Action Potential Upstroke Velocity Research Articles

Regulation of the cardiac sodium channel Nav1.5 by utrophin in dystrophin-deficient mice

Duchenne muscular dystrophy (DMD) is a severe striated muscle disease due to the absence of dystrophin. Dystrophin deficiency results in dysfunctional sodium channels and conduction abnormalities in hearts of mdx mice. Disease progression in the mdx mouse only modestly reflects that of DMD patients, possibly due to utrophin up-regulation. Here, we investigated mice deficient in both dystrophin and utrophin [double knockout (DKO)] to assess the role of utrophin in the regulation of the cardiac sodium channel (Na(v)1.5) in mdx mice. Co-immunoprecipitation studies in HEK293 cells showed that utrophin interacts with Na(v)1.5 via syntrophin proteins, an interaction abolished by deletion of the PDZ (PSD-95, Dlg, and Zona occludens) domain-binding motif of Na(v)1.5. We also provide evidence for such interaction in mouse heart using Na(v)1.5 C-terminus fusion proteins. In hearts of DKO mice, Na(v)1.5 protein levels were decreased by 25 ± 8%, together with a 42 ± 12% reduction of syntrophins compared with mdx, where utrophin was up-regulated by 52 ± 9% compared with C57BL/10 control mice. Sodium current was found to be reduced by 41 ± 5% in DKO cardiomyocytes compared with mdx, representing a loss of 63 ± 3% when compared with C57BL/10 wild-type control mice. Decreased Na(v)1.5 protein and current in DKO were reflected in a significant slowing of 27 ± 6% of maximal upstroke velocity of the cardiac action potential compared with mdx. Utrophin plays a central role in the regulation of Na(v)1.5 in mdx mice. These findings provide support for therapeutic strategies aimed at overexpressing utrophin in the hopes of reducing cardiac pathology in DMD patients.

Cardiac expression of skeletal muscle sodium channels increases longitudinal conduction velocity in the canine 1-week myocardial infarction

Skeletal muscle sodium channel (Nav1.4) expression in border zone myocardium increases action potential upstroke velocity in depolarized isolated tissue. Because resting membrane potential in the 1-week canine infarct is reduced, we hypothesized that conduction velocity (CV) is greater in Nav1.4 dogs compared with in control dogs. The purpose of this study was to measure CV in the infarct border zone border in dogs with and without Nav1.4 expression. Adenovirus was injected in the infarct border zone in 34 dogs. The adenovirus incorporated the Nav1.4- and a green fluorescent protein (GFP) gene (Nav1.4 group, n = 16) or only GFP (n = 18). After 1 week, upstroke velocity and CV were measured by sequential microelectrode recordings at 4 and 7 mM [K(+)] in superfused epicardial slabs. High-density in vivo epicardial activation mapping was performed in a subgroup (8 Nav1.4, 6 GFP) at three to four locations in the border zone. Microscopy and antibody staining confirmed GFP or Nav1.4 expression. Infarct sizes were similar between groups (30.6% +/- 3% of left ventricle mass, mean +/- standard error of the mean). Longitudinal CV was greater in Nav1.4 than in GFP sites (58.5 +/- 1.8 vs. 53.3 +/- 1.2 cm/s, 20 and 15 sites, respectively; P <.05). Transverse CV was not different between the groups. In tissue slabs, dV/dt(max) was higher and CV was greater in Nav1.4 than in control at 7 mM [K(+)] (P <.05). Immunohistochemical Nav1.4 staining was seen at the longitudinal ends of the myocytes. Nav1.4 channels in myocardium surviving 1 week infarction increases longitudinal but not transverse CV, consistent with the increased dV/dt(max) and with the cellular localization of Nav1.4.

Streptozotocin-induced diabetes modulates action potentials and ion channel currents from the rat atrioventricular node.

