Sodium current inhibition by nanosecond pulsed electric field (nsPEF)--fact or artifact?

In two recent publications in Bioelectromagnetics it has been demonstrated that the voltage-gated sodium current (I(Na)) is inhibited in response to a nanosecond pulsed electric field (nsPEF). At the same time, there was an increase in a non-inactivating "leak" current (I(leak)), which was attributed to the formation of nanoelectropores or larger pores in the plasma membrane. We demonstrate that the increase in I(leak), in combination with a residual series resistance, leads to an error in the holding potential in the patch clamp experiments and an unanticipated inactivation of the sodium channels. We conclude that the observed inhibition of I(Na) may be largely, if not fully, artifactual.

HCN4 current during human sinoatrial node-like action potentials.

BACKGROUND: Despite the many studies carried out over the past 40 years, the contribution of the HCN4 encoded hyperpolarization-activated 'funny' current (If) to pacemaker activity in the mammalian sinoatrial node (SAN), and the human SAN in particular, is still controversial and not fully established. OBJECTIVE: To study the contribution of If to diastolic depolarization of human SAN cells and its dependence on heart rate, cAMP levels, and atrial load. METHODS: HCN4 channels were expressed in human cardiac myocyte progenitor cells (CMPCs) and HCN4 currents assessed using perforated patch-clamp in traditional voltage clamp mode and during action potential clamp with human SAN-like action potential waveforms with 500-1500 ms cycle length, in absence or presence of forskolin to mimic ß-adrenergic stimulation and a -15 mV command potential offset to mimic atrial load. RESULTS: Forskolin significantly increased the fully-activated HCN4 current density at -140 mV by 14% and shifted the steady-state activation curve by +7.4 mV without affecting its slope. In addition, forskolin significantly accelerated current activation but slowed deactivation. The HCN4 current did not completely deactivate before the subsequent diastolic depolarization during action potential clamp. The amplitude of HCN4 current increased with increasing cycle length, was significantly larger in the presence of forskolin at all cycle lengths, and was significantly increased upon the negative offset to the command potential. CONCLUSIONS: If is active during a human SAN action potential waveform and its amplitude is modulated by heart rate, ß-adrenergic stimulation, and diastolic voltage range, such that If is under delicate control.

Neurokinin-3 receptor activation selectively prolongs atrial refractoriness by inhibition of a background K+ channel.

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.

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, Scn5a(1798insD/+) heterozygous mice showed significantly lower heart rates, more bradycardic episodes (pauses > or = 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.

Dietary fish oil reduces the incidence of triggered arrhythmias in pig ventricular myocytes.

BACKGROUND: Fish oil reduces the incidence of sudden cardiac death in postmyocardial infarction patients. Triggered activity is the principal mechanism of arrhythmogenesis under these conditions. OBJECTIVE: The purpose of this study was to test whether dietary fish oil in pigs inhibits Ca2+ overload-induced triggered activity. METHODS: Pigs were fed a diet of fish oil or sunflower oil for 8 weeks. Ventricular myocytes (omega3: fish oil, n = 11; control: sunflower oil, n = 8) were isolated by enzymatic dissociation and used for patch clamp studies and intracellular Ca2+ recordings. Triggered activity was induced by rapid pacing in the presence of norepinephrine. RESULTS: Dietary fish oil reduced the incidence of triggered action potentials and delayed afterdepolarizations compared to control (9.1% in omega3 and 84.6% in control, P <.05), concomitant with a reduction in spontaneous Ca2+ release. Dietary fish oil prevented Ca2+ overload and reduced action potential prolongation in response to norepinephrine (DeltaAPD(90): 23.2 +/- 8.5 ms in omega3 and 107.4 +/- 15.9 in control, P <.05). omega3 myocytes displayed decreased sarcoplasmic reticulum Ca2+ content, reduced L-type Ca2+ current (I(Ca,L)), and less recruitment of the Na+/Ca2+ exchange current (I(NCX)) in response to norepinephrine compared to control. In the absence of norepinephrine, the slow component of the delayed rectifier current (I(Ks)) was larger in omega3 myocytes. In the presence of norepinephrine, I(Ks) increased to the same level in omega3 and control myocytes. CONCLUSION: Dietary fish oil reduces the incidence of triggered activity and prevents Ca2+ overload and AP prolongation in response to norepinephrine. Fish oil may prevent arrhythmias in patients with heart failure.

