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Remifentanil is a selective mu-opioid receptor agonist characterized by a rapid onset and ultrashort predictable duration of action providing intense analgesia without prolonged respiratory depression. Remifentanil has been implicated in the causation of intraoperative bradyarrhythmias and asystole both in adults and in pediatric patients. Electrophysiological studies in humans and animals show that remifentanil provokes a dose-dependent depressor effect on sinus and AV node function, manifested by a significant prolongation of sinus node recovery time, sino-atrial conduction time and Wenckebach cycle length. These electrophysiologic effects of remifentanil suggest that it should be used with attention in vulnerable patients with predisposition to bradiarritmias during anesthesia.

Cardiac pacemaking is a complex phenomenon that is still not completely understood. Together with experimental studies, numerical modeling has been traditionally used to acquire mechanistic insights in this research area. This review summarizes the present state of numerical modeling of the cardiac pacemaker, including approaches to resolve present paradoxes and controversies. Specifically we discuss the requirement for realistic modeling to consider symmetrical importance of both intracellular and cell membrane processes (within a recent "coupled-clock" theory). Promising future developments of the complex pacemaker system models include the introduction of local calcium control, mitochondria function, and biochemical regulation of protein phosphorylation and cAMP production. Modern numerical and theoretical methods such as multi-parameter sensitivity analyses within extended populations of models and bifurcation analyses are also important for the definition of the most realistic parameters that describe a robust, yet simultaneously flexible operation of the coupled-clock pacemaker cell system. The systems approach to exploring cardiac pacemaker function will guide development of new therapies such as biological pacemakers for treating insufficient cardiac pacemaker function that becomes especially prevalent with advancing age.

Background: The cardiac conduction system consists of the sinus node, nodal extensions, atrioventricular (AV) node, penetrating bundle, bundle branches, and Purkinje fibers. Node-like AV ring tissue also exists at the AV junctions, and the right and left rings unite at the retroaortic node. The study aims were to (1) construct a 3-dimensional anatomical model of the AV rings and retroaortic node, (2) map electrical activation in the right ring and study its action potential characteristics, and (3) examine gene expression in the right ring and retroaortic node., Methods and Results: Three-dimensional reconstruction (based on magnetic resonance imaging, histology, and immunohistochemistry) showed the extent and organization of the specialized tissues (eg, how the AV rings form the right and left nodal extensions into the AV node). Multiextracellular electrode array and microelectrode mapping of isolated right ring preparations revealed robust spontaneous activity with characteristic diastolic depolarization. Using laser microdissection gene expression measured at the mRNA level (using quantitative PCR) and protein level (using immunohistochemistry and Western blotting) showed that the right ring and retroaortic node, like the sinus node and AV node but, unlike ventricular muscle, had statistically significant higher expression of key transcription factors (including Tbx3, Msx2, and Id2) and ion channels (including HCN4, Cav3.1, Cav3.2, Kv1.5, SK1, Kir3.1, and Kir3.4) and lower expression of other key ion channels (Nav1.5 and Kir2.1)., Conclusions: The AV rings and retroaortic node possess gene expression profiles similar to that of the AV node. Ion channel expression and electrophysiological recordings show the AV rings could act as ectopic pacemakers and a source of atrial tachycardia.

The pacemaker activity of mammalian sinoatrial node (SAN) of the heart plays a fundamental role in the integration of vital functions. Studying factors such as drugs that influence pacemaker activity of SAN has its significance. In this study, we isolated sinus venosus, SAN from toads (Bufo gargarizans), and analysed its electronic signal, histological characteristics and the influence of acetylcholine (ACh) and ivabradine on its pacemaker activity using PowerLab® and Chart® 5.0 software. We found that when isolated sinus venosus was treated with ACh, its histological distribution was disorganized and inter-beat (RR) interval was also broadened. The high frequency normalized unit (HFnu) and Poincaré plot of heart rate variability (HRV) of the isolated sinus venosus was also altered upon ACh treatment in a time-dependent and dose-dependent manner. When treated with ivabradine, these parameters of HRV such as square root of the mean of the squared differences between adjacent NN intervals (RMSSD) and HFnu were in the upward tendency, but low frequency normalized unit and low frequency/high frequency were in the opposite tendency. Taken together, we have developed a new model for studying the influences of drugs on autorhythmicity using isolated sinus venosus of the toad. With this model, we showed that ACh and ivabradine may affect the pacemaker activity by stimulating muscarinic receptor or inhibiting If current, respectively., (Copyright © 2013 John Wiley & Sons, Ltd.)

The origin of sinoatrial node (SAN) pacemaker activity in the heart is controversial. The leading candidates are diastolic depolarization by "funny" current (If) through HCN4 channels (the "Membrane Clock" hypothesis), depolarization by cardiac Na-Ca exchange (NCX1) in response to intracellular Ca cycling (the "Calcium Clock" hypothesis), and a combination of the two ("Coupled Clock"). To address this controversy, we used Cre/loxP technology to generate atrial-specific NCX1 KO mice. NCX1 protein was undetectable in KO atrial tissue, including the SAN. Surface ECG and intracardiac electrograms showed no atrial depolarization and a slow junctional escape rhythm in KO that responded appropriately to β-adrenergic and muscarinic stimulation. Although KO atria were quiescent they could be stimulated by external pacing suggesting that electrical coupling between cells remained intact. Despite normal electrophysiological properties of If in isolated patch clamped KO SAN cells, pacemaker activity was absent. Recurring Ca sparks were present in all KO SAN cells, suggesting that Ca cycling persists but is uncoupled from the sarcolemma. We conclude that NCX1 is required for normal pacemaker activity in murine SAN.

