Rat Atrial Myocytes Research Articles

Abstract 292: The Role of the ATF6 Branch of the ER Stress Response in the Endocrine Heart

Rationale: Atrial natriuretic peptide (ANP) is stored in the heart in large dense core granules of atrial myocytes as a biologically inactive precursor, pro-ANP. Hemodynamic stress and atrial stretch stimulate coordinate secretion and proteolytic cleavage of pro-ANP to its bioactive form, ANP, which promotes renal salt excretion and vasodilation, which, together contribute to decreasing blood pressure. While the ATF6 branch of the ER stress response has been studied in ventricular tissue mouse models of myocardial ischemia and pathological hypertrophy, roles for ATF6 and ER stress on the endocrine function of atrial myocytes have not been studied. Objective/Methods: To address this gap in our knowledge, we knocked down ATF6 in primary cultured neonatal rat atrial myocytes (NRAMs) using a chemical inhibitor of the proteolytic cleavage site enabling ATF6 activation and siRNA and measured ANP expression and secretion basally and in response to alpha- adrenergic agonist stimulation using phenylephrine. We also compared the ANP secretion from wild- type mice and ATF6 knockout mice in an ex vivo Langendorff model of the isolated perfused heart. Results: ATF6 knockdown in NRAMs significantly impaired basal and phenylephrine-stimulated ANP secretion. ATF6 knockout mice displayed lower levels of ANP in atrial tissue at baseline as well as after phenylephrine treatment. Similarly, in the ex vivo isolated perfused heart model, less ANP was detected in effluent of ATF6 knockout hearts compared to wild-type hearts. Conclusions: The ATF6 branch of the ER stress response is necessary for efficient co-secretional processing of pro-ANP to ANP and for agonist-stimulated ANP secretion from atrial myocytes. As ANP is secreted in a regulated manner in response to a stimulus and pro-ANP is synthesized and packaged through the classical secretory pathway, we posit that ATF6 is required for adequate expression, folding, trafficking, processing and secretion of biologically active ANP from the endocrine heart.

Effect of ethanol at clinically relevant concentrations on atrial inward rectifier potassium current sensitive to acetylcholine.

Alcohol intoxication tends to induce arrhythmias, most often the atrial fibrillation. To elucidate arrhythmogenic mechanisms related to alcohol consumption, the effect of ethanol on main components of the ionic membrane current is investigated step by step. Considering limited knowledge, we aimed to examine the effect of clinically relevant concentrations of ethanol (0.8-80mM) on acetylcholine-sensitive inward rectifier potassium current I K(Ach). Experiments were performed by the whole-cell patch clamp technique at 23 ± 1°C on isolated rat and guinea-pig atrial myocytes, and on expressed human Kir3.1/3.4 channels. Ethanol induced changes of I K(Ach) in the whole range of concentrations applied; the effect was not voltage dependent. The constitutively active component of I K(Ach) was significantly increased by ethanol with the maximum effect (an increase by ∼100%) between 8 and 20mM. The changes were comparable in rat and guinea-pig atrial myocytes and also in expressed human Kir3.1/3.4 channels (i.e., structural correlate of I K(Ach)). In the case of the acetylcholine-induced component of I K(Ach), a dual ethanol effect was apparent with a striking heterogeneity of changes in individual cells. The effect correlated with the current magnitude in control: the current was increased by eth-anol in the cells showing small current in control and vice versa. The average effect peaked at 20mM ethanol (an increase of the current by ∼20%). Observed changes of action potential duration agreed well with the voltage clamp data. Ethanol significantly affected both components of I K(Ach) even in concentrations corresponding to light alcohol consumption.

Shear stress activates monovalent cation channel transient receptor potential melastatin subfamily 4 in rat atrial myocytes via type 2 inositol 1,4,5-trisphosphate receptors and Ca(2+) release.

