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Functional human atrial myocytes derived from foetal atrial tissue can be massively expanded via a conditional cell-immortalization method, and used in in vitro models of atrial fibrillation.The lack of a scalable and robust source of well-differentiated human atrial myocytes constrains the development of in vitro models of atrial fibrillation (AF). Here we show that fully functional atrial myocytes can be generated and expanded one-quadrillion-fold via a conditional cell-immortalization method relying on lentiviral vectors and the doxycycline-controlled expression of a recombinant viral oncogene in human foetal atrial myocytes, and that the immortalized cells can be used to generate in vitro models of AF. The method generated 15 monoclonal cell lines with molecular, cellular and electrophysiological properties resembling those of primary atrial myocytes. Multicellular in vitro models of AF generated using the immortalized atrial myocytes displayed fibrillatory activity (with activation frequencies of 6-8 Hz, consistent with the clinical manifestation of AF), which could be terminated by the administration of clinically approved antiarrhythmic drugs. The conditional cell-immortalization method could be used to generate functional cell lines from other human parenchymal cells, for the development of in vitro models of human disease.

Purpose Several studies have indicated a potential role for SCN10A/NaV1.8 in modulating cardiac electrophysiology and arrhythmia susceptibility. However, by which mechanism SCN10A/NaV1.8 impacts on cardiac electrical function is still a matter of debate. To address this, we here investigated the functional relevance of NaV1.8 in atrial and ventricular cardiomyocytes (CMs), focusing on the contribution of NaV1.8 to the peak and late sodium current (INa) under normal conditions in different species. Methods The effects of the NaV1.8 blocker A-803467 were investigated through patch-clamp analysis in freshly isolated rabbit left ventricular CMs, human left atrial CMs and human-induced pluripotent stem cell-derived CMs (hiPSC-CMs). Results A-803467 treatment caused a slight shortening of the action potential duration (APD) in rabbit CMs and hiPSC-CMs, while it had no effect on APD in human atrial cells. Resting membrane potential, action potential (AP) amplitude, and AP upstroke velocity were unaffected by A-803467 application. Similarly, INa density was unchanged after exposure to A-803467 and NaV1.8-based late INa was undetectable in all cell types analysed. Finally, low to absent expression levels of SCN10A were observed in human atrial tissue, rabbit ventricular tissue and hiPSC-CMs. Conclusion We here demonstrate the absence of functional NaV1.8 channels in non-diseased atrial and ventricular CMs. Hence, the association of SCN10A variants with cardiac electrophysiology observed in, e.g. genome wide association studies, is likely the result of indirect effects on SCN5A expression and/or NaV1.8 activity in cell types other than CMs.

BACKGROUND: Monosodium glutamate (MSG), also referred to as Vetsin or E621, is a flavour enhancer frequently used in Asian cuisine and abundantly present in the famous Chinese dish Peking duck. MSG is notorious for triggering the onset of the so-called 'Chinese restaurant syndrome' (CRS), a complex of unpleasant symptoms, which might include flushing, sweating and the onset of atrial fibrillation (AF). This study aims to determine the effects of MSG on the occurrence of AF. METHODS: We conducted a placebo self-controlled single-arm study in the Academic Medical Centre in Amsterdam. We included paroxysmal AF patients who reported a consistent onset of AF upon MSG intake. During three admissions, participants were subsequently administered: placebo, 1.5 g and 3 g MSG. If AF was recorded after the dose of 1.5 g MSG, patients were given another placebo instead of 3 g MSG. The primary outcome was the onset of AF registered by 24-hour Holter monitoring. The secondary outcomes were any other arrhythmia and the onset of CRS defined as two or more symptoms of CRS after MSG intake. RESULTS: Six men participated in the study. Both 1.5 g and 3 g MSG were unrelated to CRS, arrhythmias or AF occurrence. CONCLUSION: Peking duck can be put on the Christmas menu without risking guests to be admitted to the emergency department with new episodes of AF.

Aims Selective inhibition of cardiac late sodium current (INaL) is an emerging target in the treatment of ventricular arrhythmias. We investigated the electrophysiological effects of GS-458967 (GS967), a potent, selective inhibitor of INaL, in an overlap syndrome model of both gain and loss of sodium channel function, comprising cardiomyocytes derived from both human SCN5A-1795insD+/- induced pluripotent stem cells (hiPSC-CMs) and mice carrying the homologous mutation Scn5a-1798insD+/-. Methods and results On patch-clamp analysis, GS967 (300 nmol/l) reduced INaL and action potential (AP) duration in isolated ventricular myocytes from wild type and Scn5a-1798insD+/- mice, as well as in SCN5A-1795insD+/- hiPSC-CMs. GS967 did not affect the amplitude of peak INa, but slowed its recovery, and caused a negative shift in voltage-dependence of INa inactivation. GS967 reduced AP upstroke velocity in Scn5a-1798insD+/- myocytes and SCN5A-1795insD+/- hiPSC-CMs. However, the same concentration of GS967 did not affect conduction velocity in Scn5a-1798insD+/- mouse isolated hearts, as assessed by epicardial mapping. GS967 decreased the amplitude of delayed after depolarizations and prevented triggered activity in mouse Scn5a-1798insD+/- cardiomyocytes. Conclusion The INaL inhibitor GS967 decreases repolarization abnormalities and has anti-arrhythmic effects in the absence of deleterious effects on cardiac conduction. Thus, selective inhibition of INaL constitutes a promising pharmacological treatment of cardiac channelopathies associated with enhanced INaL. Our findings furthermore implement hiPSC-CMs as a valuable tool for assessment of novel pharmacological approaches in inherited sodium channelopathies.

Atrial fibrillation (AF), the most common sustained heart rhythm disorder worldwide, is linked to dysfunction of the intrinsic cardiac autonomic nervous system (ICNS). The role of ICNS damage occurring during catheter-based treatment of AF, which is the therapy of choice for many patients, remains controversial. We show here that the neuronal injury marker S100B is expressed in cardiac glia throughout the ICNS and is released specifically upon catheter ablation of AF. Patients with higher S100B release were more likely to be AF free during follow-up. Subsequent in vitro studies revealed that murine intracardiac neurons react to S100B with diminished action potential firing and increased neurite growth. This suggests that release of S100B from cardiac glia upon catheter-based treatment of AF is a hallmark of acute neural damage that contributes to nerve sprouting and can be used to assess ICNS damage.

The cardiac autonomic nervous system (ANS) controls normal atrial electrical function. The cardiac ANS produces various neuropeptides, among which the neurokinins, whose actions on atrial electrophysiology are largely unknown. We here demonstrate that the neurokinin substance-P (Sub-P) activates a neurokinin-3 receptor (NK-3R) in rabbit, prolonging action potential (AP) duration through inhibition of a background potassium current. In contrast, ventricular AP duration was unaffected by NK-3R activation. NK-3R stimulation lengthened atrial repolarization in intact rabbit hearts and consequently suppressed arrhythmia duration and occurrence in a rabbit isolated heart model of atrial fibrillation (AF). In human atrial appendages, the phenomenon of NK-3R mediated lengthening of atrial repolarization was also observed. Our findings thus uncover a pathway to selectively modulate atrial AP duration by activation of a hitherto unidentified neurokinin-3 receptor in the membrane of atrial myocytes. NK-3R stimulation may therefore represent an anti-arrhythmic concept to suppress re-entry-based atrial tachyarrhythmias, including AF., The cardiac autonomic nervous system produces various neuropeptides, such as neurokinin substance-P (Sub-P), whose function remains largely unclear. Here, authors show that Sub-P causes a receptor-mediated prolongation of the atrial action potential through a reduced background potassium current, and prevents atrial fibrillation.

