Rabbit Atrial Myocytes Research Articles

Role of Mitochondrial ROS for Calcium Alternans in Atrial Myocytes.

Atrial calcium transient (CaT) alternans is defined as beat-to-beat alternations in CaT amplitude and is causally linked to atrial fibrillation (AF). Mitochondria play a significant role in cardiac excitation-contraction coupling and Ca signaling through redox environment regulation. In isolated rabbit atrial myocytes, ROS production is enhanced during CaT alternans, measured by fluorescence microscopy. Exogenous ROS (tert-butyl hydroperoxide) enhanced CaT alternans, whereas ROS scavengers (dithiothreitol, MnTBAP, quercetin, tempol) alleviated CaT alternans. While the inhibition of cellular NADPH oxidases had no effect on CaT alternans, interference with mitochondrial ROS (ROSm) production had profound effects: (1) the superoxide dismutase mimetic MitoTempo diminished CaT alternans and shifted the pacing threshold to higher frequencies; (2) the inhibition of cyt c peroxidase by SS-31, and inhibitors of ROSm production by complexes of the electron transport chain S1QEL1.1 and S3QEL2, decreased the severity of CaT alternans; however (3) the impairment of mitochondrial antioxidant defense by the inhibition of nicotinamide nucleotide transhydrogenase with NBD-Cl and thioredoxin reductase-2 with auranofin enhanced CaT alternans. Our results suggest that intact mitochondrial antioxidant defense provides crucial protection against pro-arrhythmic CaT alternans. Thus, modulating the mitochondrial redox state represents a potential therapeutic approach for alternans-associated arrhythmias, including AF.

Increased Risk for Atrial Alternans in Rabbit Heart Failure: The Role of Ca2+/Calmodulin-Dependent Kinase II and Inositol-1,4,5-trisphosphate Signaling.

Heart failure (HF) increases the probability of cardiac arrhythmias, including atrial fibrillation (AF), but the mechanisms linking HF to AF are poorly understood. We investigated disturbances in Ca2+ signaling and electrophysiology in rabbit atrial myocytes from normal and failing hearts and identified mechanisms that contribute to the higher risk of atrial arrhythmias in HF. Ca2+ transient (CaT) alternans-beat-to-beat alternations in CaT amplitude-served as indicator of increased arrhythmogenicity. We demonstrate that HF atrial myocytes were more prone to alternans despite no change in action potentials duration and only moderate decrease of L-type Ca2+ current. Ca2+/calmodulin-dependent kinase II (CaMKII) inhibition suppressed CaT alternans. Activation of IP3 signaling by endothelin-1 (ET-1) and angiotensin II (Ang II) resulted in acute, but transient reduction of CaT amplitude and sarcoplasmic reticulum (SR) Ca2+ load, and lowered the alternans risk. However, prolonged exposure to ET-1 and Ang II enhanced SR Ca2+ release and increased the degree of alternans. Inhibition of IP3 receptors prevented the transient ET-1 and Ang II effects and by itself increased the degree of CaT alternans. Our data suggest that activation of CaMKII and IP3 signaling contribute to atrial arrhythmogenesis in HF.

Does the small conductance Ca2+-activated K+ current ISK flow under physiological conditions in rabbit and human atrial isolated cardiomyocytes?

BackgroundThe small conductance Ca2+-activated K+ current (ISK) is a potential therapeutic target for treating atrial fibrillation. AimTo clarify, in rabbit and human atrial cardiomyocytes, the intracellular [Ca2+]-sensitivity of ISK, and its contribution to action potential (AP) repolarisation, under physiological conditions. MethodsWhole-cell-patch clamp, fluorescence microscopy: to record ion currents, APs and [Ca2+]i; 35–37°C. ResultsIn rabbit atrial myocytes, 0.5 mM Ba2+ (positive control) significantly decreased whole-cell current, from −12.8 to −4.9 pA/pF (P < 0.05, n = 17 cells, 8 rabbits). By contrast, the ISK blocker apamin (100 nM) had no effect on whole-cell current, at any set [Ca2+]i (∼100–450 nM). The ISK blocker ICAGEN (1 μM: ≥2 x IC50) also had no effect on current over this [Ca2+]i range. In human atrial myocytes, neither 1 μM ICAGEN (at [Ca2+]i ∼ 100–450 nM), nor 100 nM apamin ([Ca2+]i ∼ 250 nM) affected whole-cell current (5–10 cells, 3–5 patients/group). APs were significantly prolonged (at APD30 and APD70) by 2 mM 4-aminopyridine (positive control) in rabbit atrial myocytes, but 1 μM ICAGEN had no effect on APDs, versus either pre-ICAGEN or time-matched controls. High concentration (10 μM) ICAGEN (potentially ISK-non-selective) moderately increased APD70 and APD90, by 5 and 26 ms, respectively. In human atrial myocytes, 1 μM ICAGEN had no effect on APD30–90, whether stimulated at 1, 2 or 3 Hz (6–9 cells, 2–4 patients/rate). ConclusionISK does not flow in human or rabbit atrial cardiomyocytes with [Ca2+]i set within the global average diastolic-systolic range, nor during APs stimulated at physiological or supra-physiological (≤3 Hz) rates.

