Action Potential In Ventricular Myocytes Research Articles

Facilitation of hERG Activation by Its Blocker: A Mechanism to Reduce Drug-Induced Proarrhythmic Risk.

Modulation of the human Ether-à-go-go-Related Gene (hERG) channel, a crucial voltage-gated potassium channel in the repolarization of action potentials in ventricular myocytes of the heart, has significant implications on cardiac electrophysiology and can be either antiarrhythmic or proarrhythmic. For example, hERG channel blockade is a leading cause of long QT syndrome and potentially life-threatening arrhythmias, such as torsades de pointes. Conversely, hERG channel blockade is the mechanism of action of Class III antiarrhythmic agents in terminating ventricular tachycardia and fibrillation. In recent years, it has been recognized that less proarrhythmic hERG blockers with clinical potential or Class III antiarrhythmic agents exhibit, in addition to their hERG-blocking activity, a second action that facilitates the voltage-dependent activation of the hERG channel. This facilitation is believed to reduce the proarrhythmic potential by supporting the final repolarizing of action potentials. This review covers the pharmacological characteristics of hERG blockers/facilitators, the molecular mechanisms underlying facilitation, and their clinical significance, as well as unresolved issues and requirements for research in the fields of ion channel pharmacology and drug-induced arrhythmias.

AFM is used to study the biophysics of hypertension-induced tachyarrhythmia.

Patients with long-lasting hypertension often suffer from atrial or ventricular arrhythmias. Evidence suggests that mechanical stimulation can change the refractory period and dispersion of the ventricular myocyte action potential through stretch-activated ion channels (SACs) and influence cellular calcium transients, thus increasing susceptibility to ventricular arrhythmias. However, the specific pathogenesis of hypertension-induced arrhythmias is unknown. In this study, through clinical data, we found that a short-term increase in blood pressure leads to a rise in tachyarrhythmias in patients with clinical hypertension. We investigated the mechanism of this phenomenon using a combined imaging system(AC) of atomic force microscopy (AFM) and laser scanning confocal microscopy. After mechanical distraction to stimulate ventricular myocytes isolated from Wistar Kyoto rats (WKY) and spontaneously hypertensive rats (SHR), we synchronously monitored cardiomyocyte stiffness and intracellular calcium changes. This method can reasonably simulate cardiomyocytes' mechanics and ion changes when blood pressure rises rapidly. Our results indicated that the stiffness value of cardiomyocytes in SHR was significantly more extensive than that of normal controls, and cardiomyocytes were more sensitive to mechanical stress; In addition, intracellular calcium increased rapidly and briefly in rats with spontaneous hypertension. After intervention with streptomycin, a SAC blocker, ventricular myocytes are significantly less sensitive to mechanical stimuli. Thus, SAC is involved in developing and maintaining ventricular arrhythmias induced by hypertension. The increased stiffness of ventricular myocytes caused by hypertension leads to hypersensitivity of cellular calcium flow to mechanical stimuli is one of the mechanisms that cause arrhythmias. The AC system is a new research method to study the mechanical properties of cardiomyocytes. This study provides new techniques and ideas for developing new anti-arrhythmic drugs. HIGHLIGHT: The mechanism of hypertension-induced tachyarrhythmia is not precise. Through this study, it is found that the biophysical properties of myocardial abnormalities, the myocardium is excessively sensitive to mechanical stimulation, and the calcium flow appears to transient explosive changes, leading to tachyarrhythmia.

Reconstructing action potential and ionic current dynamics of a mammalian ventricular cell using data assimilation.