During each contraction and haemodynamic disturbance, cardiac myocytes are subjected to fluid shear stress as a result of blood flow and the relative movement of sheets of myocytes. The present study aimed to characterize the shear stress-sensitive membrane current in atrial myocytes using the whole-cell patch clamp technique, combined with pressurized fluid flow, as well as pharmacological and genetic interventions of specific proteins. The data obtained suggest that shear stress indirectly activates the monovalent cation current carried by transient receptor potential melastatin subfamily 4 channels via type 2 inositol 1,4,5-trisphosphate receptor-mediated Ca(2+) release in subsarcolemmal domains of atrial myocytes. Ca(2+) -mediated interactions between these two proteins under shear stress may be an important mechanism by which atrial cells measure mechanical stress and translate it to alter their excitability. Atrial myocytes are subjected to shear stress during the cardiac cycle under physiological or pathological conditions. The ionic currents regulated by shear stress remain poorly understood. We report the characteristics, molecular identity and activation mechanism of the shear stress-sensitive current (Ishear ) in rat atrial myocytes. A shear stress of ∼16dyn cm(-2) was applied to single myocytes using a pressurized microflow system, and the current was measured by whole-cell patch clamp. In symmetrical CsCl solutions with minimal concentrations of internal EGTA, Ishear showed an outwardly rectifying current-voltage relationship (reversal at -2mV). The current was conducted primarily (∼80%) by monovalent cations but not Ca(2+) . It was suppressed by intracellular Ca(2+) buffering at a fixed physiological level, inhibitors of transient receptor potential melastatin subfamily 4 (TRPM4), intracellular introduction of TRPM4 antibodies or knockdown of TRPM4 expression, suggesting that TRPM4 carries most of this current. A notable reduction in Ishear occurred upon inhibition of Ca(2+) release through the ryanodine receptors or inositol 1,4,5-trisphosphate receptors (IP3 R) and upon depletion of sarcoplasmic reticulum Ca(2+) . In type 2 IP3 R (IP3 R2) knockout atrial myocytes, Ishear was 10-20% of that in wild-type myocytes. Immunocytochemistry and proximity ligation assays revealed that TRPM4 and IP3 R2 were expressed at peripheral sites with co-localization, although they are not localized within 40nm. Peripheral localization of TRPM4 was intact in IP3 R2 knockout cells. The data obtained in the present study suggest that shear stress activates TRPM4 current by triggering Ca(2+) release from the IP3 R2 in the peripheral domains of atrial myocytes.

The MAPK pathway is involved in the regulation of rapid pacing-induced ionic channel remodeling in rat atrial myocytes

Alterations to the expression L‑type calcium channels (LTCCs) and Kv4.3 potassium channels form the possible basis of atrial electrical remodeling during rapid pacing. The mitogen‑activated protein kinase (MAPK) pathway is affected by increases in cytoplasmic Ca2+, and therefore represents an attractive candidate for the regulation and mediation of Ca2+‑induced ion channel remodeling. The present study aimed to investigate alterations to the ion channel‑MAPK axis, and to determine its influence on ion channel remodeling during atrial fibrillation. Rat atrial myocytes were isolated, cultured, and in vitro rapid pacing was established. Intracellular Ca2+ signals were monitored using the Fluo‑3/AM Ca2+ indicator. Verapamil, PD98058 and SB203580 were added to the culture medium of various groups at specific time‑points. The mRNA expression levels of LTCC‑α1c and Kv4.3 potassium channels were detected by reverse transcription‑polymerase chain reaction. Western blotting was performed to determine the expression levels of channel and signaling proteins. The results demonstrated that fast pacing significantly increased the intracellular Ca2+ concentration in atrial myocytes, whereas treatment with verapamil markedly inhibited this increase. In addition, verapamil significantly antagonized the rapid pacing‑induced activation of extracellular signal‑regulated kinase (ERK) and p38MAPK. These results indicated that the MAPK pathway may have an important role in the opening of LTCCs, and alterations to MAPK molecule expression could affect the expression and remodeling of ion channels.

Microdomain-Specific Remodelling of Autonomic Regulation of L-Type Calcium Channels Revealed by Super-Resolution Scanning Patch Clamp in Rat Atrial Myocytes in Heart Failure

