Alterations In Action Potential Duration Research Articles

Brain-to-heart cholinergic synapse-calcium signaling mediates ischemic stroke-induced atrial fibrillation.

Background: Stroke-related cardiovascular diseases have attracted considerable attention, with atrial fibrillation (AF) being among the most frequent complications. Despite increasing clinical evidence, experimental models of stroke-induced AF are still lacking, hindering mechanistic discoveries and the development of adequate therapeutics targeting this stroke-heart syndrome (SHS). This study aims to create a rat model of ischemic stroke-induced AF (ISIAF) and to explore the efficacy and mechanism of Wenxin Keli (WK), an antiarrhythmic Chinese medicine. Method: The middle cerebral artery occlusion/reperfusion model was adapted to create subacute brain ischemia in rats with normal cardiac function. Invasive electrophysiologic studies and ex vivo optical mapping were performed to evaluate the altered electrophysiological parameters and Ca2+ handling properties. RNA-seq analysis, RT-PCR, and immunohistochemistry (IHC) with immunofluorescence (IF) were employed to assess the SHS model and elucidate the mechanisms of ISIAF and the effects of WK. UPLC/Q-TOF-MS, molecular docking, and whole-cell patch recordings were used to identify the active components of WK for SHS. Results: Ischemic stroke aggravated atrial electrical instability, altered action potential duration (APD), Ca2+ transient duration (CaT), conduction heterogeneity, and spatially discordant alternans in SHS rat hearts. These abnormalities were alleviated by WK. RNA-seq analysis revealed that M3-mediated cholinergic synapse signaling and L-type calcium channel (LTCCs)-mediated Ca2+ signaling play prominent roles in ISIAF development and its reversal by WK. UPLC/Q-TOF-MS analysis identified 19 WK components as the main components in plasma after WK treatment. Molecular docking screening identified Dioscin as the major active component of WK. WK and Dioscin reduced ICa-L in a concentration-dependent manner with a half-maximal inhibitory concentration of 24.254 ± 2.051 mg/mL and 8.666 ± 0.777 µmol/L, respectively. Conclusion: This study established an experimental model of ISIAF capable of characterizing clinically relevant atrial electrophysiological changes post-cerebral ischemia. Molecular mechanistic studies revealed that the cholinergic-calcium signaling pathway is central to this brain-heart syndrome. Ischemic stroke-induced atrial fibrillation is partially reversible by the Chinese medicine Wenxin Keli, which acts via regulation of the cholinergic-calcium signaling pathway, with its active component Dioscin directly binding to IKM3 and inhibiting ICa-L.

Prediction of drug pro-arrhythmic cardiotoxicity using a semi-physiologically based pharmacokinetic model linked to cardiac ionic currents inhibition

Drug-induced torsades de pointes (TdP) risks are responsible for the withdrawal of many drugs from the market. Nowadays, assessments of drug-induced TdP risks are mainly based on maximum effective free therapeutic plasma concentration (EFTPCmax) and cardiac ionic current inhibitions using the human ventricular myocytes model (Tor-ORd model). Myocytes are targets of drug-induced TdP. The TdP risks may be directly linked to myocyte drug concentrations. We aimed to develop a semi-physiologically based pharmacokinetic (Semi-PBPK) model linked to cardiac ionic current inhibition (pharmacodynamics, PD) (Semi-PBPK-PD) to simultaneously predict myocyte drug concentrations and their TdP risks in humans. Alterations in action potential duration (ΔAPD90) were simulated using the Tor-ORd model and ionic current inhibition parameters based on myocyte or plasma drug concentrations. The predicted ΔAPD90 values were translated into in vivo alterations in QT interval(ΔQTc) induced by moxifloxacin, dofetilide, or sotalol. Myocyte drug concentrations of moxifloxacin, dofetilide, and sotalol gave better predictions of ΔQTc than plasma. Following validating the developed semi-PBPK-PD model, TdP risks of 37 drugs were assessed using ΔAPD90 and early afterdepolarization occurrence, which were estimated based on 10 × EFTPCmax and 10 × EFTMCmax (maximum effective free therapeutic myocyte concentration). 10 × EFTMCmax gave more sensitive and accurate predictions of pro-arrhythmic cardiotoxicity and the predicted TdP risks were also closer to clinic practice than 10 × EFTPCmax. In conclusion, pharmacokinetics and TdP risks of 37 drugs were successfully predicted using the semi-PBPK-PD model. Myocyte drug concentrations gave better predictions of ΔQTc and TdP risks than plasma.

