Guinea Pig Ventricular Myocytes Research Articles

New drug discovery of cardiac anti-arrhythmic drugs: insights in animal models

Cardiac rhythm regulated by micro-macroscopic structures of heart. Pacemaker abnormalities or disruptions in electrical conduction, lead to arrhythmic disorders may be benign, typical, threatening, ultimately fatal, occurs in clinical practice, patients on digitalis, anaesthesia or acute myocardial infarction. Both traditional and genetic animal models are: In-vitro: Isolated ventricular Myocytes, Guinea pig papillary muscles, Patch-Clamp Experiments, Porcine Atrial Myocytes, Guinea pig ventricular myocytes, Guinea pig papillary muscle: action potential and refractory period, Langendorff technique, Arrhythmia by acetylcholine or potassium. Acquired arrhythmia disorders: Transverse Aortic Constriction, Myocardial Ischemia, Complete Heart Block and AV Node Ablation, Chronic Tachypacing, Inflammation, Metabolic and Drug-Induced Arrhythmia. In-Vivo: Chemically induced arrhythmia: Aconitine antagonism, Digoxin-induced arrhythmia, Strophanthin/ouabain-induced arrhythmia, Adrenaline-induced arrhythmia, and Calcium-induced arrhythmia. Electrically induced arrhythmia: Ventricular fibrillation electrical threshold, Arrhythmia through programmed electrical stimulation, sudden coronary death in dogs, Exercise ventricular fibrillation. Genetic Arrhythmia: Channelopathies, Calcium Release Deficiency Syndrome, Long QT Syndrome, Short QT Syndrome, Brugada Syndrome. Genetic with Structural Heart Disease: Arrhythmogenic Right Ventricular Cardiomyopathy/Dysplasia, Dilated Cardiomyopathy, Hypertrophic Cardiomyopathy, Atrial Fibrillation, Sick Sinus Syndrome, Atrioventricular Block, Preexcitation Syndrome. Arrhythmia in Pluripotent Stem Cell Cardiomyocytes. Conclusion: Both traditional and genetic, experimental models of cardiac arrhythmias’ characteristics and significance help in development of new antiarrhythmic drugs.

Extracellular Perinexal Separation Is a Principal Determinant of Cardiac Conduction.

Cardiac conduction is understood to occur through gap junctions. Recent evidence supports ephaptic coupling as another mechanism of electrical communication in the heart. Conduction via gap junctions predicts a direct relationship between conduction velocity (CV) and bulk extracellular resistance. By contrast, ephaptic theory is premised on the existence of a biphasic relationship between CV and the volume of specialized extracellular clefts within intercalated discs such as the perinexus. Our objective was to determine the relationship between ventricular CV and structural changes to micro- and nanoscale extracellular spaces. Conduction and Cx43 (connexin43) protein expression were quantified from optically mapped guinea pig whole-heart preparations perfused with the osmotic agents albumin, mannitol, dextran 70 kDa, or dextran 2 MDa. Peak sodium current was quantified in isolated guinea pig ventricular myocytes. Extracellular resistance was quantified by impedance spectroscopy. Intercellular communication was assessed in a heterologous expression system with fluorescence recovery after photobleaching. Perinexal width was quantified from transmission electron micrographs. CV primarily in the transverse direction of propagation was significantly reduced by mannitol and increased by albumin and both dextrans. The combination of albumin and dextran 70 kDa decreased CV relative to albumin alone. Extracellular resistance was reduced by mannitol, unchanged by albumin, and increased by both dextrans. Cx43 expression and conductance and peak sodium currents were not significantly altered by the osmotic agents. In response to osmotic agents, perinexal width, in order of narrowest to widest, was albumin with dextran 70 kDa; albumin or dextran 2 MDa; dextran 70 kDa or no osmotic agent, and mannitol. When compared in the same order, CV was biphasically related to perinexal width. Cardiac conduction does not correlate with extracellular resistance but is biphasically related to perinexal separation, providing evidence that the relationship between CV and extracellular volume is determined by ephaptic mechanisms under conditions of normal gap junctional coupling.