It has recently become evident that discrete clusters of L-type Ca2+ channels (LTCCs) exist along the cardiomyocyte sarcolemma in association with distinct membrane structures and receptors forming macromolecular signalling complexes. Disruption in subcellular targeting of Ca2+ signaling complexes secondary to changes in cardiomyocyte structure, may contribute to the pathophysiology of heart failure (HF) altering cardiomyocyte function. Here, we used Ca2+ imaging and super-resolution scanning patch-clamp to examine microdomain-specific regulation of LTCCs by β1 adrenergic (β1ARs) and adenosine A1 receptors (AdoA1Rs) in atrial myocytes isolated from control and 16-weeks post-MI HF rats. In control, β1ARs stimulation (ISO 100 nM and β2AR antagonist ICI 50 nM) enhanced spontaneous Ca2+ activity which was abolished by AdoA1Rs stimulation (2′-MeCCPA 200 nM). Accordingly, activation of single LTCCs in both t-tubules and on the crest of sarcolemma from non-localized β1ARs stimulation (ISO 2 µM and ICI 50 nM in external solution) was also abolished by local AdoA1R stimulation (10 µM). In HF, both β1ARs and AdoA1R effects on LTCCs were preserved in t-tubules and lost on the crest. Similar, the effect of β1AR stimulation on spontaneous Ca2+ activities was significantly reduced (28% vs 134% in HF vs control, P<0.01) and the anti-adrenergic effect of AdoA1R was lost (8% vs 84% in HF vs control, P<0.01). In addition, the effects of both β1ARs and AdoA1Rs on normalized cell shortening were significantly reduced in HF. Our findings provide the first direct evidence for microdomain-specific remodeling of LTCC regulation by adrenergic and adenosine receptors in HF which could be linked to local disruption in the interactions between LTCCs and their cellular microenvironment.

ATP Release via GAP Junction Hemichannels in Rat Atrial Myocytes under Shear Stress

We have previously shown that shear stress (∼16 dyn/cm2) induces longitudinal Ca2+ propagation wave with a 0.2- to 3-s delay in atrial myocytes through the activation of P2Y1 purinergic signaling, and that the shear-mediated wave generation is inhibited by the blockade of gap junction hemichannels (J Physiol 2015 http://dx.doi.org/10.1113/JP271016). In the present study, we further examined whether atrial cells release ATP via the gap junction hemichannels. ATP releases of cells were directly measured with 2-s intervals as chemiluminescence emitted (at 562 nm) during ATP-driven luciferin-luciferase reaction. Shear stress was applied to fields of cells in a laminar flow chamber attached to the flow regulator. ATP was released from atrial myocytes by the application of shear stress in the range of 1-16 dyn/cm2 in a strength-dependent manner. The shear-induced ATP release occurred transiently with a maximal release (∼1.3x10−16 moles/μm2) at about 2 s and 90% decay at 4-6 s after the shear application (∼16 dyn/cm2 for 10 s). Removal of external Ca2+ to enhance the activity of the gap junction hemichannels increased the shear-mediated ATP release to ∼320% of control. In cells pre-treated with carbenoxolone (50 μM for 10 min) or La3+ (2 mM for 5 min), the hemichannel blockers, ATP release by shear application was suppressed by ∼50% and ∼90%, respectively. The shear-induced ATP release was not affected by the pre-treatment of 50 μM Gd3+ (5 min) or 1 mM 9-anthracenecarboxylic acid (10 min), suggesting no role of maxi anion channel and Cl− channels in shear-induced ATP release in these myocytes. Our data suggest that shear stress induces ATP release through the gap junction hemichannels in atrial myocytes.

Down Regulation of L-Type Calcium Current in Rat Atrial Myocytes during Heart Failure

Heart failure causes down-regulation of the atrial L-type Ca2+ current (ICaL) that has consequences on contractile function and susceptibility to atrial fibrillation. The reduction in atrial ICaL density has been suggested to involve changes in both the expression and phosphorylation of channel protein. The objective of this project was to examine the mechanism of the decrease in atrial ICaL in a surgical model of heart failure. Heart failure was induced in adult male Wistar rats by surgical ligation of the left anterior descending coronary artery (CAL) under general anesthesia. Control animals were subject to equivalent sham operation (Sham). Left atrial myocytes were isolated ∼18 weeks after surgery and superfused with a Tyrode's solution (37 °C). Whole-cell L-type Ca currents (ICaL) were recorded during depolarisation to 0 mV from a holding potential of −80 mV and normalised to whole-cell capacitance, which was also used as an index of cell size. CAL cells were hypertrophied (94.7±5.2 pF, n=63) compared to Sham (56.6±2.1 pF, n=47; P≤0.0001). ICaL density at 0 mV was reduced in CAL compared to Sham (3.3±0.4 pA/pF; n=31 vs 7.3±0.7 pA/pF; n=33; P≤0.001), but the response of ICaL to 1 μmol/L norepinephrine was greater in CAL (x6.8) than in Sham (x3.2) so that there was no difference in ICaL density between the two groups in the presence of norepinephrine. These data indicate a change in channel protein phosphorylation without a change in channel density. The regulation of L-type Ca2+ channel phosphorylation may be a potential therapeutic target for atrial arrhythmias in heart failure.This work was supported by the British Heart Foundation

TBX5 mutations contribute to early-onset atrial fibrillation in Chinese and Caucasians.