Background— Hypercholesterolemia protects against ventricular fibrillation in patients with myocardial infarction. We hypothesize that hypercholesterolemia protects against ischemia-induced reentrant arrhythmias because of altered ion channel function. Methods and Results— ECGs were measured in low-density lipoprotein receptor knockout (LDLr −/− ), apolipoprotein A1 knockout (ApoA1 −/− ), and wild-type (WT) mice. Action potentials, calcium handling, and ion currents were recorded in ventricular myocytes. Gene expression was determined by quantitative polymerase chain reaction and Western blot. In isolated perfused hearts, regional ischemia was induced and arrhythmia inducibility was tested. Serum low-density lipoprotein (LDL) cholesterol was higher in LDLr −/− mice than in WT mice (2.6 versus 0.4 mmol/L), and high-density lipoprotein cholesterol was significantly lower in ApoA1 −/− mice than in WT mice (0.3 versus 1.8 mmol/L). LDLr −/− and ApoA1 −/− myocytes contained more cholesterol than WT (34.4±2.8 and 36.5±2.4 versus 25.5±0.4 μmol/g protein). The major potassium currents were not different in LDLr −/− and ApoA1 −/− compared with WT mice. The L-type calcium current ( I Ca ), however, was larger in LDLr −/− and ApoA1 −/− than in WT (12.1±0.7 and 12.8±0.8 versus 9.4±1.1 pA/pF). Calcium transient amplitude and fractional sarcoplasmic reticulum calcium release were larger and action potential and QTc duration longer in LDLr −/− and ApoA1 −/− than in WT mice (action potential duration at 90% of repolarization: 102±4 and 106±3 versus 84±3.1 ms; QTc: 50.9±1.3 and 52.8±0.8 versus 43.5±1.2 ms). During ischemia, ventricular tachycardia/ventricular fibrillation inducibility was larger in WT than in LDLr −/− and ApoA1 −/− hearts. Expression of sodium channel and Ca-handling genes were not significantly different between groups. Conclusions— Dyscholesterolemia is associated with action potential prolongation because of increased I Ca and reduces occurrence of reentrant arrhythmias during ischemia.

In their editorial, “Matter of Fat: Are Lipids Antiarrhythmic?,” Drs Wan and Boyden1 comment on our article, “Dyscholesterolemia protects against ischemia-induced ventricular arrhythmias.”2 In our article, we have presented evidence that increased sarcolemmal cholesterol content provides an antiarrhythmic effect mediated through an increased Ca2+ current and a resulting prolongation of the ventricular action potential. In their editorial comment, the authors unduly attribute some statements to us and we feel the need to correct the authors on some of these points. First, Wan and Boyden state that we suggest that the observed …

Treatment with the anticancer drug taxol (TXL), which polymerizes the cytoskeleton protein tubulin, may evoke cardiac arrhythmias based on reduced human cardiac sodium channel (Na(v)1.5) function. Therefore, we investigated whether enhanced tubulin polymerization by TXL affects Na(v)1.5 function and expression and whether these effects are beta(1)-subunit-mediated. Human embryonic kidney (HEK293) cells, transfected with SCN5A cDNA alone (Na(v)1.5) or together with SCN1B cDNA (Na(v)1.5 + beta(1)), and neonatal rat cardiomyocytes (NRCs) were incubated in the presence and in the absence of 100 mu M TXL. Sodium current (I-Na) characteristics were studied using patch-clamp techniques. Na(v)1.5 membrane expression was determined by immunocytochemistry and confocal microscopy. Pre-treatment with TXL reduced peak I-Na amplitude nearly two-fold in both Na(v)1.5 and Na(v)1.5 + beta(1), as well as in NRCs, compared with untreated cells. Accordingly, HEK293 cells and NRCs stained with anti-Na(v)1.5 antibody revealed a reduced membrane-labelling intensity in the TXL-treated groups. In addition, TXL accelerated I-Na decay of Na(v)1.5 + beta(1), whereas I-Na decay of Na(v)1.5 remained unaltered. Finally, TXL reduced the fraction of channels that slow inactivated from 31% to 18%, and increased the time constant of slow inactivation by two-fold in Na(v)1.5. Conversely, slow inactivation properties of Na(v)1.5 + beta(1) were unchanged by TXL. Enhanced tubulin polymerization reduces sarcolemmal Na(v)1.5 expression and I-Na amplitude in a beta(1)-subunit-independent fashion and causes I-Na fast and slow inactivation impairment in a beta(1)-subunit-dependent way. These changes may underlie conduction-slowing-dependent cardiac arrhythmias under conditions of enhanced tubulin polymerization, e.g. TXL treatment and heart failure

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

Lethal ventricular arrhythmias are increasingly considered an important cause of sudden death in relatively young individuals. A genetic predisposition has been recognised in many cases, and research in the last decade has focused on underlying inherited mutations in cardiac ion channels.

Background— Patients carrying the cardiac sodium channel ( SCN5A ) mutation 1795insD show sudden nocturnal death and signs of multiple arrhythmia syndromes including bradycardia, conduction delay, QT prolongation, and right precordial ST-elevation. We investigated the electrophysiological characteristics of a transgenic model of the murine equivalent mutation 1798insD. Methods and Results— On 24-hour continuous telemetry and surface ECG recordings, Scn5a 1798insD/+ heterozygous mice showed significantly lower heart rates, more bradycardic episodes (pauses ≥500 ms), and increased PQ interval, QRS duration, and QTc interval compared with wild-type mice. The sodium channel blocker flecainide induced marked sinus bradycardia and/or sinus arrest in the majority of Scn5a 1798insD/+ mice, but not in wild-type mice. Epicardial mapping using a multielectrode grid on excised, Langendorff-perfused hearts showed preferential conduction slowing in the right ventricle of Scn5a 1798insD/+ hearts. On whole-cell patch-clamp analysis, ventricular myocytes isolated from Scn5a 1798insD/+ hearts displayed action potential prolongation, a 39% reduction in peak sodium current density and a similar reduction in action potential upstroke velocity. Scn5a 1798insD/+ myocytes displayed a slower time course of sodium current decay without significant differences in voltage-dependence of activation and steady-state inactivation, slow inactivation, or recovery from inactivation. Furthermore, Scn5a 1798insD/+ myocytes showed a larger tetrodotoxin-sensitive persistent inward current compared with wild-type myocytes. Conclusions— Mice carrying the murine equivalent of the SCN5A -1795insD mutation display bradycardia, right ventricular conduction slowing, and QT prolongation, similar to the human phenotype. These results demonstrate that the presence of a single SCN5A mutation is indeed sufficient to cause an overlap syndrome of cardiac sodium channel disease.