Investigations on Na+, K+-ATPase energy consumption in ion flow of hydrophilic pores by THz unipolar stimulation

Terahertz science and technology has recently shown new application prospects in artificial intelligence. It is found that terahertz unipolar stimulation can activate cell membrane hydrophilic pores. However, the behaviors of Na+, K+-ATPase and energy consumption during this period remain unknown. This paper investigates these behaviors by Na+, K+-ATPase and electroporation models, based on the interaction theory between terahertz fields and ions at the cellular level. The effective diameters of life ions are considered in the aqueous solution. From results, Na+, K+-ATPases can be activated and stay for a while before close after the stimulation. Their life ion flows are far lower than the flows via the pores. And their power dissipation is as low as 10-11 W in both rat neostriatal neurons and guinea pig ventricular myocytes. The results keep tenable in 0.1-1.2 THz. These lay the basis for investigations of information communication mechanisms in cells under terahertz stimulation.

Conductance Changes of Na+ Channels during the Late Na+ Current Flowing under Action Potential Voltage Clamp Conditions in Canine, Rabbit, and Guinea Pig Ventricular Myocytes.

Late sodium current (INa,late) is an important inward current contributing to the plateau phase of the action potential (AP) in the mammalian heart. Although INa,late is considered as a possible target for antiarrhythmic agents, several aspects of this current remained hidden. In this work, the profile of INa,late, together with the respective conductance changes (GNa,late), were studied and compared in rabbit, canine, and guinea pig ventricular myocytes using the action potential voltage clamp (APVC) technique. In canine and rabbit myocytes, the density of INa,late was relatively stable during the plateau and decreased only along terminal repolarization of the AP, while GNa,late decreased monotonically. In contrast, INa,late increased monotonically, while GNa,late remained largely unchanged during the AP in guinea pig. The estimated slow inactivation of Na+ channels was much slower in guinea pig than in canine or rabbit myocytes. The characteristics of canine INa,late and GNa,late were not altered by using command APs recorded from rabbit or guinea pig myocytes, indicating that the different shapes of the current profiles are related to genuine interspecies differences in the gating of INa,late. Both INa,late and GNa,late decreased in canine myocytes when the intracellular Ca2+ concentration was reduced either by the extracellular application of 1 µM nisoldipine or by the intracellular application of BAPTA. Finally, a comparison of the INa,late and GNa,late profiles induced by the toxin of Anemonia sulcata (ATX-II) in canine and guinea pig myocytes revealed profound differences between the two species: in dog, the ATX-II induced INa,late and GNa,late showed kinetics similar to those observed with the native current, while in guinea pig, the ATX-II induced GNa,late increased during the AP. Our results show that there are notable interspecies differences in the gating kinetics of INa,late that cannot be explained by differences in AP morphology. These differences must be considered when interpreting the INa,late results obtained in guinea pig.

Mechanistic insights into spontaneous transition from cellular alternans to ventricular fibrillation.

T-wave alternans (TWA) has been used for predicting the risk of malignant cardiac arrhythmias and sudden cardiac death (SCD) in multiple clinical settings; however, possible mechanism(s) underlying the spontaneous transition from cellular alternans reflected by TWA to arrhythmias in impaired repolarization remains unclear. The healthy guinea pig ventricular myocytes under E-4031 blocking IKr (0.1μM, N=12; 0.3μM, N=10; 1μM, N=10) were evaluated using whole-cell patch-clamp. The electrophysiological properties of isolated perfused guinea pig hearts under E-4031 (0.1μM, N=5; 0.3μM, N=5; 1μM, N=5) were evaluated using dual- optical mapping. The amplitude/threshold/restitution curves of action potential duration (APD) alternans and potential mechanism(s) underlying the spontaneous transition of cellular alternans to ventricular fibrillation (VF) were examined. There were longer APD80 and increased amplitude and threshold of APD alternans in E-4031 group compared with baseline group, which was reflected by more pronounced arrhythmogenesis at the tissue level, and were associated with steep restitution curves of the APD and the conduction velocity (CV). Conduction of AP alternans augmented tissue's functional spatiotemporal heterogeneity of regional AP/Ca alternans, as well as the AP/Ca dispersion, leading to localized uni-directional conduction block that spontaneous facilitated the formation of reentrant excitation waves without the need for additional premature stimulus. Our results provide a possible mechanism for the spontaneous transition from cardiac electrical alternans in cellular action potentials and intercellular conduction without the involvement of premature excitations, and explain the increased susceptibility to ventricular arrhythmias in impaired repolarization. In this study, we implemented voltage-clamp and dual-optical mapping approaches to investigate the underlying mechanism(s) for the arrhythmogenesis of cardiac alternans in the guinea pig heart at cellular and tissue levels. Our results demonstrated a spontaneous development of reentry from cellular alternans, arising from a combined actions of restitution properties of action potential duration, conduction velocity of excitation wave and interplay between alternants of action potential and the intracellular Ca handling. We believe this study provides new insights into underlying the mechanism, by which cellular cardiac alternans spontaneously evolves into cardiac arrhythmias.

