The Contribution of Ionic Currents to Rate-Dependent Action Potential Duration and Pattern of Reentry in a Mathematical Model of Human Atrial Fibrillation

Persistent atrial fibrillation (PeAF) in humans is characterized by shortening of action potential duration (APD) and attenuation of APD rate-adaptation. However, the quantitative influences of particular ionic current alterations on rate-dependent APD changes, and effects on patterns of reentry in atrial tissue, have not been systematically investigated. Using mathematical models of human atrial cells and tissue and performing parameter sensitivity analysis, we evaluated the quantitative contributions to action potential (AP) shortening and APD rate-adaptation of ionic current remodeling seen with PeAF. Ionic remodeling in PeAF was simulated by reducing L-type Ca²⁺ channel current (I_CaL), increasing inward rectifier K⁺ current (I_K1) and modulating five other ionic currents. Parameter sensitivity analysis, which quantified how each ionic current influenced APD in control and PeAF conditions, identified interesting results, including a negative effect of Na⁺/Ca²⁺ exchange on APD only in the PeAF condition. At high pacing rate (2 Hz), electrical remodeling in I_K1 alone accounts for the APD reduction of PeAF, but at slow pacing rate (0.5 Hz) both electrical remodeling in I_CaL alone (-70%) and I_K1 alone (+100%) contribute equally to the APD reduction. Furthermore, AP rate-adaptation was affected by I_Kur in control and by I_NaCa in the PeAF condition. In a 2D tissue model, a large reduction (-70%) of I_CaL becomes a dominant factor leading to a stable spiral wave in PeAF. Our study provides a quantitative and unifying understanding of the roles of ionic current remodeling in determining rate-dependent APD changes at the cellular level and spatial reentry patterns in tissue.     

M3 cholinoreceptors alter electrical activity of rat left atrium via suppression of L-type Ca<Superscript>2+</Superscript> current without affecting K<Superscript>+</Superscript> conductance

Electrophysiological effects produced by selective activation of M3 cholinoreceptors were studied in isolated left atrium preparations from rat using the standard sharp glass microelectrode technique. The stimulation of M3 receptors was obtained by application of muscarinic agonist pilocarpine (10^?5 M) in the presence of selective M2 antagonist methoctramine (10^?7 M). Stimulation of M3 receptors induced marked reduction of action potential duration by 14.4 ± 2.4% and 16.1 ± 2.5% of control duration measured at 50 and 90% of repolarization, respectively. This effect was completely abolished by selective M3 blocker 4-DAMP (10^?8 M). In isolated myocytes obtained from the rat left atrium, similar pharmacological stimulation of M3 receptors led to suppression of peak L-type calcium current by 13.9 ± 2.6% of control amplitude (measured at +10 mV), but failed to affect K⁺ currents I _to, I _Kur, and I _Kir. In the absence of M2 blocker methoctramine, pilocarpine (10^?5 M) produced stronger attenuation of I _CaL and induced an increase in I _Kir. This additive inward rectifier current could be abolished by highly selective blocker of K_ir3.1/3.4 channels tertiapin-Q (10^?6 M) and therefore was identified as I _KACh. Thus, in the rat atrial myocardium activation of M3 receptors leads to shortening of action potentials via suppression of I _CaL, but does not enhance the major potassium currents involved in repolarization. Joint stimulation of M2 and M3 receptors produces stronger action potential shortening due to M2-mediated activation of I _KACh.     

Mechanisms of Transition from Normal to Reentrant Electrical Activity in a Model of Rabbit Atrial Tissue: Interaction of Tissue Heterogeneity and Anisotropy

