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Cell culture of cardiac tissue analog is becoming increasingly interesting for regenerative medicine (cell therapy and tissue engineering) and is widely used for high throughput cardiotoxicity. As a cost-effective approach to rapidly discard new compounds with high toxicity risks, cardiotoxicity evaluation is firstly done in vitro requiring cells/tissue with physiological/pathological characteristics (close to in vivo properties). Studying multicellular electrophysiological and contractile properties is needed to assess drug effects. Techniques favoring process automation which could help in simplifying screening drug candidates are thus of central importance. A lot of effort has been made to ameliorate in vitro models including several in vitro platforms for engineering neonatal rat cardiac tissues. However, most of the initial evaluation is done by studying the rate of activity. In this study, we present new approaches that use the videomicroscopy video of monolayer activity to study contractile properties of beating cells in culture. Two new variables are proposed which are linked to the contraction dynamics and are dependent on the rhythm of activity. Methods for evaluation of regional synchronicity within the image field of view are also presented that can rapidly determine regions with abnormal activity or heterogeneity in contraction dynamics.

International audience; The atrial-specific ultrarapid delayed rectifier K+ current (IKur) inactivates slowly but completely at depolarized voltages. The consequences for IKur rate-dependence have not been analyzed in detail and currently available mathematical action-potential (AP) models do not take into account experimentally observed IKur inactivation dynamics. Here, we developed an updated formulation of IKur inactivation that accurately reproduces time-, voltage-, and frequency-dependent inactivation. We then modified the human atrial cardiomyocyte Courtemanche AP model to incorporate realistic IKur inactivation properties. Despite markedly different inactivation dynamics, there was no difference in AP parameters across a wide range of stimulation frequencies between the original and updated models. Using the updated model, we showed that, under physiological stimulation conditions, IKur does not inactivate significantly even at high atrial rates because the transmembrane potential spends little time at voltages associated with inactivation. Thus, channel dynamics are determined principally by activation kinetics. IKur magnitude decreases at higher rates because of AP changes that reduce IKur activation. Nevertheless, the relative contribution of IKur to AP repolarization increases at higher frequencies because of reduced activation of the rapid delayed-rectifier current IKr. Consequently, IKur block produces dose-dependent termination of simulated atrial fibrillation (AF) in the absence of AF-induced electrical remodeling. The inclusion of AF-related ionic remodeling stabilizes simulated AF and greatly reduces the predicted antiarrhythmic efficacy of IKur block. Our results explain a range of experimental observations, including recently reported positive rate-dependent IKur-blocking effects on human atrial APs, and provide insights relevant to the potential value of IKur as an antiarrhythmic target for the treatment of AF.

A new open-hardware bioreactor capable of applying electrical field stimulation in conjunction with static or cyclic stretch is presented. Stretch is applied to cells by a specially designed elastomeric membrane with a central seeding region. The main interest of our approach is the fine control of the characteristics of stimulations in regard to timing and amplitude in a simple design based on affordable, easy to find components and 3D printable parts. Our approach opens the way to more complex protocols for electrical and/or mechanical stimulations, which are known important regulators of cardiac phenotypes.

BackgroundOptical mapping technology is an important tool to study cardiac electrophysiology. Transmembrane fluorescence signals from voltage-dependent dyes need to be preprocessed before analysis to improve the signal-to-noise ratio. Fourier analysis, based on spectral properties of stationary signals, cannot directly provide information on the spectrum changes with respect to time. Fourier filtering has the disadvantage of causing degradation of abrupt waveform changes such as those in action potential signals. Wavelet analysis has the ability to offer simultaneous localization in time and frequency domains, suitable for the analysis and reconstruction of irregular, non-stationary signals like the fast action-potential upstroke, and better than conventional filters for denoising. MethodsWe applied discrete wavelet transformation for temporal processing of optical mapping signals and wavelet packet analysis approaches to process activation maps from simulated and experimental optical mapping data from canine right atrium. We compared the results obtained with the wavelet approach to a variety of other methods (Fast Fourier Transformation (FFT) with finite or infinite response filtering, and Gaussian filters).Results: Temporal wavelet analysis improved signal-to-noise ratio (SNR) better than FFT filtering for 5-10dB SNR, and caused less distortion of the action potential waveform over the full range of simulated noise (5-20dB). Spatial wavelet filtering produced more efficient denoising and/or more accurate conduction velocity estimates than Gaussian filtering. Propagation patterns were also best revealed by wavelet filtering.Conclusions: Wavelet analysis is a promising tool, facilitating accurate action potential characterization, activation map formation, and conduction velocity estimation. Optical mapping (OM) technology is important to study cardiac electrophysiology.Wavelet analysis offers simultaneous localization in time and frequency domains.Wavelet analysis improved the signal quality compared to Fourier-based filtering.Spatial wavelet filtering produced more accurate conduction velocity estimates.Wavelet analysis is a promising tool for OM electrophysiological characterization.

Fibroblasts are activated in heart failure (HF) and produce fibrosis, which plays a role in maintaining atrial fibrillation (AF). The effect of HF on fibroblast ion currents and its potential role in AF are unknown. Here, we used a patch-clamp technique to investigate the effects of HF on atrial fibroblast ion currents, and mathematical computation to assess the potential impact of this remodeling on atrial electrophysiology and arrhythmogenesis. Atrial fibroblasts were isolated from control and tachypacing-induced HF dogs. Tetraethylammonium-sensitive voltage-gated fibroblast current (IKv,fb) was significantly downregulated (by ∼44%), whereas the Ba2+-sensitive inward rectifier current (IKir,fb) was upregulated by 79%, in HF animals versus controls. The fibroblast resting membrane potential was hyperpolarized (−53 ± 2 mV vs. −42 ± 2 mV in controls) and the capacitance was increased (29.7 ± 2.2 pF vs. 17.8 ± 1.4 pF in controls) in HF. These experimental findings were implemented in a mathematical model that included cardiomyocyte-fibroblast electrical coupling. IKir,fb upregulation had a profibrillatory effect through shortening of the action potential duration and hyperpolarization of the cardiomyocyte resting membrane potential. IKv,fb downregulation had the opposite electrophysiological effects and was antifibrillatory. Simulated pharmacological blockade of IKv,fb successfully terminated reentry under otherwise profibrillatory conditions. We conclude that HF induces fibroblast ion-current remodeling with IKv,fb downregulation and IKir,fb upregulation, and that, assuming cardiomyocyte-fibroblast electrical coupling, this remodeling has a potentially important effect on atrial electrophysiology and arrhythmogenesis, with the overall response depending on the balance of pro- and antifibrillatory contributions. These findings suggest that fibroblast K+-current remodeling is a novel component of AF-related remodeling that might contribute to arrhythmia dynamics.

BackgroundObstructive sleep apnea (OSA) importantly contributes to the occurrence of atrial fibrillation (AF) in humans, but the mechanisms are poorly understood. Experimental research has provided insights into AF promotion by acute OSA episodes. However, patients with OSA usually have frequent nocturnal episodes for some time before manifesting AF.ObjectivesThe goal of this study was to test the hypothesis that repetitive OSA causes cardiac remodeling that predisposes to AF.MethodsWe mimicked OSA by using a mechanical ventilator and closing the airway at end-expiration with a 3-way stopcock (OSA rats). Matched control groups included rats with the ventilator stopped but airway left open (open airway rats) and continuously ventilated rats (sham rats). OSA rats were exposed to 20 consecutive 2-min cycles of 40 s of apnea/80 s of ventilation per day, 5 days per week for 4 weeks.ResultsOSA significantly increased the duration of AF from (median [interquartile range]) 2.6 s [1.9 s to 8.9 s] (shams) and 16 s [1.8 s to 93 s] (open airway) to 49s [34 s to 444 s]. AF inducibility increased to 56% (9 of 16) of OSA rats; this is up from 15% (2 of 13) and 13% (2 of 15) in open airway and sham rats, respectively (p < 0.05). OSA rats exhibited substantial atrial conduction slowing on optical mapping, along with connexin-43 down-regulation on both quantitative immunofluorescence (expression reduced by 58% vs sham rats) and Western blot (reduced by 38%), as well as increased atrial fibrous tissue content (by 71%). OSA also caused left ventricular hypertrophy, dilation, and diastolic dysfunction and enhanced AF inducibility during superimposed acute OSA episodes to 82.4% of rats.ConclusionsChronically repeated OSA episodes cause AF-promoting cardiac remodeling, with conduction abnormalities related to connexin dysregulation and fibrosis playing a prominent role. This novel animal model provides mechanistic insights into an important clinical problem and may be useful for further exploration of underlying mechanisms and therapeutic approaches.