Review on Sodium Calcium Exchanger (Na+-Ca2+X) Electrophysiological Characteristics and its Role in Cardiac Arrhythmias

Cardiac arrhythmias is one of the cardiovascular diseases that cause more death in today’s industrial world. Among the generation of arrhythmias in cardiac myocytes, Early Afterdepolarization (EAD) and Delayed Afterdepolarization (DAD) is most significant one. Sodium -Calcium Exchanger (Na+- Ca2+ X) dysfunction is one of major source of DAD and EAD. Na+- Ca2+ X which exact mechanism is responsible for promoting EAD and DAD still not derived clearly from any cardiac models investigation. It is very important to understand the electrophysiological characteristics of Na+- Ca2+ X and its role in cardiac arrhythmias to clearly detect such arrhythmias effects. In this research article the aim is to build a strong fundamental foundation of Na+- Ca2+ X mechanism in terms of electrophysiological molecular segment (internal structure) arrangements and various Na+- Ca2+ X investigations on cardiac arrhythmias. Na+-Ca2+X is one which belongs to low chemical attraction, high transport capacity and energetically driven by the Na+ ions. The second is an L-type Ca2+ channel, which has a high affinity to Ca2+. Na+-Ca2+X eject nearly thirty (30%) of Ca2+ is required to activate the myofilaments in rabbit, guinea pig and human ventricles and a very small amount (7%) of Ca2+ is required in rat and mouse ventricles. Na+-Ca2+X transport Ca2+ up to 5000 per second (≈6μM/L), which is much greater than SR Ca2+ ATP Ca2+ transport (100 to 150 per second at ≈0.3μM/L). By using voltage patch clamp, Na+-Ca2+X current is measured in guinea pig ventricular myocyte, from that density of Na+-Ca2+X is to be found, 250 to 400 Na+-Ca2+X/μm2 . Na+-Ca2+X current (INaCa) at level [Ca2+]o = 0.50mM, Na+-Ca2+X mechanism of Ca2+ ions removal is very quickly, why because Calcium Induced Calcium Release (CICR) process does increase the cytosolic Ca2+ concentration very early. Enhanced activity of Na+-Ca2+X or increased Na+-Ca2+X channel current reduces the Action Potential (AP) and AP Duration (APD), and triggers rapid firing within cardiac pulmonary veins. Minimum intracellular calcium levels in SR is responsible for systolic dysfunction in failure heart. Similarly it increases the inward Na+-Ca2+X current, life threatening arrhythmias like DAD happen by sudden release of calcium in SR. Finally increased or enhanced or hypertrophy conditions of Na+-Ca2+X is produces EAD and DAD.

Pacing Dynamics Determines the Arrhythmogenic Mechanism of the CPVT2-Causing CASQ2G112+5X Mutation in a Guinea Pig Ventricular Myocyte Computational Model.

Calsequestrin Type 2 (CASQ2) is a high-capacity, low-affinity, Ca2+-binding protein expressed in the sarcoplasmic reticulum (SR) of the cardiac myocyte. Mutations in CASQ2 have been linked to the arrhythmia catecholaminergic polymorphic ventricular tachycardia (CPVT2) that occurs with acute emotional stress or exercise can result in sudden cardiac death (SCD). CASQ2G112+5X is a 16 bp (339-354) deletion CASQ2 mutation that prevents the protein expression due to premature stop codon. Understanding the subcellular mechanisms of CPVT2 is experimentally challenging because the occurrence of arrhythmia is rare. To obtain an insight into the characteristics of this rare disease, simulation studies using a local control stochastic computational model of the Guinea pig ventricular myocyte investigated how the mutant CASQ2s may be responsible for the development of an arrhythmogenic episode under the condition of β-adrenergic stimulation or in the slowing of heart rate afterward once β-adrenergic stimulation ceases. Adjustment of the computational model parameters based upon recent experiments explore the functional changes caused by the CASQ2 mutation. In the simulation studies under rapid pacing (6 Hz), electromechanically concordant cellular alternans appeared under β-adrenergic stimulation in the CPVT mutant but not in the wild-type nor in the non-β-stimulated mutant. Similarly, the simulations of accelerating pacing from slow to rapid and back to the slow pacing did not display alternans but did generate early afterdepolarizations (EADs) during the period of second slow pacing subsequent acceleration of rapid pacing.

