Antoni C.G. van Ginneken

Human SCN5A gene mutations alter cardiac sodium channel kinetics and are associated with the Brugada syndrome.

BACKGROUND Primary dysrhythmias other than those associated with the long QT syndrome, are increasingly recognized. One of these are represented by patients with a history of resuscitation from cardiac arrest but without any structural heart disease. These patients exhibit a distinct electrocardiographic (ECG) pattern consisting of a persistent ST-segment elevation in the right precordial leads often but not always accompanied by a right bundle branch block (Brugada syndrome). This syndrome is associated with a high mortality rate and has been shown to display familial occurrence. METHODS AND RESULTS Pharmacological sodium channel blockade elicits or worsens the electrocardiographic features associated with this syndrome. Hence, a candidate gene approach directed towards SCN5A, the gene encoding the alpha-subunit of the cardiac sodium channel, was followed in six affected individuals. In two patients missense mutations were identified in the coding region of the gene: R1512W in the DIII-DIV cytoplasmic linker and A1924T in the C-terminal cytoplasmic domain. In two other patients mutations were detected near intron/exon junctions. To assess the functional consequences of the R1512W and A1924T mutations, wild-type and mutant sodium channel proteins were expressed in Xenopus oocytes. Both missense mutations affected channel function, most notably a 4-5 mV negative voltage shift of the steady-state activation and inactivation curves in R1512W and a 9 mV negative voltage shift of the steady-state activation curve in A1924T, measured at 22 degrees C. Recovery from inactivation was slightly prolonged for R1512W channels. The time dependent kinetics of activation and inactivation at -20 mV were not significantly affected by either mutation. CONCLUSIONS Two SCN5A mutations associated with the Brugada syndrome, significantly affect cardiac sodium channel characteristics. The alterations seem to be associated with an increase in inward sodium current during the action potential upstroke.

Overlap Syndrome of Cardiac Sodium Channel Disease in Mice Carrying the Equivalent Mutation of Human SCN5A-1795insD

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, Scn5a1798insD/+ 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 Scn5a1798insD/+ 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 Scn5a1798insD/+ hearts. On whole-cell patch-clamp analysis, ventricular myocytes isolated from Scn5a1798insD/+ hearts displayed action potential prolongation, a 39% reduction in peak sodium current density and a similar reduction in action potential upstroke velocity. Scn5a1798insD/+ 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, Scn5a1798insD/+ 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.

Effects of Delayed Rectifier Current Blockade by E-4031 on Impulse Generation in Single Sinoatrial Nodal Myocytes of the Rabbit

The role of the delayed rectifier current (IK) in impulse generation was studied in single sinoatrial nodal myocytes of the rabbit. We used the class III antiarrhythmic drug E-4031, which blocks IK in rabbit ventricular myocytes. In single sinoatrial nodal cells, E-4031 (0.1 mumol/L) significantly prolonged cycle length and action potential duration, depolarized maximum diastolic potential, and reduced both the upstroke velocity of the action potential and the diastolic depolarization rate. Half of the cells were arrested completely. At higher concentrations (1 and 10 mumol/L), spontaneous activity ceased in all cells. Three ionic currents fundamental for pacemaking, ie, IK, the long-lasting inward calcium current (ICa,L), and the hyperpolarization-activated current (I(f)), were studied by using the whole-cell and amphotericin-perforated patch technique. E-4031 blocked part of the outward current during depolarizing steps as well as the tail current upon subsequent repolarization (ITD) in a dose-dependent manner. E-4031 (10 mumol/L) depressed ITD (88 +/- 4%) (n = 6), reduced peak ICa,L at 0 mV (29 +/- 15%) (n = 4), but did not affect I(f). Lower concentrations did not affect ICa,L. Additional use of 5 mumol/L nifedipine demonstrated that ITD is carried in part by a calcium-sensitive current. Interestingly, complete blockade of IK and ICa,L unmasked the presence of a background current component with a reversal potential of -32 +/- 5.4 mV (n = 8) and a conductance of 39.5 +/- 5.6 pS/pF, which therefore can contribute both to the initial part of repolarization and to full diastolic depolarization.(ABSTRACT TRUNCATED AT 250 WORDS)

