Transient Outward Current Research Articles

Electrogenic Steps Associated with Substrate Binding to the Neuronal Glutamate Transporter EAAC1

Glutamate transporters actively take up glutamate into the cell, driven by the co-transport of sodium ions down their transmembrane concentration gradient. It was proposed that glutamate binds to its binding site and is subsequently transported across the membrane in the negatively charged form. With the glutamate binding site being located partially within the membrane domain, the possibility has to be considered that glutamate binding is dependent on the transmembrane potential and, thus, is electrogenic. Experiments presented in this report test this possibility. Rapid application of glutamate to the wild-type glutamate transporter subtype EAAC1 (excitatory amino acid carrier 1) through photo-release from caged glutamate generated a transient inward current, as expected for the electrogenic inward movement of co-transported Na(+) In contrast, glutamate application to a transporter with the mutation A334E induced transient outward current, consistent with movement of negatively charged glutamate into its binding site within the dielectric of the membrane. These results are in agreement with electrostatic calculations, predicting a valence for glutamate binding of -0.27. Control experiments further validate and rule out other possible explanations for the transient outward current. Electrogenic glutamate binding can be isolated in the mutant glutamate transporter because reactions, such as glutamate translocation and/or Na(+) binding to the glutamate-bound state, are inhibited by the A334E substitution. Electrogenic glutamate binding has to be considered together with other voltage-dependent partial reactions to cooperatively determine the voltage dependence of steady-state glutamate uptake and glutamate buffering at the synapse.

X‐ROS Signaling in Cerebral Artery Smooth Muscle

Mechanically induced generation of reactive oxygen species (ROS) by NADPH oxidase (NOX), referred to as X‐ROS, influences the contractility of cardiac and skeletal muscle, but the impact of this signaling pathway in smooth muscle has not been reported. Here we investigate the effect of X‐ROS on the excitability and contractility of cerebral artery smooth muscle cells (SMC). In these cells, spatially and temporally limited release of Ca2+ from the sarcoplasmic reticulum via ryanodine receptors (RyR) generate microdomains of high intracellular [Ca2+] known as Ca2+ sparks. Ca2+ sparks are functionally coupled to multiple large‐conductance Ca2+‐activated K+ (BK) channels and activate macroscopic Spontaneous Transient Outward Currents (STOCs) that cause membrane hyperpolarization and vasodilation. Several sites subject to redox modification are present on RyRs that influence activity. Therefore, we hypothesized that X‐ROS elevates Ca2+ spark and STOC activity, leading to membrane hyperpolarization and vasodilation. Experiments were carried out using cerebral pial arteries and freshly isolated cerebral artery SMC from C57/Bl6 mice. Ca2+ sparks were recorded from cerebral SMC loaded with the fast Ca2+ indicator fluo‐4AM using high‐speed, high‐resolution confocal microscopy. To determine if the production of O2− was required for generation of Ca2+ sparks, superoxide dismutase (SOD) was applied to the bath. Membrane permeable PEG‐SOD (100 U.ml−1, n = 5) nearly abolished Ca2+ spark activity. To further characterize this response, SMC were patch‐clamped using the amphotericin B perforated‐patch whole cell method to record STOCs. PEG‐SOD (100 U.ml−1, n = 6) attenuated STOC frequency, whereas non‐membrane permeable SOD (100 U.ml−1, n = 7) had no effect. The membrane permeable SOD mimetic TEMPO (1 mM, n = 5) also, attenuated STOC activity. Catalase (750 U.ml−1, n = 5) or membrane permeable PEG‐catalase (750 U.ml−1, n = 6) had no effect on STOC frequency, suggesting that H2O2 intermediates are not involved in this signaling pathway. Inhibition of NOX2 with apocynin (30 μM, n = 7) or gp91ds‐tat (1 μM, n = 6) greatly attenuated STOC frequency, whereas application of exogenous NADPH to increase NOX2 activity resulted in a concentration‐dependent increase in STOC frequency, with an EC50 of 61 nM (n = 3–8). These data indicate that intracellular generation of O2− by NOX2 is necessary for Ca2+ spark and STOC activity. Pressure myography experiments were performed to determine the functional significance of the X‐ROS pathway in SMC. Endothelium denuded posterior cerebral arteries were pressurized to 50 mmHg to generate spontaneous myogenic tone and then treated with the substrate for NOX, NADPH (10 μM). NADPH caused vasodilation that was reversed by the BK blocker paxilline (1 μM, n = 5), suggesting that NOX activity dilates cerebral arteries by activating BK channels. We conclude that generation of intracellular O2− by NOX2 stimulates Ca2+ spark and STOC activity, resulting in cerebral arteries dilation. These data highlight a novel ROS‐dependent signaling pathway that may critically influence cerebral blood flow regulation.Support or Funding InformationSupport: R01HL091905 (SE), AHA15POST2472002 (PWP).

Experimentally-Based Computational Investigation into Beat-To-Beat Variability in Ventricular Repolarization and Its Response to Ionic Current Inhibition.