The Role of Ca2+ Sparks in Force Frequency Relationships in Guinea Pig Ventricular Myocytes.

Calcium sparks are the elementary Ca2+ release events in excitation-contraction coupling that underlie the Ca2+ transient. The frequency-dependent contractile force generated by cardiac myocytes depends upon the characteristics of the Ca2+ transients. A stochastic computational local control model of a guinea pig ventricular cardiomyocyte was developed, to gain insight into mechanisms of force-frequency relationship (FFR). This required the creation of a new three-state RyR2 model that reproduced the adaptive behavior of RyR2, in which the RyR2 channels transition into a different state when exposed to prolonged elevated subspace [Ca2+]. The model simulations agree with previous experimental and modeling studies on interval-force relations. Unlike previous common pool models, this local control model displayed stable action potential trains at 7 Hz. The duration and the amplitude of the [Ca2+]myo transients increase in pacing rates consistent with the experiments. The [Ca2+]myo transient reaches its peak value at 4 Hz and decreases afterward, consistent with experimental force-frequency curves. The model predicts, in agreement with previous modeling studies of Jafri and co-workers, diastolic sarcoplasmic reticulum, [Ca2+]sr, and RyR2 adaptation increase with the increased stimulation frequency, producing rising, rather than falling, amplitude of the myoplasmic [Ca2+] transients. However, the local control model also suggests that the reduction of the L-type Ca2+ current, with an increase in pacing frequency due to Ca2+-dependent inactivation, also plays a role in the negative slope of the FFR. In the simulations, the peak Ca2+ transient in the FFR correlated with the highest numbers of SR Ca2+ sparks: the larger average amplitudes of those sparks, and the longer duration of the Ca2+ sparks.

Modulation of IKs channel-PIP2 interaction by PRMT1 plays a critical role in the control of cardiac repolarization.

Recent studies have shown that protein arginine methyltransferase 1 (PRMT1) is highly expressed in the human heart, and loss of PRMT1 contributes to cardiac remodeling in the heart failure. However, the functional importance of PRMT1 in cardiac ion channels remains uncertain. The slow activating delayed rectifier K+ (I Ks) channel is a cardiac K+ channel composed of KCNQ1 and KCNE1 subunits and is a new therapeutic target for treating lethal arrhythmias in many cardiac pathologies, especially heart failure. Here, we demonstrate that PRMT1 is a critical regulator of the I Ks channel and cardiac rhythm. In the guinea pig ventricular myocytes, treatment with furamidine, a PRMT1‐specific inhibitor, prolonged the action potential duration (APD). We further show that this APD prolongation was attributable to I Ks reduction. In HEK293T cells expressing human KCNQ1 and KCNE1, inhibiting PRMT1 via furamidine reduced I Ks and concurrently decreased the arginine methylation of KCNQ1, a pore‐forming α‐subunit. Evidence presented here indicates that furamidine decreased I Ks mainly by lowering the affinity of I Ks channels for the membrane phospholipid, phosphatidylinositol 4,5‐bisphosphate (PIP2), which is crucial for pore opening. Finally, applying exogenous PIP2 to cardiomyocytes prevented the furamidine‐induced I Ks reduction and APD prolongation. Taken together, these results indicate that PRMT1 positively regulated I Ks activity through channel–PIP2 interaction, thereby restricting excessive cardiac action potential.

Macrophage-Dependent Interleukin-6-Production and Inhibition of IK Contributes to Acquired QT Prolongation in Lipotoxic Guinea Pig Heart.

In the heart, the delayed rectifier K current, IK, composed of the rapid (IKr) and slow (IKs) components contributes prominently to normal cardiac repolarization. In lipotoxicity, chronic elevation of pro-inflammatory cytokines may remodel IK, elevating the risk for ventricular arrythmias and sudden cardiac death. We investigated whether and how the pro-inflammatory interleukin-6 altered IK in the heart, using electrophysiology to evaluate changes in IK in adult guinea pig ventricular myocytes. We found that palmitic acid (a potent inducer of lipotoxicity), induced a rapid (~24 h) and significant increase in IL-6 in RAW264.7 cells. PA-diet fed guinea pigs displayed a severely prolonged QT interval when compared to low-fat diet fed controls. Exposure to isoproterenol induced torsade de pointes, and ventricular fibrillation in lipotoxic guinea pigs. Pre-exposure to IL-6 with the soluble IL-6 receptor produced a profound depression of IKr and IKs densities, prolonged action potential duration, and impaired mitochondrial ATP production. Only with the inhibition of IKr did a proarrhythmic phenotype of IKs depression emerge, manifested as a further prolongation of action potential duration and QT interval. Our data offer unique mechanistic insights with implications for pathological QT interval in patients and vulnerability to fatal arrhythmias.