The species-specific determinants of repolarization are poorly understood. This study compared the contribution of various currents to cardiac repolarization in canine and human ventricle. Conventional microelectrode, whole-cell patch-clamp, molecular biological and mathematical modelling techniques were used. Selective IKr block (50-100 nmol l(-1) dofetilide) lengthened AP duration at 90% of repolarization (APD90) >3-fold more in human than dog, suggesting smaller repolarization reserve in humans. Selective IK1 block (10 μmol l(-1) BaCl2) and IKs block (1 μmol l(-1) HMR-1556) increased APD90 more in canine than human right ventricular papillary muscle. Ion current measurements in isolated cardiomyocytes showed that IK1 and IKs densities were 3- and 4.5-fold larger in dogs than humans, respectively. IKr density and kinetics were similar in human versus dog. ICa and Ito were respectively ~30% larger and ~29% smaller in human, and Na(+)-Ca(2+) exchange current was comparable. Cardiac mRNA levels for the main IK1 ion channel subunit Kir2.1 and the IKs accessory subunit minK were significantly lower, but mRNA expression of ERG and KvLQT1 (IKr and IKs α-subunits) were not significantly different, in human versus dog. Immunostaining suggested lower Kir2.1 and minK, and higher KvLQT1 protein expression in human versus canine cardiomyocytes. IK1 and IKs inhibition increased the APD-prolonging effect of IKr block more in dog (by 56% and 49%, respectively) than human (34 and 16%), indicating that both currents contribute to increased repolarization reserve in the dog. A mathematical model incorporating observed human-canine ion current differences confirmed the role of IK1 and IKs in repolarization reserve differences. Thus, humans show greater repolarization-delaying effects of IKr block than dogs, because of lower repolarization reserve contributions from IK1 and IKs, emphasizing species-specific determinants of repolarization and the limitations of animal models for human disease.

The atrial-specific ultrarapid delayed rectifier K+ current (IKur) inactivates slowly but completely at depolarized voltages. The consequences for IKur rate-dependence have not been analyzed in detail and currently available mathematical action-potential (AP) models do not take into account experimentally observed IKur inactivation dynamics. Here, we developed an updated formulation of IKur inactivation that accurately reproduces time-, voltage-, and frequency-dependent inactivation. We then modified the human atrial cardiomyocyte Courtemanche AP model to incorporate realistic IKur inactivation properties. Despite markedly different inactivation dynamics, there was no difference in AP parameters across a wide range of stimulation frequencies between the original and updated models. Using the updated model, we showed that, under physiological stimulation conditions, IKur does not inactivate significantly even at high atrial rates because the transmembrane potential spends little time at voltages associated with inactivation. Thus, channel dynamics are determined principally by activation kinetics. IKur magnitude decreases at higher rates because of AP changes that reduce IKur activation. Nevertheless, the relative contribution of IKur to AP repolarization increases at higher frequencies because of reduced activation of the rapid delayed-rectifier current IKr. Consequently, IKur block produces dose-dependent termination of simulated atrial fibrillation (AF) in the absence of AF-induced electrical remodeling. The inclusion of AF-related ionic remodeling stabilizes simulated AF and greatly reduces the predicted antiarrhythmic efficacy of IKur block. Our results explain a range of experimental observations, including recently reported positive rate-dependent IKur-blocking effects on human atrial APs, and provide insights relevant to the potential value of IKur as an antiarrhythmic target for the treatment of AF.

Fluctuations of the cardiac pacemaker cell membrane voltage can affect the regularity of the heartbeat. Understanding the effects of stochastic membrane variations on the period of spontaneous activity would help determine the stability and robustness of pacemaker cells. The specific dependency of these effects and the rate of spontaneous activity in presence of a bias parameter were studied in the Luo-Rudy model and a Fitzhugh-Nagumo type model. Important differences between models are found with interesting acceleration or deceleration effects and pauses of activity. These results highlight the need for further evaluation of more realistic ionic model.

Background— Atrial tissue fibrosis is often an important component of the atrial fibrillation (AF) substrate. Small noncoding microRNAs are important mediators in many cardiac remodeling paradigms. MicroRNA-21 (miR-21) has been suggested to be important in ventricular fibrotic remodeling by downregulating Sprouty-1, a protein that suppresses fibroblast proliferation. The present study examined the potential role of miR-21 in the atrial AF substrate resulting from experimental heart failure after myocardial infarction (MI). Methods and Results— Large MIs (based on echocardiographic left ventricular wall motion score index) were created by left anterior descending coronary artery ligation in rats. Changes induced by MI versus sham controls were first characterized with echocardiography, histology, biochemistry, and in vivo electrophysiology. Additional MI rats were then randomized to receive anti–miR-21 (KD21) or scrambled control sequence (Scr21) injections into the left atrial myocardium. Progressive left ventricular enlargement, hypocontractility, left atrial dilation, fibrosis, refractoriness prolongation, and AF promotion occurred in MI rats versus sham controls. Atrial tissues of MI rats showed upregulation of miR-21, along with dysregulation of the target genes Sprouty-1, collagen-1, and collagen-3. KD21 treatment reduced atrial miR-21 expression levels in MI rats to values in sham rats, decreased AF duration from 417 (69–1595; median [Q1–Q3]) seconds to 3 (2–16) seconds (8 weeks after MI; P P Conclusions— MI-induced heart failure leads to AF-promoting atrial remodeling in rats. Atrial miR-21 knockdown suppresses atrial fibrosis and AF promotion, implicating miR-21 as an important signaling molecule for the AF substrate and pointing to miR-21 as a potential target for molecular interventions designed to prevent AF.

Atrial fibrillation recurs in ∼30%-40% of patients after pulmonary vein (PV) isolation (PVI) procedures, often because of restored PV-left atrial (LA) conduction. Adenosine or isoproterenol are used clinically to reveal dormant PV conduction and guide additional ablation.The purpose of this study was to assess the differential efficacy of adenosine and/or isoproterenol in revealing dormant PV conduction.In 25 patients undergoing PVI, dormant conduction was assessed sequentially in response to intravenous adenosine, isoproterenol, and adenosine plus isoproterenol in 100 PVs. To study mechanisms, PVs were isolated by radiofrequency ablation in coronary-perfused canine LA-PV preparations. After PVI, resting membrane potential from PV cells was recorded before and after 1 mM adenosine, 1 μM isoproterenol, 1 μM isoproterenol plus 1 mM adenosine, or no drug (controls).Clinical PVI was successful in all 100 PVs, with dormant conduction in 31. Sensitivity for dormant conduction was isoproterenol 10%; adenosine 87% (P.001 vs. isoproterenol); and isoproterenol + adenosine 100% (P = .13 vs. adenosine). Dormant PV conduction in vitro was revealed with adenosine (53%) and adenosine + isoproterenol (60%) but not with isoproterenol alone or in controls (P.01). Radiofrequency lesions producing PVI depolarized resting membrane potential, causing inexcitability. Postablation, resting membrane potential hyperpolarized after both adenosine and isoproterenol, but adenosine-induced changes were greater (9.1 ± 0.6 mV, vs. 3.8 ± 0.6 mV; P0.001), with no significant additional effect when isoproterenol was added to adenosine.Adenosine is superior to isoproterenol in revealing dormant PVs clinically and experimentally because of more effective adenosine-induced hyperpolarization. Adding isoproterenol to adenosine had no significant additional value.