In the last ten years, the incidences of metabolic syndrome and supraventricular arrhythmias have greatly increased. The metabolic syndrome is a cluster of alterations, which include obesity, hypertension, hypertriglyceridemia, glucose intolerance and insulin resistance, that increase the risk of developing, among others, atrial and nodal arrhythmias. The aim of this study is to demonstrate that metabolic syndrome induces electrical remodeling of the sinus node and produces arrhythmias. We induced metabolic syndrome in 2-month-old male Wistar rats by administering 20% sucrose in the drinking water. Eight weeks later, the rats were anesthetized and the electrocardiogram was recorded, revealing the presence of arrhythmias only in treated rats. Using conventional microelectrode and voltage clamp techniques, we analyzed the electrical activity of the sinoatrial node. We observed that in the sinoatrial node of "metabolic syndrome rats", compared to controls, the spontaneous firing of all cells decreased, while the slope of the diastolic depolarization increased only in latent pacemaker cells. Accordingly, the pacemaker currents If and Ist increased. Furthermore, histological analysis showed a large amount of fat surrounding nodal cardiomyocytes and a rise in the sympathetic innervation. Finally, Poincaré plot denoted irregularity in the R-R and P-P ECG intervals, in agreement with the variability of nodal firing potential recorded in metabolic syndrome rats. We conclude that metabolic syndrome produces a dysfunction SA node by disrupting normal architecture and the electrical activity, which could explain the onset of arrhythmias in rats.

The pacemaker tissues of the heart are a complex set of specialized cells that initiate the rhythmic heartbeat. The sinoatrial node (SAN) serves as the primary pacemaker, whereas the atrioventricular node can serve as a subsidiary pacemaker in cases of SAN failure or block. The elucidation of genetic networks regulating the development of these tissues is crucial for understanding the mechanisms underlying arrhythmias and for the design of targeted therapies. Here we report temporal and spatial self-organized formation of the pacemaker and contracting tissues in three-dimensional aggregate cultures of mouse embryonic stem cells termed embryoid bodies (EBs). Using genetic marker expression and electrophysiological analyses we demonstrate that in EBs the pacemaker potential originates from a localized population of cells and propagates into the adjacent contracting region forming a functional syncytium. When Shox2, a major determinant of the SAN genetic pathway, was ablated we observed substantial slowing of spontaneous contraction rates and an altered gene expression pattern including downregulation of HCN4, Cx45, Tbx2, Tbx3, and bone morphogenetic protein 4 (BMP4); and upregulation of Cx40, Cx43, Nkx2.5, and Tbx5. This phenotype could be rescued by adding BMP4 to Shox2 knockout EBs in culture from days 6 to 16 of differentiation. When wild-type EBs were treated with Noggin, a potent BMP4 inhibitor, we observed a phenotype consistent with the Shox2 knockout EB. Altogether, we have generated a reproducible in vitro model that will be an invaluable tool for studying the molecular pathways regulating the development of cardiac pacemaker tissues.

Duration of the plateau phase (phase 2) of action potentials in pacemaker cells in mouse sinoatrial node characterized by low upstroke rate during the rapid depolarization (dV/dt max <7 V/sec, phase 0) and in atrial-like cells with dV/dt max <100 V/sec were prolonged by 50% with 4-aminopyridine in concentrations of 0.7 and 0.1 mM, respectively. This blocker did not lengthen the terminal repolarization (phase 3). The above changes were accompanied by a 20-80% decrease in dV/dt max in comparison to the control. It is hypothesized that the short-term (transient) outward current Ito takes a part in the plateau formation and modulates the duration of the rapid depolarization phase of action potentials in mouse sinoatrial node cells.

An inexorable decline in maximum heart rate (mHR) progressively limits human aerobic capacity with advancing age. This decrease in mHR results from an age-dependent reduction in "intrinsic heart rate" (iHR), which is measured during autonomic blockade. The reduced iHR indicates, by definition, that pacemaker function of the sinoatrial node is compromised during aging. However, little is known about the properties of pacemaker myocytes in the aged sinoatrial node. Here, we show that depressed excitability of individual sinoatrial node myocytes (SAMs) contributes to reductions in heart rate with advancing age. We found that age-dependent declines in mHR and iHR in ECG recordings from mice were paralleled by declines in spontaneous action potential (AP) firing rates (FRs) in patch-clamp recordings from acutely isolated SAMs. The slower FR of aged SAMs resulted from changes in the AP waveform that were limited to hyperpolarization of the maximum diastolic potential and slowing of the early part of the diastolic depolarization. These AP waveform changes were associated with cellular hypertrophy, reduced current densities for L- and T-type Ca(2+) currents and the "funny current" (If), and a hyperpolarizing shift in the voltage dependence of If. The age-dependent reduction in sinoatrial node function was not associated with changes in β-adrenergic responsiveness, which was preserved during aging for heart rate, SAM FR, L- and T-type Ca(2+) currents, and If. Our results indicate that depressed excitability of individual SAMs due to altered ion channel activity contributes to the decline in mHR, and thus aerobic capacity, during normal aging.

Normal heart function requires generation of a regular rhythm by sinoatrial pacemaker cells and the alteration of this spontaneous heart rate by the autonomic input to match physiological demand. However, the molecular mechanisms that ensure consistent periodicity of cardiac contractions and fine tuning of this process by autonomic system are not completely understood. Here we examined the contribution of the m2R-I(KACh) intracellular signaling pathway, which mediates the negative chronotropic effect of parasympathetic stimulation, to the regulation of the cardiac pacemaking rhythm. Using isolated heart preparations and single-cell recordings we show that the m2R-I(KACh) signaling pathway controls the excitability and firing pattern of the sinoatrial cardiomyocytes and determines variability of cardiac rhythm in a manner independent from the autonomic input. Ablation of the major regulator of this pathway, Rgs6, in mice results in irregular cardiac rhythmicity and increases susceptibility to atrial fibrillation. We further identify several human subjects with variants in the RGS6 gene and show that the loss of function in RGS6 correlates with increased heart rate variability. These findings identify the essential role of the m2R-I(KACh) signaling pathway in the regulation of cardiac sinus rhythm and implicate RGS6 in arrhythmia pathogenesis.