The dynamics of the cardiac ventricular myocyte action potential (AP) is regulated by intricate and nonlinear interactions between the cell transmembrane potential, and ionic currents and concentrations. Present technology limits the ability to simultaneously measuring multiple ionic currents and concentration during an AP in in vitro single cell experiments, which limits the scope of experimental data to provide a complete snapshot of the cardiac myocyte system state. This limitation presents an obstacle for understanding how cellular perturbations can trigger arrhythmogenic conditions. In this study, we applied a data assimilation approach, which combines limited noisy observations with predictions from a computational model, paired with a parameter estimation to minimize the effects of parameter uncertainty and model error. We performed a series of in silico experiments, in which a forecasting system was tasked with reconstructing AP dynamics of a virtual ventricular myocyte in the presence varying degrees of parameter and model error. With appropriate protocol design, we find that a data assimilation approach can successfully reconstruct and predict the dynamics of not only the directly observed state variables (such as the transmembrane potential and a selected ionic current), but also unobserved state variables, such as unmeasured ionic currents and concentrations. As an extension of the results of these series of in silico experiments, we generated simulations to assess which ionic currents, when measured, offer greater predictive ability using the data assimilation algorithm. This can offer valuable information for the design of single cardiac in vitro experiments, as it is not feasible to measure all ionic currents and concentrations across time within the same cell during an AP. Future work will investigate the longer-term accuracy of this approach predicting potentially pro-arrhythmic perturbations for arrhythmia risk assessment.

A novel mutation in KCNH2 yields loss-of-function of hERG potassium channel in long QT syndrome 2.

Mutations in hERG (human ether-à-go-go-related gene) potassium channel are closely associated with long QT syndromes. By direct Sanger sequencing, we identified a novel KCNH2 mutation W410R in the patient with long QT syndrome 2 (LQT2). However, the electrophysiological functions of this mutation remain unknown. In comparison to hERGWT channels, hERGW410R channels have markedly decreased total and surface expressions. W410R mutation dramatically reduces hERG channel currents (IKr) and shifts its steady-state activation curve to depolarization. Moreover, hERGW410R channels make dominant-negative effects on hERGWT channels. Significantly, we find hERG channel blocker E-4031 could partially rescue the function of hERGW410R channels by increasing the membrane expression. By using in silico model, we reveal that hERGW410R channels obviously elongate the repolarization of human ventricular myocyte action potentials. Collectively, W410R mutation decreases the currents of hERG channel, because of diminished membrane expression of mutant channels, that subsequently leads to elongated repolarization of cardiomyocyte, which might induce the pathogenesis of LQT2. Furthermore, E-4031 could partially rescue the decreased activity of hERGW410R channels. Thus, our work identifies a novel loss-of-function mutation in KCNH2 gene, which might provide a rational basis for the management of LQT2.

Pro-arrhythmic risk assessment with a population model of human ventricular myocyte action potentials

Pro-arrhythmic risk assessment with a population model of human ventricular myocyte action potentials

PKA phosphorylation underlies functional recruitment of sarcolemmal SK2 channels in ventricular myocytes from hypertrophic hearts.

Key points Small‐conductance Ca2+‐activated K+ (SK) channels expressed in ventricular myocytes are dormant in health, yet become functional in cardiac disease.SK channels are voltage independent and their gating is controlled by intracellular [Ca2+] in a biphasic manner. Submicromolar [Ca2+] activates the channel via constitutively‐bound calmodulin, whereas higher [Ca2+] exerts inhibitory effect during depolarization.Using a rat model of cardiac hypertrophy induced by thoracic aortic banding, we found that functional upregulation of SK2 channels in hypertrophic rat ventricular cardiomyocytes is driven by protein kinase A (PKA) phosphorylation. Using site‐directed mutagenesis, we identified serine‐465 as the site conferring PKA‐dependent effects on SK2 channel function.PKA phosphorylation attenuates I SK rectification by reducing the Ca2+/voltage‐dependent inhibition of SK channels without changing their sensitivity to activating submicromolar [Ca2+]i.This mechanism underlies the functional recruitment of SK channels not only in cardiac disease, but also in normal physiology, contributing to repolarization under conditions of enhanced adrenergic drive. Small‐conductance Ca2+‐activated K+ (SK) channels expressed in ventricular myocytes (VMs) are dormant in health, yet become functional in cardiac disease. We aimed to test the hypothesis that post‐translational modification of SK channels under conditions accompanied by enhanced adrenergic drive plays a central role in disease‐related activation of the channels. We investigated this phenomenon using a rat model of hypertrophy induced by thoracic aortic banding (TAB). Western blot analysis using anti‐pan‐serine/threonine antibodies demonstrated enhanced phosphorylation of immunoprecipitated SK2 channels in VMs from TAB rats vs. Shams, which was reversible by incubation of the VMs with PKA inhibitor H89 (1 μmol L–1). Patch clamped VMs under basal conditions from TABs but not Shams exhibited outward current sensitive to the specific SK inhibitor apamin (100 nmol L–1), which was eliminated by inhibition of PKA (1 μmol L–1). Beta‐adrenergic stimulation (isoproterenol, 100 nmol L–1) evoked I SK in VMs from Shams, resulting in shortening of action potentials in VMs and ex vivo optically mapped Sham hearts. Using adenoviral gene transfer, wild‐type and mutant SK2 channels were overexpressed in adult rat VMs, revealing serine‐465 as the site that elicits PKA‐dependent phosphorylation effects on SK2 channel function. Concurrent confocal Ca2+ imaging experiments established that PKA phosphorylation lessens rectification of I SK via reduction Ca2+/voltage‐dependent inhibition of the channels at high [Ca2+] without affecting their sensitivity to activation by Ca2+ in the submicromolar range. In conclusion, upregulation of SK channels in diseased VMs is mediated by hyperadrenergic drive in cardiac hypertrophy, with functional effects on the channel conferred by PKA‐dependent phosphorylation at serine‐465.

