hemichannel inhibition using La3+, Gap19, or knock-down of Cx43 expression. The level of phosphorylated Cx43 at Ser386 (p-Cx43Ser368), but not total Cx43, was higher in LA versus RA myocytes. Most LA cells (~70%) developed L-waves, whereas most RA myocytes (~80%) presented T-waves. Shear-stress-induced T-waves were completely removed by inhibition of P2X4R, which were most abundant in rat atrial cells. Expression of P2X4R was higher in RA than LA myocytes, whereas expression of P2Y1R, the mediator of L-waves, was higher in LA than RA myocytes. ATP release mainly triggers L-waves in LA myocytes and T-waves in RA myocytes under the same shear force, partly because of the differential expression of P2Y1R and P2X4R between LA and RA myocytes. Higher ATP release in LA myocytes under shear stress may not contribute to determination of the wave pattern.

Chronic consumption of a high-fat diet rich in corn oil activates intrinsic cell death pathway and induces several ultrastructural changes in the atria of healthy and type 1 diabetic rat.

This study investigates the effect of chronic consumption of a high-fat diet rich in corn oil (CO-HFD) on atrial cells ultrastructure, antioxidant levels and markers of intrinsic cell death of both control and type 1 diabetes mellitus (T1DM)-induced rats. Adult male rats (10 rats/group) were divided into four groups: control fed standard diet (STD) (3.82kcal/g, 9.4% fat), CO-HFD (5.4kcal/g, 40% fat), T1DM fed STD, and T1DM+CO-HFD. CO-HFD and T1DM alone or in combination impaired systolic and diastolic functions of rats and significantly reduced levels of GSH and the activity of SOD, enhanced lipid peroxidation, increased protein levels of P53, Bax, cleaved caspase-3, and ANF and decreased levels of Bcl-2 in their atria. Concomitantly, atrial cells exhibited fragmentation of the myofibrils, disorganized mitochondria, decreased number of atrionatriuretic factor (ANF) granules, and loss of gap junctions accompanied by changes in capillary walls. Among all treatments, the severity of all these findings was more severe in T1DM and most profound in the atria of T1DM+CO-HFD. In conclusion, chronic consumption of CO-HFD by T1DM-induced rats elicits significant biochemical and ultrastructural damage to rat atrial cells accompanied by elevated oxidative stress and mitochondria-mediated cell death.

Rat atrial engineered heart tissue: a new in vitro model to study atrial biology.

Engineered heart tissue (EHT) from rat cells is a useful tool to study ventricular biology and cardiac drug safety. Since atrial and ventricular cells differ significantly, EHT and other 3D cell culture formats generated from ventricular cells have been of limited value to study atrial biology. To date, reliable in vitro models that reflect atrial physiology are lacking. Therefore, we established a novel EHT model using rat atrial cells (atrial EHT, aEHT) to assess atrial physiology, contractility and drug response. The tissue constructs were characterized with regard to gene expression, histology, electrophysiology, and the response to atrial-specific drugs. We observed typical functional properties of atrial tissue in our model such as more regular spontaneous beating with lower force, shorter action potential duration, and faster contraction and relaxation compared to ventricular EHT (vEHT). The expression of atrial-specific genes and proteins was high, whereas ventricle-specific transcripts were virtually absent. The atrial-selective drug carbachol had a strong negative inotropic and chronotropic effect on aEHT only. Taken together, the results demonstrate the feasibility of aEHT as a novel atrial 3D model and as a benchmark for tissue engineering with human induced pluripotent stem cell-derived atrial-like cardiomyocytes. Atrial EHT faithfully recapitulates atrial physiology and shall be useful to study atrial molecular physiology in health and disease as well as drug response.