Determining the role of uremic toxins on cardiac electrophysiology and pro-arrhythmia during chronic kidney disease

Abstract Funding Acknowledgements Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): Young Talent Programme, CVON Introduction Chronic kidney disease (CKD) is represented by a diminished filtration capacity of the kidneys. End stage renal disease patients need dialysis treatment to remove waste and toxins from the circulation. However, endogenously produced uremic toxins (UTs) cannot always be filtered during dialysis. UTs are one of the CKD-related factors already linked to maladaptive and pathophysiological remodeling of the heart. Importantly, 50% of the deaths in dialysis patients is cardiovascular related, with sudden cardiac death predominating. However, the mechanisms responsible still remain poorly understood. Purpose To assess the potentially increased pro-arrhythmic risk of pre-identified UTs at clinically relevant concentrations: 1) acutely in isolated adult dog ventricular cardiomyocytes (ADCMs) and human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs), 2) chronically in iPSC-CMs and 3) both conditions in human embryonic kidney (HEK) cells transfected with constructs that generate different ion channels, using optical and manual electrophysiological in vitro approaches. Methods and results Using the voltage sensitive membrane dye FluoVolt and manual patch clamp, action potential duration (APD) prolongation, considered as a pro-arrhythmic parameter, was assessed in ADCMs and iPSC-CMs. Acute exposure of indoxyl sulfate (IS), kynurenine (KYN) or kynurenic acid (KYNA) induced no alterations in APD in ADCMs and iPSC-CMs. iPSC-CMs, although having rather immature electrophysiological characteristics, showed significant APD prolongation when exposed chronically (48h) to those UTs. Manual patch clamp on HEK293 cells was performed to investigate individual ion currents to reveal underlying mechanisms. The repolarizing current IKr, often most sensitive and responsible for APD alterations, was not acutely affected by IS, KYN, or KYNA. However, chronic exposure (48h) to IS or KYNA significantly decreased IKr at clinically relevant concentrations. Finally, the protein level of the ion channel conducting IKr was determined in HEK293 cells, to further unveil the mechanism behind the effects of UTs on channels conducting IKr. Chronic exposure (48-72h) of the three UTs decreased total ion channel expression at pathological concentrations, which could be responsible for the decreased IKr and prolonged APD. Conclusion The results show the proof of concept to identify potentially pro-arrhythmogenic UTs and their mode of action, allowing the provision of a clinically relevant overview of (patho)physiological concentrations of UTs in both acute and chronic exposure.

Thyroid Hormone Diminishes Ca2+ Overload Induced by Hypoxia/Reoxygenation in Cardiomyocytes by Inhibiting Late Sodium Current and Reverse-Na+/Ca2+ Exchange Current

Background and Purpose: Intracellular calcium concentration ([Ca²⁺]i) overload occurs in myocardial ischemia and ­reperfusion. The augmentation of the late sodium current (I_NaL) causes intracellular Na⁺ accumulation and subsequent [Ca²⁺]i overload via the reverse mode of the Na⁺/Ca²⁺ exchange current (reverse-I_NCX), which can lead to arrhythmia and cardiac dysfunction. Thus, inhibition of I_NaL is a potential therapeutic approach for ischemic heart disease. The aim of this study was to investigate the effects of thyroid hormone on augmented I_NaL, reverse-I_NCX, altered action potential duration (APD), and [Ca²⁺]i concentration in hypoxia/reoxygenation (H/R)-induced ventricular myocytes in vitro. Methods: The transient Na⁺ current (I_NaT), I_NaL, reverse-I_NCX, and APs were recorded using a whole-cell patch-clamp technique in neonatal mouse ventricular myocytes. [Ca²⁺]i concentration alteration were, respectively, observed by confocal microscopy and flow cytometry. Results: Triiodothyronine (T3) pretreatment decreased the I_NaL in a concentration-dependent manner. H/R injury aggravated the I_NaL, I_NaT, and reverse-I_NCX in cardiomyocytes and increased the continuous accumulation of [Ca²⁺]i (p < 0.05). The application of T3 prior to H/R injury significantly decreased the increased I_NaL, I_NaT, and reverse-I_NCX and blunted the [Ca²⁺]i increase. Furthermore, T3 pretreatment shortened the APD induced by H/R injury. Conclusion: T3 inhibited H/R-increased I_NaL and reverse I_NCX augmentation, shortened the APD, and diminished [Ca²⁺]i overload, indicating a potential therapeutic use of T3 as an I_NaL inhibitor to maintain Ca²⁺ homeostasis and protect cardiomyocytes against H/R injury.