The present study was conducted to evaluate whether experimentally induced type 1 diabetes results in alterations to atrioventricular nodal (AVN) electrophysiology at the cellular level. Spontaneous action potentials (APs) and ionic currents were recorded from AVN myocytes isolated from the hearts of control rats and from those with streptozotocin-induced diabetes. Perforated patch-clamp recordings were used to assess changes in cellular AP parameters and in ionic currents. Type 1 diabetes significantly increased AP duration, whilst reducing AP firing rate, upstroke velocity and rate of diastolic depolarization. The diabetes-induced changes in AP parameters were accompanied by a significant leftward shift in the zero current potential under voltage clamp, a reduction in peak L-type Ca(2+) current density and reduced amplitude of delayed rectifier and hyperpolarization-activated currents. These findings demonstrate that experimentally induced type 1 diabetes can lead to remodelling of AVN cellular electrophysiology.

Variable Nav1.5 Protein Expression from the Wild-Type Allele Correlates with the Penetrance of Cardiac Conduction Disease in the Scn5a+/− Mouse Model

BackgroundLoss-of-function mutations in SCN5A, the gene encoding Nav1.5 Na+ channel, are associated with inherited cardiac conduction defects and Brugada syndrome, which both exhibit variable phenotypic penetrance of conduction defects. We investigated the mechanisms of this heterogeneity in a mouse model with heterozygous targeted disruption of Scn5a (Scn5a +/− mice) and compared our results to those obtained in patients with loss-of-function mutations in SCN5A.Methodology/Principal FindingsBased on ECG, 10-week-old Scn5a +/− mice were divided into 2 subgroups, one displaying severe ventricular conduction defects (QRS interval>18 ms) and one a mild phenotype (QRS≤18 ms; QRS in wild-type littermates: 10–18 ms). Phenotypic difference persisted with aging. At 10 weeks, the Na+ channel blocker ajmaline prolonged QRS interval similarly in both groups of Scn5a +/− mice. In contrast, in old mice (>53 weeks), ajmaline effect was larger in the severely affected subgroup. These data matched the clinical observations on patients with SCN5A loss-of-function mutations with either severe or mild conduction defects. Ventricular tachycardia developed in 5/10 old severely affected Scn5a +/− mice but not in mildly affected ones. Correspondingly, symptomatic SCN5A–mutated Brugada patients had more severe conduction defects than asymptomatic patients. Old severely affected Scn5a +/− mice but not mildly affected ones showed extensive cardiac fibrosis. Mildly affected Scn5a +/− mice had similar Nav1.5 mRNA but higher Nav1.5 protein expression, and moderately larger INa current than severely affected Scn5a +/− mice. As a consequence, action potential upstroke velocity was more decreased in severely affected Scn5a +/− mice than in mildly affected ones.Conclusions Scn5a +/− mice show similar phenotypic heterogeneity as SCN5A-mutated patients. In Scn5a +/− mice, phenotype severity correlates with wild-type Nav1.5 protein expression.

Constitutive CaMKII activity regulates Na + channel in rat ventricular myocytes

The cardiac voltage-gated Na + channel controls the upstroke of action potential and membrane excitability. The Na + channel associates with Ca 2+/CaM-dependent protein kinase (CaMKII), but the role of CaMKII on Na + channel activity in the resting state is not clear. In this report, we investigated whether CaMKII constitutively regulates Na + currents ( I Na), independent of Ca 2+ influx in rat ventricular myocytes using patch clamp technique. CaMKII inhibition (by KN93 or autocamtide-related inhibitory peptide) caused a negative shift in I Na steady-state inactivation and delayed recovery from slow inactivation, limiting channel availability. The reduction of I Na was 29.47 ± 3.01% at a holding potential ( V h) of − 120 mV and it increased to 77.70 ± 7.92% when V h was − 70 mV, suggesting that near the resting membrane potential, three-quarters of I Na depends on CaMKII action. CaMKII inhibition also enhanced intermediate inactivation, as well as delayed recovery from fast inactivation, and decreased late I Na. KN92, an inactive analog of KN93, had no effect on I Na. Using an antibody against phosphorylated (activated) CaMKII, we found that constitutively active CaMKII co-immunoprecipitated with Na + channels under resting conditions. CaMKII inhibitors reduced the level of phosphorylated CaMKII, which correlated with the degree of reduction in channel availability. These data suggest that CaMKII in an active form contributes to regulating I Na. Finally, we observed a drastic reduction in the upstroke velocity of action potentials upon CaMKII inhibition. In conclusion, CaMKII constitutively regulates cardiac Na + channel and this regulatory mechanism is important for the maintenance of Na + channel characteristics under physiological conditions.