Calcium- and voltage-driven atrial alternans: Insight from [Ca]i and Vm asynchrony.

Cardiac alternans is defined as beat-to-beat alternations in contraction strength, action potential duration (APD), and Ca transient (CaT) amplitude. Cardiac excitation-contraction coupling relies on the activity of two bidirectionally coupled excitable systems, membrane voltage (Vm ) and Ca release. Alternans has been classified as Vm - or Ca-driven, depending whether a disturbance of Vm or [Ca]i regulation drives the alternans. We determined the primary driver of pacing induced alternans in rabbit atrial myocytes, using combined patch clamp and fluorescence [Ca]i and Vm measurements. APD and CaT alternans are typically synchronized; however, uncoupling between APD and CaT regulation can lead to CaT alternans in the absence of APD alternans, and APD alternans can fail to precipitate CaT alternans, suggesting a considerable degree of independence of CaT and APD alternans. Using alternans AP voltage clamp protocols with extra APs showed that most frequently the pre-existing CaT alternans pattern prevailed after the extra-beat, indicating that alternans is Ca-driven. In electrically coupled cell pairs, dyssynchrony of APD and CaT alternans points to autonomous regulation of CaT alternans. Thus, with three novel experimental protocols, we collected evidence for Ca-driven alternans; however, the intimately intertwined regulation of Vm and [Ca]i precludes entirely independent development of CaT and APD alternans.

The 'Reverse FDUF' Mechanism of Atrial Excitation-Contraction Coupling Sustains Calcium Alternans-A Hypothesis.

Cardiac calcium alternans is defined as beat-to-beat alternations of Ca transient (CaT) amplitude and has been linked to cardiac arrhythmia, including atrial fibrillation. We investigated the mechanism of atrial alternans in isolated rabbit atrial myocytes using high-resolution line scan confocal Ca imaging. Alternans was induced by increasing the pacing frequency until stable alternans was observed (1.6-2.5 Hz at room temperature). In atrial myocytes, action potential-induced Ca release is initiated in the cell periphery and subsequently propagates towards the cell center by Ca-induced Ca release (CICR) in a Ca wave-like fashion, driven by the newly identified 'fire-diffuse-uptake-fire' (FDUF) mechanism. The development of CaT alternans was accompanied by characteristic changes of the spatio-temporal organization of the CaT. During the later phase of the CaT, central [Ca]i exceeded peripheral [Ca]i that was indicative of a reversal of the subcellular [Ca]i gradient from centripetal to centrifugal. This gradient reversal resulted in a reversal of CICR propagation, causing a secondary Ca release during the large-amplitude alternans CaT, thereby prolonging the CaT, enhancing Ca-release refractoriness and reducing Ca release on the subsequent beat, thus enhancing the degree of CaT alternans. Here, we propose the 'reverse FDUF' mechanism as a novel cellular mechanism of atrial CaT alternans, which explains how the uncoupling of central from peripheral Ca release leads to the reversal of propagating CICR and to alternans.

Adrenoceptor sub-type involvement in Ca2+ current stimulation by noradrenaline in human and rabbit atrial myocytes

Atrial fibrillation (AF) from elevated adrenergic activity may involve increased atrial L-type Ca2+ current (ICaL) by noradrenaline (NA). However, the contribution of the adrenoceptor (AR) sub-types to such ICaL-increase is poorly understood, particularly in human. We therefore investigated effects of various broad-action and sub-type-specific α- and β-AR antagonists on NA-stimulated atrial ICaL. ICaL was recorded by whole-cell-patch clamp at 37 °C in myocytes isolated enzymatically from atrial tissues from consenting patients undergoing elective cardiac surgery and from rabbits. NA markedly increased human atrial ICaL, maximally by ~ 2.5-fold, with EC75 310 nM. Propranolol (β1 + β2-AR antagonist, 0.2 microM) substantially decreased NA (310 nM)-stimulated ICaL, in human and rabbit. Phentolamine (α1 + α2-AR antagonist, 1 microM) also decreased NA-stimulated ICaL. CGP20712A (β1-AR antagonist, 0.3 microM) and prazosin (α1-AR antagonist, 0.5 microM) each decreased NA-stimulated ICaL in both species. ICI118551 (β2-AR antagonist, 0.1 microM), in the presence of NA + CGP20712A, had no significant effect on ICaL in human atrial myocytes, but increased it in rabbit. Yohimbine (α2-AR antagonist, 10 microM), with NA + prazosin, had no significant effect on human or rabbit ICaL. Stimulation of atrial ICaL by NA is mediated, based on AR sub-type antagonist responses, mainly by activating β1- and α1-ARs in both human and rabbit, with a β2-inhibitory contribution evident in rabbit, and negligible α2 involvement in either species. This improved understanding of AR sub-type contributions to noradrenergic activation of atrial ICaL could help inform future potential optimisation of pharmacological AR-antagonism strategies for inhibiting adrenergic AF.