Mechanisms of gefitinib-induced QT prolongation

Gefitinib, a tyrosine kinase inhibitor, was the first targeted therapy for non-small cell lung cancer (NSCLC). Gefitinib could block human Ether-à-go-go-Related Gene (hERG) channel, an important target in drug-induced long QT syndrome. However, it is unclear whether gefitinib could induce QT interval prolongation. Here, whole-cell patch-clamp technique was used for evaluating the effect of gefitinib on rapidly-activating delayed rectifier K+ current (IKr), slowly-activating delayed rectifier K+ current (IKs), transient outward potassium current (Ito), inward rectifier K+ current (IK1) and on action potentials in guinea pig ventricular myocytes. The Langendorff heart perfusion technique was used to determine drug effect on the ECG. Gefitinib depressed IKr by binding to open and closed hERG channels in a concentration-dependent way (IC50: 1.91 μM). The inhibitory effect of gefitinib on wildtype hERG channels was reduced at the hERG mutants Y652A, S636A, F656V and S631A (IC50: 8.51, 13.97, 18.86, 32.99 μM), indicating that gefitinib is a pore inhibitor of hERG channels. In addition, gefitinib accelerated hERG channel inactivation and decreased channel steady-state inactivation. Gefitinib also decreased IKs with IC50 of 23.8 μM. Moreover, gefitinib increased action potential duration (APD) in guinea pig ventricular myocytes and the corrected QT interval (QTc) in isolated perfused guinea pig hearts in a concentration-dependent way (1–30 μM). These findings indicate that gefitinib could prolong QTc interval by potently blocking hERG channel, modulating kinetic properties of hERG channel. Partial block of KCNQ1/KCNE1 could also contribute to delayed repolarization and prolonged QT interval. Thus, caution should be taken when gefitinib is used for NSCLC treatment.

B-PO04-012 ACUTE TESTOSTERONE ADMINISTRATION DOES NOT SHORTEN ACTION POTENTIAL DURATION IN LANGENDORFF PERFUSED RABBIT VENTRICLES

A previous study showed that testosterone (T) rapidly shortens the action potential duration (APD) with EC50=2.1 to 8.7 nmol/L in guinea pig ventricular myocytes due to non-transcriptional enhancement of IKs and suppression of ICa,L. These findings have not been reproduced in intact rabbit ventricles. To test the hypothesis that T and dihydroxytestosterone (D) acutely shorten epicardial APD in rabbit ventricles. We performed optical mapping studies in 6 female and 7 male Langendorff-perfused rabbit hearts. Blebbistatin (10-20 μM) was used to suppress motion. Right atrium was paced during mapping. After baseline recording, mapping was done with T (N=10) or D (N=3). In T group, either high (3 μM) or low (1 to 100 nM) doses were applied to 5 and 4 hearts respectively, and 1 received both low and high doses. In D group, 1 received only low (3 to 100 nM), and 2 received low and then high dose (1 μM). In one heart we determined the QT interval recorded with widely spaced bipolar electrodes by using Tyrode’s solution only, without optical dye (RH237) or blebbistatin. At pacing cycle length (PCL) 350 ms, 300 ms, and 250 ms, there were no significant differences of APD (ms) at baseline vs high dose T (182 ± 15 vs 186 ± 5, p=0.73; 160 ± 24 vs 159 ± 20, p=0.95, 152 ± 23 vs 149 ± 15 ms, p=0.71, respectively). High-dose D tends to lengthen APD (ms) in 2 hearts at PCL 350 ms (161 vs 179 and 143 vs 153), 300 ms (152 vs 167 and 139 vs 148), and 250 ms (139 vs 158 and 129 vs 137). APDs (ms) at baseline vs low-dose T were (144 ± 9 vs 147 ± 8 at 1nM, p=0.23 and 150 ± 8 at 10 nM, p=0.05). APDs at baseline vs low dose D were 146 ± 9 vs 153 ± 10 at 3 nM (p=0.34), 162 ± 15 at 10 nM (p=0.34), and 160 ± 11 at 100 nM (p=0.19) at PCL 300 ms. APD significantly lengthened after 100 nM of T (154 ± 12 ms at 100 nM, p=0.01). In one rabbit heart without RH237 or blebbistatin, QT interval (ms) at PCL 280 ms did not shorten after T (170 at baseline, 172 at 10 nM, and 176 at 100 nM). When separately analyzed, neither male nor female hearts showed APD shortening with T or D. T and D do not shorten and may lengthen ventricular repolarization in normal male or female rabbit hearts. These findings do not support non-transcriptional regulation of cardiac ion currents by T or D to shorten APD.