Background: Omega-3 polyunsaturated fatty acids (W-PUFAs) from fish oil reduce the risk of sudden death presumably by preventing life-threatening arrhythmias. Acutely administered omega 3-PUFAs modulate the activity of several cardiac ion channels, but the chronic effects of a diet enriched with fish oil leading to omega 3-PUFA-incorporation into the sarcolemma on membrane currents are unknown. Methods: Pigs received a diet either rich in W-PUFAs or in omega 9-fatty acids for 8 weeks. Ventricular myocytes (VMs) were isolated and used for patch-clamp studies. Results: omega 3-VMs contained higher amounts of omega 3-PUFAs and had a shorter action potential (AP) with a more negative plateau than control VM. In omega 3 VMs, L-type Ca2+ current (I-Ca,I-L) and Na+-Ca2+ exchange current (I-NCX) were reduced by approximately 20% and 60%, respectively, and inward rectifier K+ current (I-KI) and slow delayed rectifier K+ current (I-Ks) were increased by approximately 50% and 70%, respectively, compared to control. Densities of rapid delayed rectifier K+ current, Ca2+-activated Cl- current, and Na+ current (I-Na) were unchanged, although voltage-dependence of IN,, inactivation was more negative in omega 3 VMs. Conclusions: A fish oil diet increases omega 3-PUFA content in the ventricular sarcolemma, decreases I-Ca,I-L and I-NCX, and increases I-KI and I-Ks, resulting in AP shortening. Incorporation of W-PUFAs in the sarcolemma may have consequences for arrhythmias independent of circulating omega 3-PUFAs. (c) 2006 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.

Background and Aim: Selective inhibition of cardiac late sodium current (I Na,L ) is an emerging target in the treatment of ventricular arrhythmias. The electrophysiological effects of GS967, a potent I Na,L inhibitor, were investigated in an overlap syndrome model of both gain and loss of sodium channel function, comprising cardiomyocytes derived from human SCN5A -1795insD induced pluripotent stem cells (iPSC-CMs) and mice carrying the homologous mutation Scn5a -1798insD. Methods and Results: On patch-clamp analysis, isolated mouse Scn5a -1798insD cardiomyocytes and human SCN5A -1795insD iPSC-CMs showed decreased peak I Na and action potential (AP) upstroke velocity (Vmax) and increased I Na,L and AP duration at 90% repolarization (APD 90 ) as compared to wild-type. GS967 (50-300 nM) significantly decreased APD 90 in mouse Scn5a -1798insD cardiomyocytes by 8±2% (mean±SEM) at 50 nM (n=7), 13±3% at 100 nM (n=11) and 20±5% at 300 nM (n=6) (all p NaL in mouse Scn5a -1798insD cardiomyocytes (GS967-sensitive current of 0.7±0.1 pA/pF, n=6), but had no effect on peak I Na . Furthermore, GS967 (100 nM) suppressed fast (5 Hz) pacing-induced afterpotentials and triggered activity. In human SCN5A -1795insD iPSC-CMs (n=6), GS967 (300 nM) significantly reduced APD 90 without affecting the resting membrane potential or Vmax. In Langendorff-perfused, isolated mouse Scn5a -1798insD hearts (n=5), GS967 (300 nM) had no effect on ventricular activation time or conduction velocity (as assessed by epicardial mapping). Conclusion: Selective inhibition of I NaL by GS967 attenuated AP prolongation and prevented pro-arrhythmic activity in mouse Scn5a -1798insD cardiomyocytes and human SCN5A-1795insD iPSC-CMs, thus suppressing the gain-of-function features of this overlap syndrome mutation. Importantly, these beneficial actions of GS967 occurred in the absence of deleterious effects on sodium channel availability or cardiac conduction, despite a pre-existing decrease in peak I Na . Thus, selective inhibition of I Na,L constitutes a promising pharmacological treatment of cardiac channelopathies associated with enhanced I NaL , even in overlap syndromes whereby peak I Na is decreased.

Background. - Mutations in the gene encoding the human cardiac sodium channel (SCN5A) have been associated with three distinct cardiac arrhythmia disorders: the long QT syndrome, the Brugada syndrome and cardiac conduction disease. Here we report the biophysical features of a novel sodium channel mutation, E161K, which we identified in individuals of two non-related families with symptoms of bradycardia. sinus node dysfunction, generalized conduction disease and Brugada syndrome, or combinations thereof. Methods and results. - Wild-type (WT) or E 161 K sodium channel alpha-subunit and beta-subunit were cotransfected into tsA201 cells to study the functional consequences of mutant sodium channels. Characterization of whole-cell sodium current (IN.) using the whole cell patch-clamp technique revealed that the E 16 1 K mutation caused an almost threefold reduction in current density (P

Gender disparities in ECG variables and susceptibility to arrhythmia exist. The basis of these sex-related distinctions in cardiac electrophysiology has been extensively studied in various species, but is virtually unexplored in humans. The aim of this study was to clarify the cellular basis of electrophysiological gender disparities in human cardiac myocytes. Human midmyocardial left ventricular myocytes were isolated from explanted hearts of male and female patients in end-stage heart failure at the time of cardiac transplantation. The action potentials, sarcolemmal ion currents, and susceptibility to the generation of early afterdepolarizations were studied using whole-cell patch-clamp methodology. The functional effects of gender disparities in sarcolemmal ion currents were assessed by computer simulations using the Priebe-Beuckelmann or the ten Tusscher-Noble-Noble-Panfilov human ventricular cell models. Female myocytes had significantly longer action potentials and greater susceptibility to early afterdepolarizations than male myocytes. All other action potential parameters (resting membrane potential, amplitude, plateau level, upstroke velocity, maximal velocity of phase-1 and phase-3 repolarization) had similar values for both genders. In female myocytes, the transient outward potassium current (I(to1)) tended to be smaller, while the L-type calcium current (I(Ca,L)) and quasi-steady state current (I(QSS)) tended to be larger. Computer simulations showed that these subtle differences in sarcolemmal ion currents may conspire to cause the observed gender disparities in action potential properties. Female failing myocytes have longer action potentials and a greater susceptibility to early afterdepolarizations than male failing myocytes. These gender disparities may be due to slightly larger depolarizing I(Ca,L) in conjunction with slightly smaller repolarizing I(QSS) and I(to1) in female myocytes.

The human ether-a-go-go-related gene (HERG) encodes the rapid component of the cardiac delayed rectifier potassium current (I(Kr)). Per-Arnt-Sim domain mutations of the HERG channel are linked to type 2 long-QT syndrome. We studied wild-type and/or type 2 long-QT syndrome-associated mutant (R56Q) HERG current (I(HERG)) in HEK-293 cells, at both 23 and 36 degrees C. Conventional voltage-clamp analysis revealed mutation-induced changes in channel kinetics. To assess functional implication(s) of the mutation, we introduce the dynamic action potential clamp technique. In this study, we effectively replace the native I(Kr) of a ventricular cell (either a human model cell or an isolated rabbit myocyte) with I(HERG) generated in a HEK-293 cell that is voltage-clamped by the free-running action potential of the ventricular cell. Action potential characteristics of the ventricular cells were effectively reproduced with wild-type I(HERG), whereas the R56Q mutation caused a frequency-dependent increase of the action potential duration in accordance with the clinical phenotype. The dynamic action potential clamp approach also revealed a frequency-dependent transient wild-type I(HERG) component, which is absent with R56Q channels. This novel electrophysiological technique allows rapid and unambiguous determination of the effects of an ion channel mutation on the ventricular action potential and can serve as a new tool for investigating cardiac channelopathies.