Rationale: A recent study published in Circulation Research by Gao et al used sinoatrial node (SAN)-targeted, incomplete Ncx1 knockout in mice to explore the role of the Na(+)/Ca(2+) exchanger (NCX) in cardiac pacemaker. The authors concluded that NCX is required for increasing sinus rates, but not for maintaining resting heart rate. This conclusion was based, in part, on numeric model simulations performed by Gao et al that reproduced their experimental results of unchanged action potentials in the knockout SAN cells. The authors, however, did not simulate the NCX current (INCX), that is, the subject of the study., Objective: We extended numeric examinations to simulate INCX in their incomplete knockout SAN cells that is crucial to interpret the study results., Methods and Results: INCX and Ca(2+) dynamics were simulated using different contemporary numeric models of SAN cells. We found that minimum diastolic Ca(2+) levels and INCX amplitudes generated by remaining NCX molecules (only 20% of control) remained almost unchanged. Simulations using a new local Ca(2+) control model indicate that these powerful compensatory mechanisms involve complex local cross-talk of Ca(2+) cycling proteins and NCX. Specifically, lower NCX expression facilitates Ca(2+)-induced Ca(2+) release and larger local Ca(2+) releases that stabilize diastolic INCX. Further reduction of NCX expression results in arrhythmia and halt of automaticity., Conclusions: Remaining NCX molecules in the incomplete knockout model likely produce almost the same diastolic INCX as in wild-type cells. INCX contribution is crucially important for both basal automaticity of SAN cells and during the fight-or-flight reflex.

Background: Electro-anatomical remodeling of the atria has been reported to be associated with sinus node dysfunction in patients with atrial fibrillation (AF). We hypothesized that post-shock sinus node recovery time (PS-SNRT: the time from cardioversion to the earliest sinus node activation) is related to the degree of left atrial (LA) remodeling and the clinical outcome of radiofrequency catheter ablation (RFCA) in patients with longstanding persistent AF (L-PeAF)., Methods and Results: We included 117 patients with L-PeAF (82.0% males, 55.4 ± 10.7 years old) who underwent RFCA. PS-SNRTs were measured after internal cardioversion (serial shocks 2, 3, 5, 7, 10, and 15J) before RFCA. All patients underwent the same ablation design, and we compared regional LA volume (3D-CT imaging) and LA voltage (NavX)., Results: 1. During the 13.5 ± 5.8-month follow-up period, it was noted that the patients with recurrent AF 3 months after RFCA (n=31) had longer PS-SNRTs (1622.90 ± 1196.92 ms vs. 1112.53 ± 690.68 ms, p=0.005) and greater anterior LA volume (p=0.032) than those who remained in sinus rhythm. 2. The patients with PS-SNRT ≥ 1100 ms showed lower AF-free rates (58.3%) compared to those with PS-SNRT <1100 ms (89.5%, p<0.001). However, shock energy, number of cardioversion, and LA volume were not different between two groups. 3. Multivariate Cox regression analysis demonstrated PS-SNRT ≥ 1100 ms was a significant predictor of clinical recurrence of AF (HR 5.426, 95% CI 2.099-14.028, p<0.001)., Conclusion: In patients with L-PeAF, prolonged PS-SNRT is an independent predictor of clinical recurrence of AF after RFCA, but not closely associated with electro-anatomical remodeling of LA., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Cardiovascular diseases are a leading cause of mortality worldwide. Terminally differentiated adult cardiomyocytes lack the innate ability to regenerate. Cell- and gene-based therapies are emerging as exciting new experimental strategies for myocardial repair and treatment of a variety of cardiovascular diseases. The potential advantages and shortcomings of each strategy for electrophysiological disorders are discussed. Since the first description of human induced pluripotent stem cell-derived cardiomyocytes, these cells have garnered tremendous interest for their potential use in patient-specific analysis and therapy. However, a full understanding of their electrophysiological and contractile function is necessary before this potential can be realized. This review focuses on the mechanisms by which stem cell therapy may function as an antiarrhythmic treatment and early clinical results.

Whether intracellular Ca(2+) cycling dynamics regulate cardiac pacemaker cell function on a beat-to-beat basis remains unknown. Here we show that under physiological conditions, application of low concentrations of caffeine (2-4 mM) to isolated single rabbit sinoatrial node cells acutely reduces their spontaneous action potential cycle length (CL) and increases Ca(2+) transient amplitude for several cycles. Numerical simulations, using a modified Maltsev-Lakatta coupled-clock model, faithfully reproduced these effects, and also the effects of CL prolongation and dysrhythmic spontaneous beating (produced by cytosolic Ca(2+) buffering) and an acute CL reduction (produced by flash-induced Ca(2+) release from a caged Ca(2+) buffer), which we had reported previously. Three contemporary numerical models (including the original Maltsev-Lakatta model) failed to reproduce the experimental results. In our proposed new model, Ca(2+) releases acutely change the CL via activation of the Na(+)/Ca(2+) exchanger current. Time-dependent CL reductions after flash-induced Ca(2+) releases (the memory effect) are linked to changes in Ca(2+) available for pumping into sarcoplasmic reticulum which, in turn, changes the sarcoplasmic reticulum Ca(2+) load, diastolic Ca(2+) releases, and Na(+)/Ca(2+) exchanger current. These results support the idea that Ca(2+) regulates CL in cardiac pacemaker cells on a beat-to-beat basis, and suggest a more realistic numerical mechanism of this regulation., (Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Acetylcholine (ACh) rapidly increases cardiac K(+) currents (IKACh) by activating muscarinic K(+) (KACh) channels followed by a gradual amplitude decrease within seconds. This phenomenon is called short-term desensitization and its precise mechanism and physiological role are still unclear. We constructed a mathematical model for IKACh to examine the conditions required to reconstitute short-term desensitization. Two conditions were crucial: two distinct muscarinic receptors (m2Rs) with different affinities for ACh, which conferred an IKACh response over a wide range of ACh concentrations, and two distinct KACh channels with different affinities for the G-protein βγ subunits, which contributed to reconstitution of the temporal behavior of IKACh. Under these conditions, the model quantitatively reproduced several unique properties of short-term desensitization observed in myocytes: 1), the peak and quasi-steady states with 0.01-100 μM [ACh]; 2), effects of ACh preperfusion; and 3), recovery from short-term desensitization. In the presence of 10 μM ACh, the IKACh model conferred recurring spontaneous firing after asystole of 8.9 s and 10.7 s for the Demir and Kurata sinoatrial node models, respectively. Therefore, two different populations of KACh channels and m2Rs may participate in short-term desensitization of IKACh in native myocytes, and may be responsible for vagal escape at nodal cells., (Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Loss of function and gain of function mutations of the sodium channel were investigated using an intact two-dimensional rabbit sinoatrial node (SAN) and atrial cell model. The effects of three external stimuli (acetylcholine secretion by the vagal nerve, acid-base concentration, and tissue temperature) on cardiac pacemaker function and conduction were studied. Our results show that these two groups of mutations have different effects on pacemaker function and conduction. Furthermore, we found that the negative effects of these mutations could be altered by external stimuli. The bradycardic effects of mutations were magnified by an increase in acetylcholine level. Changes in acid-base concentration and tissue temperature increased the ability of the SAN to recover its pacemaker function. The results of this study increase our understanding of sodium channel disorders, and help to advance research on the treatment of these conditions.