Atrial fibrillation (AF) is a common arrhythmia with an important heritable aspect. The genetic factors underlying AF have not been fully elucidated. We screened six candidate genes (CAV1, KCNJ2, KCNQ1, NKX2.5, PITX2, and TBX5) for novel mutations in 139 patients of Chinese descent with early-onset AF and 576 controls. Four missense TBX5 mutations, p.R355C, p.Q376R, p.A428S, and p.S372L, were identified in evolutionarily conserved regions. We did not find any mutations in CAV1, KCNJ2, KCNQ1, NKX2.5, and PITX2. These mutations increased the expression of atrial natriuretic peptide (ANP) and connexin-40 (CX40) in the primarily cultured rat atrial myocytes but did not alter the expression of cardiac structural genes, atrial myosin heavy chain-α (MHC-α) and myosin light chain-2α (MLC-2α). Overexpression of p.R355C developed an atrial arrhythmia suggestive of paroxysmal AF in the zebrafish model. To replicate our findings, we screened TBX5 in 527 early-onset AF cases from the Massachusetts General Hospital AF study. A novel TBX5 deletion (ΔAsp118, p.D118del) was identified, while no TBX5 mutations were identified in 1176 control subjects. Our results provide both genetic and functional evidence to support the contribution of TBX5 gene in the pathogenesis of AF. The potential mechanism of arrhythmia may be due in part to the disturbed expression of ANP and CX40.

Direct Evidence for Microdomain-Specific Localization and Remodeling of Functional L-Type Calcium Channels in Rat and Human Atrial Myocytes.

Distinct subpopulations of L-type calcium channels (LTCCs) with different functional properties exist in cardiomyocytes. Disruption of cellular structure may affect LTCC in a microdomain-specific manner and contribute to the pathophysiology of cardiac diseases, especially in cells lacking organized transverse tubules (T-tubules) such as atrial myocytes (AMs). Isolated rat and human AMs were characterized by scanning ion conductance, confocal, and electron microscopy. Half of AMs possessed T-tubules and structured topography, proportional to cell width. A bigger proportion of myocytes in the left atrium had organized T-tubules and topography than in the right atrium. Super-resolution scanning patch clamp showed that LTCCs distribute equally in T-tubules and crest areas of the sarcolemma, whereas, in ventricular myocytes, LTCCs primarily cluster in T-tubules. Rat, but not human, T-tubule LTCCs had open probability similar to crest LTCCs, but exhibited ≈ 40% greater current. Optical mapping of Ca(2+) transients revealed that rat AMs presented ≈ 3-fold as many spontaneous Ca(2+) release events as ventricular myocytes. Occurrence of crest LTCCs and spontaneous Ca(2+) transients were eliminated by either a caveolae-targeted LTCC antagonist or disrupting caveolae with methyl-β-cyclodextrin, with an associated ≈ 30% whole-cell ICa,L reduction. Heart failure (16 weeks post-myocardial infarction) in rats resulted in a T-tubule degradation (by ≈ 40%) and significant elevation of spontaneous Ca(2+) release events. Although heart failure did not affect LTCC occurrence, it led to ≈ 25% decrease in T-tubule LTCC amplitude. We provide the first direct evidence for the existence of 2 distinct subpopulations of functional LTCCs in rat and human AMs, with their biophysical properties modulated in heart failure in a microdomain-specific manner.