Monensin-Induced Increase in Intracellular Na+ Induces Changes in Na+ and Ca2+ Currents and Regulates Na+-K+ and Na+-Ca2+ Transport in Cardiomyocytes

Background/Aims: Monensin, an Na ionophore, increases intracellular Na ([Na]i). Alteration of [Na]i influences ion transport through the sarcolemmal membrane. So far, the effects of monensin on ventricular myocytes have not been examined in detail. The main objective of this study was to elucidate the mechanism via which monensin-evoked increases in [Na]i affect the membrane potential and currents in ventricular myocytes of guinea pigs. Methods: Membrane potentials and currents were measured using the whole-cell patch-clamp technique in single myocytes. The concentration of intracellular Ca ([Ca]i) was evaluated by measuring fluorescence intensity of Fluo-4. Results: Monensin (10⁻⁵M) shortened the action potential duration (APD) and reduced the amplitude of the plateau phase. In addition, monensin decreased the sodium current (I_Na) and shifted the inactivation curve to the hyperpolarized direction. Moreover, it decreased the L-type calcium current (I_Ca). However, this effect was attenuated by increasing the buffering capacity of [Ca]i. The Na-Ca exchange current (I_Na-Ca) was activated particularly in the reverse mode. Na-K pump current (I_Na-K) was also activated. Notably, the inward rectifying K current (I_K1) was not affected, and the change in the delayed outward K current (I_K) was not evident. Conclusion: These results suggest that the monensin-induced shortened APD and reduced amplitude of the plateau phase are primarily due to the decrease in the I_Ca, the activation of the reverse mode of I_Na-Ca, and the increased I_Na-K, and second due to the decreased I_Na. The I_K and the I_K1 may not be associated with the abovementioned changes induced by monensin. The elevation of [Na]i can exert multiple influences on electrophysiological phenomena in cardiac myocytes.

Inhibition of the INa/K and the activation of peak INa contribute to the arrhythmogenic effects of aconitine and mesaconitine in guinea pigs.

Aconitine (ACO), a main active ingredient of Aconitum, is well-known for its cardiotoxicity. However, the mechanisms of toxic action of ACO remain unclear. In the current study, we investigated the cardiac effects of ACO and mesaconitine (MACO), a structurally related analog of ACO identified in Aconitum with undocumented cardiotoxicity in guinea pigs. We showed that intravenous administration of ACO or MACO (25 μg/kg) to guinea pigs caused various types of arrhythmias in electrocardiogram (ECG) recording, including ventricular premature beats (VPB), atrioventricular blockade (AVB), ventricular tachycardia (VT), and ventricular fibrillation (VF). MACO displayed more potent arrhythmogenic effect than ACO. We conducted whole-cell patch-clamp recording in isolated guinea pig ventricular myocytes, and observed that treatment with ACO (0.3, 3 μM) or MACO (0.1, 0.3 μM) depolarized the resting membrane potential (RMP) and reduced the action potential amplitude (APA) and durations (APDs) in a concentration-dependent manner. The ACO- and MACO-induced AP remodeling was largely abolished by an INa blocker tetrodotoxin (2 μM) and partly abolished by a specific Na+/K+ pump (NKP) blocker ouabain (0.1 μM). Furthermore, we observed that treatment with ACO or MACO attenuated NKP current (INa/K) and increased peak INa by accelerating the sodium channel activation with the EC50 of 8.36 ± 1.89 and 1.33 ± 0.16 μM, respectively. Incubation of ventricular myocytes with ACO or MACO concentration-dependently increased intracellular Na+ and Ca2+ concentrations. In conclusion, the current study demonstrates strong arrhythmogenic effects of ACO and MACO resulted from increasing the peak INa via accelerating sodium channel activation and inhibiting the INa/K. These results may help to improve our understanding of cardiotoxic mechanisms of ACO and MACO, and identify potential novel therapeutic targets for Aconitum poisoning.

