Sodium-calcium Exchange Current Research Articles

Effects of Acute Hypernatremia on the Electrophysiology of Single Human Ventricular Cardiomyocytes: An In Silico Study.

Clinical and experimental data on the cardiac effects of acute hypernatremia are scarce and inconsistent. We aimed to determine and understand the effects of different levels of acute hypernatremia on the human ventricular action potential. We performed computer simulations using two different, very comprehensive models of the electrical activity of a single human ventricular cardiomyocyte, i.e., the Tomek-Rodriguez model following the O'Hara-Rudy dynamic (ORd) model and the Bartolucci-Passini-Severi model as published in 2020 (known as the ToR-ORd and BPS2020 models, respectively). Mild to extreme levels of hypernatremia were introduced into each model based on experimental data on the effects of hypernatremia on cell volume and individual ion currents. In both models, we observed an increase in the intracellular sodium and potassium concentrations, an increase in the peak amplitude of the intracellular calcium concentration, a hyperpolarization of the resting membrane potential, a prolongation of the action potential, an increase in the maximum upstroke velocity, and an increase in the threshold stimulus current at all levels of hypernatremia and all stimulus rates tested. The magnitude of all of these effects was relatively small in the case of mild to severe hypernatremia but substantial in the case of extreme hypernatremia. The effects on the action potential were related to an increase in the sodium-potassium pump current, an increase in the sodium-calcium exchange current, a decrease in the rapid and slow delayed rectifier potassium currents, and an increase in the fast and late sodium currents. The effects of mild to severe hypernatremia on the electrical activity of human ventricular cardiomyocytes are relatively small. In the case of extreme hypernatremia, the effects are more pronounced, especially regarding the increase in threshold stimulus current.

Increase in Ca2+-Activated cAMP/PKA Signaling Prevents Hydroxychloroquine-Induced Bradycardia of the Cardiac Pacemaker

Bradycardia or tachycardia are known side effects of drugs that limit their clinical use. The heart pacemaker function which control the heart rate under normal conditions is determined by coupled clock system. Thus, interfering with specific clock mechanism will affect other clock mechanisms through changes in interconnected signaling and can lead to rhythm disturbance. However, upregulation of a different clock components can compensate for this change. We focus here on hydroxychloroquine (HCQ), which has been shown effective in treating COVID-19 patients, however its bradycardic side effect limits its clinical use. We aim to decipher the mechanisms underlying the effect of HCQ on pacemaker automaticity, to identify a potential drug that will eliminate the bradycardia. We used isolated rabbit sinoatrial node (SAN) cells, human-induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) and mouse SAN cells residing in SAN tissue. Further, we employed SAN cell computational model to suggest mechanistic insights of the effect of HCQ on pacemaker function. HCQ increased mean spontaneous beat interval and variability in all three models in parallel to slower intracellular kinetics. The computational model suggested that HCQ affects the pacemaker (funny) current (If), L-type Ca2+ current (ICa,L), transient outward potassium (Ito) and due to changes in Ca2+ kinetics, the sodium-calcium exchanger current (INCX). Co-application of 3’-isobutylmethylxanthine (IBMX) and HCQ prevented the increase in beat interval and variability in all three experimental models. The HCQ-induced increase in rabbit and mice SAN cell and hiPSC-CM spontaneous beat interval, can be prevented by a phosphodiester inhibitor that restores automaticity due to slower intracellular Ca2+ kinetics.

PDE5 Inhibition Suppresses Ventricular Arrhythmias by Reducing SR Ca2+ Content.

[Figure: see text].

Zacopride Exerts an Antiarrhythmic Effect by Specifically Stimulating the Cardiac Inward Rectifier Potassium Current in Rabbits: Exploration of a New Antiarrhythmic Strategy.