Electrophysiological properties of embryonic stem cells during differentiation into cardiomyocyte-like cell types.

The method described here to differentiate mouse embryonic stem (ES) cells into cardiomyocytes is adapted from Maltsev et al. and results in a high percentage of spontaneously beating cardiomyocyte-like cells. In order to determine to what extent the differentiating ES cells resemble true cardiomyocytes, the cells were electrophysiologically characterized during differentiation, using the whole-cell variant of the patch-clamp technique. Action potentials (APs) and membrane currents were recorded and analyzed off-line to determine electrophysiological changes during development.

Cardiac channelopathies studied with the dynamic action potential-clamp technique.

The cardiac long QT syndrome (LQTS) is characterized by a delayed repolarization of the ventricular myocytes, resulting in prolongation of the QT interval on the electrocardiogram and increased propensity to cardiac arrhythmias. Congenital LQTS has been linked to mutations in genes encoding ion channel subunits. For a better understanding of LQTS and associated arrhythmias, insight into the nature of ion channel (dys)function is indispensable. Conventionally, voltage-clamp analysis and subsequent mathematical modeling are used to study cardiac channelopathies and to link a certain genetic defect to its cellular phenotype. The recently introduced "dynamic action potential clamp" (dAPC) technique represents an alternative approach, in which a selected native ionic current of the ventricular myocyte can effectively be replaced with wild-type (WT) or mutant current recorded from a human embryonic kidney (HEK)-293 cell that is voltage clamped by the free-running action potential (AP) of the myocyte. Both a computed model of the human ventricular cell and a freshly isolated myocyte can effectively be used in dAPC experiments, resulting in rapid and unambiguous determination of the effect(s) of an ion channel mutation on the ventricular AP. The dAPC technique represents a promising new tool to study various cardiac ion channels and may also prove useful in related fields of research, for example, in neurophysiology.

Incorporated sarcolemmal fish oil fatty acids shorten pig ventricular action potentials.

BACKGROUND: Omega-3 polyunsaturated fatty acids (omega3-PUFAs) from fish oil reduce the risk of sudden death presumably by preventing life-threatening arrhythmias. Acutely administered omega3-PUFAs modulate the activity of several cardiac ion channels, but the chronic effects of a diet enriched with fish oil leading to omega3-PUFA-incorporation into the sarcolemma on membrane currents are unknown. METHODS: Pigs received a diet either rich in omega3-PUFAs or in omega9-fatty acids for 8 weeks. Ventricular myocytes (VMs) were isolated and used for patch-clamp studies. RESULTS: omega3-VMs contained higher amounts of omega3-PUFAs and had a shorter action potential (AP) with a more negative plateau than control VM. In omega3 VMs, L-type Ca(2+) current (I(Ca,L)) and Na(+)-Ca(2+) exchange current (I(NCX)) were reduced by approximately 20% and 60%, respectively, and inward rectifier K(+) current (I(K1)) 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, Ca(2+)-activated Cl(-) current, and Na(+) current (I(Na)) were unchanged, although voltage-dependence of I(Na) inactivation was more negative in omega3 VMs. CONCLUSIONS: A fish oil diet increases omega3-PUFA content in the ventricular sarcolemma, decreases I(Ca,L) and I(NCX), and increases I(K1) and I(Ks), resulting in AP shortening. Incorporation of omega3-PUFAs in the sarcolemma may have consequences for arrhythmias independent of circulating omega3-PUFAs.