Experimental evidence suggests that regional differences in action potential (AP) morphology can provide a substrate for initiation and maintenance of reentrant arrhythmias in the right atrium (RA), but the relationships between the complex electrophysiological and anatomical organization of the RA and the genesis of reentry are unclear. In this study, a biophysically detailed three-dimensional computer model of the right atrial tissue was constructed to study the role of tissue heterogeneity and anisotropy in arrhythmogenesis. The model of Lindblad et al. for a rabbit atrial cell was modified to incorporate experimental data on regional differences in several ionic currents (primarily, I_Na, I_CaL, I_K1, I_to, and I_sus) between the crista terminalis and pectinate muscle cells. The modified model was validated by its ability to reproduce the AP properties measured experimentally. The anatomical model of the rabbit RA (including tissue geometry and fiber orientation) was based on a recent histological reconstruction. Simulations with the resultant electrophysiologically and anatomically detailed three-dimensional model show that complex organization of the RA tissue causes breakdown of regular AP conduction patterns at high pacing rates (>11.75 Hz): as the AP in the crista terminalis cells is longer, and electrotonic coupling transverse to fibers of the crista terminalis is weak, high-frequency pacing at the border between the crista terminalis and pectinate muscles results in a unidirectional conduction block toward the crista terminalis and generation of reentry. Contributions of the tissue heterogeneity and anisotropy to reentry initiation mechanisms are quantified by measuring action potential duration (APD) gradients at the border between the crista terminalis and pectinate muscles: the APD gradients are high in areas where both heterogeneity and anisotropy are high, such that intrinsic APD differences are not diminished by electrotonic interactions. Thus, our detailed computer model reconstructs complex electrical activity in the RA, and provides new insights into the mechanisms of transition from focal atrial tachycardia into reentry.     

In silico investigation of pro-arrhythmic effects of azithromycin on the human ventricle

The macrolide antibiotic azithromycin (AZM) is widely used for respiratory infections and has been suggested to be a possible treatment for the Coronavirus Disease of 2019 (COVID-19). However, AZM-associated QT interval prolongation and arrhythmias have been reported. Integrated mechanistic information on AZM actions on human ventricular excitation and conduction is lacking. Therefore, this study was undertaken to investigate the actions of AZM on ventricular cell and tissue electrical activity. The O'Hara- Virag-Varro-Rudy dynamic (ORd) model of human ventricular cells was modified to incorporate experimental data on the concentration-dependent actions of AZM on multiple ion channels, including I_Na, I_CaL, I_Kr, I_Ks, I_K1 and I_NaL in both acute and chronic exposure conditions. In the single cell model, AZM prolonged the action potential duration (APD) in a concentration-dependent manner, which was predominantly attributable to I_Kr reduction in the acute condition and potentiated I_NaL in the chronic condition. High concentrations of AZM also increased action potential (AP) triangulation (determined as an increased difference between APD₃₀ and APD₉₀) which is a marker of arrhythmia risk. In the chronic condition, the potentiated I_NaL caused a modest intracellular Na ⁺ concentration accumulation at fast pacing rates. At the 1D tissue level, the AZM-prolonged APD at the cellular level was reflected by an increased QT interval in the simulated pseudo-ECG, consistent with clinical observations. Additionally, AZM reduced the conduction velocity (CV) of APs in the acute condition due to a reduced I_Na, and it augmented the transmural APD dispersion of the ventricular tissue, which is also pro-arrhythmic. Such actions were markedly augmented when the effects of chronic exposure of AZM were also considered, or with additional I_Kr block, as may occur with concurrent use of other medications. This study provides insights into the ionic mechanisms by which high concentrations of AZM may modulate ventricular electrophysiology and susceptibility to arrhythmia.     

Systematic characterization of the ionic basis of rabbit cellular electrophysiology using two ventricular models

Several mathematical models of rabbit ventricular action potential (AP) have been proposed to investigate mechanisms of arrhythmias and excitation-contraction coupling. Our study aims at systematically characterizing how ionic current properties modulate the main cellular biomarkers of arrhythmic risk using two widely-used rabbit ventricular models, and comparing simulation results using the two models with experimental data available for rabbit. A sensitivity analysis of AP properties, Ca²⁺ and Na⁺ dynamics, and their rate dependence to variations (±15% and ±30%) in the main transmembrane current conductances and kinetics was performed using the Shannon et al. (2004) and the [Mahajan et?al., 2008a] and [Mahajan et?al., 2008b] AP rabbit models. The effects of severe transmembrane current blocks (up to 100%) on steady-state AP and calcium transients, and AP duration (APD) restitution curves were also simulated using both models. Our simulations show that, in both virtual rabbit cardiomyocytes, APD is significantly modified by most repolarization currents, AP triangulation is regulated mostly by the inward rectifier K⁺ current (I_K1) whereas APD rate adaptation as well as [Na⁺]_i rate dependence is influenced by the Na⁺/K⁺ pump current (I_NaK). In addition, steady-state [Ca²⁺]_i levels, APD restitution properties and [Ca²⁺]_i rate dependence are strongly dependent on I_NaK, the L-Type Ca²⁺ current (I_CaL) and the Na⁺/Ca²⁺ exchanger current (I_NaCa), although the relative role of these currents is markedly model dependent. Furthermore, our results show that simulations using both models agree with many experimentally-reported electrophysiological characteristics. However, our study shows that the Shannon et al. model mimics rabbit electrophysiology more accurately at normal pacing rates, whereas Mahajan et al. model behaves more appropriately at faster rates. Our results reinforce the usefulness of sensitivity analysis for further understanding of cellular electrophysiology and validation of cardiac AP models.     