Cardiac conduction disease (CCD) is a serious disorder of the heart. The pathophysiological mechanisms underlying CCD are diverse. In the last decade the genes responsible for several inherited cardiac diseases associated with CCD have been identified. If CCD is of an inherited nature (ICCD), its underlying mechanism can be either structural, functional or there can be overlap between these two mechanisms. If ICCD is structural in nature, it is often secondary to anatomical or histological abnormalities of the heart. Functional ICCD is frequently found as a "primary electrical disease" of the heart, i.e. resulting from functionally abnormal, or absent proteins encoded by mutated genes, often cardiac ion channel proteins involved in impulse formation. It can thus be hypothesised that patients with inherited structural or functional ICCD suffer from fundamentally different diseases. It is worthwhile to consider this hypothesis, since it could have implications for diagnosis, treatment, prognosis and, possibly, for the patient's relatives. In this review we aim to find evidence for the idea that functional and structural ICCD are fundamentally different diseases and, if so, whether this has diagnostic and clinical consequences. (c) 2005 Published by Elsevier Ltd on behalf of The European Society of Cardiology

Objective: Congenital long QT syndrome type 3 (LQT3) is an inherited cardiac arrhythmia disorder due to mutations in the cardiac sodium channel gene, SCN5A. Although most LQT3 mutations cause a persistent sodium current, increasing diversity in the disease mechanism is shown. Here we present the electrophysiological properties of the A1330T sodium channel mutation (DIIIS4-S5 linker). Like the A1330P, LQT3 mutation, A1330T, causes LQT3 in the absence of a persistent current. Methods: A1330T, A1330P and wild-type sodium channels were expressed in HEK-293 cells and characterized using the whole-cell configuration of the patch-clamp technique. Results: The A1330T mutation shifts positively the voltage-dependence of inactivation and speeds recovery from inactivation. Measurements of sodium window (I-Na,I- window) currents revealed a positive shift of the I-Na,I- window voltage range for both 1330 mutants, with in addition an increase in I-Na,I- window magnitude for the A1330P mutant. Action potential (AP) clamp experiments revealed that these changes in I-Na,I- window properties cause an increased inward current during the initial part of phase 4 repolarization of the AP. Conclusions: Our findings indicate that the alanine at position 1330 in the DIIIS4-S5 linker of the cardiac sodium channel has a role in channel fast inactivation. Substitution by a threonine shifts the voltage range of I-Na,I- window activity to more positive potentials. Here the counter-acting effect of outward K+ current is reduced and may delay AP repolarization, explaining the LQT3 phenotype. (C) 2005 European Society of Cardiology. Published by Elsevier B.V. All rights reserved

One variant of the long-QT syndrome (LQT3) is caused by mutations in the human cardiac sodium channel gene. In addition to the characteristic QT prolongation, LQT3 carriers regularly present with bradycardia and sinus pauses. Therefore, we studied the effect of the 1795insD Na + channel mutation on sinoatrial (SA) pacemaking. The 1795insD channel was previously characterized by the presence of a persistent inward current ( I pst ) at −20 mV and a negative shift in voltage dependence of inactivation. In the present study, we first additionally characterized I pst over the complete voltage range of the SA node action potential (AP) by measuring whole-cell Na + currents ( I Na ) in HEK-293 cells expressing either wild-type or 1795insD channels. I pst for 1795insD channels varied between 0.8±0.2% and 1.9±0.8% of peak I Na . Activity of 1795insD channels during SA node pacemaking was confirmed by AP clamp experiments. Next, I pst and the negative shift were implemented into SA node AP models. The −10-mV shift decreased sinus rate by decreasing diastolic depolarization rate, whereas I pst decreased sinus rate by AP prolongation, despite a concomitant increase in diastolic depolarization rate. In combination, moderate I pst (1% to 2%) and the shift reduced sinus rate by ≈10%. An additional increase in I pst could result in plateau oscillations and failure to repolarize completely. Thus, Na + channel mutations displaying an I pst or a negative shift in inactivation may account for the bradycardia seen in LQT3 patients, whereas SA node pauses or arrest may result from failure of SA node cells to repolarize under conditions of extra net inward current.

OBJECTIVE: The delayed phase of ventricular arrhythmias during acute ischemia (phase-1b arrhythmia) is associated with depletion of catecholamines and cell-to-cell uncoupling between depressed depolarized intramural ischemic region and surviving cells in subepicardium and subendocardium. In the present study we determined the effects of uncoupling and catecholamines on development of proarrhythmic afterdepolarizations. METHODS: Depressed depolarized ischemic region was simulated by a passive electronic circuit with a potential of -73, -53, -33 or -13 mV. Using patch-clamp methodology, single sheep Purkinje and ventricular cells were coupled to the simulated ischemic region via a variable conductance. By varying coupling conductance, we were able to selectively study the effects of various degrees of uncoupling. RESULTS: At strong coupling, cells were inexcitable and depolarized to potentials near those of the simulated ischemic region. Excitability, action potential duration and resting potential increased with progressive uncoupling. In a critical range of uncoupling, ventricular and "high-plateau" Purkinje cells developed early afterdepolarizations when the potential of the simulated ischemic region was -13 mV. Norepinephrine (1 microM) frequently induced early and delayed afterdepolarizations in both ventricular and Purkinje cells, but these afterdepolarizations were only present during uncoupling when the potential of the simulated ischemic region was -33 mV or more positive. CONCLUSIONS: In a critical range of uncoupling, afterdepolarizations were present when the potential of the simulated ischemic region was -33 or -13 mV, suggesting that triggered activity plays a role in phase-1b arrhythmias when surviving layers uncouple from a highly depolarized intramural ischemic region

Background Animal studies have shown that the Ca 2+ -activated Cl − current ( I Cl(Ca) ) and the Na + /Ca 2+ exchange current ( I Na/Ca ) contribute to the transient inward current ( I ti ). I ti is responsible for the proarrhythmic delayed afterdepolarizations (DADs). We investigated the ionic mechanism of I ti and DADs in human cardiac cells. Methods and Results Human ventricular cells were enzymatically isolated from explanted hearts of patients with end-stage heart failure and studied with patch-clamp methodology. I ti s were elicited in the presence of 1 μmol/L norepinephrine by trains of repetitive depolarizations from −80 to +50 mV. DADs were induced in the presence of 1 μmol/L norepinephrine at a stimulus frequency of 1 Hz. I ti currents were inwardly directed over the voltage range between −110 and + 50 mV. Neither the Cl − channel blocker 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid nor changes in [Cl − ] i affected I ti or DAD amplitude. This excludes an important role for I Cl(Ca) . Blockade of Na + /Ca 2+ exchange by substitution of all extracellular Na + by Li + , conversely, completely inhibited I ti . In rabbit, I Cl(Ca) density in ventricular cells isolated from control hearts did not differ significantly from that in ventricular cells isolated from failing hearts. Conclusions In contrast to many animal species, I ti and DADs in human ventricular cells from failing hearts consist only of I Na/Ca . In rabbits, heart failure per se does not alter I Cl(Ca) density, suggesting that I Cl(Ca) may also be absent during DADs in nonfailing human ventricular cells.