Zacopride, a potent antagonist of 5-HT3 receptors and an agonist of 5-HT4 receptors, is a gastrointestinal prokinetic agent. In a previous study, we discovered that zacopride selectively stimulated the inward rectifier potassium current (IK1) in the rat and that agonizing IK1 prevented or eliminated aconitine-induced arrhythmias in rats. Our aims were to confirm that the antiarrhythmic effects of zacopride are mediated by selectively enhancing IK1 in rabbits. The effects of zacopride on the function of the main ion channels were investigated using a whole-cell patch-clamp technique in rabbits. Effects of zacopride on cardiac arrhythmias were also explored experimentally both in vivo and in vitro. Zacopride moderately enhanced cardiac IK1 but had no apparent action on voltage-gated sodium current (INa), L- type calcium current (ICa-L), sodium-calcium exchange current (INa/Ca), transient outward potassium current (Ito), or delayed rectifier potassium current (IK) in rabbits. Zacopride also had a marked antiarrhythmic effect in vivo and in vitro. We proved that the resting membrane potential (RMP) was hyperpolarized in the presence of 1 μmol/L zacopride, and the action potential duration (APD) at 90% repolarization (APD90) was shortened by zacopride (0.1-10 μmol/L) in a concentration- dependent manner. Furthermore, zacopride at 1 μmol/L significantly decreased the incidence of drug-induced early afterdepolarization (EAD) in rabbit ventricular myocytes. Zacopride is a selective agonist of rabbit cardiac IK1 and that IK1 enhancement exerts potential antiarrhythmic effects.

Simulation model of CA1 pyramidal neurons reveal opposing roles for the Na+/Ca2+ exchange current and Ca2+-activated K+ current during spike-timing dependent synaptic plasticity.

Sodium Calcium exchanger (NCX) proteins utilize the electrochemical gradient of Na+ to generate Ca2+ efflux (forward mode) or influx (reverse mode). In mammals, there are three unique NCX encoding genes—NCX1, NCX2, and NCX3, that comprise the SLC8A family, and mRNA from all three exchangers is expressed in hippocampal pyramidal cells. Furthermore, mutant ncx2-/- and ncx3-/- mice have each been shown to exhibit altered long-term potentiation (LTP) in the hippocampal CA1 region due to delayed Ca2+ clearance after depolarization that alters synaptic transmission. In addition to the role of NCX at the synapse of hippocampal subfields required for LTP, the three NCX isoforms have also been shown to localize to the dendrite of hippocampal pyramidal cells. In the case of NCX1, it has been shown to localize throughout the basal and apical dendrite of CA1 neurons where it helps compartmentalize Ca2+ between dendritic shafts and spines. Given the role for NCX and calcium in synaptic plasticity, the capacity of NCX splice-forms to influence backpropagating action potentials has clear consequences for the induction of spike-timing dependent synaptic plasticity (STDP). To explore this, we examined the effect of NCX localization, density, and allosteric activation on forward and back propagating signals and, next employed a STDP paradigm to monitor the effect of NCX on plasticity using back propagating action potentials paired with EPSPs. From our simulation studies we identified a role for the sodium calcium exchange current in normalizing STDP, and demonstrate that NCX is required at the postsynaptic site for this response. We also screened other mechanisms in our model and identified a role for the Ca2+ activated K+ current at the postsynapse in producing STDP responses. Together, our data reveal opposing roles for the Na+/Ca2+ exchanger current and the Ca2+ activated K+ current in setting STDP.

Enhanced Depolarization Drive in Failing Rabbit Ventricular Myocytes: Calcium-Dependent and β-Adrenergic Effects on Late Sodium, L-Type Calcium, and Sodium-Calcium Exchange Currents.

Heart failure (HF) is characterized by electrophysiological remodeling resulting in increased risk of cardiac arrhythmias. Previous reports suggest that elevated inward ionic currents in HF promote action potential (AP) prolongation, increased short-term variability of AP repolarization, and delayed afterdepolarizations. However, the underlying changes in late Na+ current (INaL), L-type Ca2+ current, and NCX (Na+/Ca2+ exchanger) current are often measured in nonphysiological conditions (square-pulse voltage clamp, slow pacing rates, exogenous Ca2+ buffers). We measured the major inward currents and their Ca2+- and β-adrenergic dependence under physiological AP clamp in rabbit ventricular myocytes in chronic pressure/volume overload-induced HF (versus age-matched control). AP duration and short-term variability of AP repolarization were increased in HF, and importantly, inhibition of INaL decreased both parameters to the control level. INaL was slightly increased in HF versus control even when intracellular Ca2+ was strongly buffered. But under physiological AP clamp with normal Ca2+ cycling, INaL was markedly upregulated in HF versus control (dependent largely on CaMKII [Ca2+/calmodulin-dependent protein kinase II] activity). β-Adrenergic stimulation (often elevated in HF) further enhanced INaL. L-type Ca2+ current was decreased in HF when Ca2+ was buffered, but CaMKII-mediated Ca2+-dependent facilitation upregulated physiological L-type Ca2+ current to the control level. Furthermore, L-type Ca2+ current response to β-adrenergic stimulation was significantly attenuated in HF. Inward NCX current was upregulated at phase 3 of AP in HF when assessed by combining experimental data and computational modeling. Our results suggest that CaMKII-dependent upregulation of INaL in HF significantly contributes to AP prolongation and increased short-term variability of AP repolarization, which may lead to increased arrhythmia propensity, and is further exacerbated by adrenergic stress.