Effects and pharmacological mechanism of Zhigancao Decoction on electrical and structural remodeling of the atrium of rabbits induced by rapid atrial pacing.

Zhigancao decoction (ZD) has a long history in China as a traditional Chinese medicine compound for the treatment of tachyarrhythmias. This study mainly explored the pharmacological mechanism of Zhigancao Decoction in preventing atrial fibrillation by altering the electrical and structural remodeling of the atrial in rabbits. In total, 30 male New Zealand white rabbits were randomly divided into 3 groups (ten rabbits for each). The first group was sham-operated (control group). The second group was intervened by the rapid right atrium pacing (RAP) to induce atrial fibrillation (AF group), while the third group was given ZD gavage and RAP (AF + ZD group). All rabbits were anesthetized before two monophasic action potential (MAP) catheters were sequentially inserted into the right atrium. After 8h of rapid right atrial pacing, the electrophysiological indexes and the induction rate of atrial fibrillation were observed in the three groups of rabbits, and the left atrial myocardium samples were taken to observe the ultrastructure. Single atrial myocytes were separated by enzymolysis, and the L-type calcium current (ICa-L) of atrial myocytes in different experimental groups was observed by whole-cell patch clamp technique. The fluorescence intensity of Ca2+ in atrial myocytes was observed after Fluo-3/AM fluorescent staining. The main components of ZD were identified by liquid chromatography-mass spectrometry-mass spectrometry (LC-MS/MS) method. Compared with the AF group, the maximum ascent rate (Max dV/dt) and plateau potential were significantly reduced in the ZD group, the action potential duration at 10% and 20% (APD10, APD20) were significantly shortened (P < 0.01), action potential duration at 50%, 70%, and 90% (APD50, APD70, APD90) were significantly prolonged, and atrial effective refractory period (AERP) was significantly prolonged (P < 0.01) in the ZD group. In the ZD group, the ICa-L amplitudes of rabbit atrial myocytes under each clamping voltage were significantly smaller than those in the AF group (P < 0.01) and the control group (P < 0.05). The Ca2+ fluorescence intensity in the rabbit atrial myocytes in the ZD group was significantly weaker than that in the AF group (P < 0.01) and the control group (P < 0.05). Electron microscopy displayed that the control group had neatly arranged atrial tissue myofilaments and intact mitochondria. However, the ultrastructural damage of the AF group was severe compared with that of the ZD group. LC-MS/MS analysis confirmed that ZD contained several antiarrhythmic compounds including ginsenoside, isoliensinine, catalpol, glycyrrhizinate and hesperetin. Rapid atrial pacing (RAP) could cause the electrical and structural remodeling of rabbit atrial myocytes. ZD might reverse the atrial electrical remodeling but could have little effect on structural remodeling, which might be the mechanism of ZD treatment on atrial fibrillation.

Mechanism of carvedilol induced action potential and calcium alternans

ABSTRACT Carvedilol is a nonspecific β-blocker clinically used for the treatment of cardiovascular diseases but has also been shown to have profound effects on excitation-contraction coupling and Ca signaling at the cellular level. We investigate the mechanism by which carvedilol facilitates Ca transient (CaT) and action potential duration (APD) alternans in rabbit atrial myocytes. Carvedilol lowered the frequency threshold for pacing-induced CaT alternans and facilitated alternans in a concentration-dependent manner. Carvedilol prolonged the sarcoplasmic reticulum (SR) Ca release refractoriness by significantly increasing the time constant τ of recovery of SR Ca release; however, no changes in L-type calcium current recovery from inactivation or SR Ca load were found after carvedilol treatment. Carvedilol enhanced the degree of APD alternans nearly two-fold. Carvedilol slowed the APD restitution kinetics and steepened the APD restitution curve at the pacing frequency (2 Hz) where alternans were elicited. No effect on the CaT or APD alternans ratios was observed in experiments with a different β-blocker (metoprolol), excluding the possibility that the carvedilol effect on CaT and APD alternans was determined by its β-blocking properties. These data suggest that carvedilol contributes to the generation of CaT and APD alternans in atrial myocytes by modulating the restitution of CaT and APD.