Background —The ionic mechanism underlying the transient inward current ( I ti ), the current responsible for delayed afterdepolarizations (DADs), appears to be different in ventricular myocytes and Purkinje fibers. In ventricular myocytes, I ti was ascribed to a Na + -Ca 2+ exchange current, whereas in Purkinje fibers, it was additionally ascribed to a Cl − current and a nonselective cation current. If Cl − current contributes to I ti and thus to DADs, Cl − current blockade may be potentially antiarrhythmogenic. In this study, we investigated the ionic nature of I ti in single sheep Purkinje and ventricular myocytes and the effects of Cl − current blockade on DADs. Methods and Results —In whole-cell patch-clamp experiments, I ti was induced by repetitive depolarizations from −93 to +37 mV in the presence of 1 μmol/L norepinephrine. In both Purkinje and ventricular myocytes, I ti was inward at negative potentials and outward at positive potentials. The anion blocker 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) blocked outward I ti completely but inward I ti only slightly. The DIDS-sensitive component of I ti was outwardly rectifying, with a reversal close to the reversal potential of Cl − currents. Blockade of Na + -Ca 2+ exchange by substitution of extracellular Na + by equimolar Li + abolished the DIDS-insensitive component of I ti . DIDS reduced both DAD amplitude and triggered activity based on DADs. Conclusions —In both Purkinje and ventricular myocytes, I ti consists of 2 ionic mechanisms: a Cl − current and a Na + -Ca 2+ exchange current. Blockade of the Cl − current may be potentially antiarrhythmogenic by lowering DAD amplitude and triggered activity based on DADs.

OBJECTIVE: Injury current (I(injury)) and afterdepolarizations are thought to play an important role in arrhythmias that occur during acute ischemia. However, little is known about the effects of I(injury) on afterdepolarizations. The present study was designed to study the effect of I(injury) on afterdepolarizations and action potentials in single human ventricular cells. METHODS: The patch-clamp technique was used to record action potentials and to apply I(injury) to human ventricular cells. In these cells, early and delayed afterdepolarizations (EADs and DADs) were induced by 1 microM norepinephrine. I(injury) was simulated by coupling cells via a variable coupling resistance to a passive resistance circuit with a potential of 0, -20, or -40 mV, mimicking a depolarized ischemic region. RESULTS: At all potentials, I(injury) induced depolarization of the resting membrane potential and action potential shortening. Flowing from 0 mV, I(injury) induced EADs by itself and aggravated the EADs and DADs that were induced by norepinephrine. Flowing from -40 mV, I(injury) abolished the noradrenaline-induced EADs and DADs. CONCLUSIONS: Our results demonstrate that I(injury) may either prevent or promote the occurrence of afterdepolarizations in human ventricle. The latter holds if conduction is slowed to such an extent that it permits flow of current from depolarized ischemic cells at plateau level to cells in phase 3 or phase 4

Membrane potentials and currents of isolated sheep Purkinje and ventricular cells were compared using patch-clamp and microelectrode techniques. In ∼50% of Purkinje cells, we observed action potentials that showed a prominent phase 1 repolarization and relatively negative plateau (LP cells). Action potential configuration of the remaining Purkinje cells was characterized by little phase 1 repolarization and relatively positive plateau (HP cells). Microelectrode impalement of Purkinje strands also revealed these two types of action potential configuration. In LP cells, the density of L-type Ca2+ current ( I Ca,L) was lower, whereas the density of transient outward K+ current was higher, than in HP cells. Action potentials of HP cells strongly resembled those of ventricular cells. Densities of inward rectifier current and I Ca,L were significantly higher in ventricular cells compared with densities in both LP and HP Purkinje cells. Differences in current densities explain the striking differences in action potential configuration and the stimulus frequency dependency thereof that we observed in LP, HP, and ventricular cells. We conclude that LP Purkinje cells, HP Purkinje cells, and ventricular cells of sheep each have a unique action potential configuration.

Testing cardiac gene and cell therapies in vitro requires a tissue substrate that survives for several days in culture while maintaining its physiological properties. The purpose of this study was to test whether culture of intact cardiac tissue of neonatal rat ventricles (organ explant culture) may be used as a model to study gene and cell therapy. We compared (immuno) histology and electrophysiology of organ explant cultures to both freshly isolated neonatal rat ventricular tissue and monolayers. (Immuno) histologic studies showed that organ explant cultures retained their fiber orientation, and that expression patterns of α-actinin, connexin-43, and α-smooth muscle actin did not change during culture. Intracellular voltage recordings showed that spontaneous beating was rare in organ explant cultures (20%) and freshly isolated tissue (17%), but common (82%) in monolayers. Accordingly, resting membrane potential was -83.9±4.4 mV in organ explant cultures, −80.5±3.5 mV in freshly isolated tissue, and −60.9±4.3 mV in monolayers. Conduction velocity, measured by optical mapping, was 18.2±1.0 cm/s in organ explant cultures, 18.0±1.2 cm/s in freshly isolated tissue, and 24.3±0.7 cm/s in monolayers. We found no differences in action potential duration (APD) between organ explant cultures and freshly isolated tissue, while APD of monolayers was prolonged (APD at 70% repolarization 88.8±7.8, 79.1±2.9, and 134.0±4.5 ms, respectively). Organ explant cultures and freshly isolated tissue could be paced up to frequencies within the normal range for neonatal rat (CL 150 ms), while monolayers could not. Successful lentiviral (LV) transduction was shown via Egfp gene transfer. Co-culture of organ explant cultures with spontaneously beating cardiomyocytes increased the occurrence of spontaneous beating activity of organ explant cultures to 86%. We conclude that organ explant cultures of neonatal rat ventricle are structurally and electrophysiologically similar to freshly isolated tissue and a suitable new model to study the effects of gene and cell therapy.