Aims Previous studies suggested that T-type Ca2+-current (ICaT)-blockers improve cardiac remodelling, but all available ICaT-blockers have non-specific actions on other currents and/or functions. To clarify the role of ICaT in cardiac remodelling, we studied mice with either of the principal cardiac ICaT-subunits (Cav3.1 or Cav3.2) knocked out. Methods and results Adult male Cav3.1- or Cav3.2-knockout (Cav3.1−/−, Cav3.2−/−) mice and respective wild-type (WT) littermate controls were subjected to left anterior descending coronary artery ligation to create myocardial infarction (MI). Echocardiography and programmed electrical stimulation were performed at baseline and 4 weeks post-MI. At baseline, Cav3.1−/− mice had slowed heart rates and longer PR intervals vs. WT, but no other electrophysiological and no haemodynamic differences. Cav3.2−/− showed no differences vs. WT. Contractile indices (left ventricular fractional shortening and ejection fraction) decreased more post-MI in Cav3.1−/− mice than in Cav3.1+/+ (e.g. by 34 and 29% for WT; 50 and 45% for Cav3.1−/−, respectively; P < 0.05 for each). Cav3.1−/− mice had increased ventricular tachycardia (VT) inducibility post-MI (9 of 11, 82%) vs. WT (3 of 10, 30%; P < 0.05). Cav3.2−/− mice were not different in cardiac function or VT inducibility vs. WT. Quantitative polymerase chain reaction showed that Cav3.1 is the major ICaT-subunit and that no compensatory Cav3.2 up-regulation occurs in Cav3.1−/− mice. Cav3.1−/− and Cav3.2−/− mice had no mRNA expression for the knocked-out gene, at baseline or post-MI. Conclusion Our findings suggest that, contrary to suggestions from previous studies with (imperfectly selective) pharmacological agents having T-type Ca2+-channel-blocking actions, elimination of Cav3.1 expression leads to impaired cardiac function and enhanced arrhythmia vulnerability post-MI, whereas Cav3.2 elimination has no effect.

Background— Coronary artery disease predisposes to atrial fibrillation (AF), but the effects of chronic atrial ischemia/infarction on AF-related substrates are unknown. Methods and Results— Regional right atrial myocardial infarction (MI) was created in 40 dogs by ligating an artery that supplies the right atrial free wall and not the ventricles; 35 sham dogs with the same artery isolated but not ligated were controls. Dogs were observed 8 days after MI and subjected to open-chest study, in vitro optical mapping, and/or cell isolation for patch-clamp and Ca 2+ imaging on day 8. Holter ECGs showed more spontaneous atrial ectopy in MI dogs (eg, 662±281 on day 7 versus 34±25 ectopic complexes per day at baseline; 52±21 versus 1±1 atrial tachycardia episodes per day). Triggered activity was increased in MI border zone cells, which had faster decay of caffeine-evoked Ca 2+ transients and enhanced (by ≈73%) Na + -Ca 2+ exchange current. Spontaneous Ca 2+ sparks (confocal microscopy) occurred under β-adrenergic stimulation in more MI dog cells (66±9%) than in control cells (29±4%; P Conclusions— Chronic atrial ischemia/infarction creates substrates for both spontaneous ectopy (Ca 2+ -release events, increased Na + -Ca 2+ exchange current) and sustained reentry (conduction abnormalities that anchor reentry). Thus, chronic atrial infarction in dogs promotes both AF triggers and the substrate for AF maintenance. These results provide novel insights into potential AF mechanisms in patients with coronary artery disease.

The biological pacemaker approach is an alternative to cardiac electronic pacemakers. Its main objective is to create pacemaking activity from added or modified distribution of spontaneous cells in the myocardium. This paper aims to assess how automaticity strength of pacemaker cells (i.e. their ability to maintain robust spontaneous activity with fast rate and to drive neighboring quiescent cells) and structural linear anisotropy, combined with density and spatial distribution of pacemaker cells, may affect the macroscopic behavior of the biological pacemaker. A stochastic algorithm was used to randomly distribute pacemaker cells, with various densities and spatial distributions, in a semi-continuous mathematical model. Simulations of the model showed that stronger automaticity allows onset of spontaneous activity for lower densities and more homogeneous spatial distributions, displayed more central foci, less variability in cycle lengths and synchronization of electrical activation for similar spatial patterns, but more variability in those same variables for dissimilar spatial patterns. Compared to their isotropic counterparts, in silico anisotropic monolayers had less central foci and displayed more variability in cycle lengths and synchronization of electrical activation for both similar and dissimilar spatial patterns. The present study established a link between microscopic structure and macroscopic behavior of the biological pacemaker, and may provide crucial information for optimized biological pacemaker therapies., Author summary Implantation of electronic pacemakers is a standard treatment to pathologically slow heart rhythm. Despite improving quality of life, those devices display many shortcomings. Bioengineered tissue pacemakers may be a therapeutic alternative, but associated design methods usually lack control of the way cells with spontaneous activity are scattered throughout the tissue. Our study is the first to use a mathematical model to rigorously define and thoroughly characterize how pacemaker cells scattering at the microscopic level may affect macroscopic behaviors of the bioengineered tissue pacemaker. Automaticity strength (ability of pacemaker cell to drive its non-pacemaker neighbors) and anisotropy (preferential orientation of cell shape) are also implemented and give unparalleled insights on how effects of uncontrollable scattered pacemaker cells may be modulated by available experimental techniques. Our model is a powerful tool to aid in optimized bioengineered pacemaker therapies.

Background— Adenosine acutely reconnects pulmonary veins (PVs) after radiofrequency application, revealing “dormant conduction” and identifying PVs at risk of reconnection, but the underlying mechanisms are unknown. Methods and Results— Canine PV and left-atrial (LA) action potentials were recorded with standard microelectrodes and ionic currents with whole-cell patch clamp before and after adenosine perfusion. PVs were isolated with radiofrequency current application in coronary-perfused LA-PV preparations. Adenosine abbreviated action potential duration similarly in PV and LA but significantly hyperpolarized resting potential (by 3.9±0.5%; P max (by 34±10%) only in PV. Increased dV/dt max was not due to direct effects on I Na , which was reduced similarly by adenosine in LA and PV but correlated with resting-potential hyperpolarization ( r =0.80). Adenosine induced larger inward rectifier K + current (I KAdo ) in PV (eg, −2.28±0.04 pA/pF; −100 mV) versus LA (−1.28±0.16 pA/pF). Radiofrequency ablation isolated PVs by depolarizing resting potential to voltages positive to −60 mV. Adenosine restored conduction in 5 dormant PVs, which had significantly more negative resting potentials (−57±6 mV) versus nondormant (−46±5 mV, n=6; P P Conclusions— Adenosine selectively hyperpolarizes canine PVs by increasing I KAdo . PVs with dormant conduction show less radiofrequency-induced depolarization than nondormant veins, allowing adenosine-induced hyperpolarization to restore excitability by removing voltage-dependent I Na inactivation and explaining the restoration of conduction in dormant PVs.

L’electrocardiogramme (ECG) resulte de la propagation dans le torse des courants electriques generes par le tissu cardiaque. Le potentiel d’action produit par les cardiomyocytes, qui prelude a leur contraction, est la source premiere de ces courants. L’echange de ces courants intra- et extracellulaires permet a l’activation de se propager d’une cellule a l’autre et de provoquer des differences de potentiels sur le torse. La modelisation de l’activite electrique peut donc se faire a plusieurs echelles : de la cellule isolee a l’organisme entier. Quelques exemples illustrent comment la nature du probleme a analyser determine l’echelle, le niveau de complexite minimale a considerer et le type de resultats qui peuvent etre atteints.

Rationale: Although connexin changes are important for the ventricular arrhythmic substrate in congestive heart failure (CHF), connexin alterations during CHF-related atrial arrhythmogenic remodeling have received limited attention. Objective: To analyze connexin changes and their potential contribution to the atrial fibrillation (AF) substrate during the development and reversal of CHF. Methods and Results: Three groups of dogs were studied: CHF induced by 2-week ventricular tachypacing (240 bpm, n=15); CHF dogs allowed a 4-week nonpaced recovery interval after 2-week tachypacing (n=16); and nonpaced sham controls (n=19). Left ventricular (LV) end-diastolic pressure and atrial refractory periods increased with CHF and normalized on CHF recovery. CHF caused abnormalities in atrial conduction indexes and increased the duration of burst pacing-induced AF (DAF, from 22±7 seconds in control to 1100±171 seconds, P P P Conclusions: CHF causes atrial connexin changes, but these are not essential for CHF-related conduction disturbances and AF promotion, which are rather related primarily to fibrotic interruption of muscle bundle continuity.