Excitation of murine cardiac myocytes by nanosecond pulsed electric field.

Opening of voltage-gated sodium channels takes tens to hundreds of microseconds, and mechanisms of their opening by nanosecond pulsed electric field (nsPEF) stimuli remain elusive. This study was aimed at uncovering the mechanisms of how nsPEF elicits action potentials (APs) in cardiomyocytes. Fluorescent imaging of optical APs (FluoVolt) and Ca2+ -transients (Fluo-4) was performed in enzymatically isolated murine ventricular cardiomyocytes stimulated by 200-nanosecond trapezoidal pulses. nsPEF stimulation evoked tetrodotoxin-sensitive APs accompanied or preceded by slow sustained depolarization (SSD) and, in most cells, by transient afterdepolarization waves. SSD threshold was lower than the AP threshold (1.26 ± 0.03 vs 1.34 ± 0.03 kV/cm, respectively, P < 0.001). Inhibition of l-type calcium and sodium-calcium exchanger currents reduced the SSD amplitude and increased the AP threshold ( P < 0.05). The threshold for Ca 2+ -transients (1.40 ± 0.04 kV/cm) was not significantly affected by a tetrodotoxin-verapamil cocktail, suggesting the activation of a Ca 2+ entry pathway independent from the opening of Na + or Ca 2+ voltage-gated channels. Removal of external Ca 2+ decreased the SSD amplitude ( P = 0.004) and blocked Ca 2+ -transients but not APs. The incidence of transient afterdepolarization waves was decreased by verapamil and by removal of external Ca 2+ ( P = 0.002). The study established that nsPEF stimulation caused calcium entry into cardiac myocytes (including routes other than voltage-gated calcium channels) and SSD. Tetrodotoxin-sensitive APs were mediated by SSD, whose amplitude depended on the calcium entry. Plasma membrane electroporation was the most likely primary mechanism of SSD with additional contribution from l-type calcium and sodium-calcium exchanger currents.

Ionic and cellular mechanisms underlying TBX5/PITX2 insufficiency-induced atrial fibrillation: Insights from mathematical models of human atrial cells

Transcription factors TBX5 and PITX2 involve in the regulation of gene expression of ion channels and are closely associated with atrial fibrillation (AF), the most common cardiac arrhythmia in developed countries. The exact cellular and molecular mechanisms underlying the increased susceptibility to AF in patients with TBX5/PITX2 insufficiency remain unclear. In this study, we have developed and validated a novel human left atrial cellular model (TPA) based on the ten Tusscher-Panfilov ventricular cell model to systematically investigate how electrical remodeling induced by TBX5/PITX2 insufficiency leads to AF. Using our TPA model, we have demonstrated that spontaneous diastolic depolarization observed in atrial myocytes with TBX5-deletion can be explained by altered intracellular calcium handling and suppression of inward-rectifier potassium current (IK1). Additionally, our computer simulation results shed new light on the novel cellular mechanism underlying AF by indicating that the imbalance between suppressed outward current IK1 and increased inward sodium-calcium exchanger current (INCX) resulted from SR calcium leak leads to spontaneous depolarizations. Furthermore, our simulation results suggest that these arrhythmogenic triggers can be potentially suppressed by inhibiting sarcoplasmic reticulum (SR) calcium leak and reversing remodeled IK1. More importantly, this study has clinically significant implications on the drugs used for maintaining SR calcium homeostasis, whereby drugs such as dantrolene may confer significant improvement for the treatment of AF patients with TBX5/PITX2 insufficiency.