L-type Ca2+ channel recovery from inactivation in rabbit atrial myocytes.

Adaptation of the myocardium to varying workloads critically depends on the recovery from inactivation (RFI) of L‐type Ca2+ channels (LCCs) which provide the trigger for cardiac contraction. The goal of the present study was a comprehensive investigation of LCC RFI in atrial myocytes. The study was performed on voltage‐clamped rabbit atrial myocytes using a double pulse protocol with variable diastolic intervals in cells held at physiological holding potentials, with intact intracellular Ca2+ release, and preserved Na+ current and Na+/Ca2+ exchanger (NCX) activity. We demonstrate that the kinetics of RFI of LCCs are co‐regulated by several factors including resting membrane potential, [Ca2+]i, Na+ influx, and activity of CaMKII. In addition, activation of CaMKII resulted in increased I Ca amplitude at higher pacing rates. Pharmacological inhibition of NCX failed to have any significant effect on RFI, indicating that impaired removal of Ca2+ by NCX has little effect on LCC recovery. Finally, RFI of intracellular Ca2+ release was substantially slower than LCC RFI, suggesting that inactivation kinetics of LCC do not significantly contribute to the beat‐to‐beat refractoriness of SR Ca2+ release. The study demonstrates that CaMKII and intracellular Ca2+ dynamics play a central role in modulation of LCC activity in atrial myocytes during increased workloads that could have important consequences under pathological conditions such as atrial fibrillations, where Ca2+ cycling and CaMKII activity are altered.

A Computational Study of the Effects of Tachycardia-Induced Remodeling on Calcium Wave Propagation in Rabbit Atrial Myocytes.

In atrial cardiomyocytes without a well-developed T-tubule system, calcium diffuses from the periphery toward the center creating a centripetal wave pattern. During atrial fibrillation, rapid activation of atrial myocytes induces complex remodeling in diffusion properties that result in failure of calcium to propagate in a fully regenerative manner toward the center; a phenomenon termed “calcium silencing.” This has been observed in rabbit atrial myocytes after exposure to prolonged rapid pacing. Although experimental studies have pointed to possible mechanisms underlying calcium silencing, their individual effects and relative importance remain largely unknown. In this study we used computational modeling of the rabbit atrial cardiomyocyte to query the individual and combined effects of the proposed mechanisms leading to calcium silencing and abnormal calcium wave propagation. We employed a population of models obtained from a newly developed model of the rabbit atrial myocyte with spatial representation of intracellular calcium handling. We selected parameters in the model that represent experimentally observed cellular remodeling which have been implicated in calcium silencing, and scaled their values in the population to match experimental observations. In particular, we changed the maximum conductances of ICaL, INCX, and INaK, RyR open probability, RyR density, Serca2a density, and calcium buffering strength. We incorporated remodeling in a population of 16 models by independently varying parameters that reproduce experimentally observed cellular remodeling, and quantified the resulting alterations in calcium dynamics and wave propagation patterns. The results show a strong effect of ICaL in driving calcium silencing, with INCX, INaK, and RyR density also resulting in calcium silencing in some models. Calcium alternans was observed in some models where INCX and Serca2a density had been changed. Simultaneously incorporating changes in all remodeled parameters resulted in calcium silencing in all models, indicating the predominant role of decreasing ICaL in the population phenotype.

Two-hit mechanism of cardiac arrhythmias in diabetic hyperglycaemia: reduced repolarization reserve, neurohormonal stimulation, and heart failure exacerbate susceptibility.

Diabetic hyperglycaemia is associated with increased arrhythmia risk. We aimed to investigate whether hyperglycaemia alone can be accountable for arrhythmias or whether it requires the presence of additional pathological factors. Action potentials (APs) and arrhythmogenic spontaneous diastolic activities were measured in isolated murine ventricular, rabbit atrial, and ventricular myocytes acutely exposed to high glucose. Acute hyperglycaemia increased the short-term variability (STV) of action potential duration (APD), enhanced delayed afterdepolarizations, and the inducibility of APD alternans during tachypacing in both murine and rabbit atrial and ventricular myocytes. Hyperglycaemia also prolonged APD in mice and rabbit atrial cells but not in rabbit ventricular myocytes. However, rabbit ventricular APD was more strongly depressed by block of late Na+ current (INaL) during hyperglycaemia, consistent with elevated INaL in hyperglycaemia. All the above proarrhythmic glucose effects were Ca2+-dependent and abolished by CaMKII inhibition. Importantly, when the repolarization reserve was reduced by pharmacological inhibition of K+ channels (either Ito, IKr, IKs, or IK1) or hypokalaemia, acute hyperglycaemia further prolonged APD and further increased STV and alternans in rabbit ventricular myocytes. Likewise, when rabbit ventricular myocytes were pretreated with isoproterenol or angiotensin II, hyperglycaemia significantly prolonged APD, increased STV and promoted alternans. Moreover, acute hyperglycaemia markedly prolonged APD and further enhanced STV in failing rabbit ventricular myocytes. We conclude that even though hyperglycaemia alone can enhance cellular proarrhythmic mechanisms, a second hit which reduces the repolarization reserve or stimulates G protein-coupled receptor signalling greatly exacerbates cardiac arrhythmogenesis in diabetic hyperglycaemia.