Long QT syndrome caveolin-3 mutations differentially modulate Kv 4 and Cav 1.2 channels to contribute to action potential prolongation.

Mutations in the caveolae scaffolding protein, caveolin-3 (Cav3), have been linked to the long QT type 9 inherited arrhythmia syndrome (LQT9) and the cause of underlying action potential duration prolongation is incompletely understood. In the present study, we show that LQT9 Cav3 mutations, F97C and S141R, cause mutation-specific gain of function effects on Cav 1.2-encoded L-type Ca2+ channels responsible for ICa,L and also cause loss of function effects on heterologously expressed Kv 4.2 and Kv 4.3 channels responsible for Ito . A computational model of the human ventricular myocyte action potential suggests that the major ionic current change causing action potential duration prolongation in the presence of Cav3-F97C is the slowly inactivating ICa,L but, for Cav3-S141R, both increased ICa,L and increased late Na+ current contribute equally to action potential duration prolongation. Overall, the LQT9 Cav3-F97C and Cav3-S141R mutations differentially impact multiple ionic currents, highlighting the complexity of Cav3 regulation of cardiac excitability and suggesting mutation-specific therapeutic approaches. Mutations in the CAV3 gene encoding caveolin-3 (Cav3), a scaffolding protein integral to caveolae in cardiomyocytes, have been associated with the congenital long-QT syndrome (LQT9). Initial studies demonstrated that LQT9-associated Cav3 mutations, F97C and S141R, increase late sodium current as a potential mechanism to prolong action potential duration (APD) and cause LQT9. Whether these Cav3 LQT9 mutations impact other caveolae related ion channels remains unknown. We used the whole-cell, patch clamp technique to characterize the effect of Cav3-F97C and Cav3-S141R mutations on heterologously expressed Cav 1.2+Cav β2cN4 channels, as well as Kv 4.2 and Kv 4.3 channels, in HEK 293 cells. Expression of Cav3-S141R increased ICa,L density without changes in gating properties, whereas expression of Cav3-F97C reduced Ca2+ -dependent inactivation of ICa,L without changing current density. The Cav3-F97C mutation reduced current density and altered the kinetics of IKv4.2 and IKv4.3 and also slowed recovery from inactivation. Cav3-S141R decreased current density and also slowed activation kinetics and recovery from inactivation of IKv4.2 but had no effect on IKv4.3 . Using the O'Hara-Rudy computational model of the human ventricular myocyte action potential, the Cav3 mutation-induced changes in Ito are predicted to have negligible effect on APD, whereas blunted Ca2+ -dependent inactivation of ICa,L by Cav3-F97C is predicted to be primarily responsible for APD prolongation, although increased ICa,L and late INa by Cav3-S141R contribute equally to APD prolongation. Thus, LQT9 Cav3-associated mutations, F97C and S141R, produce mutation-specific changes in multiple ionic currents leading to different primary causes of APD prolongation, which suggests the use of mutation-specific therapeutic approaches in the future.