Cardiomyocytes derived from embryonic stem cells resemble cardiomyocytes of the embryonic heart tube

OBJECTIVE After formation of the linear heart tube a chamber-specific program of gene expression becomes active that underlies the formation of the chamber myocardium. To assess whether this program is recapitulated in in vitro differentiated embryonic stem cells, we performed qualitative and quantitative analyses of cardiogenesis in vivo and in vitro. METHODS Gene expression profiles were made by in situ hybridisation and real-time PCR and electrophysiological profiles by patch clamp analyses of cardiomyocytes derived from time series of differentiating HM1 mouse embryonic stem cells and from embryonic and adult mouse hearts. RESULTS In embryoid bodies the in situ patterns of expression of alpha-myosin heavy chain, myosin light chain 2a and sarcoendoplasmic reticulum calcium ATPase 2a were similar to that of the heart muscle-specific marker gene cardiac troponin I. Myosin light chain 2v was expressed in part of the cardiac troponin I-expressing area, indicating heterogeneity within the cardiac cell population. Atrial natriuretic factor expression, indicative of the chamber-type program, could only very occasionally be detected by in situ hybridisation. Quantitative reverse transcriptase PCR showed that all cardiac genes, most notably atrial natriuretic factor, were expressed at relatively low levels, similar to those in embryonic hearts at embryonic day 8.75-9. Analysis of the electrophysiological characteristics of embryonic stem cell-derived cardiomyocytes showed an increase of the upstroke velocity and a shorter duration of the action potential during prolonged differentiation in vitro. When embryonic mouse heart compartments of embryonic day 12.5 were used as a reference, the electrophysiological characteristics of a substantial part of the embryonic stem cell-derived cardiomyocytes were most reminiscent to those observed in the embryonic outflow tract. CONCLUSION Together, these data suggest that most cardiomyocytes acquired by differentiation of embryonic stem cells maintain a phenotype reminiscent of that of the cardiomyocytes of the primary heart tube, and hardly any myocytes develop a chamber myocardial phenotype.

Contribution of L-type Ca2+current to electrical activity in sinoatrial nodal myocytes of rabbits

The role of L-type calcium current ( I Ca,L) in impulse generation was studied in single sinoatrial nodal myocytes of the rabbit, with the use of the amphotericin-perforated patch-clamp technique. Nifedipine, at a concentration of 5 μM, was used to block I Ca,L. At this concentration, nifedipine selectively blocked I Ca,L for 81% without affecting the T-type calcium current ( I Ca,T), the fast sodium current, the delayed rectifier current ( I K), and the hyperpolarization-activated inward current. Furthermore, we did not observe the sustained inward current. The selective action of nifedipine on I Ca,L enabled us to determine the activation threshold of I Ca,L, which was around -60 mV. As nifedipine (5 μM) abolished spontaneous activity, we used a combined voltage- and current-clamp protocol to study the effects of I Ca,L blockade on repolarization and diastolic depolarization. This protocol mimics the action potential such that the repolarization and subsequent diastolic depolarization are studied in current-clamp conditions. Nifedipine significantly decreased action potential duration at 50% repolarization and reduced diastolic depolarization rate over the entire diastole. Evidence was found that recovery from inactivation of I Ca,L occurs during repolarization, which makes I Ca,L available already early in diastole. We conclude that I Ca,L contributes significantly to the net inward current during diastole and can modulate the entire diastolic depolarization.