Simulation study of the ionic mechanisms underlying Torsade de Pointes in a 2D cardiac tissue

BackgroundTo understand the ionic mechanism behind the genesis of Torsade de Pointes (TdP) occurring with long QT syndrome 2 (LQTS2) in a remodelled transmural tissue. MethodsThe TP06 model is used to simulate the electrical activity of cells in a 2D transmural ventricular model. LQTS2 is realised by reducing the potassium current (IKr) to 0.5 in each cell. Each cell of the tissue is remodelled by increasing the conductance of calcium current (GCaL). The above two factors make the cells prone to early after depolarizations (EADs) development. The rise in GCaL that can develop a sustained TdP at normal pacing rate is determined from this study. A look at the calcium dynamics, sodium-calcium exchanger current (INaCa) and slow delayed rectifier potassium current (IKs) distribution maps of the tissue helps us in analysing the mechanism of TdP generation. ResultsA TdP type pattern at normal pacing rate is generated when GCaL is more than 3.5 times the control parameter. From the M-cell island, an adequate number of cells spontaneously release calcium from their sarcoplasmic reticulum leading to increased intracellular calcium and inward sodium current through the sodium calcium exchanger current (INaCa). These contribute to the development of EADs which create a depolarising wavefront that triggers TdP in the tissue. When GCaL is less than 3.5 times the control value, premature ventricular complexes (PVC) occur interspersed between normal beats. ConclusionNormal pacing rates can induce a multi focal TdP when sufficient number of M-cells simultaneously undergo spontaneous calcium release (SCR) events.

Genomic upregulation of cardiac Cav1.2\u03b1 and NCX1 by estrogen in women

BackgroundWomen have a higher risk of lethal arrhythmias than men in long QT syndrome type 2 (LQTS2), but the mechanisms remain uncertain due to the limited availability of healthy control human tissue. We have previously reported that in female rabbits, estrogen increases arrhythmia risk in drug-induced LQTS2 by upregulating L-type Ca2+ (ICa,L) and sodium-calcium exchange (INCX) currents at the base of the epicardium by a genomic mechanism. This study investigates if the effects of estrogen on rabbit ICa,L and INCX apply to human hearts.MethodsPostmortem human left ventricular tissue samples were probed with selective antibodies for regional heterogeneities of ion channel protein expression and compared to rabbit myocardium. Functionally, ICa,L and INCX were measured from female and male cardiomyocytes derived from human induced pluripotent stem cells (iPS-CMs) with the voltage-clamp technique from control and estrogen-treated iPS-CMs.ResultsIn women (n = 12), Cav1.2α (primary subunit of the L-type calcium channel protein 1) and NCX1 (sodium-calcium exchange protein) levels were higher at the base than apex of the epicardium (40 ± 14 and 81 ± 30%, respectively, P < 0.05), but not in men (n = 6) or postmenopausal women (n = 6). Similarly, in cardiomyocytes derived from female human iPS-CMs, estrogen (1 nM, 1–2 days) increased ICa,L (31%, P < 0.05) and INCX (7.5-fold, − 90 mV, P < 0.01) and their mRNA levels (P < 0.05). Moreover, in male human iPS-CMs, estrogen failed to alter ICa,L and INCX.ConclusionsThe results show that estrogen upregulates cardiac ICa,L and INCX in women through genomic mechanisms that account for sex differences in Ca2+ handling and spatial heterogeneities of repolarization due to base-apex heterogeneities of Cav1.2α and NCX1. By analogy with rabbit studies, these effects account for human sex-difference in arrhythmia risk.

B-Type Natriuretic Peptide Modulates Pulmonary Vein Arrhythmogenesis: A Novel Potential Contributor to the Genesis of Atrial Tachyarrhythmia in Heart Failure.

Heart failure (HF) plays a critical role in the genesis of atrial fibrillation (AF). A high B-type natriuretic peptide (BNP) level occurs in patients with HF and in patients with AF. However, the role of BNP in the pathophysiology of AF is not clear. The purposes of this study were to evaluate the effects of BNP on pulmonary vein (PV) arrhythmogenesis. Whole-cell patch clamp and fluorescence were used to study the action potential, ionic currents, and calcium homeostasis in isolated single rabbit PV cardiomyocytes before and after a BNP infusion, with or without ODQ (10 μM), milrinone (50 μM), or ouabain (1 μM). BNP increased PV spontaneous activity by 28.2 ± 7.5% at 100 nM and by 23.8 ± 9.1% at 300 nM. Similar to those with BNP, milrinone 50 μM increased the PV beating rate from 3.0 ± 0.2 to 3.6 ± 0.3 Hz (P < 0.0005, n = 7). In the presence of ODQ application, BNP didn't change PV spontaneous activity. BNP (100 nM) increased calcium transients (F/F0 from 1.6 ± 0.1 to 1.9 ± 0.2, n = 20, P < 0.05) and increased the pacemaker current (0.4 ± 0.1 to 1.0 ± 0.2 pA/pF, n = 17, P < 0.0005) in PV cardiomyocytes. Moreover, BNP (100 nM) increased the transient inward current, sodium currents, sodium-calcium exchanger currents, and L-type calcium current; but reduced late sodium currents and the Na-K pump in PV cardiomyocytes. BNP increases PV arrhythmogenesis, which may contribute to the genesis of atrial tachyarrhythmogenesis in HF. Cyclic GMP activation, phosphodiesterase 3 inhibition and Na+ /K+ -ATPase inhibition might participate in the BNP modulation of PV electrophysiology.