Beat-to-beat variability in repolarization (BVR) has been proposed as an arrhythmic risk marker for disease and pharmacological action. The mechanisms are unclear but BVR is thought to be a cell level manifestation of ion channel stochasticity, modulated by cell-to-cell differences in ionic conductances. In this study, we describe the construction of an experimentally-calibrated set of stochastic cardiac cell models that captures both BVR and cell-to-cell differences in BVR displayed in isolated canine action potential measurements using pharmacological agents. Simulated and experimental ranges of BVR are compared in control and under pharmacological inhibition, and the key ionic currents determining BVR under physiological and pharmacological conditions are identified. Results show that the 4-aminopyridine-sensitive transient outward potassium current, Ito1, is a fundamental driver of BVR in control and upon complete inhibition of the slow delayed rectifier potassium current, IKs. In contrast, IKs and the L-type calcium current, ICaL, become the major contributors to BVR upon inhibition of the fast delayed rectifier potassium current, IKr. This highlights both IKs and Ito1 as key contributors to repolarization reserve. Partial correlation analysis identifies the distribution of Ito1 channel numbers as an important independent determinant of the magnitude of BVR and drug-induced change in BVR in control and under pharmacological inhibition of ionic currents. Distributions in the number of IKs and ICaL channels only become independent determinants of the magnitude of BVR upon complete inhibition of IKr. These findings provide quantitative insights into the ionic causes of BVR as a marker for repolarization reserve, both under control condition and pharmacological inhibition.

Allicin inhibits transient outward potassium currents in mouse ventricular myocytes.

Allicin is the active constituent of garlic, a widely used spice and food. The remedial properties of garlic have also been extensively researched and it has been demonstrated that allicin is able to inhibit the transient outward potassium current (Ito) in atrial myocytes. However, the direct effect of allicin on Ito in ventricular myocytes has yet to be elucidated. In the present study, the effects of allicin on Ito in ventricular myocytes isolated from mice were investigated, using the whole-cell patch recording technique. The results revealed that Ito current was not significantly suppressed by allicin in the low-dose group (10 µmol/l; P>0.05). However, Ito was significantly inhibited by higher doses of allicin (30, 100 and 300 µmol/l; P<0.05 vs. control; n=6) in a concentration-dependent manner (IC50=41.6 µmol/l). In addition, a high concentration of allicin (≥100 µmol/l) was able to accelerate the voltage-dependent inactivation of Ito in mouse ventricular myocytes. In conclusion, the present study revealed that allicin inhibited the Ito in mouse ventricular myocytes, which may be the mechanism through which allicin exerts its antiarrhythmic effect.

ZFHX3 knockdown increases arrhythmogenesis and dysregulates calcium homeostasis in HL-1 atrial myocytes

BackgroundZFHX3 plays an important role in the genesis of atrial fibrillation. However, the atrial electrophysiological effects of ZFHX3 are not clear. This study sought to investigate roles of ZFHX3 in atrial electrophysiology and calcium homeostasis by using HL-1 atrial myocytes knocked-down with ZFHX3. MethodsPatch clamp, confocal fluorescence microscopy and Western blot were used to study electrical activity, ionic currents, calcium homeostasis and protein expressions in stable ZFHX3 shRNA cells. ResultsAs compared to control, ZFHX3 shRNA cells with 28% decline of ZFHX3 protein had a larger sarcoplasmic reticulum Ca2+ content by 62%, Ca2+ transient by 20%, and calcium leak by 75%. ZFHX3 shRNA cells (n=35) had shorter action potential duration (APD) at 50% (14.7±0.9 versus 20.3±1.4ms, P<0.005), and 20% (6.1±0.3 versus 8.3±0.8ms, P<0.005) repolarization than control cells (n=30). ZFHX3 shRNA cells (n=10) had larger amplitudes of isoproterenol (1μM)-induced delayed afterdepolarization (14.1±0.9 versus 7.2±0.2mV, P<0.05) than control cells (n=10). Besides, acetylcholine (3μM) shortened APD at 90% repolarization to a greater extent (19±4% versus 7±2%, P<0.01) in ZFHX3 shRNA cells (n=11) than in control cells (n=12). In addition, ZFHX3 shRNA cells had increased expressions of SERCA2a, ryanodine receptor, Kv1.4, Kv1.5 and Kir3.4. Moreover, ZFHX3 shRNA cells had a larger SERCA2a activity, ultra-rapid delayed rectifier potassium currents, transient outward currents and acetylcholine-sensitive potassium currents. ConclusionsZFHX3 knock-down in atrial myocytes dysregulated calcium homeostasis and increased atrial arrhythmogenesis, which may contribute to the occurrence of AF.