Atrial fibrillation and electrophysiology in transgenic mice with cardiac-restricted overexpression of FKBP12.

Cardiomyocyte-restricted overexpression of FK506-binding protein 12 transgenic (αMyHC-FKBP12) mice develop spontaneous atrial fibrillation (AF). The aim of the present study is to explore the mechanisms underlying the occurrence of AF in αMyHC-FKBP12 mice. Spontaneous AF was documented by telemetry in vivo and Langendorff-perfused hearts of αMyHC-FKBP12 and littermate control mice in vitro. Atrial conduction velocity was evaluated by optical mapping. The patch-clamp technique was applied to determine the potentially altered electrophysiology in atrial myocytes. Channel protein expression levels were evaluated by Western blot analyses. Spontaneous AF was recorded in four of seven αMyHC-FKBP12 mice but in none of eight nontransgenic (NTG) controls. Atrial conduction velocity was significantly reduced in αMyHC-FKBP12 hearts compared with NTG hearts. Interestingly, the mean action potential duration at 50% but not 90% was significantly prolonged in αMyHC-FKBP12 atrial myocytes compared with their NTG counterparts. Consistent with decreased conduction velocity, average peak Na+ current ( INa) density was dramatically reduced and the INa inactivation curve was shifted by approximately +7 mV in αMyHC-FKBP12 atrial myocytes, whereas the activation and recovery curves were unaltered. The Nav1.5 expression level was significantly reduced in αMyHC-FKBP12 atria. Furthermore, we found increases in atrial Cav1.2 protein levels and peak L-type Ca2+ current density and increased levels of fibrosis in αMyHC-FKBP12 atria. In summary, cardiomyocyte-restricted overexpression of FKBP12 reduces the atrial Nav1.5 expression level and mean peak INa, which is associated with increased peak L-type Ca2+ current and interstitial fibrosis in atria. The combined electrophysiological and structural changes facilitated the development of local conduction block and altered action potential duration and spontaneous AF. NEW & NOTEWORTHY This study addresses a long-standing riddle regarding the role of FK506-binding protein 12 in cardiac physiology. The work provides further evidence that FK506-binding protein 12 is a critical component for regulating voltage-gated sodium current and in so doing has an important role in arrhythmogenic physiology, such as atrial fibrillation.

Chronic in vivo angiotensin II administration differentially modulates the slow delayed rectifier channels in atrial and ventricular myocytes

In the heart, slow delayed rectifier channels provide outward currents (IKs) for action potential (AP) repolarization in a region- and context-dependent manner. In diseased hearts, chronic elevation of angiotensin II (Ang II) may remodel IKs in a region-dependent manner, contributing to atrial and ventricular arrhythmias of different mechanisms. The purpose of this study was to study whether/how chronic invivo Ang II administration remodels IKs in atrial and ventricular myocytes. We used the guinea pig (GP) model whose myocytes express robust IKs. GPs were implanted with minipumps containing Ang II or vehicle. Treatment continued for 4-6 weeks. We used patch clamp, immunofluorescence/confocal microscopy, and immunoblots to evaluate changes in IKs function and to explore the underlying mechanisms. We confirmed the pathologic state of the heart after chronic Ang II treatment. IKs density was increased in atrial myocytes but decreased in ventricular myocytes in Ang II- vs vehicle-treated animals. The former was correlated with an increase in KCNQ1/KCNE1 colocalization in myocyte periphery, whereas thelatter was correlated with a decrease in KCNQ1 protein level. Interestingly, these changes in IKs were not translated into expected alterations in AP duration or plateau voltage, indicating that other currents were involved. In atrial myocytes from Ang II-treated animals, the L-type Ca channel current was increased, contributing to AP plateau elevation and AP duration prolongation. IKs is differentially modulated by chronic invivo AngII administration between atrial and ventricular myocytes. Other currents remodeled by Ang II treatment also contribute to changes in action potentials.

Restitution analysis of alternans using dynamic pacing and its comparison with S1S2 restitution in heptanol-treated, hypokalaemic Langendorff-perfused mouse hearts.