Parasympathetic regulation of sinoatrial node (SAN) pacemaker activity modulates multiple ion channels to temper heart rate. The functional role of the G-protein-activated K(+) current (IKACh) in the control of SAN pacemaking and heart rate is not completely understood. We have investigated the functional consequences of loss of IKACh in cholinergic regulation of pacemaker activity of SAN cells and in heart rate control under physiological situations mimicking the fight or flight response. We used knockout mice with loss of function of the Girk4 (Kir3.4) gene (Girk4(-/-) mice), which codes for an integral subunit of the cardiac IKACh channel. SAN pacemaker cells from Girk4(-/-) mice completely lacked IKACh. Loss of IKACh strongly reduced cholinergic regulation of pacemaker activity of SAN cells and isolated intact hearts. Telemetric recordings of electrocardiograms of freely moving mice showed that heart rate measured over a 24-h recording period was moderately increased (10%) in Girk4(-/-) animals. Although the relative extent of heart rate regulation of Girk4(-/-) mice was similar to that of wild-type animals, recovery of resting heart rate after stress, physical exercise, or pharmacological β-adrenergic stimulation of SAN pacemaking was significantly delayed in Girk4(-/-) animals. We conclude that IKACh plays a critical role in the kinetics of heart rate recovery to resting levels after sympathetic stimulation or after direct β-adrenergic stimulation of pacemaker activity. Our study thus uncovers a novel role for IKACh in SAN physiology and heart rate regulation.

Aims: Pulmonary vein ganglia (PVG) are targets for atrial fibrillation ablation. However, the functional relevance of PVG to the normal heart rhythm remains unclear. Our aim was to investigate whether PVG can modulate sinoatrial node (SAN) function., Methods and Results: Forty-nine C57BL and seven Connexin40+/EGFP mice were studied. We used tyrosine-hydroxylase (TH) and choline-acetyltransferase immunofluorescence labelling to characterize adrenergic and cholinergic neural elements. PVG projected postganglionic nerves to the SAN, which entered the SAN as an extensive, mesh-like neural network. PVG neurones were adrenergic, cholinergic, and biphenotypic. Histochemical characterization of two human embryonic hearts showed similarities between mouse and human neuroanatomy: direct neural communications between PVG and SAN. In Langendorff perfused mouse hearts, PVG were stimulated using 200-2000 ms trains of pulses (300 μs, 400 µA, 200 Hz). PVG stimulation caused an initial heart rate (HR) slowing (36 ± 9%) followed by acceleration. PVG stimulation in the presence of propranolol caused HR slowing (43 ± 13%) that was sustained over 20 beats. PVG stimulation with atropine progressively increased HR. Time-course effects were enhanced with 1000 and 2000 ms trains (P < 0.05 vs. 200 ms). In optical mapping, PVG stimulation shifted the origin of SAN discharges. In five paroxysmal AF patients undergoing pulmonary vein ablation, application of radiofrequency energy to the PVG area during sinus rhythm produced a decrease in HR similar to that observed in isolated mouse hearts., Conclusion: PVG have functional and anatomical biphenotypic characteristics. They can have significant effects on the electrophysiological control of the SAN.

The study was aimed to assess the effect of Klotho gene and sinoatrial node pacing channel gene (HCN4 and HCN2) for studying sick sinus syndrome, with Klotho gene under the interference of Plasmid-mediated short hairpin RNA. Twenty-five C57BL/6J mice were divided into four groups, i. e, plasmid shRNA 24h group, plasmid shRNA 12h group, sodium chloride 24h group and sodium chloride 12h group. Plasmid shRNA 50microL (1microg/microL) and sodium chloride 50microl were respectively injected according to mice vena caudalis into those in plasmid shRNA group and sodium chloride group. After 12h or 24h respectively, all mice were executed and their sinoatrial node tissues were cut. The mRNA of Klotho, HCN4 and HCN2 gene were detected by RT-PCR. The results of RT-PCR showed that Klotho, HCN4 and HCN2 mRNA levels were lower compared with those in sodium chloride 12h group after 12h interference interval. The results indicated that there might be the a certain relationship between Klotho gene and sinoatrial node pacing channel gene.

SCN1A mutations are found in up to 80 % of patients with Dravet syndrome (DS), and the sudden unexpected death in epilepsy (SUDEP) rate is higher in DS than in most forms of severe epilepsy. The aim of this study is to examine the autonomic cardiac function and the risk of arrhythmia in DS patients by evaluating QT and P wave dispersion and heart rate variability (HRV) using standard electrocardiography (ECG) and 24-h ECG. The study group consisted of 15 patients (9 boys and 6 girls aged 3.5-17 years) who were genetically diagnosed with DS. The control group comprised 20 healthy subjects, 13 boys and 7 girls aged 4-17 years. P wave dispersion (44.6 ± 3.5 ms), QT dispersion (58.8 ± 7.5 ms) and QTc dispersion (70.8 ± 7.4 ms) were significantly higher in DS patients as compared to the control group (p < 0.001 for all values). However, there was no significant difference in PR, QT or QTc length between the groups. 24-h Holter ECG showed that all HRV parameters were significantly lower in patients with DS. The decreased HRV and increased P wave and QT dispersion seen in DS patients are important signs of autonomic dysfunction with increased adrenergic tone. To determine whether autonomic dysfunction is correlated with SUDEP in DS, long-term electrocardiographic monitoring and wider prospective studies are necessary.