Distinctive property and pharmacology of voltage-gated sodium current in rat atrial vs ventricular myocytes

Several mammalian species display distinct biophysical properties between atrial and ventricular voltage-gated sodium current (INa); however, the potential mechanism behind this phenomenon is unknown. The purpose of this study was to investigate the potential molecular identities of the different INa in atrial and ventricular myocytes of rat hearts. Whole-cell patch voltage-clamp and molecular biology techniques were used in the study. Ventricular INa exhibited a slower inactivation, more positive potential of inactivation, and quicker recovery from inactivation compared to atrial INa. Real-time polymerase chain reaction and western blot analysis revealed that mRNA and protein levels of NaVβ2 and NaVβ4 subunits, but not NaV1.5, were greater in ventricular myocytes than in atrial myocytes. INa in heterologous HEK 293 cell expression system with coexpressing hNaV1.5 and hNaVβ2/hNaVβ4 showed similar biophysical properties to ventricular INa. Greater protein expression of NaVβ2 and NaVβ4 subunits was also observed in human ventricles. Interestingly, pharmacologic study revealed that the antiarrhythmic drug dronedarone (10 μM) inhibited atrial INa more (by 73%) than ventricular INa (by 42%), and shifted its inactivation to more negative voltages (-4.6 mV) compared to ventricular INa. The results of this study demonstrate the novel information that the distinctive biophysical properties of INa in atrial and ventricular myocytes can be attributed to inhomogeneous expression of NaVβ2 and NaVβ4 subunits, and that atrial INa is more sensitive to inhibition by dronedarone.

Abstract 11905: Loss of Adrenergic and Adenosine Regulation of Extradyadic L-type Calcium Channels in Rat Atrial Myocytes in Heart Failure

It has recently become evident that discrete clusters of L-type Ca2+ channels (LTCCs) exist along the cardiomyocyte sarcolemma in association with distinct membrane structures. Such microdomain-specific localization impact channel function and regulation by a variety of neurohormonal pathways, including adrenergic and adenosine. Disruption in their subcellular targeting may contribute to the pathophysiology of cardiac diseases, including heart failure (HF). We used Ca2+ imaging and super-resolution scanning patch clamp to examine microdomain-specific regulation of LTCCs in atrial myocytes isolated from control and 16-weeks post-MI HF rats. In control, β1 adrenergic receptors (β1ARs) stimulation (ISO 100 nM and β2AR antagonist ICI 50 nM) enhanced spontaneous Ca2+ release events while the following adenosine A1 receptors (AdoA1Rs) stimulation (2'-MeCCPA 200 nM) abolished β1ARs effects. Non-localized β1ARs stimulation (ISO 2 μM and ICI 50 nM in external solution) activated single LTCCs in both t-tubules and on the crest of sarcolemma, increasing LTCC occurrence and enhancing their open probability and amplitude. Local AdoA1R stimulation through the pipette (10 μM) on β1ARs-pretreated cells completely abolished β1ARs effects. In HF, both β1ARs and AdoA1R effects on LTCCs were preserved in t-tubules and lost on the crest. Similar, the effect of β1AR stimulation on spontaneous Ca2+ release events was significantly reduced in HF (28% vs 134% in HF vs control, P&lt;0.01) and the anti-adrenergic effect of AdoA1R was lost (8% vs 84% in HF vs control, P&lt;0.01), despite up-regulation of β1 and AdoA1Rs mRNA. No changes in caveolae density were observed in HF (4.6±0.4 caveolae/μm vs 3.7±0.5 caveolae/μm for HF vs control, P=0.167). Our findings provide the first direct evidence for microdomain-specific remodeling of LTCC regulation by adrenergic and adenosine receptors in HF which could be linked to local disruption in the interactions between LTCCs and their cellular microenvironment. This extends beyond the classical concept of electrical remodeling, stressing the involvement of alteration of spatial compartmentation of ion channels and receptors in addition to classical concepts of changes in protein expression and post-translational modifications.

Shear stress induces a longitudinal Ca(2+) wave via autocrine activation of P2Y1 purinergic signalling in rat atrial myocytes.