Homocysteine induces calcium overload in neonatal rat atrial cells through activation of sodium current and CaMKⅡδ

Objective: To investigate the effect and related mechanism of homocysteine (Hcy) on calcium overload in neonatal rat atrial cells (NRICs). Methods: NRICs were assigned to 9 groups after culture for 3 days: (1) control group; (2) Hcy group (0, 50, 100, 200, 500 μmol/L for 48 hours); (3) antioxidant group (NAC, 10 μmol/L for 24 hours); (4) Hcy+NAC group (500 μmol/L Hcy for 48 hours, then treated with 10 μmol/L NAC for 24 hours); (5) calcium/calmodulin dependent protein kinase Ⅱδ (CaMKⅡδ) inhibitor group (KN-93, 3 μmol/L KN-93 for 5 hours); (6) specific sodium current inhibitor group (ELE, 1 μmol/L ELE for 5 hours); (7) Hcy+KN-93 group (500 μmol/L Hcy for 48 hours, then treated with 3 μmol/L KN-93 for 5 hours); (8) Hcy+ELE group (500 μmol/L Hcy for 48 hours, then treated with 1 μmol/L ELE for 5 hours; (9) Hcy+KN-93+ELE group (500 μmol/L Hcy for 48 hours, then treated with 3 μmol/L KN-93 and 1 μmol/L ELE for 5 hours). Moreover, NRICs were also treated with CaMKⅡδ-siRNA lentivirus, and Nav1.5-siRNA lentivirus, negative lentivirus carrier containing green fluorescent protein (GFP) for 24 hours. The MOI values of the three groups were 10. Infection efficiency of lentivirus was determined by observing the percentage of GFP fluorescence under inverted fluorescence microscope after transfection for 24 hours, and cultured regularly with simultaneous Puro screening, then cells were grouped as Hcy+CaMKⅡδ-siRNA group, Hcy+Nav1.5-siRNA group and Hcy+negative group. The concentration of Ca(2+) in NRICs ([Ca(2+)]i) of various groups was detected through Fluo-4/AM fluorescence probe, then 2', 7'- two chlorofluorescein diacetate (DCFH-DA) was used as a probe to detect reactive oxygen species (ROS) in NRICs by flow cytometry. The malondialdehyde (MDA) was detected by the activity of superoxide dismutase (SOD) and xanthine oxidase was detected by thiobarbituric acid colorimetry. The protein and mRNA expression level of CaMKⅡδ and Nav1.5 in NRICs were detected by Western blot and quantitative real-time PCR. Results: (1) ROS, MDA and SOD were similar between NAC group and control group, ROS and MDA were significantly increased, while SOD was significantly reduced in Hcy group in a concentration-dependent manner. (2) [Ca(2+)]i: The level of [Ca(2+)]i was (155.57+7.25), (187.43+13.07), (248.98+27.22) and (307.36+15.09) nmol/L in 50, 100, 200 and 500 μmol/L Hcy groups, which was significantly higher than that in the control group ((123.18+7.24) nmol/L, P<0.01). In addition, the level of [Ca(2+)]i in Hcy+NAC group ((222.87+23.71)nmol/L) was significantly lower than that in Hcy 500 μmol/L group ((305.15+39.45) nmol/L, P<0.05), while [Ca(2+)]i level was similar between NAC group and the control group. (3) The protein expression of CaMKⅡδ and Nav1.5 was significantly upregulated in Hcy groups than in the control group. The protein expression level of CaMKⅡδ-Thr287 was significantly lower in NAC group than in Hcy 500 μmol/L group (P<0.01), however, there was no significant difference on the protein expression levels of CaMKⅡδ-Thr287 and Nav1.5 between NAC group and control group (all P>0.05). (4) The protein expression levels of CaMKⅡδ-Thr287 and the concentration of [Ca(2+)]i were significantly lower in Hcy+KN-93 group and Hcy+KN-93+ELE group than in Hcy 500 μmol/L group (P<0.05). [Ca(2+)]i concentration was significantly lower in Hcy+KN-93 group, Hcy+ELE group and KN-93+ELE+Hcy group than in Hcy 500 μmol/L group (P<0.05). (5) The mRNA and protein expression levels of CaMKⅡδ and Nav1.5 in each group infected with lentivirus: the GFP expression was ideal post lentivirus transfection for 24 hours (up to 90%), which was significantly lower in the CaMKⅡδ-siRNA group and Nav1.5-siRNA group than in the negative infection group (all P<0.05), which was similar between negative infection group and control group (P>0.05). Moreover, the mRNA and protein expression levels of CaMKⅡδ and CaMKⅡδ-Thr287 was significantly lower in Hcy+Nav1.5-siRNA group than in Hcy+negative infection group (P<0.05). The protein and mRNA levels of Nav1.5 were similar between Hcy+CaMKⅡδ-siRNA group and Hcy+negative infection group (P>0.05). Conclusions: Hcy can induce calcium overload in NRICs by increasing oxidative stress, upregulating the sodium channel protein, and activating the late sodium current and phosphorylating CaMKⅡδ.