Cardiac fibroblast paracrine factors alter impulse conduction and ion channel expression of neonatal rat cardiomyocytes

The pathological proliferation of cardiac fibroblasts (CFs) in response to heart injury results in fibrosis, which correlates with arrhythmia generation and heart failure. Here we systematically examined the effect of fibroblast-derived paracrine factors on electrical propagation in cardiomyocytes. Neonatal rat cardiac monolayers were exposed for 24 h to media conditioned by CFs. Optical mapping, sharp microelectrode recordings, quantitative RT-PCR, and immunostaining were used to assess the changes in the propagation and shape of the action potential and underlying changes in gene and protein expression. The fibroblast paracrine factors produced a 52% reduction in cardiac conduction velocity, a 217% prolongation of action potential duration, a 64% decrease of maximum capture rate, a 21% increase in membrane resting potential, and an 80% decrease of action potential upstroke velocity. These effects were dose dependent and partially reversible with removal of the conditioned media. No fibroblast proliferation, cardiomyocyte apoptosis, or decreased connexin-43 expression, phosphorylation, and function were found in conditioned cardiac cultures. In contrast, the expression of the fast sodium, inward rectifying potassium, and transient outward potassium channels were, respectively, reduced 3.8-, 6.6-fold, and to undetectable levels. The expression of beta-myosin heavy chain increased 17.4-fold. No electrophysiological changes were observed from media conditioned by CFs in the presence of cardiomyocytes. Paracrine factors from neonatal CFs alone produced significant electrophysiological changes in neonatal rat cardiomyocytes resembling those found in several cardiac pathologies.

Glucagon‐like peptide‐1 prolongs cardiac action potential duration

Glucagon‐like peptide‐1 (GLP‐1) is a hormone predominately synthesized and secreted by intestinal L cells. The biological effects of GLP‐1 are most likely via stimulation of GLP‐1 receptors. Animal studies have shown that GLP‐1 may protect the heart from ischemic damage and improve cardiac function in heart failure. However, the effects of GLP‐1 on myocyte electrophysiology have not been investigated. In this study we assessed the effects of GLP‐1 on cardiac action potentials. Extracellular application of GLP‐1 (7‐36) at 5 nM significantly prolonged the duration of action potentials in isolated canine ventricular myocytes. The duration at 50% repolarization was prolonged from 124 ± 24 ms for control to 252 ± 30 ms for GLP‐1 (n = 6, p &lt; 0.01). Such prolongation accompanied with an increase in L‐type Ca2+ currents. Other parameters, such as amplitude, threshold, maximum upstroke velocity of action potentials, were not changed by GLP‐1. The effect of action potential prolongation was reversible after washout of GLP‐1. Lower concentrations (&lt;0.05 nM) of GLP‐1 had no such effect. Adding GLP‐1 (7‐36) at 30 nM to a culture medium for 24 hours significantly increased the beating rate of cultured neonatal rat ventricular cells. Our data demonstrate that GLP‐1 prolongs cardiac action potential duration and increases beating rate of cardiomyocytes. These effects probably relate to GLP‐1‐induced increase in Ca2+ currents.

The cardiac sodium channel displays differential distribution in the conduction system and transmural heterogeneity in the murine ventricular myocardium