There are many similarities between atrial effects of atrial tachycardia remodeling (ATR) and vagal nerve stimulation (VS): both promote atrial fibrillation (AF), reduce atrial effective refractory period (AERP) and AERP rate accommodation, enhance AERP heterogeneity, and increase inward-rectifier K+ current.This study sought to compare the consequences of ATR and VS at similar levels of AERP abbreviation in dogs.ATR dogs (n = 6) were subjected to 7-day atrial tachypacing at 400 beats/min, with radiofrequency-induced atrioventricular block and ventricular demand pacing (80 beats/min) to control ventricular response. VS was applied in 6 matched dogs with stimulation parameters selected to produce similar mean AERP values to ATR dogs.ATR and VS produced similarly short AERPs (79 +/- 12 and 80 +/- 12 ms, respectively), AERP rate-adaptation loss, and AERP heterogeneity increases. Although both ATR and VS increased AF duration, VS was significantly more effective in AF promotion, with mean AF duration of 992 +/- 134 seconds, versus 440 +/- 240 seconds (P.05) under ATR. The greater AF-promoting effect of VS was associated with greater mean dominant frequency values during AF (11.7 +/- 1.8 versus 10.0 +/- 1.3 Hz ATR, P.05). VS greatly enhanced the spatial dominant frequency variability, increasing the coefficient of variation to 15.2 +/- 1.9 Hz, versus 8.9 +/- 1.5 Hz for ATR (P.05), primarily by increasing the per-dog maximum dominant frequency (15.4 +/- 0.6 Hz versus 12.5 +/- 0.6 for ATR, P.01).For matched AERP values, VS promotes AF more strongly than ATR. Despite similar AERP changes, VS produces considerably greater increases in dominant frequencies, particularly maximum values, consistent with previous suggestions that inward-rectifier K+ current enhancement is particularly effective at accelerating and stabilizing spiral wave rotors that maintain AF.

Background— Pulmonary vein (PV) –encircling radiofrequency ablation frequently is effective in vagal atrial fibrillation (AF), and there is evidence that PVs may be particularly prone to cholinergically induced arrhythmia mechanisms. However, PV ablation procedures also can affect intracardiac autonomic ganglia. The present study examined the relative role of PVs versus peri-PV autonomic ganglia in an experimental vagal AF model. Methods and Results— Cholinergic AF was studied under carbachol infusion in coronary perfused canine left atrial PV preparations in vitro and with cervical vagal stimulation in vivo. Carbachol caused dose-dependent AF promotion in vitro, which was not affected by excision of all PVs. Sustained AF could be induced easily in all dogs during vagal nerve stimulation in vivo both before and after isolation of all PVs with encircling lesions created by a bipolar radiofrequency ablation clamp device. PV elimination had no effect on atrial effective refractory period or its responses to cholinergic stimulation. Autonomic ganglia were identified by bradycardic and/or tachycardic responses to high-frequency subthreshold local stimulation. Ablation of the autonomic ganglia overlying all PV ostia suppressed the effective refractory period–abbreviating and AF-promoting effects of cervical vagal stimulation, whereas ablation of only left- or right-sided PV ostial ganglia failed to suppress AF. Dominant-frequency analysis suggested that the success of ablation in suppressing vagal AF depended on the elimination of high-frequency driver regions. Conclusions— Intact PVs are not needed for maintenance of experimental cholinergic AF. Ablation of the autonomic ganglia at the base of the PVs suppresses vagal responses and may contribute to the effectiveness of PV-directed ablation procedures in vagal AF.

Background— The development of effective and safe antiarrhythmic drugs for atrial fibrillation (AF) rhythm control is an unmet clinical need. Multichannel blockers are believed to have advantages over single-channel blockers for AF, but their development has been completely empirical to date. We tested the hypothesis that adding K + -channel blockade improves the atrium-selective electrophysiological profile and anti-AF effects of optimized Na + -channel blockers. Methods and Results— Realistic cardiomyocyte-, tissue-, and state-dependent Na + -channel block mathematical models, optical mapping, and action potential recording were used to study the effect of Na + -current ( I Na ) blockade with or without concomitant inhibition of the rapid or ultrarapid delayed-rectifier K + currents ( I Kr and I Kur , respectively). In the mathematical model, maximal AF selectivity was obtained with an inactivated-state Na + -channel blocker. Combining optimized Na + -channel blocker with I Kr block increased rate-dependent and atrium-selective peak I Na reduction, increased AF selectivity, and more effectively terminated AF compared with optimized Na + -channel blocker alone. Combining optimized Na + -channel blocker with I Kur block had similar effects but without I Kr block–induced ventricular action potential prolongation. Consistent with the mathematical model, in coronary-perfused canine hearts, the addition of dofetilide (selective I Kr blocker) to pilsicainide (selective I Na blocker) produced enhanced atrium-selective effects on maximal phase 0 upstroke and conduction velocity. Furthermore, pilsicainide plus dofetilide had higher AF termination efficacy than pilsicainide alone. Pilsicainide alone had no statistically significant effect on AF inducibility, whereas pilsicainide plus dofetilide rendered AF noninducible. Conclusions— K + -channel block potentiates the AF-selective anti-AF effects obtainable with optimized Na + -channel blockade. Combining optimized Na + -channel block with blockade of atrial K + currents is a potentially valuable AF-selective antiarrhythmic drug strategy.

Background Autoantibodies directed against various cardiac receptors have been implicated in cardiomyopathy and heart rhythm disturbances. In a previous study among patients with dilated cardiomyopathy, autoantibodies targeting the cardiac voltage-gated KCNQ1 K + channel were associated with shortened corrected QT intervals (QTc). However, the electrophysiologic actions of KCNQ1 autoimmunity have not been assessed experimentally in a direct fashion. Objective The purpose of this study was to investigate the cardiac electrophysiologic effects of KCNQ1 autoantibody production induced by vaccination in a rabbit model. Methods Rabbits were immunized with KCNQ1 channel peptide. ECG recordings were obtained during a 1-month follow-up period. Rabbits then underwent in vivo electrophysiologic study, after which cardiomyocytes were isolated for analysis of slow delayed rectifier current (I Ks ) and action potential properties via patch-clamp. Results KCNQ1-immunized rabbits exhibited shortening of QTc compared to sham-immunized controls. Reduced ventricular effective refractory periods and increased susceptibility to ventricular tachyarrhythmia induction were noted in KCNQ1-immunized rabbits upon programmed ventricular stimulation. Action potential durations were shortened in cardiomyocytes isolated from KCNQ1-immunized rabbits compared to the sham group. I Ks step and tail current densities were enhanced after KCNQ1 immunization. Functional and structural changes of the heart were not observed. The potential therapeutic significance of KCNQ1 immunization was then explored in a dofetilide-induced long QT rabbit model. KCNQ1 immunization prevented dofetilide-induced QTc prolongation and attenuated long QT–related arrhythmias. Conclusion Induction of KCNQ1 autoimmunity accelerates cardiac repolarization and increases susceptibility to ventricular tachyarrhythmia induction through I Ks enhancement. On the other hand, vaccination against KCNQ1 ameliorates drug-induced QTc prolongation and might be useful therapeutically to enhance repolarization reserve in long QT syndrome.

Angiotensin II-induced arterial hypertension (AngII-AH) is associated with an endothelial dysfunction characterized by a Ca2+ dyshomeostasis. Alteration of the delicate balance between reactive oxy...