Action potential duration (APD) and conduction velocity restitution explain the dependence of these parameters on the previous diastolic interval (DI). It is considered to be an adaptive mechanism for preserving diastole at fast heart rates. Hypokalaemia is known to induce ventricular arrhythmias that could be prevented by heptanol, the gap junction uncoupler, mediated through increases in ventricular refractory period (VERP) without alterations in APDs. The present study investigated alternans and restitution properties during normokalaemia, hypokalaemia alone or hypokalaemia with heptanol (0.1 mM) in Langendorff-perfused mouse hearts using a dynamic pacing protocol. APD90 alternans were elicited in the epicardium and endocardium during normokalaemia. Hypokalaemia increased the amplitudes of epicardial APD90 alternans when basic cycle lengths (BCLs) were ≤65 msec, which was associated with increases in maximum APD90 restitution gradients, critical DIs and APD90 heterogeneity. Heptanol (0.1 mM) did not exacerbate or reduce the APD90 alternans or alter these restitution parameters further. By contrast, endocardial APD90 alternans did not show increases in amplitudes during hypokalaemia at short BCLs studied, and restitution parameters were also unchanged. This was true whether in the presence or absence of 0.1 mM heptanol. The study demonstrates that anti-arrhythmic effects of heptanol exerted during hypokalaemia occurred despite exacerbation of APD90 alternans. This would suggest that even in the presence of arrhythmogenic APD90 alternans, arrhythmias could still be prevented by influencing VERP alone. Restitution data obtained here by dynamic pacing were compared to previous data from S1S2 pacing.

Effects of Ticagrelor on Ionic Currents and Contractility in Rat Ventricular Myocytes

Antiplatelet therapy has been widely used for management of patients with ischaemic heart diseases or thrombotic events. Experimental studies have shown that ticlopidine and clopidogrel decreased L-type Ca(2+) currents (ICaL), altered action potential (AP) duration and thence exerted negative inotropic effects. In this study we tested if ticagrelor, a non-thienopyridine agent, has any influence on contractile and electrical properties of isolated ventricular myocytes. Cardiomyocytes were isolated from male rat hearts with an enzymatic dissociation procedure and left ventricular myocytes were used for experiments. The effects of ticagrelor (1 μM) on sarcomere shortening, ionic currents and action potentials were measured at 36 ± 1 °C. Ticagrelor significantly reduced ICaL density (~18%, p < 0.01) of ventricular myocytes and this effect was reversible. In consistence, it also decreased sarcomere shortening of electrically stimulated cardiomyocytes (13%, p < 0.05), while it did not change relaxation rates. Repolarizing K(+) currents and AP duration were unaffected by 1 μM ticagrelor application. Ticagrelor exerts a significant influence on contractile properties and membrane currents of ventricular myocytes similarly to thienopyridine agents. The impact of ticagrelor on cardiac excitation-contraction coupling elements is important, since it is widely used for the treatment of patients with heart diseases.

Manipulation of KCNE2 expression modulates action potential duration and Ito and IK in rat and mouse ventricular myocytes.

In heterologous expression systems, KCNE2 has been demonstrated to interact with multiple α-subunits of voltage-dependent cation channels and modulate their functions. However, the physiological and pathological roles of KCNE2 in cardiomyocytes are poorly understood. The present study aimed to investigate the effects of bidirectional modulation of KCNE2 expression on action potential (AP) duration (APD) and voltage-dependent K(+) channels in cardiomyocytes. Adenoviral gene delivery and RNA interference were used to either increase or decrease KCNE2 expression in cultured neonatal and adult rat or neonatal mouse ventricular myocytes. Knockdown of KCNE2 prolonged APD in both neonatal and adult myocytes, whereas overexpression of KCNE2 shortened APD in neonatal but not adult myocytes. Consistent with the alterations in APD, KCNE2 knockdown decreased transient outward K(+) current (Ito) densities in neonatal and adult myocytes, whereas KCNE2 overexpression increased Ito densities in neonatal but not adult myocytes. Furthermore, KCNE2 knockdown accelerated the rates of Ito activation and inactivation, whereas KCNE2 overexpression slowed Ito gating kinetics in neonatal but not adult myocytes. Delayed rectifier K(+) current densities were remarkably affected by manipulation of KCNE2 expression in mouse but not rat cardiomyocytes. Simulation of the AP of a rat ventricular myocyte with a mathematical model showed that alterations in Ito densities and gating properties can result in similar APD alterations in KCNE2 overexpression and knockdown cells. In conclusion, endogenous KCNE2 in cardiomyocytes is important in maintaining cardiac electrical stability mainly by regulating Ito and APD. Perturbation of KCNE2 expression may predispose the heart to ventricular arrhythmia by prolonging APD.

