Marieke W. Veldkamp

Right ventricular fibrosis and conduction delay in a patient with clinical signs of Brugada syndrome : a combined electrophysiological, genetic, histopathologic, and computational study

Background— The mechanism of ECG changes and arrhythmogenesis in Brugada syndrome (BS) patients is unknown. Methods and Results— A BS patient without clinically detected cardiac structural abnormalities underwent cardiac transplantation for intolerable numbers of implantable cardioverter/defibrillator discharges. The patient’s explanted heart was studied electrophysiologically and histopathologically. Whole-cell currents were measured in HEK293 cells expressing wild-type or mutated sodium channels from the patient. The right ventricular outflow tract (RVOT) endocardium showed activation slowing and was the origin of ventricular fibrillation without a transmural repolarization gradient. Conduction restitution was abnormal in the RVOT but normal in the left ventricle. Right ventricular hypertrophy and fibrosis with epicardial fatty infiltration were present. HEK293 cells expressing a G1935S mutation in the gene encoding the cardiac sodium channel exhibited enhanced slow inactivation compared with wild-type channels. Computer simulations demonstrated that conduction slowing in the RVOT might have been the cause of the ECG changes. Conclusions— In this patient with BS, conduction slowing based on interstitial fibrosis, but not transmural repolarization differences, caused the ECG signs and was the origin of ventricular fibrillation.

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.

Contribution of Sodium Channel Mutations to Bradycardia and Sinus Node Dysfunction in LQT3 Families

Abstract— 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 (Ipst) at −20 mV and a negative shift in voltage dependence of inactivation. In the present study, we first additionally characterized Ipst over the complete voltage range of the SA node action potential (AP) by measuring whole-cell Na+ currents (INa) in HEK-293 cells expressing either wild-type or 1795insD channels. Ipst for 1795insD channels varied between 0.8±0.2% and 1.9±0.8% of peak INa. Activity of 1795insD channels during SA node pacemaking was confirmed by AP clamp experiments. Next, Ipst and the negative shift were implemented into SA node AP models. The −10-mV shift decreased sinus rate by decreasing diastolic depolarization rate, whereas Ipst decreased sinus rate by AP prolongation, despite a concomitant increase in diastolic depolarization rate. In combination, moderate Ipst (1% to 2%) and the shift reduced sinus rate by ≈10%. An additional increase in Ipst could result in plateau oscillations and failure to repolarize completely. Thus, Na+ channel mutations displaying an Ipst 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.

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.

Functional NaV1.8 Channels in Intracardiac Neurons The Link Between SCN10A and Cardiac Electrophysiology

Rationale: The SCN10A gene encodes the neuronal sodium channel isoform NaV1.8. Several recent genome-wide association studies have linked SCN10A to PR interval and QRS duration, strongly suggesting an as-yet unknown role for NaV1.8 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 NaV1.8 labeling in dorsal root ganglia and intracardiac ganglia and only modest NaV1.8 expression within the myocardium. Immunocytochemistry further revealed substantial NaV1.8 staining in isolated neurons from murine intracardiac ganglia but no NaV1.8 expression in isolated ventricular myocytes. Patch-clamp studies demonstrated that the NaV1.8 blocker A-803467 (0.5–2 &mgr;mol/L) had no effect on either mean sodium current (INa) density or INa gating kinetics in isolated myocytes but significantly reduced INa 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 NaV1.8-based INa. 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 NaV1.8 in cardiac neural activity. Conclusions: Our findings demonstrate the functional presence of SCN10A/NaV1.8 in intracardiac neurons, indicating a novel role for this neuronal sodium channel in regulation of cardiac electric activity.Rationale: The SCN10A gene encodes the neuronal sodium channel isoform NaV1.8. Several recent genome-wide association studies have linked SCN10A to PR interval and QRS duration, strongly suggesting an as-yet unknown role for NaV1.8 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 NaV1.8 labeling in dorsal root ganglia and intracardiac ganglia and only modest NaV1.8 expression within the myocardium. Immunocytochemistry further revealed substantial NaV1.8 staining in isolated neurons from murine intracardiac ganglia but no NaV1.8 expression in isolated ventricular myocytes. Patch-clamp studies demonstrated that the NaV1.8 blocker A-803467 (0.5–2 μmol/L) had no effect on either mean sodium current (INa) density or INa gating kinetics in isolated myocytes but significantly reduced INa 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 NaV1.8-based INa. 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 NaV1.8 in cardiac neural activity. Conclusions: Our findings demonstrate the functional presence of SCN10A /NaV1.8 in intracardiac neurons, indicating a novel role for this neuronal sodium channel in regulation of cardiac electric activity. # Novelty and Significance {#article-title-33}

Genetically determined differences in sodium current characteristics modulate conduction disease severity in mice with cardiac sodium channelopathy.