In silico assessment of drug safety in human heart applied to late sodium current blockers

Drug-induced action potential (AP) prolongation leading to Torsade de Pointes is a major concern for the development of anti-arrhythmic drugs. Nevertheless the development of improved anti-arrhythmic agents, some of which may block different channels, remains an important opportunity. Partial block of the late sodium current (I_NaL) has emerged as a novel anti-arrhythmic mechanism. It can be effective in the settings of free radical challenge or hypoxia. In addition, this approach can attenuate pro-arrhythmic effects of blocking the rapid delayed rectifying K⁺ current (I_Kr). The main goal of our computational work was to develop an in-silico tool for preclinical anti-arrhythmic drug safety assessment, by illustrating the impact of I_Kr/I_NaL ratio of steady-state block of drug candidates on “torsadogenic” biomarkers. The O’Hara et al. AP model for human ventricular myocytes was used. Biomarkers for arrhythmic risk, i.e., AP duration, triangulation, reverse rate-dependence, transmural dispersion of repolarization and electrocardiogram QT intervals, were calculated using single myocyte and one-dimensional strand simulations. Predetermined amounts of block of I_NaL and I_Kr were evaluated. “Safety plots” were developed to illustrate the value of the specific biomarker for selected combinations of IC₅₀s for I_Kr and I_NaL of potential drugs. The reference biomarkers at baseline changed depending on the “drug” specificity for these two ion channel targets. Ranolazine and GS967 (a novel potent inhibitor of I_NaL) yielded a biomarker data set that is considered safe by standard regulatory criteria. This novel in-silico approach is useful for evaluating pro-arrhythmic potential of drugs and drug candidates in the human ventricle.     

Inter-Subject Variability in Human Atrial Action Potential in Sinus Rhythm versus Chronic Atrial Fibrillation

Aims

Human atrial electrophysiology exhibits high inter-subject variability in both sinus rhythm (SR) and chronic atrial fibrillation (cAF) patients. Variability is however rarely investigated in experimental and theoretical electrophysiological studies, thus hampering the understanding of its underlying causes but also its implications in explaining differences in the response to disease and treatment. In our study, we aim at investigating the ability of populations of human atrial cell models to capture the inter-subject variability in action potential (AP) recorded in 363 patients both under SR and cAF conditions.

Methods and Results

Human AP recordings in atrial trabeculae (n = 469) from SR and cAF patients were used to calibrate populations of computational SR and cAF atrial AP models. Three populations of over 2000 sampled models were generated, based on three different human atrial AP models. Experimental calibration selected populations of AP models yielding AP with morphology and duration in range with experimental recordings. Populations using the three original models can mimic variability in experimental AP in both SR and cAF, with median conductance values in SR for most ionic currents deviating less than 30% from their original peak values. All cAF populations show similar variations in G_K1, G_Kur and G_to, consistent with AF-related remodeling as reported in experiments. In all SR and cAF model populations, inter-subject variability in I_K1 and I_NaK underlies variability in APD₉₀, variability in I_Kur, I_CaL and I_NaK modulates variability in APD₅₀ and combined variability in I_to and I_Kur determines variability in APD₂₀. The large variability in human atrial AP triangulation is mostly determined by I_K1 and either I_NaK or I_NaCa depending on the model.

Conclusion

Experimentally-calibrated human atrial AP models populations mimic AP variability in SR and cAF patient recordings, and identify potential ionic determinants of inter-subject variability in human atrial AP duration and morphology in SR versus cAF.     