Mutation in the KCNQ1 gene leading to the short QT-interval syndrome.

BACKGROUND: The electrocardiographic short QT-interval syndrome forms a distinct clinical entity presenting with a high rate of sudden death and exceptionally short QT intervals. The disorder has recently been linked to gain-of-function mutation in KCNH2. The present study demonstrates that this disorder is genetically heterogeneous and can also be caused by mutation in the KCNQ1 gene. METHODS AND RESULTS: A 70-year man presented with idiopathic ventricular fibrillation. Both immediately after the episode and much later, his QT interval was abnormally short without any other physical or electrophysiological anomalies. Analysis of candidate genes identified a g919c substitution in KCNQ1 encoding the K+ channel KvLQT1. Functional studies of the KvLQT1 V307L mutant (alone or coexpressed with the wild-type channel, in the presence of IsK) revealed a pronounced shift of the half-activation potential and an acceleration of the activation kinetics leading to a gain of function in I(Ks). When introduced in a human action potential computer model, the modified biophysical parameters predicted repolarization shortening. CONCLUSIONS: We present an alternative molecular mechanism for the short QT-interval syndrome. Functional and computational studies of the KCNQ1 V307L mutation identified in a patient with this disorder favor the association of short QT with mutation in KCNQ1.

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.

Ca(2+) cycling properties are conserved despite bradycardic effects of heart failure in sinoatrial node cells.

BACKGROUND: In animal models of heart failure (HF), heart rate decreases due to an increase in intrinsic cycle length (CL) of the sinoatrial node (SAN). Pacemaker activity of SAN cells is complex and modulated by the membrane clock, i.e., the ensemble of voltage gated ion channels and electrogenic pumps and exchangers, and the Ca(2+) clock, i.e., the ensemble of intracellular Ca(2+) ([Ca(2+)]i) dependent processes. HF in SAN cells results in remodeling of the membrane clock, but few studies have examined its effects on [Ca(2+)]i homeostasis. METHODS: SAN cells were isolated from control rabbits and rabbits with volume and pressure overload-induced HF. [Ca(2+)]i concentrations, and action potentials (APs) and Na(+)-Ca(2+) exchange current (INCX) were measured using indo-1 and patch-clamp methodology, respectively. RESULTS: The frequency of spontaneous [Ca(2+)]i transients was significantly lower in HF SAN cells (3.0 ± 0.1 (n = 40) vs. 3.4 ± 0.1 Hz (n = 45); mean ± SEM), indicating that intrinsic CL was prolonged. HF slowed the [Ca(2+)]i transient decay, which could be explained by the slower frequency and reduced sarcoplasmic reticulum (SR) dependent rate of Ca(2+) uptake. Other [Ca(2+)]i transient parameters, SR Ca(2+) content, INCX density, and INCX-[Ca(2+)]i relationship were all unaffected by HF. Combined AP and [Ca(2+)]i recordings demonstrated that the slower [Ca(2+)]i transient decay in HF SAN cells may result in increased INCX during the diastolic depolarization, but that this effect is likely counteracted by the HF-induced increase in intracellular Na(+). ß-adrenergic and muscarinic stimulation were not changed in HF SAN cells, except that late diastolic [Ca(2+)]i rise, a prominent feature of the Ca(2+) clock, is lower during ß-adrenergic stimulation. CONCLUSIONS: HF SAN cells have a slower [Ca(2+)]i transient decay with limited effects on pacemaker activity. Reduced late diastolic [Ca(2+)]i rise during ß-adrenergic stimulation may contribute to an impaired increase in intrinsic frequency in HF SAN cells.

Ion channelopathies in human induced pluripotent stem cell derived cardiomyocytes: a dynamic clamp study with virtual IK1.

Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) are widely used in studying basic mechanisms of cardiac arrhythmias that are caused by ion channelopathies. Unfortunately, the action potential profile of hiPSC-CMs-and consequently the profile of individual membrane currents active during that action potential-differs substantially from that of native human cardiomyocytes, largely due to almost negligible expression of the inward rectifier potassium current (IK1). In the present study, we attempted to "normalize" the action potential profile of our hiPSC-CMs by inserting a voltage dependent in silico IK1 into our hiPSC-CMs, using the dynamic clamp configuration of the patch clamp technique. Recordings were made from single hiPSC-CMs, using the perforated patch clamp technique at physiological temperature. We assessed three different models of IK1, with different degrees of inward rectification, and systematically varied the magnitude of the inserted IK1. Also, we modified the inserted IK1 in order to assess the effects of loss- and gain-of-function mutations in the KCNJ2 gene, which encodes the Kir2.1 protein that is primarily responsible for the IK1 channel in human ventricle. For our experiments, we selected spontaneously beating hiPSC-CMs, with negligible IK1 as demonstrated in separate voltage clamp experiments, which were paced at 1 Hz. Upon addition of in silico IK1 with a peak outward density of 4-6 pA/pF, these hiPSC-CMs showed a ventricular-like action potential morphology with a stable resting membrane potential near -80 mV and a maximum upstroke velocity >150 V/s (n = 9). Proarrhythmic action potential changes were observed upon injection of both loss-of-function and gain-of-function IK1, as associated with Andersen-Tawil syndrome type 1 and short QT syndrome type 3, respectively (n = 6). We conclude that injection of in silico IK1 makes the hiPSC-CM a more reliable model for investigating mechanisms underlying cardiac arrhythmias.

Atrial fibrosis and conduction slowing in the left atrial appendage of patients undergoing thoracoscopic surgical pulmonary vein isolation for atrial fibrillation.

BACKGROUND: Atrial fibrosis is an important component of the arrhythmogenic substrate in patients with atrial fibrillation (AF). We studied the effect of interstitial fibrosis on conduction velocity (CV) in the left atrial appendage of patients with AF. METHODS AND RESULTS: Thirty-five left atrial appendages were obtained during AF surgery. Preparations were superfused and stimulated at 100 beats per minute. Activation was recorded with optical mapping. Longitudinal CV (CVL), transverse CV (CVT), and activation times (> 2 mm distance) were measured. Interstitial collagen was quantified and graded qualitatively. The presence of fibroblasts and myofibroblasts was assessed immunohistochemically. Mean CVL was 0.55 ± 0.22 m/s, mean CVT was 0.25 ± 0.15 m/s, and the mean activation time was 9.31 ± 5.45 ms. The amount of fibrosis was unrelated to CV or patient characteristics. CVL was higher in left atrial appendages with thick compared with thin interstitial collagen strands (0.77 ± 0.22 versus 0.48 ± 0.19 m/s; P = 0.012), which were more frequently present in persistent patients with AF. CVT was not significantly different (P = 0.47), but activation time was 14.93 ± 4.12 versus 7.95 ± 4.12 ms in patients with thick versus thin interstitial collagen strands, respectively (P = 0.004). Fibroblasts were abundantly present and were associated with the presence of thick interstitial collagen strands (P = 0.008). Myofibroblasts were not detected in the left atrial appendage. CONCLUSIONS: In patients with AF, thick interstitial collagen strands are associated with higher CVL and increased activation time. Our observations demonstrate that the severity and structure of local interstitial fibrosis is associated with atrial conduction abnormalities, presenting an arrhythmogenic substrate for atrial re-entry.

Dynamic clamp as a tool to study the functional effects of individual membrane currents.