Modelling the effects of quinidine, disopyramide, and E-4031 on short QT syndrome variant 3 in the human ventricles

Objective: Short QT syndrome (SQTS) is an inherited cardiac channelopathy, but at present little information is available on its pharmacological treatment. SQT3 variant (linked to the inward rectifier potassium current IK1) of SQTS, results from a gain-of-function mutation (Kir2.1 D172N) in the KCNJ2-encoded channels, which is associated with ventricular fibrillation (VF). Using biophysically-detailed human ventricular computer models, this study investigated the potential effects of quinidine, disopyramide, and E-4031 on SQT3. Approach: The ten Tusscher et al model of human ventricular myocyte action potential (AP) was modified to recapitulate the changes in IK1 due to heterozygous and homozygous forms of the D172N mutation. Wild-type (WT) and mutant WT-D172N and D172N formulations were incorporated into one-dimensional (1D) and 2D tissue models with transmural heterogeneities. Effects of drugs on channel-blocking activity were modelled using half-maximal inhibitory concentration (IC50) and Hill coefficient (nH) values. Effects of drugs on AP duration (APD), effective refractory period (ERP) and QT interval of pseudo-ECGs were quantified, and both temporal and spatial vulnerability to re-entry was measured. Re-entry was simulated in the 2D ventricular tissue. Main results: At the single cell level, the drugs quinidine, disopyramide, and E-4031 prolonged APD at 90% repolarization (APD90), and decreased maximal transmural voltage heterogeneity (δV); this caused the decreased transmural dispersion of APD90. Quinidine prolonged the QT interval and decreased the T-wave amplitude. Furthermore, quinidine increased ERP and reduced temporal vulnerability and increased spatial vulnerability, resulting in a reduced susceptibility to arrhythmogenesis in SQT3. In the 2D tissue, quinidine was effective in terminating and preventing re-entry associated with the heterozygous D172N condition. Quinidine exhibited significantly better therapeutic effects on SQT3 than disopyramide and E-4031. Significance: This study substantiates a causal link between quinidine and QT interval prolongation in SQT3 Kir2.1 mutations and highlights possible pharmacological agent quinidine for treating SQT3 patients.

Ionic Current Changes during Action Potentials in Porcine Post-MI Heart Failure Model

RATIONALE: Heart failure (HF) following chronic myocardial infarction (MI) is associated with increased susceptibility for cardiac arrhythmias. However, the complex electrophysiological changes causing cardiac arrhythmias remain incompletely understood, especially in large animal models. METHOD: In a porcine model of ischemic HF we used action potential (AP)-clamp and sequential dissection of the major ionic currents during the cardiac AP in ventricular myocytes (from infarct border and remote zones 5 months after MI induction). RESULTS: (1) Compared to healthy controls, remote zone myocytes exhibited increased late sodium current, calcium-activated chloride current and calcium-activated potassium current, decreased rapid delayed rectifier potassium current and also altered sodium/calcium exchange current dynamics. These changes combine to lengthen the AP duration (APD) in the remote zone. (2) In the border zone, the above changes also occurred, but with additional pronounced decreases in L-type calcium current and inward rectifier potassium current, and calcium-activated potassium current was further enhanced to become a major repolarizing current. These additional changes shortened the APD in the border zone. CONCLUSIONS: Ischemic HF involves complex changes in many ionic currents. The changes in each individual current are modest; however, the combined changes in multiple currents integrate to cause APD shortening in the border zone but APD lengthening in the remote zone. Changes in many ionic currents significantly increase the APD dispersion surrounding the infarct to provide a substrate for cardiac arrhythmias.

Effects of magnesium supplementation on electrophysiological remodeling of cardiac myocytes in L-NAME induced hypertensive rats.