Slow-Rapid-Slow Pacing in the Heart Having CASQ2G112+5X Gene Mutation Produces Eads as the Mechanism of CPVT During Adrenergic Stimulation

Clinical studies have observed in some patients of catecholaminergic polymorphic ventricular tachycardia (CPVT), arrhythmia occurs during post-exercise. A stochastic computational model of the Guinea pig ventricular myocyte EC-coupling was developed to probe the mechanism causing this phenomenon using data from the CASQ2G112+5x mutation. CASQ2G112+5X is a homozygous deletion mutation and like many CPVT2-causing mutations, it increases RyR open probability through modulation of luminal dependency on Ca2+. It causes disruption of CASQ2 polymerization in protein expression. The 16 bp deletion c.339-354 causes a frameshift to generate premature stop-codon after the removal of 5 amino-acids. Because of this mutation SR has almost total loss of CASQ2 buffer and to upset that loss an increase in SR volume takes place. To replicate these features in model, we increased 50% SR volume and reduced 95% SR buffer. β-adrenergic stimulation was modeled by raising LCC (40%), Ca2+ luminal dependence (90%) and SERCA activities (50%). The simulations suggest under rapid pacing (6 Hz) cellular alternans occurs under β-adrenergic stimulation in the CPVT mutant, but not in the wild-type and unstimulated mutant. Simulations accelerating pacing from 1 Hz to 5 Hz and back to 1 Hz didn’t display cellular alternans but did generate early afterdepolarizations (EADs) during the period of 1 Hz pacing after the acceleration to 5 Hz. The model suggests that the rapid pacing loads the cytosol and sarcoplasmic reticulum with Ca2+, which in CPVT mutant with increased RyR open probability can trigger spontaneous Ca2+ release which activates Na+-Ca2+ exchange resulting in AP prolongation. These studies suggest that Ca2+-overload is the underlying arrhythmic mechanism in these patients.

Prevailing Effects of Ibutilide on Fast Delayed Rectifier K+ Channel.

Effects of ibutilide, a class III antiarrhythmic drug, on delayed rectifier potassium currents (IK) in freshly isolated guinea pig ventricular myocytes were studied. Experiments were performed using the whole-cell configuration of patch-clamp technique under blockade of L-type calcium currents (Cav1). Ibutilide at concentrations ranging between 10 nM and 100µM inhibited IKr in dose-dependent manner with a half maximal effective concentration of 2.03 ± 0.74µM (n = 5-10). The amplitude of tail currents activated by prepulse to + 20mV was decreased from 253 ± 52 to 130 ± 25 pA (n = 8, p < 0.01) in the presence of 1µM ibutilide. The envelope test revealed time-dependent changes in ratio of IK-tail/ΔIK during 0.2-2s pulse durations in the absence of drug. With ibutilide, regardless of pulse duration, a relatively constant ratio was estimated, indicative of predominant involvement of IKr component. The slow IKs persisted to greater extent even at 100 μM ibutilide revealing a distinguishable selectivity toward the IKr component.

Potent hERG channel inhibition by sarizotan, an investigative treatment for Rett Syndrome

Rett Syndrome (RTT) is an X-linked neurodevelopmental disorder associated with respiratory abnormalities and, in up to ~40% of patients, with prolongation of the cardiac QTc interval. QTc prolongation calls for cautious use of drugs with a propensity to inhibit hERG channels. The STARS trial has been undertaken to investigate the efficacy of sarizotan, a 5-HT1A receptor agonist, at correcting RTT respiratory abnormalities. The present study investigated whether sarizotan inhibits hERG potassium channels and prolongs ventricular repolarization. Whole-cell patch-clamp measurements were made at 37 °C from hERG-expressing HEK293 cells. Docking analysis was conducted using a recent cryo-EM structure of hERG. Sarizotan was a potent inhibitor of hERG current (IhERG; IC50 of 183 nM) and of native ventricular IKr from guinea-pig ventricular myocytes. 100 nM and 1 μM sarizotan prolonged ventricular action potential (AP) duration (APD90) by 14.1 ± 3.3% (n = 6) and 29.8 ± 3.1% (n = 5) respectively and promoted AP triangulation. High affinity IhERG inhibition by sarizotan was contingent upon channel gating and intact inactivation. Mutagenesis experiments and docking analysis implicated F557, S624 and Y652 residues in sarizotan binding, with weaker contribution from F656. In conclusion, sarizotan inhibits IKr/IhERG, accessing key binding residues on channel gating. This action and consequent ventricular AP prolongation occur at concentrations relevant to those proposed to treat breathing dysrhythmia in RTT. Sarizotan should only be used in RTT patients with careful evaluation of risk factors for QTc prolongation.