Inward rectifying potassium currents resolved into components: modeling of complex drug actions.

Inward rectifier potassium currents (I Kir,x) belong to prominent ionic currents affecting both resting membrane voltage and action potential repolarization in cardiomyocytes. In existing integrative models of electrical activity of cardiac cells, they have been described as single current components. The proposed quantitative model complies with findings indicating that these channels are formed by various homomeric or heteromeric assemblies of channel subunits with specific functional properties. Each I Kir,x may be expressed as a total of independent currents via individual populations of identical channels, i.e., channels formed by the same combination of their subunits. Solution of the model equations simulated well recently observed unique manifestations of dual ethanol effect in rat ventricular and atrial cells. The model reflects reported occurrence of at least two binding sites for ethanol within I Kir,x channels related to slow allosteric conformation changes governing channel conductance and inducing current activation or inhibition. Our new model may considerably improve the existing models of cardiac cells by including the model equations proposed here in the particular case of the voltage-independent drug-channel interaction. Such improved integrative models may provide more precise and, thus, more physiologically relevant results.

P1078Correlation between magnitude of cardiac ionic currents and cell membrane capacity: always positive?

Introduction: The magnitude of cardiac ionic currents is generally considered as being directly proportional to the membrane area of investigated cell which may be determined by measurement of the cell membrane capacity. Therefore, the measured values of ionic currents are usually converted to the current density (by dividing measured current by estimated cell membrane capacity) to reduce differences among cells caused by their varying membrane area. This strategy is asserted in patch clamp studies on cardiomyocytes and, in most cases, also on cell lines heterogeneously expressing cardiac ionic channels. Our recent pilot data has shown that the correlation may be absent or even inverse in some cases which may lead to misinterpretation of the evaluated data. Purpose: We aimed to investigate the assumed correlation between the magnitude of cardiac ionic currents (absolute value) and membrane capacity of respective cells. Methods: Experiments were performed by the whole cell patch clamp technique on enzymatically isolated rat atrial myocytes (two inward rectifier potassium currents were investigated, namely IK1 and acetylcholine-sensitive current IK(Ach); at 23 ? 1?C) and on Chinese hamster ovary (CHO) cells transiently expressing human slowly activated delayed rectifier (IKs) channels (KCNQ1, KCNE1, Yotiao; at 37?C). Results: The significance test indicated that the Pearson’s coefficients of correlation (r) between the magnitude of inward component of IK1 or of acetylcholine-induced IK(Ach) and the capacity of respective rat atrial myocytes were significant (r = 0.79 and 0.64, n = 27 and 20, P ? 0.001 and 0.002, respectively). Surprisingly, analogical significant correlation was not apparent in the case of inward component of constitutively active IK(Ach) measured in rat atrial cells (r = 0.17, n = 56). In the case of CHO cells, the magnitude of expressed human IKs and the capacity of CHO cells did not significantly correlate as well. Furthermore, even negative correlation was indicated, i.e. the higher was the cell capacity, the smaller was this current (r = -0.22, n = 18). Conclusions: We proved the expected significant positive correlation between the magnitude of big ionic currents, namely IK1 and acetylcholine-induced IK(Ach), and the membrane area of rat atrial myocytes. On the contrary, no significant correlation was apparent in the case of small constitutively active IK(Ach). Importantly, even negative (but insignificant) correlation was observed in the case of human IKs transiently expressed in CHO cells. Hence, the usually used conversion of the current magnitude to the current density using the membrane capacity of respective cell should be used with caution, namely in the case of small ionic currents in cardiomyocytes and, especially, in the case of currents measured in cell lines. The cause of missing or even inverse proportionality between membrane current and capacity remains to be clarified.

Nkx2.5/CARP signaling pathway contributes to the regulation of ion channel remodeling induced by rapid pacing in rat atrial myocytes.