Eleutheroside B, a selective late sodium current inhibitor, suppresses atrial fibrillation induced by sea anemone toxin II in rabbit hearts.

Eleutheroside B (EB) is the main active constituent derived from the Chinese herb Acanthopanax senticosus (AS) that has been reported to possess cardioprotective effects. In this study we investigated the effects of EB on cardiac electrophysiology and its suppression on atrial fibrillation (AF). Whole-cell recording was conducted in isolated rabbit atrial myocytes. The intracellular calcium ([Ca2+]i) concentration was measured using calcium indicator Fura-2/AM fluorescence. Monophasic action potential (MAP) and electrocardiogram (ECG) synchronous recordings were conducted in Langendorff-perfused rabbit hearts using ECG signal sampling and analysis system. We showed that EB dose-dependently inhibited late sodium current (INaL), transient sodium current (INaT), and sea anemone toxin II (ATX II)-increased INaL with IC50 values of 167, 1582, and 181 μM, respectively. On the other hand, EB (800 μM) did not affect L-type calcium current (ICaL), inward rectifier potassium channel current (IK), and action potential duration (APD). Furthermore, EB (300 μM) markedly decreased ATX II-prolonged the APD at 90% repolarization (APD90) and eliminated ATX II-induced early afterdepolarizations (EADs), delayed afterdepolarizations (DADs), and triggered activities (TAs). Moreover, EB (200 μM) significantly suppressed ATX II-induced Na+-dependent [Ca2+]i overload in atrial myocytes. In the Langendorff-perfused rabbit hearts, application of EB (200 μM) or TTX (2 μM) substantially decreased ATX II-induced incidences of atrial fibrillation (AF), ventricular fibrillation (VF), and heart death. These results suggest that augmented INaL alone is sufficient to induce AF, and EB exerts anti-AF actions mainly via blocking INaL, which put forward the basis of pharmacology for new clinical application of EB.

The Role of NAADP-Mediated Endo-Lysosomal Calcium Release in the Cardiac Atria

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a highly potent calcium-mobilising second messenger which stimulates calcium release from endo-lysosomes via the opening of TPC2 channels. In cardiac ventricular myocytes, lysosomes form nano-junctions with the sarcoplasmic reticulum (SR) and mitochondria and exposure to NAADP (synthesised by CD38 upon beta-adrenergic stimulation), leads to an augmented calcium transient and SR calcium content, and contributes to the hypertrophy and arrhythmogenesis associated with chronic beta-adrenergic stress. We utilised transmission electron microscopy (TEM) and optical mapping in lapine and murine models to investigate endo-lysosomal calcium signalling in the cardiac atria. TEM of fixed, imbedded rabbit atrial myocytes or tissue (n=3 animals) confirmed that lysosomes form nano-junctions with both the SR (median distance 17nm, IQR 13-21nm, n=15 lysosomes) and mitochondria (median distance 13nm, IQR 10-57nm, n=16 lysosomes). These are close enough to have the potential for localised calcium signalling. In intact murine left atria, 10nM isoprenaline caused a 59.3±4.8% increase in calcium transient amplitude (n=6, fluorophore rhod-2). Abolishing endo-lysosomal calcium storage using 5µM Bafilomycin A1 (Baf) significantly reduced the calcium transient response to isoprenaline (to 35.3±2.7%, n=6, P<0.01). In CD38KO mice, left atrial responses were significantly reduced (35.9±2.6%, n=6) in comparison to wild-type controls (59.3±4.9%, n=6, P<0.01) and there was no longer an effect of Baf on isoprenaline response (35.9±2.6% and 33.6±4.0% in CD38KO control and Baf respectively, P>0.05, both n=6). Our data suggest that, endo-lysosomes can form nanojunctions for calcium signalling within cardiac atrial myocytes and that the endo-lysosomal NAADP pathway is functionally relevant during beta-adrenergic stimulation. The changes to and effects of this stereotyped architecture and pathway in atrial disease states are unknown and will be a vital avenue for future research.