Background: Pregnancy is associated with a faster heart rate (HR), which is a risk factor for arrhythmias. However, the underlying mechanisms for this increased HR are poorly understood. Therefore, this study was performed to gain mechanistic insight into the pregnancy-induced increase in HR., Methods and Results: Using surface ECG we observed that pregnant (P) mice have faster HR (531±14 beats per minute [bpm]) compared with nonpregnant (NP) mice (470±27 bpm; P<0.03). Results obtained with Langendorff-perfused hearts showed that this difference persisted in the absence of autonomic nervous innervation (NP, 327±16 bpm; P, 385±18 bpm; P<0.02). Spontaneous action potentials of sinoatrial node cells from pregnant mice exhibited higher automaticity (NP, 292±13 bpm; P, 330±12 bpm; P=0.047) and steeper diastolic depolarization (NP, 0.20±0.03 V/s; P, 0.40±0.06 V/s; P=0.004). Pregnancy increased the density of the hyperpolarization-activated current (If) (at -90mV: NP, -15.2±1.0 pA/pF; P, -28.6±2.9 pA/pF; P=0.0002) in sinoatrial node cells. Voltage dependence of the If activation curve and the intracellular cAMP levels were unchanged in sinoatrial node cells of pregnant mice. However, there was a significant increase in HCN2 channel protein expression with no change in HCN4 expression. Maximal depolarizing shift of the If activation curve induced by isoproterenol was attenuated in pregnancy. This reduced response to isoproterenol may be attributable to the lower cAMP sensitivity of HCN2 isoform compared with that of HCN4., Conclusions: This study shows that an increase in If current density contributes to the acceleration of sinoatrial node automaticity and explains, in part, the higher HR observed in pregnancy.

To elucidate the effects of hyperpolarization-activated current I(f) on robustness of sinoatrial node (SAN) pacemaking in connection with intracellular Na(+) concentration (Na(i)) changes, we theoretically investigated 1) the impacts of I(f) on dynamical properties of SAN model cells during inhibition of L-type Ca(2+) channel currents (I(CaL)) or hyperpolarizing loads and 2) I(f)-dependent changes in Na(i) and their effects on dynamical properties of model cells. Bifurcation analyses were performed for Na(i)-variable and Na(i)-fixed versions of mathematical models for rabbit SAN cells; equilibrium points (EPs), limit cycles (LCs), and their stability were determined as functions of model parameters. Increasing I(f) conductance (g(f)) shrank I(CaL) conductance (g(CaL)) regions of unstable EPs and stable LCs (rhythmic firings) in the Na(i)-variable system but slightly broadened that of rhythmic firings at lower g(f) in the Na(i)-fixed system. In the Na(i)-variable system, increased g(f) yielded elevations in Na(i) at EPs and during spontaneous oscillations, which caused EP stabilization and shrinkage in the parameter regions of unstable EPs and rhythmic firings. As g(f) increased, parameter regions of unstable EPs and stable LCs determined for hyperpolarizing loads shrank in the Na(i)-variable system but were enlarged in the Na(i)-fixed system. These findings suggest that 1) I(f) does not enhance but rather attenuates robustness of rabbit SAN cells via facilitating EP stabilization and LC destabilization even in physiological g(f) ranges; and 2) the enhancing effect of I(f) on robustness of pacemaker activity, which could be observed at lower g(f) when Na(i) was fixed, is actually reversed by I(f)-dependent changes in Na(i).

Cardiac pacemaker cells autonomously generate electrical impulses that initiate and maintain the rhythmic contraction of the heart. Although the majority of heart cells are thought to originate from the primary and secondary heart fields, we found that chick pacemaker cells arise from a discrete region of mesoderm outside of these fields. Shortly after gastrulation, canonical Wnts promote the recruitment of mesodermal cells within this region into the pacemaker lineage. These findings suggest that cardiac pacemaker cells are physically segregated and molecularly programmed in a tertiary heart field prior to the onset of cardiac morphogenesis.

An increase in blood pressure variability (BPV) contributes to the development of target organ damage associated with hypertension. Treatment with conventional β-blockers, such as atenolol, has been associated with an increase in BPV; however, the extrapolation of these results to third generation β-blockers with pleiotropic effects seems to be inappropriate. The cardiovascular effects of third generation β-blockers, carvedilol and nebivolol, were assessed in sinoaortic-denervated rats (SAD) and compared with the second generation β-blocker atenolol and the calcium channel blocker verapamil, with a special focus on short-term BPV. Male SAD rats were acutely treated with carvedilol, nebivolol, atenolol or verapamil at two different doses, and the effects on blood pressure and BPV were recorded. Short-term BPV was assessed by the s.d. of BP recordings. Beat-to-beat BPV was studied using spectral analysis to assess the vascular sympatholytic activity of carvedilol and nebivolol by estimating the effects of these drugs on the ratio of low frequency (LF) to high frequency (HF) BPV (LF/HF ratio). Nebivolol, carvedilol and the calcium channel blocker verapamil significantly attenuated short-term BPV at both doses in SAD animals, and there were no differences between the drugs. Conversely, atenolol did not modify baseline s.d. values at either dose. Carvedilol and nebivolol significantly reduced the LF/HF ratio in SAD rats compared with the effects of atenolol and verapamil, suggesting the ability of the third generation β-blockers to reduce vascular sympathetic activity. In conclusion, third generation β-blockers induce a marked reduction in short-term BPV in SAD rats compared to atenolol. Moreover, the ability of carvedilol and nebivolol to reduce short-term BPV in SAD rats is equivalent to that of verapamil, suggesting that these β-blockers may have an additional beneficial effect through their control of short-term variability to a similar extent to calcium channel blockers.