Hypokalaemia induces Ca²⁺ overload and Ca²⁺ waves in ventricular myocytes by reducing Na⁺,K⁺-ATPase α₂ activity.

Hypokalaemia is a risk factor for development of ventricular arrhythmias. In rat ventricular myocytes, low extracellular K(+) (corresponding to clinical moderate hypokalaemia) increased Ca(2+) wave probability, Ca(2+) transient amplitude, sarcoplasmic reticulum (SR) Ca(2+) load and induced SR Ca(2+) leak. Low extracellular K(+) reduced Na(+),K(+)-ATPase (NKA) activity and hyperpolarized the resting membrane potential in ventricular myocytes. Both experimental data and modelling indicate that reduced NKA activity and subsequent Na(+) accumulation sensed by the Na(+), Ca(2+) exchanger (NCX) lead to increased Ca(2+) transient amplitude despite concomitant hyperpolarization of the resting membrane potential. Low extracellular K(+) induced Ca(2+) overload by lowering NKA α2 activity. Triggered ventricular arrhythmias in patients with hypokalaemia may therefore be attributed to reduced NCX forward mode activity linked to an effect on the NKA α2 isoform. Hypokalaemia is a risk factor for development of ventricular arrhythmias. The aim of this study was to determine the cellular mechanisms leading to triggering of arrhythmias in ventricular myocytes exposed to low Ko. Low Ko, corresponding to moderate hypokalaemia, increased Ca(2+) transient amplitude, sarcoplasmic reticulum (SR) Ca(2+) load, SR Ca(2+) leak and Ca(2+) wave probability in field stimulated rat ventricular myocytes. The mechanisms leading to Ca(2+) overload were examined. Low Ko reduced Na(+),K(+)-ATPase (NKA) currents, increased cytosolic Na(+) concentration and increased the Na(+) level sensed by the Na(+), Ca(2+) exchanger (NCX). Low Ko also hyperpolarized the resting membrane potential (RMP) without significant alterations in action potential duration. Experiments in voltage clamped and field stimulated ventricular myocytes, along with mathematical modelling, suggested that low Ko increases the Ca(2+) transient amplitude by reducing NKA activity despite hyperpolarization of the RMP. Selective inhibition of the NKA α2 isoform by low dose ouabain abolished the ability of low Ko to reduce NKA currents, to increase Na(+) levels sensed by NCX and to increase the Ca(2+) transient amplitude. We conclude that low Ko, within the range of moderate hypokalaemia, increases Ca(2+) levels in ventricular myocytes by reducing the pumping rate of the NKA α2 isoform with subsequent Na(+) accumulation sensed by the NCX. These data highlight reduced NKA α2 -mediated control of NCX activity as a possible mechanism underlying triggered ventricular arrhythmias in patients with hypokalaemia.

Blebbistatin specifically inhibits actin-myosin interaction in mouse cardiac muscle

Blebbistatin is a powerful inhibitor of actin-myosin interaction in isolated contractile proteins. To examine whether blebbistatin acts in a similar manner in the organized contractile system of striated muscle, the effects of blebbistatin on contraction of cardiac tissue from mouse were studied. The contraction of paced intact papillary muscle preparations and shortening of isolated cardiomyocytes were inhibited by blebbistatin with inhibitory constants in the micromolar range (1.3-2.8 muM). The inhibition constants are similar to those previously reported for isolated cardiac myosin subfragments showing that blebbistatin action is similar in filamentous myosin of the cardiac contractile apparatus and isolated proteins. The inhibition was not associated with alterations in action potential duration or decreased influx through L-type Ca(2+) channels. Experiments on permeabilized cardiac muscle preparations showed that the inhibition was not due to alterations in Ca(2+) sensitivity of the contractile filaments. The maximal shortening velocity was not affected by 1 muM blebbistatin. In conclusion, we show that blebbistatin is an inhibitor of the actin-myosin interaction in the organized contractile system of cardiac muscle and that its action is not due to effects on the Ca(2+) influx and activation systems.