Remodeling of atrial electrophysiology and structure is the primary feature of atrial fibrillation (AF). Evidence suggests that abnormalities in the expression levels of embryological cardiovascular development‑associated transcription factors, including Nkx2.5, are crucial for the development of AF. Rat atrial myocardial cells (AMCs) in culture dishes were placed in an electric field and stimulated. Transmission electron microscopy was used to observe the ultrastucture prior to and following rapid pacing. The action potential durations and effective refractory periods were measured. RT‑PCR and western blotting were performed to investigate the effect of rapid pacing on the expression levels of ion channel and nuclear proteins in AMCs. Nkx2.5 short interfering RNA (siRNA) transfection was performed. Through this invitro rat AMC culture and rapid pacing model, it was demonstrated that rapid pacing shortened the action potential and downregulated the expression levels of L‑type calcium and potassium channels. Expression levels of Nkx2.5 and cardiac ankyrin repeat protein (CARP) were significantly upregulated by rapid pacing at the mRNA and protein levels. siRNA‑mediated Nkx2.5 silencing attenuated the rapid pacing‑induced downreglation of ion channel levels. These results suggest that the Nkx2.5/CARP signaling pathway contributes to the early electrical remodeling process of AF.

Dependence of cardiac transverse tubules on the BAR domain protein amphiphysin II (BIN-1).

Transverse tubules (t-tubules) regulate cardiac excitation-contraction coupling and exhibit interchamber and interspecies differences in expression. In cardiac disease, t-tubule loss occurs and affects the systolic calcium transient. However, the mechanisms controlling t-tubule maintenance and whether these factors differ between species, cardiac chambers, and in a disease setting remain unclear. To determine the role of the Bin/Amphiphysin/Rvs domain protein amphiphysin II (AmpII) in regulating t-tubule maintenance and the systolic calcium transient. T-tubule density was assessed by di-4-ANEPPS, FM4-64 or WGA staining using confocal microscopy. In rat, ferret, and sheep hearts t-tubule density and AmpII protein levels were lower in the atrium than in the ventricle. Heart failure (HF) was induced in sheep using right ventricular tachypacing and ferrets by ascending aortic coarctation. In both HF models, AmpII protein and t-tubule density were decreased in the ventricles. In the sheep, atrial t-tubules were also lost in HF and AmpII levels decreased. Conversely, junctophilin 2 levels did not show interchamber differences in the rat and ferret nor did they change in HF in the sheep or ferret. In addition, in rat atrial and sheep HF atrial cells where t-tubules were absent, junctophilin 2 had sarcomeric intracellular distribution. Small interfering RNA-induced knockdown of AmpII protein reduced t-tubule density, calcium transient amplitude, and the synchrony of the systolic calcium transient. AmpII is intricately involved in t-tubule maintenance. Reducing AmpII protein decreases t-tubule density, reduces the amplitude, and increases the heterogeneity of the systolic calcium transient.

AdipoR1/APPL1 potentiates the protective effects of globular adiponectin on angiotensin II-induced cardiac hypertrophy and fibrosis in neonatal rat atrial myocytes and fibroblasts.

Atrial hypertrophy and fibrosis are essential pathological features of atrial fibrillation. Recently, adiponectin has become a protein of interest due to its beneficial effects on cardiovascular diseases. However, the molecular mechanism of atrial structural remodeling and signaling pathways evoked by adiponectin remain unclear. In the present study, we investigated the cardioprotective effect of globular adiponectin (gAcrp) on angiotensin II-induced atrial hypertrophy and fibrosis in neonatal Sprague-Dawley rat. To further investigate the molecular mechanisms underlying the preventive effect of gAcrp, transfection of cells with siRNA was used to suppress the mRNA expression of adiponectin receptor 1 (AdipoR1) and its downstream adaptor protein APPL1. Non-silencing-Cy-3 labelled siRNA was used to determine transfection efficiency using fluorescence microscopy. The expression of atrial natriuretic peptide and procollagen type1 α-1, hypertrophy marker and fibrosis one, respectively, was detected by real-time PCR. Furthermore, the expression of adenosine monophosphate-activated protein kinase (AMPK), phosphatidylinositol 3-kinase (PI3K) and Akt was detected by western blotting. In addition, nuclear p65 translocation activity was analyzed by EMSA supershift assay. Our results showed that AdipoR1 and the adaptor protein APPL1 mediated the protective effects of gAcrp. In addition, the function of adiponectin and phosphorylation of AMPK were prominently diminished by inhibition of PI3K. Furthermore, nuclear factor-κB (NF-κB) transcription was diminished by the specific inhibition of AMPK. Taken together, AMPK pivotally interacts with NF-κB and PI3K, mediating the cardioprotective effect of adiponectin, and may serve as a therapeutic target for preventing atrial hypertrophy and fibrosis. Our present study suggests that gAcrp could ameliorate AngII-induced cardiac hypertrophy and fibrosis in rat atrial cells, which is mediated by the activation of AMPK signaling pathways. APPL1 and AdipoR1 are the key factors involved in the downstream of gAcrp approach.