Pro-arrhythmic action of autoantibodies against the second extracellular loop of β1-adrenoceptor and its underlying molecular mechanisms

ObjectivesThe incidence of arrhythmia is associated with autoantibodies against the second extracellular loop of β1-adrenergic receptor (β1-AAbs). The current study was designed to determine the mechanisms by which arrhythmia experimentally might be induced by β1-AAbs. MethodsBlood samples were collected from patients with varied arrhythmias or coronary heart disease (CHD) and healthy subjects. The titer of β1-AAbs was assessed. Passive immunization rat models with β1-AAbs were established to determine whether β1-AAbs induced arrhythmia. Conventional intracellular microelectrode technique and whole cell patch clamp were employed to record action potential duration (APD), resting potential (RP), L-type calcium current (ICa-L), sodium–calcium exchange current (INCX), transient outward potassium current (Ito), inward rectifier potassium current (Ik1) and delayed rectifier potassium current (Ik). ResultsHigh levels of β1-AAbs were found in the sera of heart disease patients, especially in ventricular arrhythmia (VA). Transfusion with β1-AAbs could induce arrhythmias in normal rats in vivo. β1-AAbs purified from the sera of active immunized rats induced triggered activity (TA), delayed after depolarization (DAD), and prolonged APD in the papillary muscles of rats. β1-AAbs prolonged QT interval, increased ICa-L and decreased IK1, Ito and INa–Ca in rat ventricular myocytes in vitro. All these effects can be inhibited by β1-AR blocker metoprolol. ConclusionsThese results demonstrate for the first time that β1-AAbs could directly induce ventricular arrhythmia by prolonging QT interval.

Identification and characterization of two ankyrin-B isoforms in mammalian heart

Excitation-contraction coupling in cardiomyocytes requires the proper targeting and retention of membrane proteins to unique domains by adaptor proteins like ankyrin-B. While ankyrin-B has been shown to interact with a variety of membrane and structural proteins located at different subcellular domains in cardiomyocytes, what regulates the specificity of ankyrin-B for particular interacting proteins remains elusive. Here, we report the identification of two novel ankyrin-B isoforms AnkB-188 and AnkB-212 in human, rat, and mouse hearts. Novel cDNAs for both isoforms were isolated by long-range PCR of reverse-transcribed mRNA isolated from human ventricular tissue. The isoforms can be discriminated based on their function and subcellular distribution in cardiomyocytes. Heterologous overexpression of AnkB-188 increases sodium-calcium exchanger (NCX) membrane expression and current, while selective knockdown of AnkB-188 in cardiomyocytes reduces NCX expression and localization in addition to causing irregular contraction rhythms. Using an isoform-specific antibody, we demonstrate that the expression of AnkB-212 is restricted to striated muscles and is localized to the M-line of cardiomyocytes by interacting with obscurin. Selective knockdown of AnkB-212 significantly attenuates the expression of endogenous ankyrin-B at the M-line but does not disrupt NCX expression at transverse tubules in cardiomyocytes. The identification and characterization of two functionally distinct ankyrin-B isoforms in heart provide compelling evidence that alternative splicing of the ANK2 gene regulates the fidelity of ankyrin-B interactions with proteins.

Regulation of calcium clock-mediated pacemaking by inositol-1,4,5-trisphosphate receptors in mouse sinoatrial nodal cells.