Effects and mechanism of digoxin on atrium electrical remodeling and susceptibility of atrial fibrillation in aged rabbits

Objective: To investigate the effects and mechanism of digoxin on atrium electrical remodeling and susceptibility of atrial fibrillation (AF) in aged rabbits. Methods: Twenty aged male New Zealand rabbits were divided into aged group and aged plus digoxin group (n=10 each). Electrical parameters including heart rate (HR), RR and QT interval, ST segment and P wave dispersion from normal Ⅱ electrocardiogram, and the maximum upstroke velocity (Max(dv/dt)), plateau potential (plateau P), action potential duration of 10%, 20% and 90% (APD(10), APD(20), APD(90)) from recording of monophasic action potential (MAP), as well as atrial effective refractory period (AERP(200)) and dispersion (dERP(200)) with 200 ms of basic cycle length (BCL), and frequency self adaptation of AERP with 300 ms and 150 ms of BCLs (fERP) were recorded and compared between the 2 groups. BCLs and inducibility of AF post programmed electrical stimulation and Burst-pacing in left atrium tissue of rabbits in vivo were also analyzed. The L-type calcium current (I(Ca-L)) in 2 groups were recorded via whole-cell patch clamp technique, and the fluorescence intensity of intracellular free Ca(2+) was detected with Flup-3/AM loading by the laser scanning confocal microscope in enzymatically dissociated single rabbit atrial myocytes. Results: Compared with aged group, the heart rate was faster, RR and QT interval were obvious shorter, ST segment was raised and P wave dispersion was significantly increased in aged plus digoxin group (all P<0.05). Moreover, compared with aged group, the Max(dv/dt) and plateau P were obviously increased, APD(10) and APD(20) were significantly prolongated, and APD(9)0 was significantly shorter in aged plus digoxin group (all P<0.01). Otherwise, the fERP was markedly increased (0.81±0.15 vs. 0.67±0.05), and the induced rate of AF was obviously higher in aged plus digoxin group than in aged group (6/8 vs. 4/9) (all P<0.01). With voltage clamp model, digoxin significantly increased I(Ca-L) of atrial myocytes of aged rabbits, When command potential was 10 mV, the current densities of I(Ca-L) were significantly higher in digoxin group than that in aged group ((15.45±2.38) pA/pF vs. (7.03±1.69) pA/pF, P<0.01). Otherwise, the I-V curve of I(Ca-L) was downward shifted of all I-V curves in digoxin perfused aged atrial cells of rabbits. Moreover, the fluorescence intensities of intracellular free Ca(2+) was significantly higher in aged plus digoxin group than in aged group ((1 748±173) μmol/L vs. (478.13±87.63) μmol/L, P<0.01). Conclusion: Digoxin could aggravate the atrial electrical remodeling in atrium of aged rabbits, facilitate susceptibility of atrial fibrillation in aged rabbit, increased current density of I(Ca-L) and concentration of intracellular free Ca(2+), followed Ca(2+) overload and oscillations might be part of the underlying mechanisms.

Dynamic clamping human and rabbit atrial calcium current: narrowing ICaL window abolishes early afterdepolarizations.

Key points Early‐afterdepolarizations (EADs) are abnormal action potential oscillations and a known cause of cardiac arrhythmias. Ventricular EADs involve reactivation of a Ca2+ current (I CaL) in its ‘window region’ voltage range. However, electrical mechanisms of atrial EADs, a potential cause of atrial fibrillation, are poorly understood.Atrial cells were obtained from consenting patients undergoing heart surgery, as well as from rabbits. I CaL was blocked with nifedipine and then a hybrid patch clamp/mathematical‐modelling technique, ‘dynamic clamping’, was used to record action potentials at the same time as injecting an artificial, modifiable, I CaL (I CaL,D‐C).Progressively widening the I CaL,D‐C window region produced EADs of various types, dependent on window width. EAD production was strongest upon moving the activation (vs. inactivation) side of the window.EADs were then induced by a different method: increasing I CaL,D‐C amplitude and/or K+ channel‐blockade (4‐aminopyridine). Narrowing of the I CaL,D‐C window by ∼10 mV abolished these EADs.Atrial I CaL window narrowing is worthy of further testing as a potential anti‐atrial fibrillation drug mechanism. Atrial early‐afterdepolarizations (EADs) may contribute to atrial fibrillation (AF), perhaps involving reactivation of L‐type Ca2+ current (I CaL) in its window region voltage range. The present study aimed (i) to validate the dynamic clamp technique for modifying the I CaL contribution to atrial action potential (AP) waveform; (ii) to investigate the effects of widening the window I CaL on EAD‐propensity; and (iii) to test whether EADs from increased I CaL and AP duration are supressed by narrowing the window I CaL. I CaL and APs were recorded from rabbit and human atrial myocytes by whole‐cell‐patch clamp. During AP recording, I CaL was inhibited (3 µm nifedipine) and replaced by a dynamic clamp model current, I CaL,D‐C (tuned to native I CaL characteristics), computed in real‐time (every 50 µs) based on myocyte membrane potential. I CaL,D‐C‐injection restored the nifedipine‐suppressed AP plateau. Widening the window I CaL,D‐C, symmetrically by stepwise simultaneous equal shifts of half‐voltages (V 0.5) of I CaL,D‐C activation (negatively) and inactivation (positively), generated EADs (single, multiple or preceding repolarization failure) in a window width‐dependent manner, as well as AP alternans. A stronger EAD‐generating effect resulted from independently shifting activation V 0.5 (asymmetrical widening) than inactivation V 0.5; for example, a 15 mV activation shift produced EADs in nine of 17 (53%) human atrial myocytes vs. 0 of 18 from inactivation shift (P < 0.05). In 11 rabbit atrial myocytes in which EADs were generated either by increasing the conductance of normal window width I CaL,D‐C or subsequent 4‐aminopyridine (2 mm), window I CaL,D‐C narrowing (10 mV) abolished EADs of all types (P < 0.05). The present study validated the dynamic clamp for I CaL, which is novel in atrial cardiomyocytes, and showed that EADs of various types are generated by widening (particularly asymmetrically) the window I CaL, as well as abolished by narrowing it. Window I CaL narrowing is a potential therapeutic mechanism worth pursuing in the search for improved anti‐AF drugs.