Atrial myocytes are exposed to shear stress during the cardiac cycle and haemodynamic disturbance. In response, they generate a longitudinally propagating global Ca(2+) wave. Here, we investigated the cellular mechanisms underlying the shear stress-mediated Ca(2+) wave, using two-dimensional confocal Ca(2+) imaging combined with a pressurized microflow system in single rat atrial myocytes. Shear stress of ∼16 dyn cm(-2) for 8 s induced ∼1.2 aperiodic longitudinal Ca(2+) waves (∼79 μm s(-1)) with a delay of 0.2-3 s. Pharmacological blockade of ryanodine receptors (RyRs) or inositol 1,4,5-trisphosphate receptors (IP3 Rs) abolished shear stress-induced Ca(2+) wave generation. Furthermore, in atrial myocytes from type 2 IP3R (IP3R2) knock-out mice, shear stress failed to induce longitudinal Ca(2+) waves. The phospholipase C (PLC) inhibitor U73122, but not its inactive analogue U73343, abolished the shear-induced longitudinal Ca(2+) wave. However, pretreating atrial cells with blockers for stretch-activated channels, Na(+)-Ca(2+) exchanger, transient receptor potential melastatin subfamily 4, or nicotinamide adenine dinucleotide phosphate oxidase did not suppress wave generation under shear stress. The P2 purinoceptor inhibitor suramin, and the potent P2Y1 receptor antagonist MRS 2179, both suppressed the Ca(2+) wave, whereas the P2X receptor antagonist, iso-PPADS, did not alter it. Suppression of gap junction hemichannels permeable to ATP or extracellular application of ATP-metabolizing apyrase inhibited the wave. Removal of external Ca(2+) to enhance hemichannel opening facilitated the wave generation. Our data suggest that longitudinally propagating, regenerative Ca(2+) release through RyRs is triggered by P2Y1-PLC-IP3R2 signalling that is activated by gap junction hemichannel-mediated ATP release in atrial myocytes under shear stress.

GW26-e2307 Acute and chronic effects of acehytisine on sodium channel in primary rat atrial myocytes

GW26-e2307 Acute and chronic effects of acehytisine on sodium channel in primary rat atrial myocytes

Fluid Shear Induces Proarrhythmic Ca2+ Wave and Alters Atrial Ca2+ Signaling: A Role of Autocrine Activations of P2-Purinergic/Type 2 Inositol Trisphosphate Receptor Signaling

Cardiac myocytes are subjected to fluid shear force (FSF) during each contraction and relaxation. Under pathological conditions, such as valve disease, heart failure and hypertension, FSF in cardiac chamber increases due to high blood volume and pressure. We have previously shown that FSF induces proarrhythmic longitudinal global Ca2+ waves and premature beat in rat atrial myocytes (Biophys J, 2014; 106 (2): 320a). In the present study, we further explored underlying cellular mechanisms for the FSF-induced longitudinal global Ca2+ waves and their effects on Ca2+ signaling in rat atrial myocytes. A FSF of ∼16 dyn/cm2 was applied onto entire single myocyte using pressurized fluid puffing. Confocal Ca2+ imaging was performed to measure local and global Ca2+ signals. Proarrhythmic longitudinal Ca2+ waves in atrial cells under FSF were resistant to the blockers for stretch-activated cation channel, Na+-Ca2+ exchange or Ca2+-activated cation channel (TRPM4), and to removal of external Ca2+. Interestingly, this FSF-induced atrial Ca2+ wave was eliminated by inositol 1,4,5-trisphosphate receptor (IP3R) blockers, tetracaine, or by phospholipase C inhibitor, and were absent in the type 2 IP3R (IP3R2) knockout cells. Furthermore, the FSF-induced atrial Ca2+ waves were suppressed by blockade of P2-purinergic receptors or by inhibition of gap junction that can secrete ATP. In field-stimulated cells, FSF immediately enlarged Ca2+ transients with larger increase in non-junctional Ca2+ release relative to junctional release. This FSF-induced enhancement in Ca2+ release was soon followed by significant reductions in Ca2+ transient and sarcoplasmic reticulum Ca2+ content. Our data suggest that IP3R2-mediated Ca2+ release, triggered by autocrine activation of P2-purinergic signaling, may generate proarrhythmic Ca2+ wave, thereby altering atrial Ca2+ signaling.