Today, the patch-clamp technique is the main technique in electrophysiology to record action potentials or membrane current from isolated cells, using a patch pipette to gain electrical access to the cell. The common recording modes of the patch-clamp technique are current clamp and voltage clamp. In the current clamp mode, the current injected through the patch pipette is under control while the free-running membrane potential of the cell is recorded. Current clamp allows for measurements of action potentials that may either occur spontaneously or in response to an injected stimulus current. In voltage clamp mode, the membrane potential is held at a set level through a feedback circuit, which allows for the recording of the net membrane current at a given membrane potential.A less common configuration of the patch-clamp technique is the dynamic clamp. In this configuration, a specific non-predetermined membrane current can be added to or removed from the cell while it is in free-running current clamp mode. This current may be computed in real time, based on the recorded action potential of the cell, and injected into the cell. Instead of being computed, this current may also be recorded from a heterologous expression system such as a HEK-293 cell that is voltage-clamped by the free-running action potential of the cell ("dynamic action potential clamp"). Thus, one may directly test the effects of an additional or mutated membrane current, a synaptic current or a gap junctional current on the action potential of a patch-clamped cell. In the present chapter, we describe the dynamic clamp on the basis of its application in cardiac cellular electrophysiology.

Slow delayed rectifier potassium current blockade contributes importantly to drug-induced long QT syndrome.

BACKGROUND: Drug-induced long QT syndrome is generally ascribed to inhibition of the cardiac rapid delayed rectifier potassium current (IKr). Effects on the slow delayed rectifier potassium current (IKs) are less recognized. Triggered by a patient who carried the K422T mutation in KCNQ1 (encoding the α-subunit of the IKs channel), who presented with excessive QT prolongation and high serum levels of norfluoxetine, we investigated the effects of fluoxetine and its metabolite norfluoxetine on IKs. METHODS AND RESULTS: ECG data from mutation carriers and noncarriers revealed that the K422T mutation per se had mild clinical effects. Patch clamp studies, performed on HEK293 cells, showed that heterozygously expressed K422T KCNQ1/KCNE1 channels had a positive shift in voltage dependence of activation and an increase in deactivation rate. Fluoxetine and its metabolite norfluoxetine both inhibited KCNQ1/KCNE1 current, with norfluoxetine being the most potent. Moreover, norfluoxetine increased activation and deactivation rates. Computer simulations of the effects of norfluoxetine on IKs and IKr demonstrated significant action potential prolongation, to which IKs block contributed importantly. Although the effects of the mutation per se were small, additional IKs blockade by norfluoxetine resulted in more prominent QTc prolongation in mutation carriers than in noncarriers, demonstrating synergistic effects of innate and drug-induced IKs blockade on QTc prolongation. CONCLUSIONS: IKs blockade contributes importantly to drug-induced long QT syndrome, especially when repolarization reserve is reduced. Drug safety tests might have to include screening for IKs blockade.

Electrophysiologic remodeling of the left ventricle in pressure overload-induced right ventricular failure.

OBJECTIVES: The purpose of this study was to analyze the electrophysiologic remodeling of the atrophic left ventricle (LV) in right ventricular (RV) failure (RVF) after RV pressure overload. BACKGROUND: The LV in pressure-induced RVF develops dysfunction, reduction in mass, and altered gene expression, due to atrophic remodeling. LV atrophy is associated with electrophysiologic remodeling. METHODS: We conducted epicardial mapping in Langendorff-perfused hearts, patch-clamp studies, gene expression studies, and protein level studies of the LV in rats with pressure-induced RVF (monocrotaline [MCT] injection, n = 25; controls with saline injection, n = 18). We also performed epicardial mapping of the LV in patients with RVF after chronic thromboembolic pulmonary hypertension (CTEPH) (RVF, n = 10; no RVF, n = 16). RESULTS: The LV of rats with MCT-induced RVF exhibited electrophysiologic remodeling: longer action potentials (APs) at 90% repolarization and effective refractory periods (ERPs) (60 ± 1 ms vs. 44 ± 1 ms; p < 0.001), and slower longitudinal conduction velocity (62 ± 2 cm/s vs. 70 ± 1 cm/s; p = 0.003). AP/ERP prolongation agreed with reduced Kcnip2 expression, which encodes the repolarizing potassium channel subunit KChIP2 (0.07 ± 0.01 vs. 0.11 ± 0.02; p < 0.05). Conduction slowing was not explained by impaired impulse formation, as AP maximum upstroke velocity, whole-cell sodium current magnitude/properties, and mRNA levels of Scn5a were unaltered. Instead, impulse transmission in RVF was hampered by reduction in cell length (111.6 ± 0.7 µm vs. 122.0 ± 0.4 µm; p = 0.02) and width (21.9 ± 0.2 µm vs. 25.3 ± 0.3 µm; p = 0.002), and impaired cell-to-cell impulse transmission (24% reduction in Connexin-43 levels). The LV of patients with CTEPH with RVF also exhibited ERP prolongation (306 ± 8 ms vs. 268 ± 5 ms; p = 0.001) and conduction slowing (53 ± 3 cm/s vs. 64 ± 3 cm/s; p = 0.005). CONCLUSIONS: Pressure-induced RVF is associated with electrophysiologic remodeling of the atrophic LV.