Endothelial Ca2+ pulsar consists in a spontaneous Ca2+ release from IP3R localized within myoendothelial projections (MEP). We recently showed CaMKII activation by Ca2+ pulsars, thus modulating endothelial function through NO production. Moreover, CaMKII is a known regulator of intracellular Ca2+ homeostasis in several cell types. Our hypothesis was that Ca2+ pulsar-activated CaMKII modulates endothelial intracellular Ca2+ dynamics. Our investigation showed that preincubation with KN-93 (10 µM), CaMKII inhibitor, significantly altered endoplasmic reticulum (ER) Ca2+ content (≍ -60%). A similar KN-93-induced alteration of ER Ca2+ levels (≍ -55%) was observed while SERCA pumps were inhibited (Thapsigargin; 1 µM). However, inhibition of IP3R (2APB; 100 µM) precluded KN-93 effect on ER Ca2+ content. Colocalization ( IP3R1>IP3R3) has been found by in situ immunofluorescence. Final...

The endothelial dysfunction associated with arterial hypertension is characterized by a Ca2+ dyshomeostasis resulting in an alteration of the delicate balance between reactive oxygen species (ROS) and nitric oxide (NO). However, the underlying mechanisms remain controversial. It has been recently suggested that CaMKII, a Ca2+-calmodulin dependent kinase plays an important role in cardiovascular diseases such as diabetes-associated hypertension. Paradoxically, CaMKII also modulates eNOS expression and activity, and is thereby involved in the regulation of endothelial function. Indeed, acting as a Ca2+ sensor, CaMKII has the unique ability to integrate oscillatory Ca2+ signals into specific outcomes. We recently provided the first evidence that endothelial CaMKII is activated by a local Ca2+ signal, the Ca2+ pulsars, localized within myoendothelial projections (MEP), and results in an increased NO production. The characterization of this novel endothelial CaMKII signalling pathway in pathophysiological conditions will strengthen our understanding of endothelial dysfunction associated with hypertension. This study aimed at the characterization of the impact of AngII-induced hypertension on the correlation between endothelial Ca2+pulsars and CaMKII in microvasculature. Real-time confocal imaging showed that acute exposure to AngII (10 μM) increases both ROS intracellular levels (52%) and Ca2+ pulsars frequency (35%). Exposure to lower AngII levels (100 nM) only increased Ca2+ pulsars frequency (49%). However, CaMKIIα and β clustering (and activation) within MEP was observed at both AngII levels. Acute AngII also stimulated NO production (42%), an increase blunted by CaMKII inhibition with KN-93 (17%). Although a significant increase in Ca2+ pulsars frequency was observed (147%) in AngII-hypertensive mice, an AngII-induce increased in endothelial ROS levels was recorded independently of blood pressure levels. Recruitment of CaMKIIα and β at the MEP by chronic AngII was also increased regardless of blood pressure (60% and 43%). Our finding strongly suggests that AngII stimulates CaMKII through both ROS and Ca2+ pulsars . This study consolidates our understanding of CaMKII as a key regulatory component of endothelial function.

The species-specific determinants of repolarization are poorly understood. This study compared the contribution of various currents to cardiac repolarization in canine and human ventricle. Conventional microelectrode, whole-cell patch-clamp, molecular biological and mathematical modelling techniques were used. Selective IKr block (50-100 nmol l(-1) dofetilide) lengthened AP duration at 90% of repolarization (APD90)3-fold more in human than dog, suggesting smaller repolarization reserve in humans. Selective IK1 block (10 μmol l(-1) BaCl2) and IKs block (1 μmol l(-1) HMR-1556) increased APD90 more in canine than human right ventricular papillary muscle. Ion current measurements in isolated cardiomyocytes showed that IK1 and IKs densities were 3- and 4.5-fold larger in dogs than humans, respectively. IKr density and kinetics were similar in human versus dog. ICa and Ito were respectively ~30% larger and ~29% smaller in human, and Na(+)-Ca(2+) exchange current was comparable. Cardiac mRNA levels for the main IK1 ion channel subunit Kir2.1 and the IKs accessory subunit minK were significantly lower, but mRNA expression of ERG and KvLQT1 (IKr and IKs α-subunits) were not significantly different, in human versus dog. Immunostaining suggested lower Kir2.1 and minK, and higher KvLQT1 protein expression in human versus canine cardiomyocytes. IK1 and IKs inhibition increased the APD-prolonging effect of IKr block more in dog (by 56% and 49%, respectively) than human (34 and 16%), indicating that both currents contribute to increased repolarization reserve in the dog. A mathematical model incorporating observed human-canine ion current differences confirmed the role of IK1 and IKs in repolarization reserve differences. Thus, humans show greater repolarization-delaying effects of IKr block than dogs, because of lower repolarization reserve contributions from IK1 and IKs, emphasizing species-specific determinants of repolarization and the limitations of animal models for human disease.

The fundamental mechanisms underlying fibrillation have long been debated (Figure 16-1). The classical notion of multiple circuit reentry (Figure 16-1C) has been challenged by recent ideas suggesting that in some cases a single high-frequency rotor (Figure 16-1B)1 or even a rapidly firing focus (Figure 16-1A)2 can underlie fibrillation. In the latter two cases, although the primary source may be firing regularly, the inability of tissues to follow 1∶1 causes wave breakup and fibrillation. Ironically, these recent ideas echo notions first put forward in the early twentieth century.3

Acute or sustained stretch of cardiac tissue is known to play a key role in arrhythmogenesis. Using a fluorescence approach, we designed a system measuring calcium transients and transmembrane potential changes in monolayers of cultured cardiomyocytes under uniaxial elongation and electrical stimulation. Cardiac myocytes are seeded on a rectangular PDMS template held and stretched by a motorized linear guide system. Electrical stimulation is performed with two parallel carbon electrodes supplied by amplified pulses from a digital-to-analog converter. The cells are stained with either voltage- or calcium-sensitive dye (di-4-ANEPPS and Fluo-4 AM respectively). The two available excitation light sources are both current-controlled LED arrays (λ = 523 ± 45 nm for di-4-ANEPPS and λ = 505 ± 15 nm for Fluo-4 AM). The filtered emitted fluorescence (λ610 nm for di-4-ANEPPS and λ = 535 ± 25 nm for Fluo-4 AM) is transduced to current with a photodiode, converted to amplified voltage signals and digitized. The design and preliminary validation results are presented.

Atrial fibrillation (AF) is the most common type of clinical arrhythmia. Currently available anti-AF drugs are limited by only moderate efficacy and an unfavorable safety profile. Thus, there is a recognized need for improved antiarrhythmic agents with actions that are selective for the fibrillating atrium. State-dependent Na+-channel blockade potentially allows for the development of drugs with maximal actions on fibrillating atrial tissue and minimal actions on ventricular tissue at resting heart rates. In this study, we applied a mathematical model of state-dependent Na+-channel blocking (class I antiarrhythmic drug) action, along with mathematical models of canine atrial and ventricular cardiomyocyte action potentials, AF, and ventricular proarrhythmia, to determine the relationship between their pharmacodynamic properties and atrial-selectivity, AF-selectivity (atrial Na+-channel block at AF rates versus ventricular block at resting rates), AF-termination effectiveness, and ventricular proarrhythmic properties. We found that drugs that target inactivated channels are AF-selective, whereas drugs that target activated channels are not. The most AF-selective drugs were associated with minimal ventricular proarrhythmic potential and terminated AF in 33% of simulations; slightly fewer AF-selective agents achieved termination rates of 100% with low ventricular proarrhythmic potential. Our results define properties associated with AF-selective actions of class-I antiarrhythmic drugs and support the idea that it may be possible to develop class I antiarrhythmic agents with optimized pharmacodynamic properties for AF treatment.

Cardiac fibrosis is an important form of pathological tissue remodeling. Fibrosis can electrically-uncouple neighboring excitable cardiomyocytes thus acting as an obstacle to electrical propagation. In this study, we investigated the effects of fibrosis spatial pattern on electrical propagation in control, decreased maximum sodium conductance, and increased intracellular resistivity conditions. Simulations were performed with a monodomain approach and a realistic canine ionic model. We found that the propagation failure is highly dependent on the spatial pattern of fibrosis for all conditions studied with maximum sensitivity for patterns with combination of small and large clusters. However, the effect is particularly sensitive to reduced sodium current condition where conduction block occurred at lower fibrosis density.