Activation of Ca2+-Dependent Cation Current by Fluid Shear Force in Atrial Myocytes

Atrial myocytes are subjected to fluid shear force (FSF) during each contraction and relaxation. Ionic currents regulated by shear force and their molecular integrity in cardiac myocytes have not been well-understood. We examined whether FSF activates specific current in atrial myocytes and underlying mechanisms for FSF-sensitive ionic current using whole-cell patch-clamp technique. A FSF of ∼16 dyne/cm2 was applied to entire single atrial myocyte using automated micro-puffing apparatus. A FSF-sensitive current (IFSF) was detected in lowly Ca2+-buffered (0.5 mM EGTA) atrial myocytes, but not in highly Ca2+-buffered (≥4 mM EGTA or 10 mM BAPTA) myocytes. The IFSF showed an outward rectification with a reversal potential of about -6 mV. The IFSF was inhibited by high concentrations (20-50 μM) of ryanodine and by replacement of external and internal cation with impermeant NMDG+, suggesting that IFSF is a Ca2+ release-dependent cation current. Application of either transient receptor potential melastatin subfamily 4 (TRPM4) inhibitor 9-phenanthrol or TRPM4-specific antibodies removed most of inward IFSF and ∼80% of outward IFSF. However, stretch-activated cation channel blocker GsMTx-4 did not affect IFSF. Interestingly, IFSF was strongly inhibited by inositol 1,4,5-trisphosphate receptor (IP3R) blockers, 2-APB (2 μM) or xestospongin C. In addition, in atrial myocytes isolated from type 2 IP3R (IP3R2) knock-out mouse, IFSF was not detected, although 9-phenanthrol-sensitive IFSF was recorded in wild-type myocytes. Co-immunostaining of TRPM4 and IP3R2 in rat atrial myocytes revealed peripheral localization of these proteins with some co-localizations. These results suggest that fluid shear stimuli may activate TRPM4 channels in atrial myocytes via Ca2+ releases triggered by the activation of nearby IP3R2.

Long-Term Modulation of Ion Channels by Aldosterone in Adult Rat Atrial Myocytes

In recent years, both aldosterone and mineralocorticoid receptor (MR) have drawn attention as important factors that promote structural remodeling of the atrium. Here, we investigated effects of chronic aldosterone treatment on both intracellular Ca2+ and ion channels. Atrial myocytes were cultured in either the absence or presence of aldosterone, and then the activity of ion channels was studied, under whole-cell patch-clamp conditions. Aldosterone increased both the cell membrane capacitance (Cm) and the maximal conductance of ion channels that give rise to IKsus, INa, ICaT, and ICaL (30-100%). Except for inactivation curves of INa, which were shifted by −10 mV, aldosterone produced no major alterations in the biophysical properties of the channels. Interestingly, at resting membrane potentials the increase in INa was cancelled by a greater fraction of inactivation. The onset and recovery of the changes -in ICaT, ICaL, and Cm- were also assessed. In general, they required 2d to be noticeable, reached their maximal value in 6d, and returned to basal values after 1-3d of aldosterone removal. The effects on both Cm and ICaL were further studied -to explore both a potential dose-response relationship and a possible implication of the MR. In fact, co-incubating with 10 µM of spironolactone (an MR antagonist) abolished both effects. Furthermore, their corresponding magnitudes fitted well with the Hill equation, being the EC50 values for Cm and ICaL 20 and 130 (nM), respectively. Interestingly, aldosterone did not alter expression levels of Cav1.2, suggesting that the action on ICaL arises from a stimulus in open probability. The hormone also produced a 40% increase in the amplitude of Ca2+ transients along with a higher proportion of arrhythmic cells (2.5-fold increase). These results contribute to understanding the role of the MR and aldosterone in atrial electrophysiology.

ASSA14-14-06 Expressions of endothelin-1 in primary cultured atrial myocytes after rapid electrical-field stimulation

<h3>Background</h3> To study the expressions of endothelin-1 at early stages of atrial fibrillation in a rapid paced primary cultured atrial myocyte model. <h3>Methods</h3> Primary rat atrial myocytes were cultured and a rapid paced cell model was established. The polymerase chain reaction (RT-PCR) and Enzyme-Linked Immunosorbent Assay (ELISA) were applied to detect the messenger ribonucleic acid (mRNA) expression and secretion of endothelin-1. <h3>Results</h3> mRNA expression of endothelin-1 increased after 6 h of rapid pacing and continued to elevate in a time dependent manner. Similarly, changes of endothelin-1 secretion were paralleled. <h3>Conclusion</h3> Expressions of endothelin-1 increased in early phase of rapid paced atrial myocytes. It implicated the endothelin-1 was implicated in pathological remodelling of atrial fibrillation.