Hypertension is one of the major risk factors of cardiac hypertrophy and magnesium deficiency is suggested to be a contributing factor in the progression of this complication. In this study, we aimed to investigate the relationship between intracellular free Mg(2+) levels and electrophysiological changes developed in the myocardium of L-NAME induced hypertensive rats. Hypertension was induced by administration of 40mg/kg of L-NAME for 6weeks, while magnesium treated rats fed with a diet supplemented with 1g/kg of MgO for the same period. L-NAME administration for 6weeks elicited a significant increase in blood pressure which was corrected with MgO treatment; thereby cardiac hypertrophy developing secondary to hypertension was prevented. Cytosolic free magnesium levels of ventricular myocytes were significantly decreased with hypertension and magnesium administration restored these changes. Hypertension significantly decreased the fractional shortening with slowing of shortening kinetics in left ventricular myocytes whereas magnesium treatment was capable of restoring hypertension-induced contractile dysfunction. Long-term magnesium treatment significantly restored the hypertension-induced prolongation in action potentials of ventricular myocytes and suppressed Ito and Iss currents. In contrast, hypertension dependent decrement in intracellular Mg(2+) level did not cause a significant change in L-type Ca(2+) currents, SR Ca(2+) content and NCX activity. Nevertheless, hypertension mediated increase in superoxide anion, hydrogen peroxide and protein oxidation mitigated with magnesium treatment. In conclusion, magnesium administration improves mechanical abnormalities observed in hypertensive rat ventricular myocytes due to reduced oxidative stress. It is likely that, changes in intracellular magnesium balance may contribute to the pathophysiology of chronic heart diseases.

Heart failure duration progressively modulates the arrhythmia substrate through structural and electrical remodeling

AimsVentricular arrhythmias are a common cause of death in patients with heart failure (HF). Structural and electrical abnormalities in the heart provide a substrate for such arrhythmias. Canine tachypacing-induced HF models of 4–6weeks duration are often used to study pathophysiology and therapies for HF. We hypothesized that a chronic canine model of HF would result in greater electrical and structural remodeling than a short term model, leading to a more arrhythmogenic substrate. Main methodsHF was induced by ventricular tachypacing for one (short-term) or four (chronic) months to study remodeling. Key findingsLeft ventricular contractility was progressively reduced, while ventricular hypertrophy and interstitial fibrosis were evident at 4month but not 1month of HF. Left ventricular myocyte action potentials were prolonged after 4 (p<0.05) but not 1month of HF. Repolarization instability and early afterdepolarizations were evident only after 4months of HF (p<0.05), coinciding with a prolonged QTc interval (p<0.05). The transient outward potassium current was reduced in both HF groups (p<0.05). The outward component of the inward rectifier potassium current was reduced only in the 4month HF group (p<0.05). The delayed rectifier potassium currents were reduced in 4 (p<0.05) but not 1month of HF. Reactive oxygen species were increased at both 1 and 4months of HF (p<0.05). SignificanceReduced Ito, outward IK1, IKs, and IKr in HF contribute to EAD formation. Chronic, but not short term canine HF, results in the altered electrophysiology and repolarization instability characteristic of end-stage human HF.

Andersen-Tawil syndrome: a review of its clinical and genetic diagnosis with emphasis on cardiac manifestations

The Andersen-Tawil syndrome is a cardiac ion channel disease that is inherited in an autosomal dominant way and is classified as type 7 of the congenital long QT syndromes. Affected gene is KCNJ2, which forms the inward rectifier potassium channel designated Kir2.1. This protein is involved in stabilizing the resting membrane potential and controls the duration of the action potential in skeletal muscle and heart. It also participates in the terminal repolarization phase of the action potential in ventricular myocytes and is a major component responsible for the correction in the potassium current during phase 3 of the action potential repolarization. Kir 2.1 channel has a predominant role in skeletal muscle, heart and brain. Alterations in this channel produce flaccid paralysis, arrhythmias, impaired skeletal development primarily in extremities and facial area. In this review we address the disease from the point of view of clinical and molecular diagnosis with emphasis on cardiac manifestations.