Cardiac sodium channels are responsible for conduction in the normal and diseased heart. We aimed to investigate regional and transmural distribution of sodium channel expression and function in the myocardium. Sodium channel Scn5a mRNA and Nav1.5 protein distribution was investigated in adult and embryonic mouse heart through immunohistochemistry and in situ hybridization. Functional sodium channel availability in subepicardial and subendocardial myocytes was assessed using patch-clamp technique. Adult and embryonic (ED14.5) mouse heart sections showed low expression of Nav1.5 in the HCN4-positive sinoatrial and atrioventricular nodes. In contrast, high expression levels of Nav1.5 were observed in the HCN4-positive and Cx43-negative AV or His bundle, bundle branches and Purkinje fibers. In both ventricles, a transmural gradient was observed, with a low Nav1.5 labeling intensity in the subepicardium as compared to the subendocardium. Similar Scn5a mRNA expression patterns were observed on in situ hybridization of embryonic and adult tissue. Maximal action potential upstroke velocity was significantly lower in subepicardial myocytes (mean ± SEM 309 ± 32 V/s; n = 14) compared to subendocardial myocytes (394 ± 32 V/s; n = 11; P < 0.05), indicating decreased sodium channel availability in subepicardium compared to subendocardium. Scn5a and Nav1.5 show heterogeneous distribution patterns within the cardiac conduction system and across the ventricular wall. This differential distribution of the cardiac sodium channel may have profound consequences for conduction disease phenotypes and arrhythmogenesis in the setting of sodium channel disease.Electronic supplementary materialThe online version of this article (doi:10.1007/s00395-009-0012-8) contains supplementary material, which is available to authorized users.

Clinical Summaries

Clinical Summaries

I K1 Heterogeneity Affects Genesis and Stability of Spiral Waves in Cardiac Myocyte Monolayers

Previous studies have postulated an important role for the inwardly rectifying potassium current (I(K1)) in controlling the dynamics of electrophysiological spiral waves responsible for ventricular tachycardia and fibrillation. In this study, we developed a novel tissue model of cultured neonatal rat ventricular myocytes (NRVMs) with uniform or heterogeneous Kir2.1expression achieved by lentiviral transfer to elucidate the role of I(K1) in cardiac arrhythmogenesis. Kir2.1-overexpressed NRVMs showed increased I(K1) density, hyperpolarized resting membrane potential, and increased action potential upstroke velocity compared with green fluorescent protein-transduced NRVMs. Opposite results were observed in Kir2.1-suppressed NRVMs. Optical mapping of uniformly Kir2.1 gene-modified monolayers showed altered conduction velocity and action potential duration compared with nontransduced and empty vector-transduced monolayers, but functional reentrant waves could not be induced. In monolayers with an island of altered Kir2.1 expression, conduction velocity and action potential duration of the locally transduced and nontransduced regions were similar to those of the uniformly transduced and nontransduced monolayers, respectively, and functional reentrant waves could be induced. The waves were anchored to islands of Kir2.1 overexpression and remained stable but dropped in frequency and meandered away from islands of Kir2.1 suppression. In monolayers with an inverse pattern of I(K1) heterogeneity, stable high frequency spiral waves were present with I(K1) overexpression, whereas lower frequency, meandering spiral waves were observed with I(K1) suppression. Our study provides direct evidence for the contribution of I(K1) heterogeneity and level to the genesis and stability of spiral waves and highlights the potential importance of I(K1) as an antiarrhythmia target.

Epicardial Border Zone Overexpression of Skeletal Muscle Sodium Channel SkM1 Normalizes Activation, Preserves Conduction, and Suppresses Ventricular Arrhythmia