Inositol-1,4,5-trisphosphate receptors (IP3 Rs) modulate pacemaking in embryonic heart, but their role in adult sinoatrial node (SAN) pacemaking is uncertain. We found that stimulation of IP3 Rs accelerates spontaneous pacing rate in isolated mouse SAN cells, whereas inhibition of IP3 Rs slows pacing. In atrial-specific sodium-calcium exchanger (NCX) knockout (KO) SAN cells, where the Ca(2+) clock is uncoupled from the membrane clock, IP3 R agonists and antagonists modulate the rate of spontaneous Ca(2+) waves, suggesting that IP3 R-mediated Ca(2+) release modulates the Ca(2+) clock. IP3 R modulation also regulates Ca(2+) spark parameters, a reflection of ryanodine receptor open probability, consistent with the effect of IP3 signalling on Ca(2+) clock frequency. Modulation of Ca(2+) clock frequency by IP3 signalling in NCX KO SAN cells demonstrates that the effect is independent of NCX. These findings support development of IP3 signalling modulators for regulation of heart rate, particularly in heart failure where IP3 Rs are upregulated. Cardiac pacemaking initiated by the sinus node is attributable to the interplay of several membrane currents. These include the depolarizing 'funny current' (If ) and the sodium-calcium exchanger current (INCX ). The latter is activated by ryanodine receptor (RyR)-mediated calcium (Ca(2+) ) release from the sarcoplasmic reticulum (SR). Another SR Ca(2+) release channel, the inositol-1,4,5-triphosphate receptor (IP3 R), has been implicated in the generation of spontaneous Ca(2+) release in atrial and ventricular cardiomyocytes. Whether IP3 R-mediated Ca(2+) release also influences SAN automaticity is controversial, in part due to the confounding influence of periodic Ca(2+) flux through the sarcolemma accompanying each beat. We took advantage of atrial-specific sodium-calcium exchanger (NCX) knockout (KO) SAN cells to study the influence of IP3 signalling on cardiac pacemaking in a system where periodic intracellular Ca(2+) cycling persists despite the absence of depolarization or Ca(2+) flux across the sarcolemma. We recorded confocal line scans of spontaneous Ca(2+) release in WT and NCX KO SAN cells in the presence or absence of an IP3 R blocker (2-aminoethoxydiphenyl borate, 2-APB), or during block of IP3 production by the phospholipase C inhibitor U73122. 2-APB and U73122 decreased the frequency of spontaneous Ca(2+) transients and waves in WT and NCX KO cells, respectively. Alternatively, increased IP3 production induced by phenylephrine increased Ca(2+) transient and wave frequency. We conclude that IP3 R-mediated SR Ca(2+) flux is crucial for initiating and modulating the RyR-mediated Ca(2+) cycling that regulates SAN pacemaking. Our results in NCX KO SAN cells also demonstrate that RyRs, but not NCX, are required for IP3 to modulate Ca(2+) clock frequency.

Histone Deacetylase Inhibition Reduces Pulmonary Vein Arrhythmogenesis through Calcium Regulation

Pulmonary veins (PVs) play a critical role in the pathophysiology of atrial fibrillation (AF). Histone deacetylases (HDACs) are vital to calcium homeostasis and AF genesis. However, the electrophysiological effects of HDAC inhibition were unclear. This study evaluated whether HDAC inhibition can regulate PV electrical activity through calcium modulation.Whole-cell patch-clamp, confocal microscopic with fluorescence, and Western blot were used to evaluate electrophysiological characteristics and Ca2+ dynamics in isolated rabbit PV cardiomyocytes with and without MPT0E014 (a pan HDAC inhibitor), MS-275 (HDAC1 and 3 inhibitor), and MC-1568 (HDAC4 and 6 inhibitor) for 5~8h. Atrial electrical activity and induced-AF (rapid atrial pacing and acetylcholine infusion) were measured in rabbits with and without MPT0E014 (10mg/kg treated for 5hours) in vivo.MPT0E014 (1μM)-treated PV cardiomyocytes (n=12) had slower beating rates (2.1±0.2 vs. 2.8±0.1Hz, p<0.05) than control PV cardiomyocytes. However, control (n=11) and MPT0E014 (1μM)-treated (n=12) SAN cardiomyocytes had similar beating rates (3.2±0.2 vs. 2.9±0.3Hz). MS-275-treated PV cardiomyocytes (n=12, 2.3±0.2Hz), but not MC-1568-treated PV cardiomyocytes (n=14, 3.1±0.3Hz) had slower beating rates than control PV cardiomocytes. MPT0E014-treated PV cardiomyocytes (n=14) had a lower frequency (2.4±0.6 vs. 0.3±0.1 spark/mm/s, p<0.05) of Ca2+ sparks than control PV (n=17) cardiomyocytes. As compared to control, MPT0E014-treated PV cardiomyocytes had reduced Ca2+ transient amplitudes, sodium-calcium exchanger currents, and ryanodine receptor expressions. Moreover, MPT0E014-treated rabbits had less AF and shorter AF duration than control rabbits.In conclusions, HDAC inhibition reduced PV arrhythmogenesis and AF inducibility with modulation on calcium homeostasis.