Inhibition of cell metabolism is associated with significant changes in action potential duration. The aim of this study was to investigate the time course of the changes in action potential duration during metabolic inhibition and to determine what changes in membrane currents are responsible. The amphotericin perforated patch clamp technique was used to study membrane currents and voltage in single rabbit and human ventricular myocytes. In all myocytes inhibition of cell metabolism, induced by hypoxia (PO2

Background Previous studies have shown that in heart there are two kinetically distinct components of delayed rectifier current: a rapidly activating component (I Kr ) and a more slowly activating component (I Ks ). The presence of I Kr and/or I Ks appears to be species dependent. We studied the nature of the delayed rectifier current in human ventricle in whole-cell and single-channel experiments. Methods and Results Ventricular myocytes were obtained from hearts of patients with ischemic or dilated cardiomyopathy. Single-channel currents and whole-cell tail currents were recorded at negative potentials directly after return from a depolarizing step. Single-channel currents were measured in the cell-attached patch configuration with 140 mmol/L K + in the pipette. In the present study, we identified a voltage-dependent channel with a single-channel conductance of 12.9±0.8 pS (mean±SEM, n=5) and a reversal potential near to the K + equilibrium potential, suggesting that the channel is selective to K + ions. Channel activity was observed only after a depolarizing step and increased with the duration and amplitude of the depolarization, indicating time- and voltage-dependent activation. Activation at +30 mV was complete within 300 milliseconds, and the time constant of activation, determined in the whole-cell configuration, was 101±25 milliseconds (mean±SEM, n=4). The voltage dependence of activation could be described by a Boltzmann equation with a half-activation potential of −29.9 mV and a slope factor of 9.5 mV. The addition of the class III antiarrhythmic drug E-4031 completely blocked channel activity in one patch. No indications for the presence of I Ks were found in these experiments. Conclusions The conformity between the properties of I Kr and those of the K + channel in the present study strongly suggests that I Kr is present in human ventricle.

Rationale: The SCN10A gene encodes the neuronal sodium channel isoform Na V 1.8. Several recent genome-wide association studies have linked SCN10A to PR interval and QRS duration, strongly suggesting an as-yet unknown role for Na V 1.8 in cardiac electrophysiology. Objective: To demonstrate the functional presence of SCN10A /Nav1.8 in intracardiac neurons of the mouse heart. Methods and Results: Immunohistochemistry on mouse tissue sections showed intense Na V 1.8 labeling in dorsal root ganglia and intracardiac ganglia and only modest Na V 1.8 expression within the myocardium. Immunocytochemistry further revealed substantial Na V 1.8 staining in isolated neurons from murine intracardiac ganglia but no Na V 1.8 expression in isolated ventricular myocytes. Patch-clamp studies demonstrated that the Na V 1.8 blocker A-803467 (0.5–2 μmol/L) had no effect on either mean sodium current (I Na ) density or I Na gating kinetics in isolated myocytes but significantly reduced I Na density in intracardiac neurons. Furthermore, A-803467 accelerated the slow component of current decay and shifted voltage dependence of inactivation toward more negative voltages, as expected for blockade of Na V 1.8-based I Na . In line with these findings, A-803467 did not affect cardiomyocyte action potential upstroke velocity but markedly reduced action potential firing frequency in intracardiac neurons, confirming a functional role for Na V 1.8 in cardiac neural activity. Conclusions: Our findings demonstrate the functional presence of SCN10A /Na V 1.8 in intracardiac neurons, indicating a novel role for this neuronal sodium channel in regulation of cardiac electric activity.

The autonomic nervous system controls heart rate and contractility through sympathetic and parasympathetic inputs to the cardiac tissue, with acetylcholine (ACh) and noradrenalin (NA) as the chemical transmitters. In recent years, it has become clear that specific Regulators of G protein Signalling proteins (RGS proteins) suppress muscarinic sensitivity and parasympathetic tone, identifying RGS proteins as intriguing potential therapeutic targets. In the present study, we have identified the effects of 1 µM ACh and 1 µM NA on the intrinsic action potentials of sinotrial (SA) nodal and atrial myocytes. Single cells were enzymatically isolated from the SA node or from the left atrium of rabbit hearts. Action potentials were recorded using the amphotericin-perforated patch-clamp technique in the absence and presence of ACh, NA or a combination of both. In SA nodal myocytes, ACh increased cycle length and decreased diastolic depolarization rate, whereas NA decreased cycle length and increased diastolic depolarization rate. Both ACh and NA increased maximum upstroke velocity. Furthermore, ACh hyperpolarized the maximum diastolic potential. In atrial myocytes stimulated at 2 Hz, both ACh and NA hyperpolarized the maximum diastolic potential, increased the action potential amplitude, and increased the maximum upstroke velocity. Action potential duration at 50 and 90% repolarization was decreased by ACh, but increased by NA. The effects of both ACh and NA on action potential duration showed a dose dependence in the range of 1–1,000 nM, while a clear-cut frequency dependence in the range of 1–4 Hz was absent. Intermediate results were obtained in the combined presence of ACh and NA in both SA nodal and atrial myocytes. Our data uncover the extent to which SA nodal and atrial action potentials are intrinsically dependent on ACh, NA or a combination of both and may thus guide further experiments with RGS proteins.

See article by Deschenes et al. [3] (pages 55–65) in this issue. Successfully resuscitated patients without structural cardiac abnormalities (despite extensive investigations) are diagnosed as suffering from “primary electrical disease”. Within this group of patients the Long QT syndrome (LQTS) and Brugada syndrome are electrocardiographically discernible, although the abnormalities may be difficult to appreciate. Both syndromes share an autosomal dominant transmission based on mutations in ion channel genes and a high incidence of sudden cardiac death [1]. The number of disease-related mutations in various genes is growing rapidly, challenging the scientific community to get insight into channel (dys-)function and its relation to these diseases. In HERG for example, different mutations, ultimately leading to functionally less repolarizing currents, appear to affect channel function in different ways [2]. Such data are important because they relate to human disease and may shed light on the pathophysiology of the relevant arrhythmia syndromes. In addition, this information will also undoubtedly advance the study of the pathophysiological basis of lethal arrhythmias in more common acquired heart disease. The manuscript of Deschenes et al. in this issue [3] describes human ion channel gene mutations, identified in individual patients with LQTS or Brugada syndrome, and their impact on channel function. Before going into the details of the study one should address the question whether the mutations identified are indeed disease-related. For a correct genetic diagnosis linkage data are pivotal. A LOD-score >3, leaving a change of

AIMS: Cardiac voltage-gated sodium channels control action potential (AP) upstroke and cell excitability. Intracellular calcium (Ca(i)(2+)) regulates AP properties by modulating various ion channels. Whether Ca(i)(2+) modulates sodium channels in ventricular myocytes, is unresolved. We studied whether Ca(i)(2+) modulates sodium channels in ventricular myocytes at Ca(i)(2+) concentrations ([Ca(i)(2+)]) present during the cardiac AP (0-500 nM), and how this modulation affects sodium channel properties in heart failure (HF), a condition in which Ca(i)(2+) homeostasis is disturbed. METHODS: Sodium current (I(Na)) and maximal AP upstroke velocity (dV/dt(max)), a measure of I(Na), were studied at 20 masculineC and 37 masculineC, respectively, in freshly isolated left ventricular myocytes of control and HF rabbits, using whole-cell patch-clamp methodology. [Ca(i)(2+)] was varied using different pipette solutions, the Ca(i)(2+) buffer BAPTA, and caffeine administration. RESULTS: Elevated [Ca(i)(2+)] reduced I(Na) density and dV/dt(max), but caused no I(Na) gating changes. Reductions in I(Na) density occurred simultaneously with increases in [Ca(i)(2+)], suggesting that these effects were due to permeation block. Accordingly, unitary sodium current amplitudes were reduced at higher [Ca(i)(2+)]. While I(Na) density and gating at fixed [Ca(i)(2+)] were not different between HF and control, reductions in dV/dt(max) upon increases in stimulation rate were larger in HF than in control; these differences were abolished by BAPTA. CONCLUSION: Ca(i)(2+) exerts acute modulation of I(Na) density in ventricular myocytes, but does not modify I(Na) gating. These effects, occurring rapidly and in the [Ca(i)(2+)] range observed physiologically, may contribute to beat-to-beat regulation of cardiac excitability in health and disease