In depolarized myocardial infarct epicardial border zones, the cardiac sodium channel (SCN5A) is largely inactivated, contributing to low action potential upstroke velocity (V(max)), slow conduction, and reentry. We hypothesized that a fast inward current such as the skeletal muscle sodium channel (SkM1) operating more effectively at depolarized membrane potentials might restore fast conduction in epicardial border zones and be antiarrhythmic. Computer simulations were done with a modified Hund-Rudy model. Canine myocardial infarcts were created by coronary ligation. Adenovirus expressing SkM1 and green fluorescent protein or green fluorescent protein alone (sham) was injected into epicardial border zones. After 5 to 7 days, dogs were studied with epicardial mapping, programmed premature stimulation in vivo, and cellular electrophysiology in vitro. Infarct size was determined, and tissues were immunostained for SkM1 and green fluorescent protein. In the computational model, modest SkM1 expression preserved fast conduction at potentials as positive as -60 mV; overexpression of SCN5A did not. In vivo epicardial border zone electrograms were broad and fragmented in shams (31.5 +/- 2.3 ms) and narrower in SkM1 (22.6 +/- 2.8 ms; P=0.03). Premature stimulation induced ventricular tachyarrhythmia/fibrillation >60 seconds in 6 of 8 shams versus 2 of 12 SkM1 (P=0.02). Microelectrode studies of epicardial border zones from SkM1 showed membrane potentials equal to that of shams and V(max) greater than that of shams as membrane potential depolarized (P<0.01). Infarct sizes were similar (sham, 30 +/- 2.8%; SkM1, 30 +/- 2.6%; P=0.86). SkM1 expression in injected epicardium was confirmed immunohistochemically. SkM1 increases V(max) of depolarized myocardium and reduces the incidence of inducible sustained ventricular tachyarrhythmia/fibrillation in canine infarcts. Gene therapy to normalize activation by increasing V(max) at depolarized potentials may be a promising antiarrhythmic strategy.

Acidosis inhibits spontaneous activity and membrane currents in myocytes isolated from the rabbit atrioventricular node

Recent evidence from intact hearts suggests that the function of cardiac nodal tissue may be particularly susceptible to acidosis. Little is currently known, however, about the effects of acidosis on the cellular electrophysiology of the atrioventricular node (AVN). This study was conducted, therefore, to determine the effect of acidosis on the spontaneous activity and membrane currents of myocytes isolated from the rabbit AVN, recorded at 35–37 °C using whole-cell patch-clamp. Reduction of extracellular pH (pH e; from 7.4 to 6.8 or 6.3) produced pH-dependent slowing of spontaneous action potential rate and upstroke velocity, and reductions in maximum diastolic potential and action potential amplitude. Ionic current recordings under voltage-clamp indicated that acidosis (pH e 6.3) decreased L-type Ca current ( I Ca,L), without significant changes in voltage-dependent activation or inactivation. Acidosis reduced the E-4031-sensitive, rapid delayed rectifier current ( I Kr) tail amplitude at − 40 mV following command pulses to between − 30 and + 50 mV, and accelerated tail-current deactivation. In contrast, the time-dependent hyperpolarisation-activated current, I f, was unaffected by acidosis. Background current insensitive to E-4031 and nifedipine was reduced by acidosis. Measurement of intracellular pH (pH i) from undialysed cells using BCECF showed a reduction in mean pH i from 7.24 to 6.45 ( n = 17) when pH e was lowered from 7.4 to 6.3. We conclude that I f is unlikely to be involved in the response of the AVN to acidosis, whilst inhibition of I Ca,L and I Kr by acidosis are likely to play a significant role in effects on AVN cellular electrophysiology.

Role of activated CaMKII in abnormal calcium homeostasis and INa remodeling after myocardial infarction: Insights from mathematical modeling

Ca2+/calmodulin-dependent protein kinase II is a multifunctional serine/threonine kinase with diverse cardiac roles including regulation of excitation contraction, transcription, and apoptosis. Dynamic regulation of CaMKII activity occurs in cardiac disease and is linked to specific disease phenotypes through its effects on ion channels, transporters, transcription and cell death pathways. Recent mathematical models of the cardiomyocyte have incorporated limited elements of CaMKII signaling to advance our understanding of how CaMKII regulates cardiac contractility and excitability. Given the importance of CaMKII in cardiac disease, it is imperative that computer models evolve to capture the dynamic range of CaMKII activity. In this study, using mathematical modeling combined with biochemical and imaging techniques, we test the hypothesis that CaMKII signaling in the canine infarct border zone (BZ) contributes to impaired calcium homeostasis and electrical remodeling. We report that the level of CaMKII autophosphorylation is significantly increased in the BZ region. Computer simulations using an updated mathematical model of CaMKII signaling reproduce abnormal Ca2+ transients and action potentials characteristic of the BZ. Our simulations show that CaMKII hyperactivity contributes to abnormal Ca2+ homeostasis and reduced action potential upstroke velocity due to effects on INa gating kinetics. In conclusion, we present a new mathematical tool for studying effects of CaMKII signaling on cardiac excitability and contractility over a dynamic range of kinase activities. Our experimental and theoretical findings establish abnormal CaMKII signaling as an important component of remodeling in the canine BZ.