Aims Increasing evidence indicates that congenital long QT syndromes (LQTSs) promote atrial fibrillation. The atrial action potential (AP) has a short plateau, and whether LQTS atrial cardiomyocytes generate triggered activity via early afterdepolarizations (EADs) is unclear. Atrial cellular arrhythmia mechanisms have not been defined in congenital LQTS. Therefore, we studied atrial cardiomyocyte electrophysiology in mice with an LQTS3 SCN5A inactivation-impairing mutation (ΔKPQ heterozygotes). Methods and results Peak and late Na+ current ( I NaP and I NaL) were measured with whole-cell patch clamp in left atrial (LA) cardiomyocytes. APs were recorded in multicellular LA preparations with floating microelectrodes. I NaL was increased by 110% in LA cardiomyocytes of ΔKPQ mice, whereas I NaP was unchanged. AP duration (APD) was prolonged over all frequencies in ΔKPQ mice, but particularly at lower frequencies [e.g. APD90 at 0.5 Hz: 197 ± 8 ms vs. wild-type (WT) 82 ± 2 ms, P < 0.001]. EADs occurred at 0.5 Hz in 10/18 ΔKPQ (56%) vs. 1/10 WT (10%) atria ( P < 0.05). EADs immediately preceded premature APs in other LA regions, suggesting triggered activity. Ranolazine preferentially inhibited I NaL (50% inhibitory concentration: 12.5 vs. 151.8 µM for I NaP) in ΔKPQ myocytes. At 10 µM, ranolazine shortened APD (e.g. APD90 at 0.5 Hz to 122 ± 4 ms, P = 0.01) without changing APD in WT and suppressed EAD occurrence and triggered activity (from 10/18 to 1/9 preparations, 11%, P < 0.05). Conclusion This study implicates increased I NaL in excessive atrial APD prolongation and arrhythmic EAD occurrence in a congenital LQTS3 mouse model. Our observations provide the first direct demonstration of atrial EADs and triggered activity in a genetically defined animal model of human LQTS and have potential clinically-relevant mechanistic and therapeutic implications.

Aims Pulmonary vein (PV)-encircling ablation, which is effective in suppressing atrial fibrillation (AF), damages autonomic ganglia near the PV ostia. This study examined the effects of PV isolation (PVI) vs. peri-PV ganglionic plexus ablation (GPA) in two discrete canine AF models: ventricular tachypacing (240 bpm, 2 weeks)-induced congestive heart failure (CHF), and atrial tachypacing (400 bpm, 1 week)-induced atrial tachycardia remodeling (ATR). Methods and results All PVs were isolated with an epicardial radiofrequency clamp in nine CHF and eight ATR dogs. Peri-PV ganglionic plexi (identified by bradycardic responses to high-frequency stimulation) were ablated in six CHF and five ATR dogs with an epicardial radiofrequency-ablation pen. Electrophysiologic measurements, including 240-electrode AF mapping, were obtained and dominant frequencies (DFs) determined. Atrial growth associated protein-43 (GAP-43) and neurofilament-M (NF-M) expression were determined immunohistologically. In CHF, neither PVI nor GPA affected AF duration, DF or the already low AF vulnerability. In ATR, PVI reduced AF vulnerability (75 ± 6% to 55 ± 11%, P < 0.05) but did not alter AF duration or DF. In contrast, GPA prolonged atrial refractory period and decreased AF vulnerability (75 ± 8 to 30 ± 10%, P < 0.05), AF duration (617 ± 246 to 39 ± 23 s, ** P < 0.01), and DF (11.4 ± 0.6 to 8.6 ± 0.3** Hz, left atrium) in ATR dogs. Both GAP-43 and NF-M expression were decreased in CHF (by 63.1** and 60.0%**) and increased in ATR (by 65.5** and 92.1%, P < 0.001) compared with control. Conclusions PVs play a minor role in experimental AF due to CHF or ATR, but autonomic ganglia are important in AF related to ATR. Differential neural remodelling may contribute to varying effects of GPA in discrete AF substrates.

Obesity increases the incidence of cardiac arrhythmias and impairs wound healing. However, it is presently unknown whether a high-fat diet affects arrhythmic risk or wound healing before the onset of overt obesity or hyperlipidemia. After 8 wk of feeding a high-fat diet to adult female rats, a nonsignificant increase in body weight was observed and associated with a normal plasma lipid profile. Following ischemia/reperfusion injury, scar length (standard diet 0.29 ± 0.09 vs. high-fat 0.32 ± 0.13 cm), thickness (standard diet 0.047 ± 0.02 vs. high-fat 0.059 ± 0.01 cm), and collagen α1 type 1 content (standard diet 0.21 ± 0.04 vs. high-fat 0.20 ± 0.04 arbitrary units/mm2) of infarcted hearts were not altered by the high-fat diet. However, the mortality rate was greatly increased 24 h postinfarction (from 5% to 46%, P < 0.01 for ischemia/reperfusion rats; from 20% to 89%, P < 0.0001, in complete-occlusion rats) in high-fat fed rats, in association with a higher prevalence of ventricular arrhythmias. Ventricular arrhythmia inducibility was also significantly increased in noninfarcted rats fed a high-fat diet. In the hearts of rats fed a high-fat diet, connexin-40 expression was absent, connexin-43 was hypophosphorylated and lateralized, and neurofilament-M immunoreactive fiber density (standard diet 2,020 ± 260 vs. high-fat diet 2,830 ± 250 μm2/mm2) and tyrosine hydroxylase protein expression were increased ( P < 0.05). Thus, in the absence of overt obesity and hyperlipidemia, sympathetic hyperinnervation and an aberrant pattern of gap junctional protein expression and regulation in the heart of female rats fed a high-fat diet may have contributed in part to the higher incidence of inducible cardiac arrhythmias.

Models of cardiac electrical activity cover a wide range of spatial scales, from the genesis of the ionic currents in individual cardiomyocytes to the generation of electrocardiograms on the torso. The level of detail that is appropriate and practicable depends on the problem investigated and the scope of the computations that are required. We briefly present three examples of modelling: the dynamics of the entrainment of a single cell, the impact of fibrosis on electrical propagation in a piece of tissue and the generation of ECG in human. In each case, the methods, results and limitations are discussed.

Purkinje fibers (PFs) play key roles in cardiac conduction and arrhythmogenesis. Congestive heart failure (CHF) causes well-characterized atrial and ventricular ion channel subunit expression changes, but effects on PF ion channel subunits are unknown. This study assessed changes in PF ion channel subunit expression (real-time PCR, immunoblot, immunohistochemistry), action potential properties, and conduction in dogs with ventricular tachypacing–induced CHF. CHF downregulated mRNA expression of subunits involved in action potential propagation (Nav1.5, by 56%; connexin [Cx]40, 66%; Cx43, 56%) and repolarization (Kv4.3, 43%, Kv3.4, 46%). No significant changes occurred in KChIP2, KvLQT1, ERG, or Kir3.1/3.4 mRNA. At the protein level, downregulation was seen for Nav1.5 (by 38%), Kv4.3 (42%), Kv3.4 (57%), Kir2.1 (26%), Cx40 (53%), and Cx43 (30%). Cx43 dephosphorylation was indicated by decreased larger molecular mass bands (pan-Cx43 antibody) and a 57% decrease in Ser368-phosphorylated Cx43 (phospho-specific antibody). Immunohistochemistry revealed reduced Cx40, Cx43, and phospho-Cx43 expression at intercalated disks. Action potential changes were consistent with observed decreases in ion channel subunits: CHF decreased phase 1 slope (by 56%), overshoot (by 32%), and phase 0 dV/dt max (by 35%). Impulse propagation was slowed in PF false tendons: conduction velocity decreased significantly from 2.2±0.1 m/s (control) to 1.5±0.1 m/s (CHF). His-Purkinje conduction also slowed in vivo, with HV interval increasing from 35.5±1.2 (control) to 49.3±3.4 ms (CHF). These results indicate important effects of CHF on PF ion channel subunit expression. Alterations in subunits governing conduction properties may be particularly important, because CHF-induced impairments in Purkinje tissue conduction, which this study is the first to describe, could contribute significantly to dyssynchronous ventricular activation, a major determinant of prognosis in CHF-patients.