Since the development of the first mathematical cardiac cell model 50 years ago, computational modelling has become an increasingly powerful tool for the analysis of data and for the integration of information related to complex cardiac behaviour. Current models build on decades of iteration between experiment and theory, representing a collective understanding of cardiac function. All models, whether computational, experimental, or conceptual, are simplified representations of reality and, like tools in a toolbox, suitable for specific applications. Their range of applicability can be explored (and expanded) by iterative combination of 'wet' and 'dry' investigation, where experimental or clinical data are used to first build and then validate computational models (allowing integration of previous findings, quantitative assessment of conceptual models, and projection across relevant spatial and temporal scales), while computational simulations are utilized for plausibility assessment, hypotheses-generation, and prediction (thereby defining further experimental research targets). When implemented effectively, this combined wet/dry research approach can support the development of a more complete and cohesive understanding of integrated biological function. This review illustrates the utility of such an approach, based on recent examples of multi-scale studies of cardiac structure and mechano-electric function.

Heart rate (HR) of mammalian species changes postnatally, i.e., HR of large animals including humans decreases, while HR in small animals such as mice and rats increases. To clarify cellular mechanisms underlying the postnatal HR changes, we performed in vivo HR measurement and electrophysiological analysis on sinoatrial node (SAN) cells in mice. The in vivo HR was ~320 beats min(-1) (bpm) immediately after birth, and increased with age to ~690 bpm at postnatal day 14. Under blockage of autonomic nervous systems, HR remained constant until postnatal day 5 and then increased day by day. The spontaneous beating rate of SAN preparation showed a similar postnatal change. The density of the L-type Ca(2+) current (LCC) was smaller in neonatal SAN cells than in adult cells, accompanied by a positive shift of voltage-dependent activation. Thus, the postnatal increase in HR is caused by both the increased sympathetic influence and the intrinsic activity of SAN cells. The different conductance and kinetics of LCC may be involved in the postnatal increase in pacemaker activity.

The heart's rhythm is initiated and regulated by a group of specialized cells in the sinoatrial node (SAN), the primary pacemaker of the heart. Abnormalities in the development of the SAN can result in irregular heart rates (arrhythmias). Although several of the critical genes important for SAN formation have been identified, our understanding of the transcriptional network controlling SAN development remains at a relatively early stage. The homeodomain transcription factor Shox2 is involved in the specification and patterning of the SAN. While the Shox2 knockout in mice results in embryonic lethality due to severe cardiac defects including improper SAN development, Shox2 knockdown in zebrafish causes a reduced heart rate (bradycardia). In order to gain deeper insight into molecular pathways involving Shox2, we compared gene expression levels in right atria of wildtype and Shox2 (-/-) hearts using microarray experiments and identified the LIM homeodomain transcription factor Islet1 (Isl1) as one of its putative target genes. The downregulation of Isl1 expression in Shox2 (-/-) hearts was confirmed and the affected region narrowed down to the SAN by whole-mount in situ hybridization. Using luciferase reporter assays and EMSA studies, we identified two specific SHOX2 binding sites within intron 2 of the ISL1 locus. We also provide functional evidence for Isl1 as a transcriptional target of Shox2 by rescuing the Shox2-mediated bradycardia phenotype with Isl1 using zebrafish as a model system. Our findings demonstrate a novel epistatic relationship between Shox2 and Isl1 in the heart with important developmental consequences for SAN formation and heart beat.

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 "Ca(2+) 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 Ca(2+) 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 ([Ca(2+)] i ) that we have acquired from these cells. The human SAN pacemaker cells have a smaller I f , a weaker [Ca(2+)] 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.

Heart rate variability (HRV) is an indirect estimator of autonomic modulation of heart rate and is considered a risk marker in critical illness, particularly in heart failure and severe sepsis. A reduced HRV has been found in critically ill patients and has been associated with neuro-autonomic uncoupling or decreased baroreflex sensitivity. However, results from human and animal experimental studies indicate that intracardiac mechanisms might also be responsible for interbeat fluctuations. These studies have demonstrated that different membrane channel proteins and especially the so-called 'funny' current (If), an hyperpolarization-activated, inward current that drives diastolic depolarization resulting in spontaneous activity in cardiac pacemaker cells, are altered during critical illness. Furthermore, membrane channels kinetics seem to have significant impact upon HRV, whose early decrease might reflect a cellular metabolic stress. In this review article we present research findings regarding intracardiac origin of HRV, at the cellular level and in both isolated sinoatrial node and whole ex vivo heart preparations. In addition, we will review results from various experimental studies that support the interrelation between If and HRV during endotoxemia. We suggest that reduced HRV during sepsis could also be associated with altered pacemaker cell membrane properties, due to ionic current remodeling.

Rationale: The sodium-calcium exchanger 1 (NCX1) is predominantly expressed in the heart and is implicated in controlling automaticity in isolated sinoatrial node (SAN) pacemaker cells, but the potential role of NCX1 in determining heart rate in vivo is unknown., Objective: To determine the role of Ncx1 in heart rate., Methods and Results: We used global myocardial and SAN-targeted conditional Ncx1 knockout (Ncx1(-/-)) mice to measure the effect of the NCX current on pacemaking activity in vivo, ex vivo, and in isolated SAN cells. We induced conditional Ncx1(-/-) using a Cre/loxP system. Unexpectedly, in vivo and ex vivo hearts and isolated SAN cells showed that basal rates in Ncx1(-/-) (retaining ≈20% of control level NCX current) and control mice were similar, suggesting that physiological NCX1 expression is not required for determining resting heart rate. However, increases in heart rate and SAN cell automaticity in response to isoproterenol or the dihydropyridine Ca(2+) channel agonist BayK8644 were significantly blunted or eliminated in Ncx1(-/-) mice, indicating that NCX1 is important for fight or flight heart rate responses. In contrast, the pacemaker current and L-type Ca(2+) currents were equivalent in control and Ncx1(-/-) SAN cells under resting and isoproterenol-stimulated conditions. Ivabradine, a pacemaker current antagonist with clinical efficacy, reduced basal SAN cell automaticity similarly in control and Ncx1(-/-) mice. However, ivabradine decreased automaticity in SAN cells isolated from Ncx1(-/-) mice more effectively than in control SAN cells after isoproterenol, suggesting that the importance of NCX current in fight or flight rate increases is enhanced after pacemaker current inhibition., Conclusions: Physiological Ncx1 expression is required for increasing sinus rates in vivo, ex vivo, and in isolated SAN cells, but not for maintaining resting heart rate.