Lysophosphatidic Acid Increases the Electrophysiological Instability of Adult Rabbit Ventricular Myocardium by Augmenting L-Type Calcium Current

Lysophosphatidic acid (LPA) has diverse actions on the cardiovascular system and is widely reported to modulate multiple ion currents in some cell types. However, little is known about its electrophysiological effects on cardiac myocytes. This study investigated whether LPA has electrophysiological effects on isolated rabbit myocardial preparations. The results indicate that LPA prolongs action potential duration at 90% repolarization (APD₉₀) in a concentration- and frequency-dependent manner in isolated rabbit ventricular myocytes. The application of extracellular LPA significantly increases the coefficient of APD₉₀ variability. LPA increased L-type calcium current (I_Ca,L) density without altering its activation or deactivation properties. In contrast, LPA has no effect on two other ventricular repolarizing currents, the transient outward potassium current (I_to) and the delayed rectifier potassium current (I_K). In arterially perfused rabbit left ventricular wedge preparations, the monophasic action potential duration, QT interval, and Tpeak-end are prolonged by LPA. LPA treatment also significantly increases the incidence of ventricular tachycardia induced by S₁S₂ stimulation. Notably, the effects of LPA on action potentials and I_Ca,L are PTX-sensitive, suggesting LPA action requires a G_i-type G protein. In conclusion, LPA prolongs APD and increases electrophysiological instability in isolated rabbit myocardial preparations by increasing I_Ca,L in a G_i protein-dependent manner.     

Positive Feedback Mechanisms among Local Ca Releases,NCX, and ICaL Ignite Pacemaker Action Potentials

Recent data suggest that cardiac pacemaker cell function is determined by numerous time-, voltage-, and Ca-dependent interactions of cell membrane electrogenic proteins (M-clock) and intracellular Ca cycling proteins (Ca-clock), forming a coupled-clock system. Many aspects of the coupled-clock system, however, remain underexplored. The key players of the system are Ca release channels (ryanodine receptors), generating local Ca releases (LCRs) from sarcoplasmic reticulum, electrogenic Na/Ca exchanger (NCX) current, and L-type Ca current (I_CaL). We combined numerical model simulations with experimental simultaneous recordings of action potentials (APs) and Ca to gain further insight into the complex interactions within the system. Our simulations revealed a positive feedback mechanism, dubbed AP ignition, which accelerates the diastolic depolarization (DD) to reach AP threshold. The ignition phase begins when LCRs begin to occur and the magnitude of inward NCX current begins to increase. The NCX current, together with funny current and T-type Ca current accelerates DD, bringing the membrane potential to I_CaL activation threshold. During the ignition phase, I_CaL-mediated Ca influx generates more LCRs via Ca-induced Ca release that further activates inward NCX current, creating a positive feedback. Simultaneous recordings of membrane potential and confocal Ca images support the model prediction of the positive feedback among LCRs and I_CaL, as diastolic LCRs begin to occur below and continue within the voltage range of I_CaL activation. The ignition phase onset (identified within the fine DD structure) begins when DD starts to notably accelerate (～0.15 V/s) above the recording noise. Moreover, the timing of the ignition onset closely predicted the duration of each AP cycle in the basal state, in the presence of autonomic receptor stimulation, and in response to specific inhibition of either the M-clock or Ca-clock, thus indicating general importance of the new coupling mechanism for regulation of the pacemaker cell cycle duration, and ultimately the heart rate.     

Cardiac action potential duration and calcium regulation in males and females

Adult women have longer QT intervals compared with men of a similar age, indicating differences in the speed of repolarisation of the ventricles. We investigate the influences of gender on ventricular electrophysiology and intracellular Ca²⁺ regulation of the guinea pig heart. Comparing sexually mature animals, females exhibited a significantly longer APD. Peak L-type Ca²⁺ current (I_CaL) was larger in females and when this current was inhibited with nifedipine the gender differences in APD were removed. APD differences also disappeared when the SR was depleted of Ca²⁺. Inactivation of I_CaL during a clamp step is faster in females but slower during an action potential and SR Ca²⁺ content is larger. We suggest that gender differences in APD result from variation in the kinetics of I_CaL stemming from alterations to Ca²⁺ release.     