Distribution of Atrial and Nodal Cells Within the Rabbit Sinoatrial Node Models of Sinoatrial Transition

BACKGROUND In the sinoatrial node (SAN) the course of the action potential gradually changes from the primary pacemaker region toward the atrium. It is not known whether this gradient results from different intrinsic characteristics of the nodal cells, from an increasing electrotonic interaction with the atrium, or from both. Therefore we have characterized the immunohistochemical, morphological, and electrophysiological correlates of this functional gradient. METHODS AND RESULTS The distribution of rabbit nodal myocytes in the SAN has been studied by immunohistochemistry. After cell isolation, the electrophysiological characteristics of different nodal cell types were measured. (1) The staining pattern of a neurofilament protein coincides with the electrophysiologically mapped pacemaker region in the SAN. (2) Enzymatic digestion of the SAN reveals three morphologically different nodal cell types and one atrial type. Of each nodal cell type, neurofilament-positive as well as neurofilament-negative myocytes are found. Atrial cells are all neurofilament-negative. (3) In contrast to previous findings, we observed atrial cells in the very center of the SAN. The relative number of atrial cells gradually increases from the central pacemaker area toward the atrium. (4) Differences in electrophysiological characteristics between individual nodal cells are not associated with differences in cell type. CONCLUSIONS (1) The expression of neurofilaments can be used to delineate the nodal area in the intact SAN but is not sufficiently sensitive for characterizing all individual isolated nodal cells. (2) A fundamentally different organization of the SAN is presented: The gradual increase in density of atrial cells from the dominant area toward the crista terminalis in the SAN causes a gradual increase of atrial electrotonic influence that may be an important cause of the gradual transition of the nodal to the atrial type of action potential.

Delayed Rectifier Channels in Human Ventricular Myocytes

BACKGROUND Previous studies have shown that in heart there are two kinetically distinct components of delayed rectifier current: a rapidly activating component (IKr) and a more slowly activating component (IKs). The presence of IKr and/or IKs 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 IKs were found in these experiments. CONCLUSIONS The conformity between the properties of IKr and those of the K+ channel in the present study strongly suggests that IKr is present in human ventricle.

Intracellular calcium modulation of voltage-gated sodium channels in ventricular myocytes

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 AND RESULTS Sodium current (I(Na)) and maximal AP upstroke velocity (dV/dt(max)), a measure of I(Na), were studied at 20 and 37 degrees C, 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. 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 increase 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.

Tubulin polymerization modifies cardiac sodium channel expression and gating

AIMS 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 beta1-subunit-mediated. METHODS AND RESULTS Human embryonic kidney (HEK293) cells, transfected with SCN5A cDNA alone (Na(v)1.5) or together with SCN1B cDNA (Na(v)1.5 + beta1), and neonatal rat cardiomyocytes (NRCs) were incubated in the presence and in the absence of 100 microM 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 + beta1, 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 + beta1, 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 + beta1 were unchanged by TXL. CONCLUSION Enhanced tubulin polymerization reduces sarcolemmal Na(v)1.5 expression and I(Na) amplitude in a beta1-subunit-independent fashion and causes I(Na) fast and slow inactivation impairment in a beta1-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.

Defective Tbx2-dependent patterning of the atrioventricular canal myocardium causes accessory pathway formation in mice

Ventricular preexcitation, a feature of Wolff-Parkinson-White syndrome, is caused by accessory myocardial pathways that bypass the annulus fibrosus. This condition increases the risk of atrioventricular tachycardia and, in the presence of atrial fibrillation, sudden death. The developmental mechanisms underlying accessory pathway formation are poorly understood but are thought to primarily involve malformation of the annulus fibrosus. Before birth, slowly conducting atrioventricular myocardium causes a functional atrioventricular activation delay in the absence of the annulus fibrosus. This myocardium remains present after birth, suggesting that the disturbed development of the atrioventricular canal myocardium may mediate the formation of rapidly conducting accessory pathways. Here we show that myocardium-specific inactivation of T-box 2 (Tbx2), a transcription factor essential for atrioventricular canal patterning, leads to the formation of fast-conducting accessory pathways, malformation of the annulus fibrosus, and ventricular preexcitation in mice. The accessory pathways ectopically express proteins required for fast conduction (connexin-40 [Cx40], Cx43, and sodium channel, voltage-gated, type V, α [Scn5a]). Additional inactivation of Cx30.2, a subunit for gap junctions with low conductance expressed in the atrioventricular canal and unaffected by the loss of Tbx2, did not affect the functionality of the accessory pathways. Our results suggest that malformation of the annulus fibrosus and preexcitation arise from the disturbed development of the atrioventricular myocardium.