Couplons in rat atria form distinct subgroups defined by their molecular partners

Standard local control theory, which describes Ca(2+) release during excitation-contraction coupling (ECC), assumes that all ryanodine receptor 2 (RyR2) complexes are equivalent. Findings from our laboratory have called this assumption into question. Specifically, we have shown that the RyR2 complexes in ventricular myocytes are different, depending on their location within the cell. This has led us to hypothesize that similar differences occur within the rat atrial cell. To test this hypothesis, we have triple-labelled enzymatically isolated fixed myocytes to examine the distribution and colocalization of RyR2, calsequestrin (Casq), voltage-gated Ca(2+) channels (Ca(v)1.2), the sodium-calcium exchanger (Ncx) and caveolin-3 (Cav3). A number of different surface RyR2 populations were identified, and one of these groups, in which RyR2, Ca(v)1.2 and Ncx colocalized, might provide the structural basis for 'eager' sites of Ca(2+) release in atria. A small percentage of the dyads containing RyR2 and Ca(v)1.2 were colocalized with Cav3, and therefore could be influenced by the signalling molecules it anchors. The majority of the RyR2 clusters were tightly linked to Ca(v)1.2, and, whereas some were coupled to both Ca 1.2 and Ncx, none were with Ncx alone. This suggests that Ca(v)1.2-mediated Ca(2+) -induced Ca(2+) release is the primary method of ECC. The two molecules studied that were found in the interior of atrial cells, RyR2 and Casq, showed significantly less colocalization and a reduced nearest-neighbour distance in the interior, compared with the surface of the cell. These differences might result in a higher excitability for RyR2 in the interior of the cells, facilitating the spread of excitation from the periphery to the centre. We also present morphometric data for all of the molecules studied, as well as for those colocalizations found to be significant.

Role of internalization of M2muscarinic receptor via clathrin-coated vesicles in desensitization of the muscarinic K+current in heart

In the heart, ACh activates the ACh-activated K(+) current (I(K,ACh)) via the M(2) muscarinic receptor. The relationship between desensitization of I(K,ACh) and internalization of the M(2) receptor has been studied in rat atrial cells. On application of the stable muscarinic agonist carbachol for 2 h, I(K,ACh) declined by approximately 62% with time constants of 1.5 and 26.9 min, whereas approximately 83% of the M(2) receptor was internalized from the cell membrane with time constants of 2.9 and 51.6 min. Transfection of the cells with beta-adrenergic receptor kinase 1 (G protein-receptor kinase 2) and beta-arrestin 2 significantly increased I(K,ACh) desensitization and M(2) receptor internalization during a 3-min application of agonist. Internalized M(2) receptor in cells exposed to carbachol for 2 h was colocalized with clathrin and not caveolin. It is concluded that a G protein-receptor kinase 2- and beta-arrestin 2-dependent internalization of the M(2) receptor into clathrin-coated vesicles could play a major role in I(K,ACh) desensitization.