Objective: Mutations in SCN5A, the gene encoding the a-subunit of the cardiac sodium channel (Na(v)1.5), have been associated with various inherited arrhythmia syndromes, including Brugada syndrome (BrS). Here, we report the functional consequences of a novel missense SCN5A mutation, G1319V, identified in a BrS patient. The G1319V mutation is located in the loop connecting transmembrane segments 4 and 5 in domain III (DIIIS4-S5), a region so far considered to be exclusively involved in fast inactivation. Methods: Whole-cell mutant (G1319V) and wild-type (WT) sodium currents (1(Na)) were studied in the Human Embryonic Kidney cell line (HEK-293) transfected with Na(v)1.5 alpha-subunit cDNA (WT or mutant) together with h beta(1)-subunit cDNA, using the patch-clamp technique. Results: Maximal peak IN and persistent sodium current were similar in WT and channel G1319V channels. The G1319V mutation shifted the potential of half-maximal (V-1/2) activation towards more positive potentials (+3.7 mV), thereby increasing the degree of depolarization required for activation. The V-1/2 of inactivation of G1319V channels was shifted by -6.0 mV compared to WT, resulting in a reduced channel availability. The change in the steady-state inactivation was completely due to a negative shift (-6.8 mV) of the voltage-dependence of slow inactivation, while the voltage-dependence of fast inactivation was unaffected. The fast component of recovery from inactivation of G1319V channels was slowed down. Finally, the G1319V mutation caused a two-fold increase in the propensity of the channels to enter the slow inactivated state. Reduction in IN, peak amplitude on repetitive depolarizations at short interpulse intervals (40 ms) was significantly more pronounced in G1319V compared to WT. Accordingly, carriers of the G1319V mutation showed marked QRS widening upon increases in heart rate during exercise testing, pointing to enhancement of slow inactivation. Conclusions: We identified the DIIIS4-S5 linker as a new region involved in slow inactivation of Na(v)1.5. The biophysical alterations of the G1319V mutation all contribute to a reduction in 1(Na), in line with the proposed mechanism underlying BrS. (c) 2007 Published by Elsevier B.V on behalf of European Society of Cardiology

Type 3 of the long-QT syndrome (LQT3) is caused by mutations in the human cardiac sodium channel gene (SCN5A). LQT3 carriers regularly present with bradycardia and sinus pauses. This has been reported for carriers of the /spl Delta/K1500, /spl Delta/KPQ, E1784K, D1790G, and 1795insD mutations, which all show a persistent inward sodium current and/or a negative shift in voltage dependence of inactivation. The 1795insD channel had previously been characterized by a -10 mV. We hypothesized that the relative bradycardia that has been reported for carriers of the 1795insD mutation is, at least in part, due to intrinsic slowing of the sinus node. Therefore, we carried out computer simulations to test the effects of the 1795insD mutation on the intrinsic pacemaker activity of sinus node cells. We found that the negative shift in inactivation reduces sinus rate by slowing diastolic depolarization, whereas the persistent inward current reduces sinus rate by an increase in action potential duration, and that these effects are almost additive. Also, sinus node cells may fail to repolarize. We conclude that sodium channel mutations may account for the relative bradycardia and sinus node dysfunction seen in LQT3 patients.

At the Purkinje (P) - ventricular (V) junction a zone of "transitional (T)" cells is found. In the present study we investigated the role of these T cells in P-to-V conduction. Using the "model clamp" technique, an experimentally recorded rabbit P cell was coupled to a phase-2 Luo and Rudy (LR) model cell, which in turn was coupled to a strand of phase-2 LR model cells. In our experiments, the single LR model represents the T cell, while the strand of LR models represents subendocardial V cells. This approach enabled us to change selectively coupling conductance (G/sub c/) between cells, presence or T cell, and relative size of cells. We demonstrated that: 1) a decrease of G/sub c/ between P-T and T-V increases the delay of V activation, 2) the delay of V activation is importantly due to conduction between T and V cells, 3) there is a critical Cc for successful conduction at the P-V junction, 4) the critical value of Cc for conduction at the P-V junction is lower in presence (11.0/spl plusmn/0.7 nS) than in absence (13.7/spl plusmn/0.8 nS) of the T cell, and 5) enlargement of the T zone size hampers successful P-to-V conduction.

AIMS: Congestive heart failure is characterized by high levels of norepinephrine which is considered to be arrhythmogenic. It is unclear whether increased norepinephrine is only a marker of the severity of heart failure or whether it directly triggers ventricular arrhythmias. METHODS AND RESULTS: Ventricular myocytes were isolated from eight explanted hearts of patients with end-stage heart failure (ischaemic or dilated cardiomyopathy). With the whole-cell configuration of the patch-clamp technique the effect of 1 micromol x l(-1)norepinephrine on action potentials and membrane currents was studied. The cells had a membrane capacitance of 256 +/- 25 pF (n = 26) and action potential duration (APD90) during control conditions was 620 +/- 45 ms at 1 Hz (n = 14). Norepinephrine induced action potential prolongation in all cells and early afterdepolarizations in 50% of them. Norepinephrine significantly increased the calcium current but had no effect on the delayed rectifier current, the inward rectifier current or the transient outward current. Norepinephrine also significantly increased the steady-state calcium window-current measured between -40 and 0 mV. CONCLUSIONS: In contrast to many animal species, norepinephrine induces action potential prolongation in ventricular myocytes from human failing hearts, as well as early afterdepolarization, by an increase in both the calcium peak current and window current. Thus norepinephrine seems to be an important arrhythmogenic factor in congestive heart failure

Background — Congenital long QT syndrome (LQTS), a cardiac ion channel disease, is an important cause of sudden cardiac death. Prolongation of the QT interval has recently been associated with sudden infant death syndrome, which is the leading cause of death among infants between 1 week and 1 year of age. Available data suggest that early onset of congenital LQTS may contribute to premature sudden cardiac death in otherwise healthy infants. Methods and Results — In an infant who died suddenly at the age of 9 weeks, we performed mutation screening in all known LQTS genes. In the surface ECG soon after birth, a prolonged QTc interval (600 ms 1/2 ) and polymorphic ventricular tachyarrhythmias were documented. Mutational analysis identified a missense mutation (Ala1330Pro) in the cardiac sodium channel gene SCN5A , which was absent in both parents. Subsequent genetic testing confirmed paternity, thus suggesting a de novo origin. Voltage-clamp recordings of recombinant A1330P mutant channel expressed in HEK-293 cells showed a positive shift in voltage dependence of inactivation, a slowing of the time course of inactivation, and a faster recovery from inactivation. Conclusions — In this study, we report a de novo mutation in the sodium channel gene SCN5A , which is associated with sudden infant death. The altered functional characteristics of the mutant channel was different from previously reported LQTS3 mutants and caused a delay in final repolarization. Even in families without a history of LQTS, de novo mutations in cardiac ion channel genes may lead to sudden cardiac death in very young infants.