Measurement and interpretation of electrocardiographic QT intervals in murine hearts

Alterations in ECG QT intervals correlate with the risk of potentially fatal arrhythmias, for which transgenic murine hearts are becoming increasingly useful experimental models. However, QT intervals are poorly defined in murine ECGs. As a consequence, several different techniques have been used to measure murine QT intervals. The present work develops a consistent measure of the murine QT interval that correlates with changes in the duration of ventricular myocyte action potentials (APs). Volume-conducted ECGs were compared with simultaneously recorded APs, obtained using floating intracellular microelectrodes in Langendorff-perfused mouse hearts. QT intervals were measured from the onset of the QRS complex. The interval, Q-APR90, measured to the time at 90% AP recovery, was compared with two measures of the QT interval. QT1 was measured to the recovery of the ECG trace to the isoelectric baseline for entirely positive T-waves or to the trough of any negative T-wave undershoot. QT2—used extensively in previous studies—was measured to the return of any ECG trough to the isoelectric baseline. QT1, but not QT2, closely correlated with changes in Q-APR90. These findings were confirmed over a range of pacing rates, in low K+ concentration solutions, and in Scn5a+/ΔKPQ hearts used to model human long QT syndrome. Application of this method in whole anesthetized mice similarly demonstrated a prolonged corrected QT (QTc) in Scn5a+/ΔKPQ hearts. We therefore describe a robust method for the determination of QT and QTc intervals that correlate with the duration of ventricular myocyte APs in murine hearts.

Profile of L-Type Ca2+ Current and Na+/Ca2+ Exchange Current during Cardiac Action Potential in Ventricular Myocytes

Objective: The L-type Ca2+ current (ICa,L) and the Na+/Ca2+ exchange current (INCX) are major inward currents that shape the cardiac action potential (AP). Previously the profiles of these currents during AP were determined from mathematical models based on data from voltage-clamp experiments that used Ca2+ buffer. In this study we aimed to obtain direct experimental measurement of these currents during cardiac AP with Ca2+ cycling. Method: A newly developed AP-clamp sequential dissection method was used to record ionic currents in guinea pig ventricular myocytes under a triad of conditions: (1) using the cell's own AP as the voltage command, (2) using internal and external solutions that mimic the cell's ionic milieu and, importantly, (3) no exogenous Ca2+ buffer was used. Results: The nifedipine-sensitive current (INIFE), which is composed of ICa,L and INCX, revealed hitherto unreported features during AP with Ca2+ cycling in the cell. We identified two peaks in the current profile followed by a long residual current extending beyond the AP, coinciding with a residual depolarization. The second peak and the residual current become apparent only when Ca2+ is not buffered. Pharmacological dissection of INIFE using SEA0400 shows that ICa,L is dominant during AP phase-1&2 whereas INCX contributes significantly to the inward current at phase-3&4. Conclusion: These data provide the first direct experimental visualization of ICa,L and INCX during cardiac AP and Ca2+ cycle. The residual current reported here can serve as a potential substrate for afterdepolarizations when increased under pathological conditions.

Arrhythmia formation in subclinical (“silent”) long QT syndrome requires multiple insults: Quantitative mechanistic study using the KCNQ1 mutation Q357R as example

In subclinical or silent long QT syndrome, the QT interval is normal under basal conditions. The hypothesis that insults to the repolarization reserve may cause arrhythmias in silent mutation carriers but not in noncarriers has been proposed as a general principle, yet crucial aspects remain descriptive, lacking quantification. To utilize accurate mathematical models of the human action potential and β-adrenergic stimulation to quantitatively investigate arrhythmia-formation mechanisms peculiar to silent long QT syndrome, using mutation Q357R in KCNQ1 (α subunit of slow-delayed rectifier I(Ks)) as a paradigm. Markov models were formulated to account for altered I(Ks) kinetics in Q357R compared with wild type and introduced into a detailed model of the human ventricular myocyte action potential. Dominant negative loss of I(Ks) available reserve accurately represents Q357R. Action potential prolongation with mutant I(Ks) was minimal, reproducing the silent phenotype. Partial block of rapid delayed rectifier current (I(Kr)) was needed in addition to fast pacing and isoproterenol application to cause early afterdepolarizations (EADs) in epicardial cells with mutant I(Ks), but this did not produce EADs in wild type. Reduced channel expression at the membrane, not I(Ks) kinetic differences, caused EADs in the silent mutant. With mutant I(Ks), isoproterenol plus partial I(Kr) block resulted in dramatic QT prolongation in the pseudo-electrocardiogram and EADs formed without I(Kr) block in mid-myocardial cells during simulated exercise onset. Multiple severe insults are needed to evince an arrhythmic phenotype in silent mutation Q357R. Reduced membrane I(Ks) expression, not kinetic changes, underlies the arrhythmic phenotype.