Re-evaluation of the action potential upstroke velocity as a measure of the Na+ current in cardiac myocytes at physiological conditions.

BACKGROUND: The SCN5A encoded sodium current (I(Na)) generates the action potential (AP) upstroke and is a major determinant of AP characteristics and AP propagation in cardiac myocytes. Unfortunately, in cardiac myocytes, investigation of kinetic properties of I(Na) with near-physiological ion concentrations and temperature is technically challenging due to the large amplitude and rapidly activating nature of I(Na), which may seriously hamper the quality of voltage control over the membrane. We hypothesized that the alternating voltage clamp-current clamp (VC/CC) technique might provide an alternative to traditional voltage clamp (VC) technique for the determination of I(Na) properties under physiological conditions. PRINCIPAL FINDINGS: We studied I(Na) under close-to-physiological conditions by VC technique in SCN5A cDNA-transfected HEK cells or by alternating VC/CC technique in both SCN5A cDNA-transfected HEK cells and rabbit left ventricular myocytes. In these experiments, peak I(Na) during a depolarizing VC step or maximal upstroke velocity, dV/dt(max), during VC/CC served as an indicator of available I(Na). In HEK cells, biophysical properties of I(Na), including current density, voltage dependent (in)activation, development of inactivation, and recovery from inactivation, were highly similar in VC and VC/CC experiments. As an application of the VC/CC technique we studied I(Na) in left ventricular myocytes isolated from control or failing rabbit hearts. CONCLUSIONS: Our results demonstrate that the alternating VC/CC technique is a valuable experimental tool for I(Na) measurements under close-to-physiological conditions in cardiac myocytes.

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.

Pacemaker activity of the human sinoatrial node: role of the hyperpolarization-activated current, I(f).

The mechanism of primary, spontaneous cardiac pacemaker activity of the sinoatrial node (SAN) has extensively been studied in several animal species, but is virtually unexplored in man. Understanding the mechanisms of human SAN pacemaker activity is important for developing new therapeutic approaches for controlling the heart rate in the sick sinus syndrome and in diseased myocardium. Here we review the functional role of the hyperpolarization-activated 'funny' current, I(f), in human SAN pacemaker activity. Despite the many animal studies performed over the years, the contribution of I(f) to pacemaker activity is still controversial and not fully established. However, recent clinical data on mutations in the I(f) encoding HCN4 gene, which is thought to be the most abundant isoform of the HCN gene family in SAN, suggest a functional role of I(f) in human pacemaker activity. These clinical findings are supported by recent experimental data from single isolated human SAN cells that provide direct evidence that I(f) contributes to human SAN pacemaker activity. Therefore, controlling heart rate in clinical practice via I(f) blockers offers a valuable approach to lowering heart rate and provides an attractive alternative to conventional treatment for a wide range of patients with confirmed stable angina, while upregulation or artificial expression of I(f) may relieve disease-causing bradycardias.