Comparison of the cardiac electrophysiological effects between doxazosin and bunazosin

In Langendorff-perfused adult rat heart with constant pressure at 80 mmHg, we found doxazosin, an alpha(1) adrenoceptor blocker, at 10 muM prolonged PR interval and induced occasional arrhythmia followed by complete inhibition of the sinus rhythm, whereas bunazosin, another alpha(1)-blocker, at same concentration did not. The results of voltage-clamp study showed that, at the concentration of 10 muM, doxazosin inhibited I (Na), I ( (Ca,L) ), I (to), and Iss without changing I (k1) but bunazosin only inhibited I ( (Ca,L) ) about 30%. Doxazosin also caused markedly negative shift of the I (Na) inactivation curve. In current-clamp study, doxazosin prolonged action potential duration in association with the decreased action potential amplitude and upstroke velocity, whereas bunazosin did not. We hypothesize that doxazosin-induced arrhythmia may result from the heterogeneous or different level of I ( (Ca,L) ) blockade of AV nodal tissue. In conclusion, the present study suggests that bunazosin is safer than doxazosin for long-term treatment in view of electrophysiological effect. However, the underlying mechanism of doxazosin induced nodal arrhythmia is still needed to be determined.

Simulation of Ca-Calmodulin-Dependent Protein Kinase II on Rabbit Ventricular Myocyte Ion Currents and Action Potentials

Ca-calmodulin-dependent protein kinase II (CaMKII) was recently shown to alter Na + channel gating and recapitulate a human Na + channel genetic mutation that causes an unusual combined arrhythmogenic phenotype in patients: simultaneous long QT syndrome and Brugada syndrome. CaMKII is upregulated in heart failure where arrhythmias are common, and CaMKII inhibition can reduce arrhythmias. Thus, CaMKII-dependent channel modulation may contribute to acquired arrhythmic disease. We developed a Markovian Na + channel model including CaMKII-dependent changes, and incorporated it into a comprehensive myocyte action potential (AP) model with Na + and Ca 2+ transport. CaMKII shifts Na + current ( I Na) availability to more negative voltage, enhances intermediate inactivation, and slows recovery from inactivation (all loss-of-function effects), but also enhances late noninactivating I Na (gain of function). At slow heart rates, with long diastolic time for I Na recovery, late I Na is the predominant effect, leading to AP prolongation (long QT syndrome). At fast heart rates, where recovery time is limited and APs are shorter, there is little effect on AP duration, but reduced availability decreases I Na, AP upstroke velocity, and conduction (Brugada syndrome). CaMKII also increases cardiac Ca 2+ and K + currents ( I Ca and I to), complicating CaMKII-dependent AP changes. Incorporating I Ca and I to effects individually prolongs and shortens AP duration. Combining I Na, I Ca, and I to effects results in shortening of AP duration with CaMKII. With transmural heterogeneity of I to and I to downregulation in heart failure, CaMKII may accentuate dispersion of repolarization. This provides a useful initial framework to consider pathways by which CaMKII may contribute to arrhythmogenesis.

Electrophysiological Modeling of Fibroblasts and their Interaction with Myocytes

Experimental studies have shown that cardiac fibroblasts are electrically inexcitable, but can contribute to electrophysiology of myocardium in various manners. The aim of this computational study was to give insights in the electrophysiological role of fibroblasts and their interaction with myocytes. We developed a mathematical model of fibroblasts based on data from whole-cell patch clamp and polymerase chain reaction (PCR) studies. The fibroblast model was applied together with models of ventricular myocytes to assess effects of heterogeneous intercellular electrical coupling. We investigated the modulation of action potentials of a single myocyte varying the number of coupled fibroblasts and intercellular resistance. Coupling to fibroblasts had only a minor impact on the myocyte's resting and peak transmembrane voltage, but led to significant changes of action potential duration and upstroke velocity. We examined the impact of fibroblasts on conduction in one-dimensional strands of myocytes. Coupled fibroblasts reduced conduction and upstroke velocity. We studied electrical bridging between ventricular myocytes via fibroblast insets for various coupling resistors. The simulations showed significant conduction delays up to 20.3 ms. In summary, the simulations support strongly the hypothesis that coupling of fibroblasts to myocytes modulates electrophysiology of cardiac cells and tissues.