The acute effects of hydrocortisone on cardiac electrocardiography, action potentials, intracellular calcium, and contraction: The role of protein kinase C

Hydrocortisone exerts adverse effects on various organs, including the heart. This study investigated the still unclear effects of hydrocortisone on electrophysiological and biochemical aspects of cardiac excitation–contraction coupling. In guinea pigs’ hearts, hydrocortisone administration reduced the QT interval of ECG and the action potential duration (APD). In guinea pig ventricular myocytes, hydrocortisone reduced contraction and Ca2+ transient amplitudes. These reductions and the effects on APD were prevented by pretreatment with the protein kinase C (PKC) inhibitor staurosporine. In an overexpression system of Xenopus oocytes, hydrocortisone increased hERG K+ currents and reduced Kv1.5 K+ currents; these effects were negated by pretreatment with staurosporine. Western blot analysis revealed dose- and time-dependent changes in PKCα/βII, PKCε, and PKCγ phosphorylation by hydrocortisone in guinea pig ventricular myocytes. Therefore, hydrocortisone can acutely affect cardiac excitation–contraction coupling, including ion channel activity, APD, ECG, Ca2+ transients, and contraction, possibly via biochemical changes in PKC.

A Local-Control Model of the Guinea Pig Ventricular Myocyte Allows Understanding of Force-Interval Relations at the Calcium Spark Level

A local control model of the guinea pig ventricular myocytes was developed to explore the mechanisms governing Ca2+ dynamics. This dynamic balance is controlled by the model features including L-type Ca2+ channels (LCC), sarcolemmal sodium calcium exchangers (NCX) and ryanodine receptors, type 2 (RyR2s). We developed a new three state RyR2 model that simulates RyR2 adaptation in which the channels accumulate in a closed state when exposed to prolonged elevated subspace [Ca2+] (Gyorke and Fill Science. 1993 260:807-9). The model was tested for agreement with previous experimental and modeling studies on force-interval relations, i.e. the changes in calcium transient and action potential morphology at different pacing frequencies. Our local control model displays stable action potential trains at 7 Hz, unlike previous common pool models. The duration of the [Ca2+]i transient and the action potential (AP) decrease with increased pacing rates consistent with experiment. With increased pacing rate, the [Ca2+]i transient peak values increases up to 3Hz and decreases afterward, consistent with experimental force-frequency curves. The model predicts, in agreement with our previous modeling studies (Jafri et al., Biophys J. 1998 Mar;74(3):1149-68), that diastolic sarcoplasmic reticulum (SR) [Ca2+] and RyR2 adaptation increases with increased stimulation frequency giving rise to rising than falling amplitude of the myoplasmic [Ca2+] transients. The model also allows us to dissect these frequency dependent changes down to the spark level giving new insight into mechanism governing cardiac calcium dynamics.

α&lt;sub&gt;1&lt;/sub&gt;-adrenergic Receptor Attenuates β&lt;sub&gt;1&lt;/sub&gt;-adrenergic Modulation of IKr by Protein Kinase C-dependent Suppression of Adenylyl Cyclase in Ventricular Myocytes

The rapid delayed rectifier K+ current (Ikr) is critical for repolarization of the cardiac action potential. Previous studies have shown activated α1-adrenergic receptor (AR) attenuates β1-adrenergic regulation of Ikr while the mechanisms involved are poorly understood. To evalutate how α1-adrenergic receptor affect β1-adrenergic modulation of Ikr, whole-cell patch-clamp recordings were peformed in isolated guinea-pig ventricular myocytes. Application of xamoterol, a selective β1-AR agonist, induced a negative shift in the activation curve and Ikr current reduction by 40.50±6.66% at the test pulse of +40 mV. Forskolin and 8-Br-cAMP also resulted in Ikr reduction by 38.17±1.50% and 24.65±3.37%, respectively. Phenylephrine, a selective α1-AR agonist, prevented the activation shift and Ikr current reduction induced by xamoterol and forskolin, but not by 8-Br-cAMP. The effect of xamoterol or forskolin on Ikr was also prevented by pretreatment with PDBu, a protein kinase C (PKC) activator, while the effect of cAMP on Ikr can not, which was similar to pretreatment with phenylephrine. When cells were pretreated with chelerythrine, a specific PKC inhibitor, phenylephrine failed to prevent Ikr reduction induced by xamoterol. Our data suggests that α1-adrenergic stimulation attenuates β1-adrenergic regulation of Ikr, through PKC-dependent downregulation of adenylyl cyclase/cyclic AMP pathway.