Abstract —The congenital long-QT syndrome (LQT3) and the Brugada syndrome are distinct, life-threatening rhythm disorders linked to autosomal dominant mutations in SCN5A , the gene encoding the human cardiac Na + channel. It is believed that these two syndromes result from opposite molecular effects: LQT3 mutations induce a gain of function, whereas Brugada syndrome mutations reduce Na + channel function. Paradoxically, an inherited C-terminal SCN5A mutation causes affected individuals to manifest electrocardiographic features of both syndromes: QT-interval prolongation (LQT3) at slow heart rates and distinctive ST-segment elevations (Brugada syndrome) with exercise. In the present study, we show that the insertion of the amino acid 1795insD has opposite effects on two distinct kinetic components of Na + channel gating (fast and slow inactivation) that render unique, simultaneous effects on cardiac excitability. The mutation disrupts fast inactivation, causing sustained Na + current throughout the action potential plateau and prolonging cardiac repolarization at slow heart rates. At the same time, 1795insD augments slow inactivation, delaying recovery of Na + channel availability between stimuli and reducing the Na + current at rapid heart rates. Our findings reveal a novel molecular mechanism for the Brugada syndrome and identify a new dual mechanism whereby single SCN5A mutations may evoke multiple cardiac arrhythmia syndromes by influencing diverse components of Na + channel gating function. The full text of this article is available at http://www.circresaha.org.

See article by Jiang et al. [1] (pages 34–43) in this issue. The paper of Jiang et al. [1] in this issue of Cardiovascular Research – ‘Delayed rectifier K currents have reduced amplitudes and altered kinetics in myocytes from infarcted canine ventricle’ – forms the coping-stone in a long series of investigations into the mechanisms underlying arrhythmias post myocardial infarction (in terms of altered ionic currents). In this paper, delayed rectifier currents were characterized in ventricular myocytes isolated from the surviving layer of epicardium overlying a 5 day-old infarct, the epicardial borderzone (EBZ). This canine model of myocardial infarction, caused by complete occlusion of the left anterior descending coronary artery, has been thoroughly studied over the past decades. It has been demonstrated that during the healing phase post infarction (days to weeks after coronary occlusion), inducible ventricular tachycardias are a consequence of reentrant excitation localized to this EBZ (see for review [2]). Previous experimental studies on whole hearts and multicellular preparations already indicated important changes in cellular electrophysiology in the EBZ besides changes in anatomy relevant for reentry. Electrophysiological properties of the surviving epicardial fibers 5 days after coronary occlusion are characterized by a reduction in resting membrane potential, action potential amplitude and Vmax of phase 0 (upstroke velocity), partly contributing to slowed conduction. Additionally, action potentials had a brief plateau phase and thereby action potential duration was decreased [2,3]. Despite the latter, prolonged effective and relative refractory periods were observed … * Tel.: +31-20-566-3269; fax: +31-20-697-5458 m.w.veldkamp{at}amc.uva.nl

OBJECTIVE: Regulation of ion channel function in heart has been shown to be affected by changes in the cellular environment. Recently it was shown that rabbit ventricular myocytes kept in primary culture, show a strong reduction in inward rectifier current (IK1). The aim of the present study was to elucidate the mechanism underlying this decrease in IK1, using single-channel measurements. In addition, we studied the effects of primary culture on the ATP-regulated K+ (K.ATP) channel, also a member of the inwardly rectifying K+ channel family. METHODS: Adult rabbit ventricular myocytes were cultured for up to 3 days in Ham's F-10 medium complemented with 1% rabbit serum and 5% glutamine. IK1 and K.ATP channel activity was studied in the inside-out patch configuration of the patch-clamp technique with equimolar K+ concentrations (140 mM K+) on the intra- and extracellular side. Single channel characteristics were determined at various times during culture and compared to those present in freshly isolated myocytes. RESULTS: IK1 channels in freshly isolated myocytes (day 0) had a single-channel conductance of 56.1 +/- 2.5 pS (mean +/- SEM) and an open probability of 0.64 +/- 0.05 (mean +/- SEM). Neither the single-channel conductance nor the open probability (Po) underwent significant changes during culture. The mean number of channels per patch, however, was drastically reduced from 1.2 +/- 0.3 (mean +/- SEM) at day 0 to 0.17 +/- 0.06 at day three. K.ATP channel density and open probability, on the other hand, were both increased with an optimum at day two. Po increased from 0.27 +/- 0.06 at day 0 to 0.63 +/- 0.06 at day three. The mean number of channels per patch was 2.29 +/- 0.57 and 3.25 +/- 0.48 at days 0 and 3 respectively. The unitary current amplitude at -50 mV remained unchanged, suggesting no change in the K.ATP single-channel conductance. CONCLUSIONS: The decrease in IK1 in rabbit ventricular myocytes as has been observed during primary culture is the result of a reduction in the number of active channels and not of altered kinetic or conductive channel properties. The increase in K.ATP channel activity under the same conditions suggests that gene expression of both channel types is differently regulated

See article by Cheng et al. ([1], pages 135–147) in this issue. In this issue of Cardiovascular Research Cheng et al. [1] demonstrate that the electrical properties of single cells isolated from apex and base are different. They show that action potentials elicited at 1 Hz are 40 ms shorter in ventricular myocytes isolated from the base than in myocytes isolated from the apex. This is in line with earlier measurements of the same group. In epicardial electrograms of Langendorff-perfused rabbits hearts they found different QT intervals between ventricular apex and base [2]. Cheng et al. [1] found that tail currents in basal myocytes were larger than in apical myocytes. This indicates that delayed rectifier current I K is larger in basal cells and it explains the shorter basal action potential. Ventricular action potentials may differ regionally. Action potentials from subendocardial myocytes are longer than those from subepicardial myocytes. This difference in action potential duration is probably caused by a higher density of the transient outward current I TO in subepicardial myocytes [3–7]. Also the different rate-dependent properties of I TO between in subepi- and subendocardial myocytes may play a role [6]. Different action potential configurations also exist between left and right epicardium [8]. The most likely cause is again a different I TO density. The delayed recifier current I K is composed of two components, a rapidly activating I Kr and a slowly activating I Ks. Cheng et al. [1] found that I Kr is the largest component of I K in apical myocytes and that in basal myocytes I Ks is the largest component. These findings show that I Kr and I Ks are regionally … * Corresponding author. Tel.: +31-20-566-4644; fax: +31-20-691-9319 a.c.vanginneken{at}amc.uva.nl

An important method to study the behavior of ion channels is the patch-clamp technique, developed by Neher and Sakmann (1976). This method allows clamping of a voltage difference across the cell membrane and, at the same time, measurement of the currents that flow through the ion channels as result of the applied voltage difference. With this technique one can either measure the sum of currents flowing through the channels in the entire cell membrane, referred to as wholecell configuration (Fig. 10.1A), or one can measure the very small currents through single channels in a patch of cell membrane (Fig. 10.1B-D). In the cell-attached configuration this patch of membrane is still attached to the intact cell (Fig. 10.1B), in the inside-out configuration and outside-out configuration the patch of membrane is excised from the cell (Fig. 10.1C, D).