Optimisation and validation of a medium-throughput electrophysiology-based hNav1.5 assay using IonWorks™

Introduction The safety implications of blocking the human cardiac Na + channel (hNav1.5) make it prudent to test for this activity early in the drug discovery process and design-out any potential liability. This needs a method with adequate throughput and a demonstrable predictive value to effects in native cardiac tissues. Here we describe the validation of a method that combines the ability to screen tens of compounds a day, with direct assessment of channel function. Methods The electrophysiological and pharmacological properties of hNav1.5 were compared using two methods: conventional, low-throughput electrophysiology and planar-array-based, medium-throughput electrophysiology (IonWorks™ HT). A pharmacological comparison was also made between IonWorks™ HT and canine cardiac Purkinje Fibre action potential upstroke data. Results Activation curve parameters for hNav1.5 in IonWorks™ HT were not statistically different ( p > 0.05) from those generated using conventional electrophysiology. IonWorks™ HT V 1/2 = − 22 ± 0.8 mV, slope = 6.9 ± 0.2 ( n = 11); conventional electrophysiology V 1/2 = − 20 ±1.6 mV, slope = 6.4 ± 0.3 ( n = 11). Potency values for a range of hNav1.5 blockers determined using IonWorks™ HT correlated closely with those obtained using conventional electrophysiology ( R = 0.967, p < 0.001). The assay was able to distinguish between highly use-dependent blockers (e.g. tetracaine) and blockers that do not display strong use-dependence (e.g. quinidine). Comparison of the degree of hNav1.5 inhibition and decrease in canine Purkinje fibre action potential upstroke velocity ( V max) showed that the IonWorks™ HT assay would have predicted the outcome in Purkinje fibres in the majority of cases, with false negative and positive rates estimated at 8 and 7%, respectively. Finally, hNav1.5 pharmacology was similar when determined using either IonWorks™ HT or IonWorks™ Quattro, although the latter yielded more consistent data. Discussion The assay described combines a functional assessment of hNav1.5 with medium-throughput. Furthermore the assay was able to reveal information on the use-dependency of compound block, as well as predicting Na + channel effects in more integrated systems such as the cardiac Purkinje fibre action potential. This makes it possible to determine quantitative potency data, and mechanistic information about use-dependence, in a timeframe short enough to influence medicinal chemistry.

Pacemaker current (If) in the human sinoatrial node

Animal studies revealed that the hyperpolarization-activated pacemaker current, I(f), contributes to action potential (AP) generation in sinoatrial node (SAN) and significantly determines heart rate. I(f) is becoming a novel therapy target to modulate heart rate. Yet, no studies have demonstrated that I(f) is functionally present and contributes to pacemaking in human SAN. We aimed to study I(f) properties in human SAN. In a patient undergoing SAN excision, we identified SAN using epicardial activation mapping. From here, we isolated myocytes and recorded APs and I(f) using patch-clamp techniques. Pacemaker cells generated spontaneous APs (cycle length 828 +/- 15 ms) following slow diastolic depolarization, maximal diastolic potential - 61.7 +/- 4.3 mV, and maximal AP upstroke velocity 4.6 +/- 1.2 V/s. They exhibited an hyperpolarization-activated inward current, blocked by external Cs(+) (2 mmol/L), characterizing it as I(f). Fully-activated conductance was 75.2 +/- 3.8 pS/pF, reversal potential - 22.1 +/- 2.4 mV, and half-maximal activation voltage and slope factor of steady-state activation - 96.9 +/- 2.7 and - 8.8 +/- 0.5 mV. Activation time constant ranged from approximately 350 ms (-130 mV) to approximately 1 s (-100 mV), deactivation time constant 156 +/- 45 ms (-40 mV). The role of I(f) in pacemaker activity was demonstrated by slowing of pacemaker cell diastolic depolarization and beating rate by Cs(+). I(f) is functionally expressed in human SAN and probably contributes to pacemaking in human SAN.