Background— Sinoatrial node (SAN) dysfunction is frequently associated with atrial tachyarrhythmias (ATs). Abnormalities in SAN pacemaker function after termination of ATs can cause syncope and require pacemaker implantation, but underlying mechanisms remain poorly understood. This study examined the hypothesis that ATs impair SAN function by altering ion channel expression. Methods and Results— SAN tissues were obtained from 28 control dogs and 31 dogs with 7-day atrial tachypacing (400 bpm). Ionic currents were measured from single SAN cells with whole-cell patch-clamp techniques. Atrial tachypacing increased SAN recovery time in vivo by ≈70% ( P 50% ( P P I Kr ) α-subunit ERG, the slow delayed-rectifier ( I Ks ) α-subunit KvLQT1, the β-subunit MiRP1, the L-type ( I CaL ) and T-type ( I CaT ) Ca 2+ -current subunits Cav1.2 and Cav3.1, and the gap-junction subunit connexin 43 (were unaffected by atrial tachypacing. Atrial tachypacing reduced densities of the HCN-related funny current ( I f ) and I Ks by ≈48% ( P P I Kr , I CaL , and I CaT were unaffected. SAN cells lacked Ba 2+ -sensitive inward-rectifier currents, irrespective of AT. SAN action potential simulations that incorporated AT-induced alterations in I f accounted for slowing of periodicity, with no additional contribution from changes in I Ks . Conclusions— AT downregulates SAN HCN2/4 and minK subunit expression, along with the corresponding currents I f and I Ks . Tachycardia-induced remodeling of SAN ion channel expression, particularly for the “pacemaker” subunit I f , may contribute to the clinically significant association between SAN dysfunction and supraventricular tachyarrhythmias.

Purkinje fibers (PFs) play a key role in cardiac conduction and arrhythmogenesis. Heart failure (HF) causes extensive electrical remodeling. HF-induced changes in atrial and ventricular ion channel subunit expression have been well characterized, but little is known about HF-induced ion channel subunit remodeling and functional consequences in PFs. This study assessed ion channel subunit expression, action potential (AP) properties and conduction in cardiac PF false tendons from control and HF dogs. HF was induced by 2 wk ventricular tachypacing (240 bpm). Control and HF PFs were fast-frozen for ion channel subunit mRNA (RT-qPCR) and protein (Western Blot, immunohistochemistry) assessment. APs were studied with standard micro-electrodes. HF significantly downregulated mRNA expression of subunits involved in AP propagation (Nav1.5, by 56%**, **Pto and I Na subunits: HF decreased phase 1 slope (by 56%**), AP overshoot (by 32%*) and dV/dt max (by 35%*). AP properties associated with unchanged subunits (eg, resting potential, overall AP duration) were unaltered. Because of consistently significant changes in subunits governing impulse propagation (Nav1.5, Cx40, Cx43), we examined HF effects on AP propagation in PF false tendons with dual microelectrodes: conduction velocity decreased from 2.2±0.1 m/s (control) to 1.5±0.1 m/s* (HF). We have characterized for the first time HF effects on ion-channel subunit expression in cardiac PFs, finding prominent alterations in a variety of important subunits that control AP repolarization and propagation. These changes in PF ion-channel subunits likely contribute to conduction disturbances and arrhythmogenesis in the failing heart.

Atrial fibrillation (AF) is the most common sustained clinical arrhythmia and is a problem of growing proportions. Recent studies have increased interest in fast-unbinding Na+ channel blockers like vernakalant (RSD1235) and ranolazine for AF therapy, but the mechanism of efficacy is poorly understood. To study how fast-unbinding INa blockers affect AF, we developed realistic mathematical models of state-dependent Na+ channel block, using a lidocaine model as a prototype, and studied the effects on simulated cholinergic AF in two- and three-dimensional atrial substrates. We then compared the results with in vivo effects of lidocaine on vagotonic AF in dogs. Lidocaine action was modeled with the Hondeghem-Katzung modulated-receptor theory and maximum affinity for activated Na+ channels. Lidocaine produced frequency-dependent Na+ channel blocking and conduction slowing effects and terminated AF in both two- and three-dimensional models with concentration-dependent efficacy (maximum ∼89% at 60 μM). AF termination was not related to increases in wavelength, which tended to decrease with the drug, but rather to decreased source Na+ current in the face of large ACh-sensitive K+ current-related sinks, leading to the destabilization of primary generator rotors and a great reduction in wavebreak, which caused primary rotor annihilations in the absence of secondary rotors to resume generator activity. Lidocaine also reduced the variability and maximum values of the dominant frequency distribution during AF. Qualitatively similar results were obtained in vivo for lidocaine effects on vagal AF in dogs, with an efficacy of 86% at 2 mg/kg iv, as well as with simulations using the guarded-receptor model of lidocaine action. These results provide new insights into the mechanisms by which rapidly unbinding class I antiarrhythmic agents, a class including several novel compounds of considerable promise, terminate AF.

Background: Vagal (VG) and atrial tachycardia remodeled (ATR) AF substrates share many features: reduced effective refractory period (ERP), increased ERP heterogeneity and some common molecular mechanisms (I KACh enhancement by acetylcholine release in VG, constitutive I KACh enhancement in ATR). This study compared VG and ATR substrates at comparable ERP abbreviation. Methods: In each of 5 VG dogs, bilateral cervical VG stimulation parameters were adjusted (mean±SD: 3.6±1.7 V and 12.2±1.5 Hz; 0.2 ms) to produce the same mean ERP (at 4 RA and 4 LA sites) as a sex and weight matched ATR dog (RA paced 400 bpm × 7 days). Mean duration of burst pacing induced AF (DAF) and local dominant frequencies (DFs, analyzed by FFT at 240 bipolar electrodes, Fig A ) were determined. Results: Mean ERP was 79±13 ms in VG and 78±13 ms in ATR dogs. DAF was greater in VG than ATR dogs (1056±323 vs 289±510 s *P control, 43±61 s). Despite matched ERPs, there were significant differences in DF distribution (Fig B ): DF was faster (mean DF: 11.8±1.1 Hz VG vs 9.7±1.3 Hz ATR*) and DF variability greater (indicated by SD: 1.8±0.6 Hz VG vs 0.8±0.5 Hz ATR*) in VG dogs. AF drivers reflected by maximum DF zones were adjacent to autonomic ganglia (over RA in 4/5) for VG dogs; in ATR dogs driver zones were less clear and showed variable location. Conclusions: For a comparable atrial ERP, VG AF is faster and more persistent than AF with an ATR substrate. These results are consist with modeling work suggesting that VG-induced hyperpolarization is an important contributor to AF-maintaining rotor stabilization and acceleration, and indicate important differences between these superficially similar AF substrates.