Action potential shortening rescues atrial calcium alternans.

Cardiac alternans refers to a beat-to-beat alternation in contraction, action potential (AP) morphology and Ca2+ transient (CaT) amplitude, and represents a risk factor for cardiac arrhythmia, including atrial fibrillation. We developed strategies to pharmacologically manipulate the AP waveform with the goal to reduce or eliminate the occurrence of CaT and contraction alternans in atrial tissue. With combined patch-clamp and intracellular Ca2+ measurements we investigated the effect of specific ion channel inhibitors and activators on alternans. In single rabbit atrial myocytes, suppression of Ca2+ -activated Cl- channels eliminated AP duration alternans, but prolonged the AP and failed to eliminate CaT alternans. In contrast, activation of K+ currents (IKs and IKr ) shortened the AP and eliminated both AP duration and CaT alternans. As demonstrated also at the whole heart level, activation of K+ conductances represents a promising strategy to suppress alternans, and thus reducing a risk factor for atrial fibrillation. At the cellular level alternans is observed as beat-to-beat alternations in contraction, action potential (AP) morphology and magnitude of the Ca2+ transient (CaT). Alternans is a well-established risk factor for cardiac arrhythmia, including atrial fibrillation. This study investigates whether pharmacological manipulation of AP morphology is a viable strategy to reduce the risk of arrhythmogenic CaT alternans. Pacing-induced AP and CaT alternans were studied in rabbit atrial myocytes using combined Ca2+ imaging and electrophysiological measurements. Increased AP duration (APD) and beat-to-beat alternations in AP morphology lowered the pacing frequency threshold and increased the degree of CaT alternans. Inhibition of Ca2+ -activated Cl- channels reduced beat-to-beat AP alternations, but prolonged APD and failed to suppress CaT alternans. In contrast, AP shortening induced by activators of two K+ channels (ML277 for Kv7.1 and NS1643 for Kv11.1) abolished both APD and CaT alternans in field-stimulated and current-clamped myocytes. K+ channel activators had no effect on the degree of Ca2+ alternans in AP voltage-clamped cells, confirming that suppression of Ca2+ alternans was caused by the changes in AP morphology. Finally, activation of Kv11.1 channel significantly attenuated or even abolished atrial T-wave alternans in isolated Langendorff perfused hearts. In summary, AP shortening suppressed or completely eliminated both CaT and APD alternans in single atrial myocytes and atrial T-wave alternans at the whole heart level. Therefore, we suggest that AP shortening is a potential intervention to avert development of alternans with important ramifications for arrhythmia prevention and therapy.

Axial Tubule Junctions Activate Atrial Ca2+ Release Across Species.