Propagation of the cardiac impulse in the diabetic rat heart: reduced conduction reserve.

Diabetes mellitus is a growing epidemic with severe cardiovascular complications. Although much is known about mechanical and electrical cardiac dysfunction in diabetes, few studies have investigated propagation of the electrical signal in the diabetic heart and the associated changes in intercellular gap junctions. This study was designed to investigate these issues, using hearts from control and diabetic rats. Diabetic conditions were induced by streptozotocin (STZ), given i.v. 7-14 days before experiments. Optical mapping with the voltage-sensitive dye di-4-ANEPPS, using hearts perfused on a Langendorff apparatus, showed little change in baseline conduction velocity in diabetic hearts, reflecting the large reserve of function. However, both the gap junction uncoupler heptanol (0.5-1 mM) and elevated potassium (9 mM, to reduce cell excitability) produced a significantly greater slowing of impulse propagation in diabetic hearts than in controls. The maximal action potential upstroke velocity (an index of the sodium current) and resting potential was similar in single ventricular myocytes from control and diabetic rats, suggesting similar electrical excitability. Immunoblotting of connexin 43 (Cx43), a major gap junction component, showed no change in total expression. However, immunofluorescence labelling of Cx43 showed a significant redistribution, apparent as enhanced Cx43 lateralization. This was quantified and found to be significantly larger than in control myocytes. Labelling of two other gap junction proteins, N-cadherin and beta-catenin, showed a (partial) loss of co-localization with Cx43, indicating that enhancement of lateralized Cx43 is associated with non-functional gap junctions. In conclusion, conduction reserve is smaller in the diabetic heart, priming it for impaired conduction upon further challenges. This can desynchronize contraction and contribute to arrhythmogenesis.

Beta-, Not Alpha-Adrenergic Stimulation Enhances Conduction Velocity in Cultures of Neonatal Cardiomyocytes

During both cardiac maturation and myopathy, elevated levels of circulating catecholamines coincide with alterations in impulse propagation. An in vitro model of cultured cardiomyocytes was used to study the effects of adrenergic stimulation on the conduction characteristics of immature heart cells. Neonatal rat cardiomyocytes were cultured on preparations designed to measure conduction velocity (CV). CV was measured on the same preparation twice at t=0 and at t=24 h. Under control conditions (n=7), CV at t=0 (30.9+/-1.9 cm/s) and t=24 (32.4+/-4.4 cm/s) was similar (p=0.70). Immunohistochemistry revealed expression of the gap junction proteins connexin (Cx) 40, Cx43 and Cx45, with Cx43 being highly predominant. Stimulation for 24 h with the beta-adrenergic agonist isoproterenol (ISO) significantly increased CV from 28.0 +/-2.0 cm/s at t=0 to 34.8+/-2.2 cm/s at t=24 (p=0.002, n=5). Microelectrode recordings showed a faster upstroke of the action potential (AP) of ISO-treated cells. Reverse transcribed-polymerase chain reactions (RT-PCR) showed that ISO increased expression of SCN5A and alpha(1c) (alpha-subunit of the cardiac sodium and L-type calcium channel, respectively). Stimulation of cells with ISO did not induce alterations in distribution or expression of Cx40, Cx43 and Cx45 (both mRNA and protein), but slightly increased the phosphorylation of Cx43. Stimulation for 24 h with the alpha-adrenergic agonist phenylephrine did neither affect CV nor the expression of the connexin isoforms, SCN5A and alpha(1c). Alpha- and beta-adrenergic stimulation differently affect propagation of the electric impulse, which is primarily not caused by a differential effect on intercellular coupling. RT-PCR analysis and an enhanced AP upstroke velocity indicate a higher functional expression level of alpha(1c) and SCN5A in beta-adrenergic stimulated cells, which may explain the observed increase in CV.