Modulation of action potential duration on myocyte hypertrophic pathways

Prolongation of the action potential duration (APD) has consistently been observed in experimental models of cardiac hypertrophy and failure as well as in humans and is partially attributed to a reduction of a hyperpolarizing current provided by the calcium-independent transient outward K(+) channel (I(to)). In the present study, we examined the effects of manipulating ion channel currents (I(to) and sodium/calcium exchanger (NCX)) and the associated alterations in action potential duration on cardiomyocyte hypertrophy and signaling induced by angiotensin II (AngII). Our aim was to examined whether distinct patterns of intracellular calcium manipulation could generate distinct patterns of MAPkinase activation and cellular hypertrophy. Cultured neonatal rat ventricular myocytes (NRVMs) were infected with Ad. beta-gal/GFP, Ad.Kv4.3, Ad.Kv4.3 antisense or Ad.NCX adenoviruses and hypertrophy induced by incubation with AngII. Overexpression of Kv4.3 increased I(to) density, shortened APD, decreased Ca(2+) influx and inhibited AngII-induced (3)H-leucine incorporation and ANF and beta-MHC expression. These hypertrophic changes were also paralleled by blockade of ERK MAP kinases activation as well as calcineurin expression. These electrical and hypertrophic changes produced by overexpression of Kv4.3 were completely and significantly reversed by Kv4.3 antisense and NCX gene transfer. Our findings indicate that AngII-mediated hypertrophy response in NRVMs can be abrogated by an enhancement of I(to) function through overexpression of Kv4.3 and that modulation of action potential duration can be important in the development of cardiac hypertrophy.

Simulation of QRST integral maps with a membrane-based computer heart model employing parallel processing.

The simulation of the propagation of electrical activity in a membrane-based realistic-geometry computer model of the ventricles of the human heart, using the governing monodomain reaction-diffusion equation, is described. Each model point is represented by the phase 1 Luo-Rudy membrane model, modified to represent human action potentials. A separate longer duration action potential was used for the M cells found in the ventricular midwall. Cardiac fiber rotation across the ventricular wall was implemented via an analytic equation, resulting in a spatially varying anisotropic conductivity tensor and, consequently, anisotropic propagation. Since the model comprises approximately 12.5 million points, parallel processing on a multiprocessor computer was used to cut down on simulation time. The simulation of normal activation as well as that of ectopic beats is described. The hypothesis that in situ electrotonic coupling in the myocardium can diminish the gradients of action-potential duration across the ventricular wall was also verified in the model simulations. Finally, the sensitivity of QRST integral maps to local alterations in action-potential duration was investigated.

1110-219 Regionally altered action potential duration restitution in swine with hibernating myocardium and sudden cardiac death

1110-219 Regionally altered action potential duration restitution in swine with hibernating myocardium and sudden cardiac death

Targeting Ca2+ cycling proteins and the action potential in heart failure by gene transfer.

Cardiomyocytes isolated from failing human hearts are characterized by contractile dysfunction including prolonged relaxation, reduced systolic force and elevated diastolic force. These contractile abnormalities are paralleled by abnormal Ca2+ homeostasis such as reduced sarcoplasmic reticulum (SR) Ca2+ release, elevated diastolic Ca2+ and reduced rate of Ca2+ removal. In addition, failing human myocardium is characterized by a frequency-dependent decrease in systolic force and Ca2+ as opposed to normal myocardium where an increase in pacing rate results in potentiation of contractility and an increase in SR Ca2+ release. In the failing heart, the decrease in SR Ca2+ load has been linked to a decrease in SR Ca2+ ATPase (SERCA2a) function. We have recently shown that overexpression of SERCA2a by adenoviral gene transfer restores contractile function in cardiac myocytes from failing human hearts. In addition, we have shown that overexpression of SERCA2a in a model of pressure-overload hypertrophy in transition to failure improves contractile function and reserve in these animals. We are currently exploring the effect of long-term expression of SERCA2a in failing animals along with the energy cost of SERCA2a expression using NMR methods. We are also using a different strategy to improve SR Ca2+ ATPase activity which involves decreasing the expression of phospholamban by antisense strategies to enhance SR Ca2+ ATPase activity. The Na/Ca exchanger is also being targeted to enhance calcium removal in failing hearts. Action potential prolongation is attributed to reductions in transient outward current (Ito) density in human heart failure. This prolongation can alter contractility but can also cause afterdepolarization. Using gene transfer of various K channels responsible for Ito, we are investigating the molecular and the ionic basis of action potential prolongation in cardiac hypertrophy and failure and we are examining how intracellular calcium handling changes in response to alterations in action potential duration. Gene transfer, which serves initially as an experimental tool, may provide a novel therapeutic approach.