Background: Previous studies suggested that T-type Ca 2+ -current (I CaT ) blockers suppress disease-related cardiac remodeling, but all available I CaT -blockers have non-specific actions on other currents and/or functions. To obtain a clearer sense of the role of I CaT in cardiac remodeling, we studied mice with the principal cardiac I CaT subunit (Cav3.1) knocked out. Methods: Adult male Cav3.1 knockout mice (KO) were compared with wild type (WT). LAD-ligation was performed to create anterior-wall myocardial infarction (MI). Echocardiography was used to evaluate cardiac performance at baseline, 2-w and 4-w post-MI. Intracardiac programmed stimulation was performed 4-w post-MI. Standard pacing protocols were used to determine atrial and ventricular electrophysiological properties and to induce arrhythmias. Results: Surface ECG parameters were examined in 10 KO and 10 WT mice prior to LAD ligation. At baseline, PQ interval was significantly longer in KO mice than in WT animals (44 ± 1 ms vs 40 ± 1 ms, respectively; p < 0.05); no significant differences were observed in RR, QRS or QTc intervals. At 10 minutes post-MI, WT (but not KO) mice showed increased heart rate (p = 0.002). Baseline echocardiography showed smaller LV fractional shortening (FS) and ejection fraction (EF) in KO mice compared with WT (34 ± 1% KO vs 42 ± 3% WT; 69 ± 2% KO vs 77 ± 3% WT; respectively, p < 0.05). At 4-w post-MI, FS and EF of KO mice were still smaller than WT mice (15.5 ± 1.6% vs 21.0 ± 1.5%; 37.4 ± 3.2% vs 48.1 ± 2.8%, respectively; p < 0.05). At baseline and 4-w post-MI, the contractility index (pressure-volume curves) was decreased in KO mice (p < 0.05). Intracardiac electrophysiological recordings revealed no differences in atrial and ventricular ERP or sinus node recovery time for WT vs KO. At 4-w post-MI, extrastimuli induced sustained atrial fibrillation (> 2 s) in 60% (6/10) KO mice, versus 17% (1/6) in WT (p = 0.05) and ventricular tachyarrhythmias in 82% (9/11) of KO mice versus 17% (1/6) of WT (p < 0.01). Conclusion: Contrary to previous studies with (imperfectly selective) pharmacological agents having T-type Ca 2+ channel blocking actions, our findings suggests that reductions in I CaT do not favorably alter post-MI remodeling and may negatively affect cardiac function and arrhythmias.

In native conditions, cardiac cells must continuously comply with diverse stimuli necessitating a perpetual adaptation. Polydimethylsiloxane (PDMS) is commonly used in cell culture to study cellular response to changes in the mechanical environment. The aim of this study was to evaluate the impact of using PDMS substrates on the properties of spontaneous activity of cardiomyocyte monolayer cultures. We compared PDMS to the gold standard normally used in culture: a glass substrate. Although mean frequency of spontaneous activity remained unaltered, incidence of reentrant activity was significantly higher in samples cultured on glass compared to PDMS substrates. Higher spatial and temporal instability of the spontaneous rate activation was found when cardiomyocytes were cultured on PDMS, and correlated with decreased connexin-43 and increased CaV3.1 and HCN2 mRNA levels. Compared to cultures on glass, cultures on PDMS were associated with the strongest response to isoproterenol and acetylcholine. These results reveal the importance of carefully selecting the culture substrate for studies involving mechanical stimulation, especially for tissue engineering or pharmacological high-throughput screening of cardiac tissue analog.

The dynamics of reentry in a model of a two-dimensional annulus of homogeneous cardiac tissue, with a Beeler-Reuter type formulation of the membrane ionic currents, is examined. The bifurcation structure of the sustained propagated solutions is described as a function of Rin and Rout, the inner and outer radii of the annulus. The transition from periodic to quasiperiodic reentry occurs at a critical Rin, which first diminishes and then saturates as Rout is increased. The reduction of the critical Rin is a consequence of the increase of the wave-front curvature. There is a range of Rin below the critical radius in which two distinct quasiperiodic solutions coexist. Each of these solutions disappears at its own specific value of Rin, and their annihilation is preceded by a new type of bifurcation leading to a regime of propagation with transient successive detachments of the wave front from the inner border of the annulus.

Spiral wave formation and disorganized activity in excitable media require the existence of broken waves and are related to partial wave block. The determinants of wave block in excitable systems are incompletely understood, especially for cardiac excitable tissue. Previous work in one-dimensional cardiac models has suggested that wave break of a premature excitation (PE) requires critical timing and that the conditions for broken waves are improbable. We analyzed the mechanism of unidirectional wave block that occurs when two consecutive PEs interact with a normal plane wave in a generic one-dimensional spatial excitable medium. A nondimensional coupled-map model built from mesoscopic characteristics of the substrate (the velocity and action potential duration restitution functions) shows that block can occur over a large interval of timing between the two PEs and leads to wave break in two-dimensional media. This mechanism may be an important determinant of spiral wave formation by the response to premature excitations.

The “leading circle model” was the first detailed attempt at understanding the mechanisms of functional reentry, and remains a widely-used notion in cardiac electrophysiology. The “spiral wave” concept was developed more recently as a result of modern theoretical analysis and is the basis for consideration of reentry mechanisms in present biophysical theory. The goal of this paper is to present these models in a way that is comprehensible to both the biophysical and electrophysiology communities, with the idea of helping clinical and experimental electrophysiologists to understand better the spiral wave concept and of helping biophysicists to understand why the leading circle concept is so attractive and widely used by electrophysiologists. To this end, the main properties of the leading circle and spiral wave models of reentry are presented. Their basic assumptions and determinants are discussed and the predictions of the two concepts with respect to pharmacological responses of arrhythmias are reviewed. A major difference between them lies in the predicted responses to Na+-channel blockade, for which the spiral wave paradigm appears more closely to correspond to the results of clinical and experimental observations. The basis of this difference is explored in the context of the fundamental properties of the models.

We present the bifurcation analysis of a revised version of the integral-delay model [Courtemanche et al., Siam J. Appl. Math. 56, 119 (1996)] of reentry in a one-dimensional ring that includes a spatial coupling in the calculation of the action potential duration. This coupling is meant to reproduce the modulation of repolarization by the diffusive current flowing through the intercellular resistance. We show that coupling modifies the criterion for the stability of the period-1 solution, which is no longer uniquely related to the action potential restitution curve, but depends also on the degree of coupling between cells and on the dispersion relation of the velocity. Coupling also changes the scenario from an infinite-dimension Hopf bifurcation to a finite sequence of Hopf bifurcations that take place at different ring lengths.

Resetting and annihilation of reentrant activity by a single stimulus pulse (S1) or a pair (S1-S2) of coupled pulses are studied in a model of one-dimensional loop of cardiac tissue using a Beeler-Reuter-type ionic model. Different modes of reentry termination are described. The classical mode of termination by unidirectional block, in which a stimulus produces only a retrograde front that collides with the activation front of the reentry, can be obtained for both S1 and S1-S2 applied over a small vulnerable window. We demonstrate that another scenario of termination-that we term collision block-can also be induced by the S1-S2 protocol. This scenario is obtained over a much wider range of S1-S2 coupling intervals than the one leading to a unidirectional block. In the collision block, S1 produces a retrograde front, colliding with the activation front of the pre-existing reentry, and an antegrade front propagating in the same direction as the initial reentry. Then, S2 also produces an antegrade and a retrograde front. However, the propagation of these fronts in the spatial profile of repolarization left by S1 leads to a termination of the reentrant activity. More complex behaviors also occur in which the antegrade fronts produced by S1 and S2 both persist for several turns, displaying a growing alternation in action potential duration ("alternans amplification") that may lead to the termination of the reentrant activity. The hypothesis that both collision block and alternans amplification depend on the interaction between the action potential duration restitution curve and the recovery curve of conduction velocity is supported by the fact that the dynamical behaviors were reproduced using an integro-delay equation based on these two properties. We thus describe two new mechanisms (collision block and alternans amplification) whereby electrical stimulation can terminate reentrant activity. (c) 2002 American Institute of Physics.

Self-organization of pacemaker (PM) activity of interconnected elements is important to the general theory of reaction–diffusion systems as well as for applications such as PM activity in cardiac tissue to initiate beating of the heart. Monolayer cultures of neonatal rat ventricular myocytes (NRVMs) are often used as experimental models in studies on cardiac electrophysiology. These monolayers exhibit automaticity (spontaneous activation) of their electrical activity. At low plated density, cells usually show a heterogeneous population consisting of PM and quiescent excitable cells (QECs). It is therefore highly probable that monolayers of NRVMs consist of a heterogeneous network of the two cell types. However, the effects of density and spatial distribution of the PM cells on spontaneous activity of monolayers remain unknown. Thus, a simple stochastic pattern formation algorithm was implemented to distribute PM and QECs in a binary-like 2D network. A FitzHugh–Nagumo excitable medium was used to simulate electrical spontaneous and propagating activity. Simulations showed a clear nonlinear dependency of spontaneous activity (occurrence and amplitude of spontaneous period) on the spatial patterns of PM cells. In most simulations, the first initiation