Rationale: Recently, abundant axial tubule (AT) membrane structures were identified deep inside atrial myocytes (AMs). Upon excitation, ATs rapidly activate intracellular Ca2+ release and sarcomeric contraction through extensive AT junctions, a cell-specific atrial mechanism. While AT junctions with the sarcoplasmic reticulum contain unusually large clusters of ryanodine receptor 2 (RyR2) Ca2+ release channels in mouse AMs, it remains unclear if similar protein networks and membrane structures exist across species, particularly those relevant for atrial disease modeling.Objective: To examine and quantitatively analyze the architecture of AT membrane structures and associated Ca2+ signaling proteins across species from mouse to human.Methods and Results: We developed superresolution microscopy (nanoscopy) strategies for intact live AMs based on a new custom-made photostable cholesterol dye and immunofluorescence imaging of membraneous structures and membrane proteins in fixed tissue sections from human, porcine, and rodent atria. Consistently, in mouse, rat, and rabbit AMs, intact cell-wide tubule networks continuous with the surface membrane were observed, mainly composed of ATs. Moreover, co-immunofluorescence nanoscopy showed L-type Ca2+ channel clusters adjacent to extensive junctional RyR2 clusters at ATs. However, only junctional RyR2 clusters were highly phosphorylated and may thus prime Ca2+ release at ATs, locally for rapid signal amplification. While the density of the integrated L-type Ca2+ current was similar in human and mouse AMs, the intracellular Ca2+ transient showed quantitative differences. Importantly, local intracellular Ca2+ release from AT junctions occurred through instantaneous action potential propagation via transverse tubules (TTs) from the surface membrane. Hence, sparse TTs were sufficient as electrical conduits for rapid activation of Ca2+ release through ATs. Nanoscopy of atrial tissue sections confirmed abundant ATs as the major network component of AMs, particularly in human atrial tissue sections.Conclusion: AT junctions represent a conserved, cell-specific membrane structure for rapid excitation-contraction coupling throughout a representative spectrum of species including human. Since ATs provide the major excitable membrane network component in AMs, a new model of atrial “super-hub” Ca2+ signaling may apply across biomedically relevant species, opening avenues for future investigations about atrial disease mechanisms and therapeutic targeting.

A Novel Computational Model of the Rabbit Atrial Myocyte Offers Insight into Calcium Wave Propagation Failure

A Novel Computational Model of the Rabbit Atrial Myocyte Offers Insight into Calcium Wave Propagation Failure

ICaL and Ito mediate rate-dependent repolarization in rabbit atrial myocytes.

Rate-dependent repolarization (RDR) of action potential (AP) in cardiomyocyte plays a critical role in the genesis of arrhythmias and RDR in atrium has been linked with atrial fibrillation. However, detailed studies focusing on the role of RDR in rabbit atrium are scant. In this study, atrial cells were isolated from rabbit heart and rate-dependent property was explored in single atrial cell to elucidate the underlying mechanism. Our results indicated that rate-dependent prolongation was evident at the action potential duration at 20% (APD20) and 50% (APD50) repolarization but not at 90% repolarization (APD90) under control condition. Using transient outward potassium current (Ito) inhibitor 4-Aminopyridine (4-AP, 2mM) effectively eliminated the changes in APD20 and APD50, and unmasked the rate-dependent reduction of APD90 which could be diminished by further adding L-type calcium current (ICaL) inhibitor nifedipine (30μM). However, using the selective late sodium current (INaL) inhibitor GS-458967 (GS967, 1μM) caused minimal effect on APD90 of atrial cells both in the absence and presence of 4-AP. In consistence with results from APs, Ito and ICaL displayed significant rate-dependent reduction because of their slow reactivation kinetics. In addition, the magnitude of INaL in rabbit atrium was so small that its rate-dependent changes were negligible. In conclusion, our study demonstrated that Ito and ICaL mediate RDR of AP in rabbit atrium, while minimal effect of INaL was seen.

A Method Sustaining the Bioelectric, Biophysical, and Bioenergetic Function of Cultured Rabbit Atrial Cells.

Culturing atrial cells leads to a loss in their ability to be externally paced at physiological rates and to maintain their shape. We aim to develop a culture method that sustains the shape of atrial cells along with their biophysical and bioenergetic properties in response to physiological pacing. We hypothesize that adding 2,3-Butanedione 2-monoxime (BDM), which inhibits contraction during the culture period, will preserve these biophysical and bioenergetic properties. Rabbit atrial cells were maintained in culture for 24 h in a medium enriched with a myofilament contraction inhibitor, BDM. The morphology and volume of the cells, including their ability to contract in response to 1–3 Hz electrical pacing, was maintained at the same level as fresh cells. Importantly, the cells could be successfully infected with a GFP adenovirus. Action potentials, Ca2+ transients, and local Ca2+ spark parameters were similar in the cultured and in fresh cells. Finally, these cultured cells' flavoprotein autofluorescence was maintained at a constant level in response to electrical pacing, a response similar to that of fresh cells. Thus, eliminating contraction during the culture period preserves the bioelectric, biophysical and bioenergetic properties of rabbit atrial myocytes. This method therefore has the potential to further improve our understanding of energetic and biochemical regulation in the atria.