Impaired Inactivation of L-Type Ca2+ Current as a Potential Mechanism for Variable Arrhythmogenic Liability of HERG K+ Channel Blocking Drugs

The proarrhythmic effects of new drugs have been assessed by measuring rapidly activating delayed-rectifier K⁺ current (I_Kr) antagonist potency. However, recent data suggest that even drugs thought to be highly specific I_Kr blockers can be arrhythmogenic via a separate, time-dependent pathway such as late Na⁺ current augmentation. Here, we report a mechanism for a quinolone antibiotic, sparfloxacin-induced action potential duration (APD) prolongation that involves increase in late L-type Ca²⁺ current (I_CaL) caused by a decrease in Ca²⁺-dependent inactivation (CDI). Acute exposure to sparfloxacin, an I_Kr blocker with prolongation of QT interval and torsades de pointes (TdP) produced a significant APD prolongation in rat ventricular myocytes, which lack I_Kr due to E4031 pretreatment. Sparfloxacin reduced peak I_CaL but increased late I_CaL by slowing its inactivation. In contrast, ketoconazole, an I_Kr blocker without prolongation of QT interval and TdP produced reduction of both peak and late I_CaL, suggesting the role of increased late I_CaL in arrhythmogenic effect. Further analysis showed that sparfloxacin reduced CDI. Consistently, replacement of extracellular Ca²⁺ with Ba²⁺ abolished the sparfloxacin effects on I_CaL. In addition, sparfloxacin modulated I_CaL in a use-dependent manner. Cardiomyocytes from adult mouse, which is lack of native I_Kr, demonstrated similar increase in late I_CaL and afterdepolarizations. The present findings show that sparfloxacin can prolong APD by augmenting late I_CaL. Thus, drugs that cause delayed I_CaL inactivation and I_Kr blockage may have more adverse effects than those that selectively block I_Kr. This mechanism may explain the reason for discrepancies between clinically reported proarrhythmic effects and I_Kr antagonist potencies.     

Atrial Tachyarrhythmia in Rgs5-Null Mice

Aims

The aim of this study was to elucidate the effects of regulator of G-protein signaling 5 (Rgs5), a negative regulator of G protein-mediated signaling, on atrial repolarization and tachyarrhythmia (ATA) in mice.

Methods and Results

In present study, the incidence of ATA were increased in Rgs5^−/− Langendorff-perfused mouse hearts during program electrical stimulation (PES) (46.7%, 7 of 15) and burst pacing (26.7%, 4 of 15) compared with wild-type (WT) mice (PES: 7.1%,1 of 14; burst:7.1%,1 of 14) (P<0.05). And the duration of ATA also shown longer in Rgs5^−/− heart than that in WT, 2 out of 15 hearts exhibited sustained ATA (>30 s) but none of them observed in WT mice. Atrial prolonged repolarization was observed in Rgs5^−/− hearts including widened P wave in surface ECG recording, increased action potential duration (APD) and atrial effective refractory periods (AERP), all of them showed significant difference with WT mice (P<0.05). At the cellular level, whole-cell patch clamp recorded markedly decreased densities of repolarizing K⁺ currents including I_Kur (at +60 mV: 14.0±2.2 pF/pA) and I_to (at +60 mV: 16.7±1.3 pA/pF) in Rgs5^−/− atrial cardiomyocytes, compared to those of WT mice (at +60 mV I_to: 20.4±2.0 pA/pF; I_kur: 17.9±2.0 pF/pA) (P<0.05).

Conclusion

These results suggest that Rgs5 is an important regulator of arrhythmogenesis in the mouse atrium and that the enhanced susceptibility to atrial tachyarrhythmias in Rgs5^−/− mice may contribute to abnormalities of atrial repolarization.     

Gating-Related Molecular Motions in the Extracellular Domain of the I<Subscript>Ks</Subscript> Channel: Implications for I<Subscript>Ks</Subscript> Channelopathy