Stretch-induced changes in arrhythmogenesis and excitability in experimentally based heart cell models.

Mechanoelectric coupling in the heart is well documented and has been suggested as a cause of arrhythmia. One hypothesized mechanism for the stretch sensitivity of cardiac muscle is the presence of stretch-activated channels (SACs). This study uses modeling to explore the influence of SACs on cardiac resting potential, excitation threshold, and action potential in the context of arrhythmia. We added a putative SAC, modeled as a linear, time-independent conductance with reversal potential of -20 or -50 mV, to guinea pig and frog ventricular membrane models. Increased stretch conductance led to resting potential depolarization, a decreased excitation threshold, altered action potential duration, and, under certain conditions, early afterdepolarizations. We conclude that stretch increases cellular excitability, making the heart prone to ectopic activity. Regional effects of stretch on action potential duration can vary and are influenced by factors such as the SAC reversal potential, ionic conditions, and baseline currents, all of which may lead to an increased dispersion of refractoriness throughout the heart and therefore an increased risk of arrhythmia.

New aspects of cardiac electrophysiology and function: effects of inhalational anaesthetics

Summary The potent inhalation anaesthetics have been shown to inhibit a number of cardiac trans-sarcolemmal and sarcoplasmic reticulum channels. These actions may lead to bradycardia, AV conduction disturbances, altered action potential duration and depression of contractility. Therefore, it is not surprising that volatile anaesthetics can exhibit both dysrhythmic (e.g. re-entrant excitation) and antidysrhythmic actions (e.g. decreased automaticity and delayed afterdepolarization-triggered sustained rhythmic activity). The cellular electrophysiological basis for the occurrence of perioperative cardiac dysrhythmias in the presence of myocardial ischaemia and catecholamines remains unclear. Anaesthetics have been shown to reduce Ca 2+ influx and subcellular Ca 2+ release and, therefore, can produce a reduction of certain cardiac dysrhythmias. Anaesthetics differ, however, in their ability to potentiate the dysrhythmogenic action of catecholamines, with halothane being the most sensitizing. The adverse interaction between halothane and catecholamines on conduction and regional refractory characteristics probably involves α 1 -adrenoceptor stimulation. Further studies of this and other electrophysiological interactions will be necessary to establish the mechanism(s) responsible for the adverse effects of volatile anaesthetics on cardiac rhythm. New techniques in the measurement and analysis of left ventricular function by ventricular pressure-dimension loops can reveal important information concerning the effects of inhalational anaesthetics on cardiac performance. These techniques need not be limited to the experimental laboratory but can also be applied to man. Over the last 10 years, an increasing awareness of the importance of left ventricular diastolic function to overall cardiac performance has evolved. The influence of inhalational anaesthetic agents on diastolic function has not been thoroughly studied and represents an important future goal of research. Although volatile anaesthetics produce decreases in contractility and alterations in diastolic function, some of these agents also have important protective effects on postischaemic, reperfused (stunned) myocardium.

The Antiarrhythmic and Antifibrillatory Effects of Calcium Antagonists

Calcium-channel antagonists are widely used in the treatment of supraventricular tachyarrhythmias. The potential benefits of these agents in the management of ventricular arrhythmias, however, are not widely appreciated. Ventricular arrhythmias result from abnormalities of impulse generation and/or impulse conduction. The calcium ion has been implicated in both mechanisms. Myocardial cytosolic calcium increases during ischemia and sympathetic nervous system activation. Elevations in cytosolic calcium provoke oscillatory depolarizations of the cardiac membrane, triggering sustained action potential generation and extrasystoles. Myocardial ischemia also results in a dispersion of refractory period, producing random re-entrant circuits and ventricular fibrillation. Alterations in action potential duration that produce this dispersion of refractory period are associated with spatial and temporal nonuniformities of intracellular calcium transients. Calcium-channel antagonists have been shown to prevent afterdepolarizations and to reduce the endocardial to epicardial dispersion of the refractory period during myocardial ischemia, and therefore could prevent malignant arrhythmias. A slow inward calcium current may, in fact, be required for the initiation and maintenance of ventricular fibrillation. Calcium antagonists reduce, whereas calcium agonists enhance, the susceptibility to ventricular fibrillation induced by the combination of exercise and acute myocardial ischemia. The cardioprotection occurs independently of actions on vascular smooth muscle. Thus, calcium antagonists that exert direct myocardial actions may prove to be particularly effective in the prevention of sudden cardiac death in patients. The challenge remains to develop calcium antagonists that selectively modulate myocardial calcium entry without compromising cardiac mechanical function.