Evidence for proteasomal degradation of Kv1.5 channel protein

Background The voltage-gated potassium channel Kv1.5 plays a critical role in the maintenance of the membrane potential. While protein degradation is one of the major mechanisms for the regulation of channel functions, little is known on the degradation mechanism of Kv1.5. Methods and results Kv1.5 was expressed in COS cells and its degradation, intracellular localization, and channel activities were assessed by pulse-chase analysis, immunofluorescence, and patch clamp techniques, respectively. Expressed Kv1.5 had a half-life time of ∼6.7 h, which was prolonged by the proteasome inhibitors of MG132, ALLN, proteasomal inhibitor 1, or lactacystine, but not by a lysosomal inhibitor chloroquine. MG132 increased the protein level of Kv1.5, as well as the level of its ubiquitinated form in a dose-dependent manner. Similar effects of MG132 on endogenous Kv1.5 were seen in cultured rat atrial cells. Within a cell, Kv1.5 was mainly localized in both the endoplasmic reticulum and Golgi apparatus. MG132 increased the immunoreactivity of Kv1.5 in these compartments and also increased Ik ur currents through the cell-surface Kv1.5. Pretreatment with either brefeldin A or colchicine abolished MG132-induced increase in Ik ur currents. Conclusion Kv1.5 is degraded by the proteasome. The inhibition of the proteasome increased Ik ur currents secondary to stabilization of the channel protein in the endoplasmic reticulum/Golgi apparatus.

Positive inotropic and negative chronotropic effects of proton pump inhibitors in isolated rat atrium

The effects of three specific H +/K +-ATPase inhibitors (omeprazole, lansoprazole and SCH 28080 (2-methyl-8-(phenylmethoxy)-imidazo[1,2-a] pyridine-3-acetonitrile)) were investigated on the mechanical and electrophysiological properties of rat atrium, in vitro. Omeprazole (100–300 μM), lansoprazole (100–300 μM) and SCH 28080 (10–100 μM) increased the amplitude of contractions and decreased the beating rate. These effects are reversible, reproducible and correlated with their order of potency as gastric H +/K +-ATPase inhibitors; SCH 28080 > omeprazole = lansoprazole. Cardiac effects of proton pump inhibitors were not inhibited with phentolamine (5 μM), propranolol (15 μM), atropine (1 μM), ouabain (2 μM), theophylline (300 μM) and milrinone (100 μM). Ouabain-induced increase in beating rate and contracture development were antagonized by H +/K +-ATPase inhibitors. Ouabain increased the positive inotropic effect of H +/K +-ATPase inhibitors. Lansoprazole (300 μM) significantly prolonged the duration of action potentials in rat atrial cells. H +/K +-ATPase may play a crucial role in the mechanical and electrophysiological properties of rat atrial myocardium.

Effect of Sodium Channel Blocker, Pilsicainide Hydrochloride, on Net Inward Current of Atrial Myocytes in Thyroid Hormone Toxicosis Rats

To investigate effect of pilsicainide hydrochloride (pilsicainide) on electrocardiogram (ECG) signals and action potentials (APs) of atrial myocytes, levo-thyroxine (T4, 500 microg/kg body weight) was daily injected into peritoneal cavity of Sprague-Dawley rats for 14 days. T4-treatment significantly shortened RR interval, P wave, and QRS complex durations on ECG. Although pilsicainide did not affect the heart rate, P wave and corrected QT interval (QTc) was increased in T4-treated rats. AP recordings revealed that AP durations at 20%, 50%, and 90% repolarization were significantly shortened and maximal rate of rise (Max dV/dt) was significantly increased in T4-treated rat atrial cells. Pilsicainide significantly decreased AP amplitude (APA) and Max dV/dt in both control and T4-treated rat atrial cells. Concentration-inhibition study demonstrated that pilsicainide significantly inhibited net inward current of T4-treated rats at lower concentration (IC50 of 29.2 microg/mL) than that of control rats (133 microg/mL). In conclusion, pilsicainide could decrease the conduction velocity in T4-treated rat atrium by decreasing the Max dV/dt and net inward current, which could be a possible treatment of thyrotoxicosis-induced arrhythmia.

Functional Characterization of a Small Conductance GIRK Channel in Rat Atrial Cells

Muscarinic K + (K ACh) channels are key determinants of the inhibitory synaptic transmission in the heart. These channels are heterotetramers consisting of two homologous subunits, G-protein-gated inwardly rectifying K + (GIRK)1 and GIRK4, and have unitary conductance of ∼35 pS with symmetrical 150 mM KCl solutions. Activation of atrial K ACh channels, however, is often accompanied by the appearance of openings with a lower conductance, suggesting a functional heterogeneity of G-protein-sensitive ion channels in the heart. Here we report the characterization of a small conductance GIRK (scGIRK) channel present in rat atria. This channel is directly activated by G βγ subunits and has a unitary conductance of 16 pS. The scGIRK and K ACh channels display similar affinities for G βγ binding and are frequently found in the same membrane patches. Furthermore, G βγ-activated scGIRK channels—like their K ACh counterparts—exhibit complex gating behavior, fluctuating among four functional modes conferred by the apparent binding of a different number of G βγ subunits to the channel. The electrogenic efficacy of the scGIRK channels, however, is negligible compared to that of K ACh channels. Thus, G βγ subunits employ the same signaling strategy to regulate two ion channels that are apparently endowed with very different functions in the atrial membrane.