A generic cardiomyocyte ionic model, whose complexity lies between a simple phenomenological formulation and a biophysically detailed ionic membrane current description, is presented. The model provides a user-defined number of ionic currents, employing two-gate Hodgkin-Huxley type kinetics. Its generic nature allows accurate reconstruction of action potential waveforms recorded experimentally from a range of cardiac myocytes. Using a multiobjective optimisation approach, the generic ionic model was optimised to accurately reproduce multiple action potential waveforms recorded from central and peripheral sinoatrial nodes and right atrial and left atrial myocytes from rabbit cardiac tissue preparations, under different electrical stimulus protocols and pharmacological conditions. When fitted simultaneously to multiple datasets, the time course of several physiologically realistic ionic currents could be reconstructed. Model behaviours tend to be well identified when extra experimental information is incorporated into the optimisation.

The effects of the specific blockers of the inward Na-current --lidocaine and tetrodotoxin (TTX) were studied with microelectrode technique on the spontaneously beating strips of the mouse sinoauricular (SA) area. Lidocaine (25 microM) and TTX (25 pM) increased the duration of the peak of the action potentials (AP) of true pacemaker cells by extending the plateau phase (phase 2 or APD 20), slowing the dV/dt max from 2.6 +/- 0.8 V/s (n = 25) to 1.4 +/- 0.3 V/s (n = 5, p < 0.05) and reducing the velocity of diastolic depolarization (DD) by 20%. The extend of the dV/dt max value decline depended on the lidocaine concentration. The experimental data fully meted to Hill equation. The lidocaine threshold concentration was 20 microM. The lidocaine effective concentration which decreased dV/dt max by 50% (EC50) was 35 microM. The TTX (25 microM) exposure decreased the dV/dt max from 1.6 V/s to 0.8 V/s and DD velocity slowed by 49%. It should be noted that TTX also increased the duration of APD20. Our data show that dV/ dt max of the true pacemaker cells was reduced by 35-45% after exposure to TTX and lidocaine. This fact confirms the involvement of Na-current in the generation of the upstroke true pacemaker cells AP.

As a vertebrate cardiovascular model, the zebrafish heart has been used extensively in physiology research to study cardiac development and human cardiac disease. Optical mapping techniques provide an effective approach to record action potential propagation in the zebrafish heart. However, manual analysis of functional time series recorded from optical mapping can be laborious and time consuming. In this paper, a novel pipeline is proposed to assist physiologists in identifying the sinoatrial node (SAN) in zebrafish heart based on functional time series. First, the original optical mapping data are enhanced and averaged. Next, the heart is divided into small regions, and representative average time series are calculated. A 'discretization of derivative' (DoD) process is performed to model physiological similarity between signals. Finally, grouping is done on the DoD transformed representation, which is found to produce physiologically meaningful classification for SAN identification.

An isolated rabbit cardiac sinoatrial node (SAN) tissue preparation was used experimentally to map activation times and conduction velocities of extracellular cardiac action potential (AP) propagation. Extracellular recordings were carried out using a two-dimensional array of unipolar Ag-AgCl microelectrodes connected to a 128-channel data acquisition system. A 20(th) order, low-pass Butterworth filter, with a cut-off frequency of 50 Hz, was used in conjunction with a Matlab algorithm to map activation times and conduction velocities. Results show an initial slow-down of the activation wavefront emanating from the SAN, followed by acceleration in some regions, particularly near the Superior Vena Cava, as it travels towards the SAN periphery.

We present a computationally efficient three-dimensional bidomain model of torso-embedded whole heart electrical activity, with spontaneous initiation of activation in the sinoatrial node, incorporating a specialized conduction system with heterogeneous action potential morphologies throughout the heart. The simplified geometry incorporates the whole heart as a volume source, with heart cavities, lungs, and torso as passive volume conductors. We placed four surface electrodes at the limbs of the torso: V R , V L , V F and V GND and six electrodes on the chest to simulate the Einthoven, Goldberger-augmented and precordial leads of a standard 12-lead system. By placing additional seven electrodes at the appropriate torso positions, we were also able to calculate the vectorcardiogram of the Frank lead system. Themodel was able to simulate realistic electrocardiogram (ECG) morphologies for the 12 standard leads, orthogonal X, Y, and Z leads, as well as the vectorcardiogram under normal and pathological heart states. Thus, simplified and easy replicable 3D cardiac bidomain model offers a compromise between computational load and model complexity and can be used as an investigative tool to adjust cell, tissue, and whole heart properties, such as setting ischemic lesions or regions of myocardial infarction, to readily investigate their effects on whole ECG morphology.

A 3D model of atrial electrical activity has been developed with spatially heterogeneous electrophysiological properties. The atrial geometry, reconstructed from the male Visible Human dataset, included gross anatomical features such as the central and peripheral sinoatrial node (SAN), intra-atrial connections, pulmonary veins, inferior and superior vena cava, and the coronary sinus. Membrane potentials of myocytes from spontaneously active or electrically paced in vitro rabbit cardiac tissue preparations were recorded using intracellular glass microelectrodes. Action potentials of central and peripheral SAN, right and left atrial, and pulmonary vein myocytes were each fitted using a generic ionic model having three phenomenological ionic current components: one time-dependent inward, one time-dependent outward, and one leakage current. To bridge the gap between the single-cell ionic models and the gross electrical behaviour of the 3D whole-atrial model, a simplified 2D tissue disc with heterogeneous regions was optimised to arrive at parameters for each cell type under electrotonic load. Parameters were then incorporated into the 3D atrial model, which as a result exhibited a spontaneously active SAN able to rhythmically excite the atria. The tissue-based optimisation of ionic models and the modelling process outlined are generic and applicable to image-based computer reconstruction and simulation of excitable tissue.