Profile of L-type Ca2+ current and Na+/Ca2+ exchange current during cardiac action potential in ventricular myocytes

The L-type Ca(2+) current (I(Ca,L)) and the Na(+)/Ca(2+) exchange current (I(NCX)) are major inward currents that shape the cardiac action potential (AP). Previously, the profile of these currents during the AP was determined from voltage-clamp experiments that used Ca(2+) buffer. In this study, we aimed to obtain direct experimental measurement of these currents during cardiac AP with Ca(2+) cycling. A newly developed AP-clamp sequential dissection method was used to record ionic currents in guinea pig ventricular myocytes under a triad of conditions: using the cell's own AP as the voltage command, using internal and external solutions that mimic the cell's ionic composition, and, importantly, not using any exogenous Ca(2+) buffer. The nifedipine-sensitive current (I(NIFE)), which is composed of I(Ca,L) and I(NCX), revealed hitherto unreported features during the AP with Ca(2+) cycling in the cell. We identified 2 peaks in the current profile followed by a long residual current extending beyond the AP, coinciding with a residual depolarization. The second peak and the residual current become apparent only when Ca(2+) is not buffered. Pharmacological dissection of I(NIFE) by using SEA0400 shows that I(Ca,L) is dominant during phases 1 and 2 whereas I(NCX) contributes significantly to the inward current during phases 3 and 4 of the AP. These data provide the first direct experimental visualization of I(Ca,L) and I(NCX) during cardiac the AP and Ca(2+) cycle. The residual current reported here can serve as a potential substrate for afterdepolarizations when increased under pathologic conditions.

Puerarin: a novel antagonist to inward rectifier potassium channel (I K1)

Puerarin, isolated from the root of pueraria, had been widely used to prevent and treat arrhythmia. We show that puerarin effectively prevents and reverses aconitine-induced arrhythmias in perfused heart in vitro and in rats in vivo. To study the mechanisms of antiarrhythmic action of puerarin, we investigated the electrophysiological actions of puerarin using whole-cell clamp in isolated rodent ventricular myocytes and two electrode voltage-clamp (TEV) in I(K1)-expressing Xenopus oocytes. Puerarin had no prominent effect on action potentials of ventricular myocytes from guinea pig. However, puerarin (1.2 mM) significantly inhibited the I(K1) current in rat ventricular cells. Consistently, puerarin blocked I(K1) expressed in Xenopus oocytes in a dose-dependent manner. Puerarin competed with barium, an open-channel blocker of I(K1), to inhibit I(K1) currents. Thus, our data demonstrated that puerarin is a novel open-channel blocker of I(K1), which may underlie the antiarrhythmic action of puerarin.

Inhibition of HCN Overexpressed in Failing Heart of Dilated Cardiomyopathy Mouse Model by Ivabradine Prevents Sudden Arrhythmic Death

Growing evidence demonstrates that the hyperpolarization-activated cyclic nucleotide gated channels (HCNs) is overexpressed in failing hearts and potentially involved in increased arrhythmogenicity. Inhibiting HCN channels could be a promising approach to preventing lethal arrhythmias associated with heart failure. Transgenic mice expressing a dominant-negative mutant of neuron-restrictive silencer factor specifically in the heart (dnNRSF-Tg) exhibit dilated cardiomyopathy and sudden arrhythmic death, accompanied with the increased ventricular HCNs expression. We examined the effects of ivabradine (Iva, 7 mg/kg/d), a specific HCN channel inhibitor on survival and arrhythmogenicity in dnNRSF-Tg and found that Iva significantly improved the survival among dnNRSF-Tg. Iva significantly reduced ventricular arrhythmias in dnNRSF-Tg in ECG telemetry analysis and isoproterenol-induced increase in spontaneous action potentials in ventricular myocytes from dnNRSF-Tg, suggesting that Iva improved the survival by preventing lethal arrhythmias. We also found that transgenic mice overexpressing HCN2 in the heart are highly susceptible to arrhythmias induced by chronic isoproterenol infusion. Our findings demonstrate the contribution of increased ventricular expression of HCNs to the increased arrhythmogenicity and define HCN inhibition by Iva as an useful therapeutic approach to preventing lethal arrhythmias.