Functional Characterization of a Small Conductance GIRK Channel in Rat Atrial Cells

Functional Characterization of a Small Conductance GIRK Channel in Rat Atrial Cells

Role of receptor kinase in long-term desensitization of the cardiac muscarinic receptor-K+ channel system.

Desensitization of the cardiac muscarinic K+ channel was studied in cultured neonatal rat atrial cells and in Chinese hamster ovary (CHO) cells transfected with muscarinic receptor (HM(2)), G protein-coupled inward rectifying K+ channels 1 and 4, and G protein-coupled receptor kinase 2. In atrial cells incubated in 10 microM carbachol for 24 h, channel activity in cell-attached patches was substantially reduced as a result of long-term desensitization. The long-term desensitization was also observed in CHO cells transfected with the wild-type receptor and receptor kinase (as well as the channel). However, long-term desensitization was greatly reduced or abolished if the cells were 1) not transfected with the receptor kinase, 2) transfected with a mutant receptor lacking phosphorylation sites (rather than the wild-type receptor), or 3) transfected with a mutant receptor kinase lacking kinase activity (rather than the wild-type receptor kinase). We suggest that long-term desensitization of the cardiac muscarinic receptor-K+ channel system to muscarinic agonist may involve phosphorylation of the receptor by receptor kinase.

Levosimendan increases L-type Ca 2+ current via phosphodiesterase-3 inhibition in human cardiac myocytes

To evaluate the potency of levosimendan, a newly developed cardiotonic agent, as a phosphodiesterase-3 inhibitor, we examined its effects on the L-type Ca 2+ current ( I Ca,L) in single human atrial cells using the whole-cell voltage-clamp method. Levosimendan significantly increased I Ca,L in a concentration-dependent manner ( E max, 139.0±1.8%; EC 50, 54±3.6 nM). The increase in I Ca,L induced by 1 μM levosimendan was significantly greater in human atrial cells (136.7±11.0%, n=8) than in rabbit atrial cells (23.5±3.5%, n=6) (depolarization to +10 mV in each case). In rat atrial and ventricular cells, I Ca,L was unaffected by 1–10 μM levosimendan. These results indicate that the selective phosphodiesterase-3 inhibitor levosimendan increases cardiac-cell I Ca,L significantly more strongly in human than in rabbit and rat. It seems likely that the positive inotropic effect of levosimendan on the human myocardium depends on an increase in I Ca,L that is modulated by adenosine 3′5′-cyclic monophosphate (cAMP)-dependent phosphorylation.

Inhibitory effect of bepridil on hKv1.5 channel current: comparison with amiodarone and E-4031

Effects of bepridil on the depolarization-activated outward K + currents ( I out) in rat atrial myocytes and the human cardiac K + (hKv1.5) channel current stably expressed in human embryonic kidney (HEK) 293 cells were examined, and compared with those of amiodarone and N-[4-[[1-[2-(6-methyl-2-pyridinyl)ethyl]-4-piperidinyl]carbonyl]phenyl] methanesulphonamide dihydrochloride dihydrate (E-4031). Membrane currents were recorded using patch-clamp techniques in enzymatically isolated rat atrial myocytes and HEK 293 cells expressing hKv1.5 channels. Bepridil potently inhibited I out elicited by depolarization pulses and prolonged the action potential in rat atrial cells. Bepridil also inhibited the hKv1.5 channel current with the IC 50 value of 6.6 μM. The inhibitory effects of bepridil on the currents in HEK 293 cells were voltage-dependent. Amiodarone weakly inhibited rat atrial I out and hKv1.5 channel current. In contrast, E-4031 at a concentration of 10 μM had little influence on these currents. Thus, bepridil inhibits hKv1.5 channel current and the inhibitory effect may be useful for the treatment of atrial fibrillation.