Background: Idiopathic atrial fibrillation (AF) refers to a clinically lacking cardiovascular or pulmonary disease generating the pathophysiologic substrate for the arrhythmia. However, because idiopathic AF is associated with an increased event rate, it could be a harbinger of as-yet undetected underlying heart disease., Objective: The purpose of this study was to determine the prevalence of coronary artery disease (CAD) in patients diagnosed with idiopathic paroxysmal AF., Methods: Of the 3243 patients who underwent cardiac computed tomographic angiography (CTA) in our center between January 2008 and March 2011, we identified a total of 115 consecutive idiopathic paroxysmal AF patients who underwent CTA before electrophysiologic ablation. Patients were compared with 275 age-, sex-, and PROCAM risk score-matched healthy controls in permanent sinus rhythm. All patients were free of hypertension, diabetes, congestive heart failure, previous known coronary artery and peripheral vascular disease, previous stroke, thyroid, pulmonary, and renal disease, and structural abnormalities on echocardiography., Results: Controls more often showed a family history of CAD (38% vs 15%, P <.001), had a higher prevalence of smoking (25% vs 14%, P = .021), higher fasting blood glucose levels (5.5 ± 0.7 mmol/L vs 5.4 ± 0.6 mmol/L, P = .025), and smaller atrial diameters (37 ± 4 mm vs 40 ± 5 mm, P <.001) compared to AF patients. Notwithstanding the above, idiopathic AF patients significantly more often suffered from subclinical CAD compared to controls (49% vs 34%, P = .008). Multivariable regression analysis revealed that beside (as expected) age and gender, a history of AF and left atrial diameter were significant predictors of underlying CAD., Conclusion: Half of patients originally diagnosed with idiopathic paroxysmal AF show concealed underlying CAD. The detection and treatment of CAD at an early stage could improve the prognosis of these patients., (Copyright © 2012 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

Background: We evaluated the viability of I(Kur) as a target for maintenance of sinus rhythm in patients with a history of atrial fibrillation through the testing of MK-0448, a novel I(Kur) inhibitor., Methods and Results: In vitro MK-0448 studies demonstrated strong inhibition of I(Kur) with minimal off-target activity. In vivo MK-0448 studies in normal anesthetized dogs demonstrated significant prolongation of the atrial refractory period compared with vehicle controls without affecting the ventricular refractory period. In studies of a conscious dog heart failure model, sustained atrial fibrillation was terminated with bolus intravenous MK-0448 doses of 0.03 and 0.1 mg/kg. These data led to a 2-part first-in-human study: Part I evaluated safety and pharmacokinetics, and part II was an invasive electrophysiological study in healthy subjects. MK-0448 was well-tolerated with mild adverse experiences, most commonly irritation at the injection site. During the electrophysiological study, ascending doses of MK-0448 were administered, but no increases in atrial or ventricular refractoriness were detected, despite achieving plasma concentrations in excess of 2 μmol/L. Follow-up studies in normal anesthetized dogs designed to assess the influence of autonomic tone demonstrated that prolongation of atrial refractoriness with MK-0448 was markedly attenuated in the presence of vagal nerve simulation, suggesting that the effects of I(Kur) blockade on atrial repolarization may be negated by enhanced parasympathetic neural tone., Conclusions: The contribution of I(Kur) to human atrial electrophysiology is less prominent than in preclinical models and therefore is likely to be of limited therapeutic value for the prevention of atrial fibrillation.

Background: There is a clear association between excessive ethanol ( EtOH ) consumption and the risk of sudden cardiac death. The hyperpolarization-activated cyclic nucleotide-gated (HCN) current, I (f) , is known to contribute to spontaneous pacemaker activity of sinoatrial (SA) node cells. However, the exact mechanisms of EtOH on arrhythmia induction are not well understood., Methods: The preparations of SA node were excised from rabbit heart, transmembrane potentials were recorded by standard glass microelectrode technique, and a whole-cell patch clamp technique was used to record I (f) in enzymatically isolated rabbit SA node pacemaker cells. Human HCN2 (hHCN2) and HCN4 channels were heterologously expressed in Xenopus oocytes and studied using 2-electrode voltage clamp technique., Results: Superfusion of EtOH increased the spontaneous firing frequency of SA node cells in a reversible fashion. Treatment with ivabradine irreversibly depressed basal firing frequency and markedly attenuated the enhancement effect of EtOH on firing. The stimulatory effects of EtOH on I (f) were concentration-dependent in the range of 1 to 100 mM, with an average EC (50) value of 20.81 ± 6.71 mM and Hill coefficient of 1.19 ± 0.10. Furthermore, EtOH reversibly enhanced the HCN currents in a concentration-dependent fashion with an EC (50) value of 18.41 ± 2.75 mM for the HCN2 channel and 21.98 ± 3.54 mM for the HCN4 channel, which was accompanied by the acceleration of activation and deactivation kinetics. In addition, EtOH , at both moderate and high doses, caused a shift in the voltage dependence of hHCN4 channel activation to more depolarizing potentials. However, superfusion of high, not moderate, concentration of EtOH caused a shift in the voltage dependence of hHCN2 channel activation to more hyperpolarizing potentials., Conclusions: This study provides insight into the molecular interaction of EtOH and the hHCN channels, which may shed light on elucidating the potentially proarrhythmic mechanism of EtOH ., (Copyright © 2012 by the Research Society on Alcoholism.)

The definition of atrial fibrillation (AF) as a functional electrical disorder does not reflect the significant underlying structural abnormalities. Atrial and Pulmonary Vein (PV) muscle sleeve microstructural remodeling is present, and establishes a vulnerable substrate for AF maintenance. In spite of an incomplete understanding of the anatomo-functional basis for AF, current evidence demonstrates that this arrhythmia usually requires a trigger for initiation and a vulnerable electrophysiological and/or anatomical substrate for maintenance. It is still unclear whether the trigger mechanisms include focal enhanced automaticity, triggered activity and/or micro re-entry from myocardial tissue. Initiation of AF can be favored by both parasympathetic and sympathetic stimulation, which also seem to play a role in maintaining AF. Finally, evolving clinical evidence demonstrates that inflammation is associated with new-onset and recurrent AF through a mechanism that possibly involves cellular degeneration, apoptosis, and subsequent atrial fibrosis.