Not All hERG Pore Domain Mutations Have a Severe Phenotype: G584S Has an Inactivation Gating Defect with Mild Phenotype Compared to G572S, Which Has a Dominant Negative Trafficking Defect and a Severe Phenotype

Mutations in the pore domain of the human ether-a-go-go-related gene (hERG) potassium channel are associated with higher risk of sudden death. However, in many kindreds clinical presentation is variable, making it hard to predict risk. We hypothesized that in vitro phenotyping of the intrinsic severity of individual mutations can assist with risk stratification. We analyzed 2 hERG pore domain mutations, G572S and G584S. Similar to 90% of hERG missense mutations, G572S-hERG subunits did not traffic to the plasma membrane but could coassemble with WT subunits and resulted in a dominant negative suppression of hERG current density. The G584S-hERG subunits traffic normally but have abnormal inactivation gating. Computer models of human ventricular myocyte action potentials (AP), incorporating Markov models of the hERG mutants, indicate that G572S-hERG channels would cause more severe AP prolongation than that seen with G584S-hERG channels. hERG-G572S and -G584S are 2 pore domain mutations that involve the same change in sidechain but have very different in vitro phenotypes; G572S causes a dominant negative trafficking defect, whereas G584S is the first hERG missense mutation where the cause of disease can be exclusively attributed to enhanced inactivation. The G572S mutation is intrinsically more severe than the G584S mutation, consistent with the overall clinical presentation in the 2 small kindreds studied here. Further investigation, involving a larger number of cohorts, to test the hypothesis that in vitro phenotyping of the intrinsic severity of a given mutation will assist with risk stratification is therefore warranted.

1-[1-Hexyl-6-(methyloxy)-1H-indazol-3-yl]-2-methyl-1-propanone, a Potent and Highly Selective Small Molecule Blocker of the Large-Conductance Voltage-Gated and Calcium-Dependent K+Channel

The large-conductance voltage-gated and calcium-dependent K(+) (BK) channels are widely distributed and play important physiological roles. Commonly used BK channel inhibitors are peptide toxins that are isolated from scorpion venoms. A high-affinity, nonpeptide, synthesized BK channel blocker with selectivity against other ion channels has not been reported. We prepared several compounds from a published patent application (Doherty et al., 2004) and identified 1-[1-hexyl-6-(methyloxy)-1H-indazol-3-yl]-2-methyl-1-propanone (HMIMP) as a potent and selective BK channel blocker. The patch-clamp technique was used for characterizing the activity of HMIMP on recombinant human BK channels (alpha subunit, alpha+beta1 and alpha+beta4 subunits). HMIMP blocked all of these channels with an IC(50) of approximately 2 nM. The inhibitory effect of HMIMP was not voltage-dependent, nor did it require opening of BK channels. HMIMP also potently blocked BK channels in freshly isolated detrusor smooth muscle cells and vagal neurons. HMIMP (10 nM) reduced the open probability significantly without affecting single BK-channel current in inside-out patches. HMIMP did not change the time constant of open states but increased the time constants of the closed states. More importantly, HMIMP was highly selective for the BK channel. HMIMP had no effect on human Na(V)1.5 (1 microM), Ca(V)3.2, L-type Ca(2+), human ether-a-go-go-related gene potassium channel, KCNQ1+minK, transient outward K(+) or voltage-dependent K(+) channels (100 nM). HMIMP did not change the action potentials of ventricular myocytes, confirming its lack of effect on cardiac ion channels. In summary, HMIMP is a highly potent and selective BK channel blocker, which can serve as an important tool in the pharmacological study of the BK channel.