Angiotensin II upregulates Kv1.5 expression through ROS-dependent transforming growth factor-beta1 and extracellular signal-regulated kinase 1/2 signalings in neonatal rat atrial myocytes

Kv1.5 potassium channel represents a promising target for atrial fibrillation (AF) therapy. During AF, the renin–angiotensin system is markedly activated. Recent evidence indicates that angiotensin II (Ang II) can upregulate Kv1.5 channel, but the mechanism remains unknown. In this study, we report that Ang II-mediated transforming growth factor-beta1 (TGF-β1)/Smad2/3 and extracellular signal-regulated kinase (ERK) 1/2 signalings are involved in atrial Kv1.5 expression. In neonatal rat atrial myocytes, quantitative PCR and Western blotting revealed that Ang II upregulated TGF-β1, synapse-associated protein 97 (SAP97) and Kv1.5 expression in a time- and concentration-dependent manner. The Ang II-induced upregulation of Kv1.5, SAP97 and phosphorylated Smad2/3 (P-Smad2/3) were reversed by the Ang II type 1 (AT1) receptor antagonist losartan, an anti-TGF-β1 antibody and the ERK 1/2 inhibitor PD98059 but not by the AT2 receptor antagonist PD123319. mRNA knockdown of either Smad2 or Smad3 blocked Ang II-induced expression of Kv1.5 and SAP97. These data suggest that AT1 receptor/TGF-β1/P-Smad2/3 and ERK 1/2 signalings are involved in Ang II-induced Kv1.5 and SAP97 expression. Flow cytometry and Western blotting revealed that losartan and the anti-TGF-β1 antibody diminished Ang II-induced reactive oxygen species (ROS) generation and that the antioxidants diphenyleneiodonium and N-acetyl cysteine inhibited Ang II-induced expression of P-Smad2/3, phosphorylated ERK (P-ERK) 1/2, Kv1.5, SAP97, suggesting that ROS participate in Kv1.5 and SAP97 regulation by modulating Ang II-induced P-Smad2/3 and P-ERK 1/2 expression. In conclusion, we demonstrate that ROS-dependent Ang II/AT1 receptor/TGF-β1/P-Smad2/3 and Ang II/ERK 1/2 signalings are involved in atrial Kv1.5 and SAP97 expression. Antioxidants would be beneficial for AF treatment through inhibiting atrial Kv1.5 expression.

Inhibition of a TREK-like K+ channel current by noradrenaline requires both β1- and β2-adrenoceptors in rat atrial myocytes.

AimsNoradrenaline plays an important role in the modulation of atrial electrophysiology. However, the identity of the modulated channels, their mechanisms of modulation, and their role in the action potential remain unclear. This study aimed to investigate the noradrenergic modulation of an atrial steady-state outward current (IKss).Methods and resultsRat atrial myocyte whole-cell currents were recorded at 36°C. Noradrenaline potently inhibited IKss (IC50 = 0.90 nM, 42.1 ± 4.3% at 1 µM, n = 7) and potentiated the L-type Ca2+ current (ICaL, EC50 = 136 nM, 205 ± 40% at 1 µM, n = 6). Noradrenaline-sensitive IKss was weakly voltage-dependent, time-independent, and potentiated by the arachidonic acid analogue, 5,8,11,14-eicosatetraynoic acid (EYTA; 10 µM), or by osmotically induced membrane stretch. Noise analysis revealed a unitary conductance of 8.4 ± 0.42 pS (n = 8). The biophysical/pharmacological properties of IKss indicate a TREK-like K+ channel. The effect of noradrenaline on IKss was abolished by combined β1-/β2-adrenoceptor antagonism (1 µM propranolol or 10 µM β1-selective atenolol and 100 nM β2-selective ICI-118,551 in combination), but not by β1- or β2-antagonist alone. The action of noradrenaline could be mimicked by β2-agonists (zinterol and fenoterol) in the presence of β1-antagonist. The action of noradrenaline on IKss, but not on ICaL, was abolished by pertussis toxin (PTX) treatment. The action of noradrenaline on ICaL was mediated by β1-adrenoceptors via a PTX-insensitive pathway. Noradrenaline prolonged APD30 by 52 ± 19% (n = 5; P < 0.05), and this effect was abolished by combined β1-/β2-antagonism, but not by atenolol alone.ConclusionNoradrenaline inhibits a rat atrial TREK-like K+ channel current via a PTX-sensitive mechanism involving co-operativity of β1-/β2-adrenoceptors that contributes to atrial APD prolongation.

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.