A monounsaturated fatty acid (oleic acid) modulates electrical activity in atrial myocytes with calcium and sodium dysregulation

BackgroundObesity and metabolic syndrome are important risk factors for atrial fibrillation. High plasma concentrations of monounsaturated fatty acids, including oleic acid (OLA), are frequently noted in obese individuals and patients with metabolic syndrome. However, it is not clear whether monounsaturated fatty acids (MUFAs) can directly modulate the electrophysiological characteristics of atrial myocytes. MethodsWhole-cell patch clamp, indo-1 fluorescence, and Western blot analyses were used to record the action potentials (APs), ionic currents, and protein expressions of HL-1 myocytes incubated with and without (control) OLA (0.5mM) for 24h. ResultsCompared to control myocytes (n=14), OLA-treated myocytes (n=16) had shorter APD90 (65±6 vs. 85±6ms, p<0.05) and APD50 (24±6 vs. 38±4ms, p<0.05) with a higher incidence of delayed afterdepolarizations (35.7% vs. 7%, p<0.05), which were suppressed by 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS, a blocker of the calcium-activated chloride current). In addition, OLA-treated myocytes (n=19) exhibited larger calcium transients (0.54±0.06 vs. 0.38±0.05 R410/485, p<0.05), and sarcoplasmic reticular calcium contents (0.91±0.05 vs. 0.64±0.08 R410/485, p<0.05) than control myocytes (n=15). OLA-treated myocytes had larger late sodium currents, smaller sodium–calcium exchanger currents, and smaller sodium–potassium pump currents. Moreover OLA-treated myocytes had higher expressions of sarcoplasmic reticular Ca2+-ATPase and calmodulin kinase II, but lower expression of the sodium–potassium ATPase protein than control myocytes. ConclusionsMUFAs can regulate atrial electrophysiological characteristics with calcium and sodium dysregulation, which may contribute to atrial arrhythmogenesis.

Intracellular Calcium and the Mechanism of the Dip in the Anodal Strength-Interval Curve in Cardiac Tissue

The strength-interval (SI) curve is an important measure of refractoriness in cardiac tissue. The anodal SI curve contains a "dip" in which the S2 threshold increases with interval. Two explanations exist for this dip: (1) electrotonic interaction between regions of depolarization and hyperpolarization; and (2) the sodium-calcium exchange (NCX) current. The goal of this study is to use mathematical modeling to determine which explanation is correct. The bidomain model represents cardiac tissue and the Luo-Rudy model describes the active membrane. The SI curve is determined by applying a threshold stimulus at different time intervals after a previous action potential. During space-clamped and equal-anisotropy-ratios simulations, anodal excitation does not occur. During unequal-anisotropy-ratios simulations, electrotonic currents, not membrane currents, are present during the few milliseconds before excitation. The dip disappears with no NCX current, but is present with 50% or 75% reduction of it. The calcium-induced-calcium-release (CICR) current has little effect on the dip. These results indicate that neither the NCX nor the CICR current is responsible for the dip in the anodal SI curve. It is caused by the electrotonic interaction between regions of depolarization and hyperpolarization following the S2 stimulus.

A new potassium ion current induced by stimulation of M2-cholinoreceptors in fish atrial myocytes

A novel potassium ion current induced by muscarinic stimulation (IKACh2) is characterized in atrial cardiomyocytes of teleost fishes (crucian carp, Carassius carassius; rainbow trout, Oncorhynchus mykiss) by means of the whole-cell patch-clamp technique. The current is elicited in atrial, but not ventricular, cells by application of carbamylcholine (CCh) in moderate to high concentrations (10(-7)-10(-4) mol l(-1)). It can be distinguished from the classic IKACh, activated by the βγ-subunit of the Gi-protein, because of its low sensitivity to Ba(2+) ions and distinct current-voltage relationship with a very small inward current component. Ni(2+) ions (5 mmol l(-1)) and KB-R7943 (10(-5) mol l(-1)), non-selective blockers of the sodium-calcium exchange current (INCX), strongly reduced and completely abolished, respectively, the IKACh2. Therefore, IKACh2 was initially regarded as a CCh-induced outward component of the INCX. However, the current is not affected by either exclusion of intracellular Na(+) or extracellular Ca(2+), but is completely abolished by intracellular perfusion with K(+)-free solution. Atropine (10(-6) mol l(-1)), a non-selective muscarinic blocker, completely eliminated the IKACh2. A selective antagonist of M2 cholinoreceptors, AF-DX 116 (2×10(-7) mol l(-1)) and an M3 antagonist, 4-DAMP (10(-9) mol l(-1)), decreased IKACh2 by 84.4% and 16.6%, respectively. Pertussis toxin, which irreversibly inhibits Gi-protein coupled to M2 receptors, reduced the current by 95%, when applied into the pipette solution. It is concluded that IKACh2, induced by stimulation of M2 cholinoceptors and subsequent Gi-protein activation, represents a new molecular target for the cardiac parasympathetic innervation.