Effects of flecainide and quinidine on human atrial action potentials. Role of rate-dependence and comparison with guinea pig, rabbit, and dog tissues.

Flecainide and other class IC antiarrhythmic drugs are effective in the prevention and termination of atrial fibrillation, but the mechanism of this action is unknown. To gain insights into potential cellular mechanisms, we evaluated the response of human atrial action potentials to equimolar therapeutic concentrations of flecainide and quinidine and compared this response to that of guinea pig, rabbit, and dog atria. Both compounds reduced Vmax more as activation rate increased, but flecainide was more potent than quinidine and had slower kinetics. The rate-dependence of Vmax reduction was similar for all species, but human tissue was more sensitive to the drugs tested. In contrast to changes in Vmax, drug-induced alterations in action potential duration showed opposite rate-dependence for the two drugs. Quinidine increased action potential duration to 95% repolarization (APD95) in human atria by 33 +/- 7% (mean +/- SD) at a cycle length of 1,000 msec, but this effect was reduced as cycle length decreased, to 12 +/- 4% (p less than 0.001) at a cycle length of 300 msec. Flecainide increased APD95 (by 6 +/- 3%) much less than quinidine at a cycle length of 1,000 msec, but its effect was increased by faster pacing, to 27 +/- 12% at a cycle length of 300 msec and 35 +/- 8% (p less than 0.001) at the shortest 1:1 cycle length. The rate-dependent response of APD to drugs was qualitatively similar but quantitatively different among species. Human tissue showed the greatest frequency-dependent drug effects on repolarization, followed by tissue from dogs and rabbits. Guinea pig atria showed the least (and statistically nonsignificant) rate-dependence of drug effect on APD. Drug-induced changes in refractoriness paralleled those in APD. We conclude that: 1) flecainide and quinidine both increase APD in human atrial tissue but with opposite rate-dependence, 2) the effects of flecainide to increase atrial APD and refractoriness are enhanced by the rapid rates typical of atrial fibrillation, and 3) animal tissues may differ importantly from human in both their sensitivity and rate-dependent response to antiarrhythmic drugs. The salutary response of atrial fibrillation to flecainide may be due to enhancement of drug action by the rapid atrial activation rates characteristic of this arrhythmia.

Glucose reverses 2,4-dinitrophenol induced changes in action potentials and membrane currents of guinea pig ventricular cells via enhanced glycolysis

The precise mechanism involved in the restoration by glucose of action potentials and membrane currents has not been established, so we studied it in single guinea pig ventricular cells under conditions of metabolic inhibition by 2,4-dinitrophenol (DNP). Application of DNP (50 mumol.litre-1) significantly shortened the action potential duration (APD) from 276.3 (SD 47.5) to 43.0(3.9) ms (n = 10). The increase in glucose concentration from 5.5 (control) to 22 mmol.litre-1 significantly restored the APD within 10 min, to 233.2(51.2) ms. In tight seal whole cell clamp studies, we found that DNP increased the steady state outward currents at potentials positive to -60 mV, and that this current was all but suppressed by 22 mmol.litre-1 glucose. The glucose induced recovery in the APD and membrane current was not seen in the presence of 2-deoxyglucose (2 mmol.litre-1), an inhibitor of glycolysis. Insulin (40 mIU.ml-1) reversed the DNP induced shortening of APD and abolished the increase in the outward current in the presence of 11 mmol.litre-1 glucose, though the application of glucose alone at this concentration failed to reverse these changes. Glucose (5.5 to 44 mmol.litre-1) did not directly affect single channel currents of ATP regulated K channels of the excised patch membrane in an outside out mode. These results suggest that the antagonising effects of glucose on the DNP induced alterations in APD and membrane currents are mediated by an increase in intracellular ATP concentration via enhanced glycolysis. Insulin might enhance this effect by facilitating the transport of glucose across the cell membrane.