Cardiac slow delayed rectifier (I_Ks) channel complex consists of KCNQ1 channel and KCNE1 auxiliary subunits. The extracellular juxtamembranous region of KCNE1 is an unstructured loop that contacts multiple KCNQ1 positions in a gating-state-dependent manner. Congenital arrhythmia-related mutations have been identified in the extracellular S1–S2 linker of KCNQ1. These mutations manifest abnormal phenotypes only when coexpressed with KCNE1, pointing to the importance of proper KCNQ1/KCNE1 interactions here in I_Ks channel function. We investigate the interactions between the KCNE1 loop (positions 36–47) and KCNQ1 S1–S2 linker (positions 140–148) by means of disulfide trapping and voltage clamp techniques. During transitions among the resting-state conformations, KCNE1 positions 36–43 make contacts with KCNQ1 positions 144, 145, and 147 in a parallel fashion. During conformational changes in the activated state, KCNE1 position 40 can make contacts with all three KCNQ1 positions, while the neighboring KCNE1 positions (36, 38, 39, and 41) can make contact with KCNQ1 position 147. Furthermore, KCNQ1 positions 143 and 146 are high-impact positions that cannot tolerate cysteine substitution. To maintain the proper I_Ks channel function, position 143 requires a small side chain with a hydroxyl group, and position 146 requires a negatively charged side chain. These data and the proposed molecular motions provide insights into the mechanisms by which mutations in the extracellular juxtamembranous region of the I_Ks channel impair its function.     

Targeting the late component of the cardiac L-type Ca2+ current to suppress early afterdepolarizations

Early afterdepolarizations (EADs) associated with prolongation of the cardiac action potential (AP) can create heterogeneity of repolarization and premature extrasystoles, triggering focal and reentrant arrhythmias. Because the L-type Ca²⁺ current (I_Ca,L) plays a key role in both AP prolongation and EAD formation, L-type Ca²⁺ channels (LTCCs) represent a promising therapeutic target to normalize AP duration (APD) and suppress EADs and their arrhythmogenic consequences. We used the dynamic-clamp technique to systematically explore how the biophysical properties of LTCCs could be modified to normalize APD and suppress EADs without impairing excitation–contraction coupling. Isolated rabbit ventricular myocytes were first exposed to H₂O₂ or moderate hypokalemia to induce EADs, after which their endogenous I_Ca,L was replaced by a virtual I_Ca,L with tunable parameters, in dynamic-clamp mode. We probed the sensitivity of EADs to changes in the (a) amplitude of the noninactivating pedestal current; (b) slope of voltage-dependent activation; (c) slope of voltage-dependent inactivation; (d) time constant of voltage-dependent activation; and (e) time constant of voltage-dependent inactivation. We found that reducing the amplitude of the noninactivating pedestal component of I_Ca,L effectively suppressed both H₂O₂- and hypokalemia-induced EADs and restored APD. These results, together with our previous work, demonstrate the potential of this hybrid experimental–computational approach to guide drug discovery or gene therapy strategies by identifying and targeting selective properties of LTCC.     

Protective effect of piperine on electrophysiology abnormalities of left atrial myocytes induced by hydrogen peroxide in rabbits

Aims

Piperine had protective effects on oxidative stress damage of ventricular myocytes by hydrogen peroxide (H₂O₂). In this study we aimed to explore the protective effect of piperine on abnormalities of the cardiac action potential (AP) and several ion currents induced by hydrogen peroxide (H₂O₂) in single rabbit left atrial myocyte.

Main methods

Conventional microelectrodes were used to record action potential duration (APD), resting membrane potential (RMP) and some ion currents (I_Ca,L,I_to,I_K1 and I_kur,ect.), before and after H₂O₂ administration with or without piperine.

Key findings

The piperine (7 μmol/L) had no significant effect on APD, I_Ca,L,I_to,I_K1 and I_kur and their channel dynamics. In the presence of 50 μmol/L H₂O₂, APD₅₀ and APD₉₀ shortened (P < 0.01), amplitude of RMP decreased (P < 0.05), the peak of I_Ca,L reduced significantly (P < 0.05). Piperine (7 μmol/L) significantly alleviated the inhibiting effect of H₂O₂ on APD and I_Ca,L (P < 0.01) and protected the changes of I_Ca,L dynamics induced by H₂O₂. The peak current of I_to was reduced significantly (P < 0.05); Piperine (7 μmol/L) significantly alleviated the inhibiting effect of H₂O₂ on I_to (P < 0.01). In addition, piperine protected the changes of I_to dynamics induced by H₂O_2. The peak current of I_K1 and I_KUr was significantly reduced (P < 0.05); Piperine (7 μmol/L) alleviated the inhibiting effect of H₂O₂ on I_K1 and I_KUr significantly (P < 0.01). In addition, piperine protected the changes of I_KUr dynamics induced by H₂O₂.