Effects of phosphodiesterase type 5A inhibition on intracellular calcium handling and its implications for cardioprotection and antiarrhythmogenesis

Phosphodiesterase type 5A inhibition with sildenafil improves cardiac function in heart failure. In addition, sildenafil in animal models of myocardial infarction has direct cardioprotective and antiarrhythmic effects. Sildenafil reduces L-type calcium current (Ica-L) and attenuates adrenergically driven inotropism, but effects on calcium handling are largely undetermined.Isolated adult rat ventricular myocytes were voltage clamped and calcium fluorescence measured with the indicator fura-2. Cells were paced at 0·5 Hz with depolarisations from −60 mV to +10 mV. Sarcoplasmic reticulum (SR) content was determined by application of caffeine (10–20 mmol/L) and integration of inward sodium-calcium exchanger current. Rate constants for calcium extrusion from the cell (Kcaff) and calcium uptake into the SR (KSERCA) were determined by fitting first order exponentials to decay phases of the respective calcium transients. Following the initial control protocol, a therapeutically relevant dose of sidenafil (1 μM) was applied. Differences were determined with student's paired t tests.Sildenafil reduced SR content by 26·5% (n=9, p<0·01). To a lesser extent, sildenafil also reduced calcium transient amplitude (by −13·6%, n=9, p<0·05); this was not accompanied by a reduction in KSERCA (–2.3% with sildenafil, p=0·97, n=5). Peak and integrated Ica-L were also reduced with sildenafil (–9·1% and −6·0%, respectively, n=9, p<0·05). The effect on Ica-L was also seen in adult dog ventricular myocytes (reducing peak and integrated Ica-L by 15·9% and 26·4%, respectively, p<0·05 and p<0·01, n=6, 23°C). These effects cannot be attributed to run-down effects.Sildenafil substantially reduced SR content with no reduction in KSERCA, and thus may be mediated through ryanodine receptor modulation. Such reduction in SR load may reduce proarrhythmic SR calcium release, indicating a novel mechanism through which sildenafil exerts an antiarrhythmic effect. Acute reductions in calcium transient amplitude and Ica-L with sildenafil indicate acute negative inotropic effects and may contribute to our understanding of its cardioprotective effects in the setting of hyperadrenergic drive in heart failure. FundingBritish Heart Foundation.

Calcium Transient and Sodium‐Calcium Exchange Current in Human versus Rabbit Sinoatrial Node Pacemaker Cells

There is an ongoing debate on the mechanism underlying the pacemaker activity of sinoatrial node (SAN) cells, focusing on the relative importance of the “membrane clock” and the “Ca2+ clock” in the generation of the small net membrane current that depolarizes the cell towards the action potential threshold. Specifically, the debate centers around the question whether the membrane clock-driven hyperpolarization-activated current, I f, which is also known as the “funny current” or “pacemaker current,” or the Ca2+ clock-driven sodium-calcium exchange current, I NaCa, is the main contributor to diastolic depolarization. In our contribution to this journal's “Special Issue on Cardiac Electrophysiology,” we present a numerical reconstruction of I f and I NaCa in isolated rabbit and human SAN pacemaker cells based on experimental data on action potentials, I f, and intracellular calcium concentration ([Ca2+]i) that we have acquired from these cells. The human SAN pacemaker cells have a smaller I f, a weaker [Ca2+]i transient, and a smaller I NaCa than the rabbit cells. However, when compared to the diastolic net membrane current, I NaCa is of similar size in human and rabbit SAN pacemaker cells, whereas I f is smaller in human than in rabbit cells.