Conduction slowing of the electric impulse that drives the heartbeat may evoke lethal cardiac arrhythmias. Mutations in SCN5A, which encodes the pore-forming cardiac sodium channel &agr; subunit, are associated with familial arrhythmia syndromes based on conduction slowing. However, disease severity among mutation carriers is highly variable. We hypothesized that genetic modifiers underlie the variability in conduction slowing and disease severity. With the aim of identifying such modifiers, we studied the Scn5a1798insD/+ mutation in 2 distinct mouse strains, FVB/N and 129P2. In 129P2 mice, the mutation resulted in more severe conduction slowing particularly in the right ventricle (RV) compared to FVB/N. Pan-genomic mRNA expression profiling in the 2 mouse strains uncovered a drastic reduction in mRNA encoding the sodium channel auxiliary subunit &bgr;4 (Scn4b) in 129P2 mice compared to FVB/N. This corresponded to low to undetectable &bgr;4 protein levels in 129P2 ventricular tissue, whereas abundant &bgr;4 protein was detected in FVB/N. Sodium current measurements in isolated myocytes from the 2 mouse strains indicated that sodium channel activation in myocytes from 129P2 mice occurred at more positive potentials compared to FVB/N. Using computer simulations, this difference in activation kinetics was predicted to explain the observed differences in conduction disease severity between the 2 strains. In conclusion, genetically determined differences in sodium current characteristics on the myocyte level modulate disease severity in cardiac sodium channelopathies. In particular, the sodium channel subunit &bgr;4 (SCN4B) may constitute a potential genetic modifier of conduction and cardiac sodium channel disease.

Single delayed rectifier channels in the membrane of rabbit ventricular myocytes.

In rabbit ventricular cells, the delayed rectifier current (IK) has not been extensively studied, and properties of single IK channels still need to be determined. In this study, we present data on a voltage-dependent channel in rabbit ventricular cells; the properties indicate that it is an IK channel. Patch-clamp experiments were carried out on cell-attached and inside-out patches of rabbit ventricular cells. Single-channel currents were recorded at negative potentials as inward currents with 150 mM K+ in the pipette. Voltage-dependent channel activity was only present after the return from a depolarizing test pulse, indicating activation on depolarization. Single-channel conductance calculated from the current-voltage relation was 13.1 pS (pooled data, n = 8). The shift in reversal potential of the unitary currents, determined at 150 and 300 mM K+ at the intracellular side of the membrane, showed that the channels were highly permeable to potassium ions. Increase of the duration or the amplitude of the depolarizing test pulse increased channel activity. The time constant for activation at +30 mV was 187 msec (pooled data, n = 4). Half-activation potential was -4.9 +/- 3.8 mV (mean +/- SD), and the slope factor was 7.2 +/- 3.7 mV (mean +/- SD). Current tails, reconstructed from averaged single-channel currents, revealed that the time course of deactivation decreased from 694 +/- 73 msec at -80 mV to 136 +/- 39 msec at -110 mV. Additional evidence that the channel was indeed an IK channel was provided by the observation that the channel was blocked by 10(-7) M E-4031, a class III antiarrhythmic agent that has been shown to block a component of the macroscopic IK in guinea pig heart.

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.

Functional NaV1.8 Channels in Intracardiac NeuronsNovelty and Significance: The Link Between SCN10A and Cardiac Electrophysiology

Rationale: The SCN10A gene encodes the neuronal sodium channel isoform NaV1.8. Several recent genome-wide association studies have linked SCN10A to PR interval and QRS duration, strongly suggesting an as-yet unknown role for NaV1.8 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 NaV1.8 labeling in dorsal root ganglia and intracardiac ganglia and only modest NaV1.8 expression within the myocardium. Immunocytochemistry further revealed substantial NaV1.8 staining in isolated neurons from murine intracardiac ganglia but no NaV1.8 expression in isolated ventricular myocytes. Patch-clamp studies demonstrated that the NaV1.8 blocker A-803467 (0.5–2 &mgr;mol/L) had no effect on either mean sodium current (INa) density or INa gating kinetics in isolated myocytes but significantly reduced INa 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 NaV1.8-based INa. 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 NaV1.8 in cardiac neural activity. Conclusions: Our findings demonstrate the functional presence of SCN10A/NaV1.8 in intracardiac neurons, indicating a novel role for this neuronal sodium channel in regulation of cardiac electric activity.Rationale: The SCN10A gene encodes the neuronal sodium channel isoform NaV1.8. Several recent genome-wide association studies have linked SCN10A to PR interval and QRS duration, strongly suggesting an as-yet unknown role for NaV1.8 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 NaV1.8 labeling in dorsal root ganglia and intracardiac ganglia and only modest NaV1.8 expression within the myocardium. Immunocytochemistry further revealed substantial NaV1.8 staining in isolated neurons from murine intracardiac ganglia but no NaV1.8 expression in isolated ventricular myocytes. Patch-clamp studies demonstrated that the NaV1.8 blocker A-803467 (0.5–2 μmol/L) had no effect on either mean sodium current (INa) density or INa gating kinetics in isolated myocytes but significantly reduced INa 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 NaV1.8-based INa. 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 NaV1.8 in cardiac neural activity. Conclusions: Our findings demonstrate the functional presence of SCN10A /NaV1.8 in intracardiac neurons, indicating a novel role for this neuronal sodium channel in regulation of cardiac electric activity. # Novelty and Significance {#article-title-33}