Significance

These results suggest that piperine effectively protects atrial myocytes from oxidative stress injury in atrial electrophysiology.     

Hydrogen Sulfide Suppresses Outward Rectifier Potassium Currents in Human Pluripotent Stem Cell-Derived Cardiomyocytes

Aim

Hydrogen sulfide (H₂S) is a promising cardioprotective agent and a potential modulator of cardiac ion currents. Yet its cardiac effects on humans are poorly understood due to lack of functional cardiomyocytes. This study investigates electrophysiological responses of human pluripotent stem cells (hPSCs) derived cardiomyocytes towards H₂S.

Methods and Results

Cardiomyocytes of ventricular, atrial and nodal subtypes differentiated from H9 embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) were electrophysiologically characterized. The effect of NaHS, a donor of H₂S, on action potential (AP), outward rectifier potassium currents (I _Ks and I _Kr), L-type Ca²⁺ currents (I _CaL) and hyperpolarization-activated inward current (I _f) were determined by patch-clamp electrophysiology and confocal calcium imaging. In a concentration-dependent manner, NaHS (100 to 300 µM) consistently altered the action potential properties including prolonging action potential duration (APD) and slowing down contracting rates of ventricular-and atrial-like cardiomyocytes derived from both hESCs and hiPSCs. Moreover, inhibitions of slow and rapid I _K (I _Ks and I _Kr), I _CaL and I _f were found in NaHS treated cardiomyocytes and it could collectively contribute to the remodeling of AP properties.

Conclusions

This is the first demonstration of effects of H₂S on cardiac electrophysiology of human ventricular-like, atrial-like and nodal-like cardiomyocytes. It reaffirmed the inhibitory effect of H₂S on I _CaL and revealed additional novel inhibitory effects on I _f, I _Ks and I _Kr currents in human cardiomyocytes.     

Comparative study of cardiac electrophysiological effects of atrial natriuretic peptide

The effects of atrial natriuretic peptide (ANP) on action potential characteristics were studied in various (human, rabbit, guinea-pig) atrial and guinea-pig right ventricular papillary muscles. ANP (1–100 nM) did not modify the resting membrane potential nor the maximum rate of depolarization phase (V_max). Up to 10 nM, ANP dose-dependently decreased the action potential amplitude both in guinea-pig atrial and ventricular muscles, but it did not affect this parameter in the other atrial preparations. ANP caused a dose-dependent, marked decrease of action potential duration (APD) in practically every cardiac preparation studied (exception of guinea-pig left atrium). The strongest effect on APD can be observed in human atrial and guinea-pig ventricular fibers. The K⁺ channel blocker 4-aminopyridine (1 mM) and the ATP-dependent K⁺ channel inhibitor glibenclamide (10Nl) prevented the effect of ANP on APD in both ventricular atrial preparations. ANP prevented the appearance of isoprenaline (0.5 M) induced slow AP in K⁺ depolarized myocardium. The present data suggest that ANP may inhibit the slow inward Ca²⁺ channel activity and facilitate the K⁺ channel activity.     

Induced pluripotent stem cells used to reveal drug actions in a long QT syndrome family with complex genetics

Understanding the basis for differential responses to drug therapies remains a challenge despite advances in genetics and genomics. Induced pluripotent stem cells (iPSCs) offer an unprecedented opportunity to investigate the pharmacology of disease processes in therapeutically and genetically relevant primary cell types in vitro and to interweave clinical and basic molecular data. We report here the derivation of iPSCs from a long QT syndrome patient with complex genetics. The proband was found to have a de novo SCN5A LQT-3 mutation (F1473C) and a polymorphism (K897T) in KCNH2, the gene for LQT-2. Analysis of the biophysics and molecular pharmacology of ion channels expressed in cardiomyocytes (CMs) differentiated from these iPSCs (iPSC-CMs) demonstrates a primary LQT-3 (Na⁺ channel) defect responsible for the arrhythmias not influenced by the KCNH2 polymorphism. The F1473C mutation occurs in the channel inactivation gate and enhances late Na⁺ channel current (I_NaL) that is carried by channels that fail to inactivate completely and conduct increased inward current during prolonged depolarization, resulting in delayed repolarization, a prolonged QT interval, and increased risk of fatal arrhythmia. We find a very pronounced rate dependence of I_NaL such that increasing the pacing rate markedly reduces I_NaL and, in addition, increases its inhibition by the Na⁺ channel blocker mexiletine. These rate-dependent properties and drug interactions, unique to the proband’s iPSC-CMs, correlate with improved management of arrhythmias in the patient and provide support for this approach in developing patient-specific clinical regimens.     

Autocrine A2 in the T-System of Ventricular Myocytes Creates Transmural Gradients in Ion Transport: A Mechanism to Match Contraction with Load?

Transmural heterogeneities in Na/K pump current (I_P), transient outward K⁺-current (I_to), and Ca²⁺-current (I_CaL) play an important role in regulating electrical and contractile activities in the ventricular myocardium. Prior studies indicated angiotensin II (A2) may determine the transmural gradient in I_to, but the effects of A2 on I_P and I_CaL were unknown. In this study, myocytes were isolated from five muscle layers between epicardium and endocardium. We found a monotonic gradient in both I_p and I_to, with the lowest currents in ENDO. When AT₁Rs were inhibited, EPI currents were unaffected, but ENDO currents increased, suggesting endogenous extracellular A2 inhibits both currents in ENDO. I_P- and I_to-inhibition by A2 yielded essentially the same K_0.5 values, so they may both be regulated by the same mechanism. A2/AT₁R-mediated inhibition of I_P or I_to or stimulation of I_CaL persisted for hours in isolated myocytes, suggesting continuous autocrine secretion of A2 into a restricted diffusion compartment, like the T-system. Detubulation brought EPI I_P to its low ENDO value and eliminated A2 sensitivity, so the T-system lumen may indeed be the restricted diffusion compartment. These studies showed that 33–50% of I_P, 57–65% of I_to, and a significant fraction of I_CaL reside in T-tubule membranes where they are transmurally regulated by autocrine secretion of A2 into the T-system lumen and activation of AT₁Rs. Increased AT₁R activation regulates each of these currents in a direction expected to increase contractility. Endogenous A2 activation of AT₁Rs increases monotonically from EPI to ENDO in a manner similar to reported increases in passive tension when the ventricular chamber fills with blood. We therefore hypothesize load is the signal that regulates A2-activation of AT₁Rs, which create a contractile gradient that matches the gradient in load.     

Atrial Natriuretic Peptide Regulates Ca2+ Channel in Early Developmental Cardiomyocytes

Background

Cardiomyocytes derived from murine embryonic stem (ES) cells possess various membrane currents and signaling cascades link to that of embryonic hearts. The role of atrial natriuretic peptide (ANP) in regulation of membrane potentials and Ca²⁺ currents has not been investigated in developmental cardiomyocytes.

Methodology/Principal Findings

We investigated the role of ANP in regulating L-type Ca²⁺ channel current (I_CaL) in different developmental stages of cardiomyocytes derived from ES cells. ANP decreased the frequency of action potentials (APs) in early developmental stage (EDS) cardiomyocytes, embryonic bodies (EB) as well as whole embryo hearts. ANP exerted an inhibitory effect on basal I_CaL in about 70% EDS cardiomyocytes tested but only in about 30% late developmental stage (LDS) cells. However, after stimulation of I_CaL by isoproterenol (ISO) in LDS cells, ANP inhibited the response in about 70% cells. The depression of I_CaL induced by ANP was not affected by either Nω, Nitro-L-Arginine methyl ester (L-NAME), a nitric oxide synthetase (NOS) inhibitor, or KT5823, a cGMP-dependent protein kinase (PKG) selective inhibitor, in either EDS and LDS cells; whereas depression of I_CaL by ANP was entirely abolished by erythro-9-(2-Hydroxy-3-nonyl) adenine (EHNA), a selective inhibitor of type 2 phosphodiesterase(PDE2) in most cells tested.

Conclusion/Significances

Taken together, these results indicate that ANP induced depression of action potentials and I_CaL is due to activation of particulate guanylyl cyclase (GC), cGMP production and cGMP-activation of PDE2 mediated depression of adenosine 3′, 5′–cyclic monophophate (cAMP)–cAMP-dependent protein kinase (PKA) in early cardiomyogenesis.