Ionic Basis of Repolarization of Atrial and Ventricular Specific Cell Types Derived from Human Induced Pluripotent Stem Cells

Cardiomyocytes derived from human induced pluripotent stem cells (hIPSC-CMs) are an innovative cellular system for understanding human cardiac pathology and physiology. However, hIPSC-CMs express low levels of inward rectifying potassium channel (IK1) relative to native cardiac cells. The lack of this current deforms the action potential (AP) and leads to a depolarized or spontaneous diastolic potential. We used electronic expression of IK1 via dynamic clamp to restore the resting membrane potential back to physiological levels. This resulted in an improved AP morphology, including a reduction in variability and a rate dependent spike and dome shape. There were several significant differences between atrial and ventricular cells including differences in cell capacitance, sodium current magnitude and kinetics and most prominently differences in the transient outward currents. The late components of outward current, cells with atrial APs had a significantly larger sustained outward IKUR or Kv1.5-like component at +50 mV than ventricular shaped APs (in pA/pF: 3.71 ± 0.55 (n=5) vs 1.00 ± 0.10 (n=16),P<0.05) but similar peak outward currents: (6.89 ± 0.50 (n=5) vs. 6.58 ± 0 .67 (n=14),P=N.S). This plateau current is strongly inhibited by 50 micro molar 4-aminopyridine(4-AP). Similarly, atrial-like APs took on a ventricular like shape when treated with 4-AP while the ventricular myocytes APs were 4-AP insensitive. A cloned Kv1.5 current was expressed in oocytes and was used through the electronic expression system to add an IKur to the ventricular myocytes. Addition of this current changed the action potential morphology from ventricle to atrial like. This strongly suggests that IKur is the major determinant of atrial action potential morphology.

Reductions in the Cardiac Transient Outward K+ Current Ito Caused by Chronic β-Adrenergic Receptor Stimulation Are Partly Rescued by Inhibition of Nuclear Factor κB

The fast transient outward potassium current (Ito,f) plays a critical role in the electrical and contractile properties of the myocardium. Ito,f channels are formed by the co-assembly of the pore-forming α-subunits, Kv4.2 and Kv4.3, together with the accessory β-subunit KChIP2. Reductions of Ito,f are common in the diseased heart, which is also associated with enhanced stimulation of β-adrenergic receptors (β-ARs). We used cultured neonatal rat ventricular myocytes to examine how chronic β-AR stimulation decreases Ito,f. To determine which downstream pathways mediate these Ito,f changes, adenoviral infections were used to inhibit CaMKIIδc, CaMKIIδb, calcineurin, or nuclear factor κB (NF-κB). We observed that chronic β-AR stimulation with isoproterenol (ISO) for 48 h reduced Ito,f along with mRNA expression of all three of its subunits (Kv4.2, Kv4.3, and KChIP2). Inhibiting either CaMKIIδc nor CaMKIIδb did not prevent the ISO-mediated Ito,f reductions, even though CaMKIIδc and CaMKIIδb clearly regulated Ito,f and the mRNA expression of its subunits. Likewise, calcineurin inhibition did not prevent the Ito,f reductions induced by β-AR stimulation despite strongly modulating Ito,f and subunit mRNA expression. In contrast, NF-κB inhibition partly rescued the ISO-mediated Ito,f reductions in association with restoration of KChIP2 mRNA expression. Consistent with these observations, KChIP2 promoter activity was reduced by p65 as well as β-AR stimulation. In conclusion, NF-κB, and not CaMKIIδ or calcineurin, partly mediates the Ito,f reductions induced by chronic β-AR stimulation. Both mRNA and KChIP2 promoter data suggest that the ISO-induced Ito,f reductions are, in part, mediated through reduced KChIP2 transcription caused by NF-κB activation.

Cellular and ionic mechanisms underlying the effects of cilostazol, milrinone, and isoproterenol to suppress arrhythmogenesis in an experimental model of early repolarization syndrome

Early repolarization syndrome (ERS) is associated with polymorphic ventricular tachycardia (PVT) and ventricular fibrillation, leading to sudden cardiac death. The present study tests the hypothesis that the transient outward potassium current (Ito)-blocking effect of phosphodiesterase-3 (PDE-3) inhibitors plays a role in reversing repolarization heterogeneities responsible for arrhythmogenesis in experimental models of ERS. Transmembrane action potentials (APs) were simultaneously recorded from epicardial and endocardial regions of coronary-perfused canine left ventricular (LV) wedge preparations, together with a transmural pseudo-electrocardiogram. The Ito agonist NS5806 (7-15 μM) and L-type calcium current (ICa) blocker verapamil (2-3 μM) were used to induce an early repolarization pattern and PVT. After stable induction of arrhythmogenesis, the PDE-3 inhibitors cilostazol and milrinone or isoproterenol were added to the coronary perfusate. All were effective in restoring the AP dome in the LV epicardium, thus abolishing the repolarization defects responsible for phase 2 reentry and PVT. Arrhythmic activity was suppressed in 7 of 8 preparations by cilostazol (10 μM), 6 of 7 by milrinone (2.5 μM), and 7 of 8 by isoproterenol (0.1-1 μM). Using voltage clamp techniques applied to LV epicardial myocytes, both cilostazol (10 μM) and milrinone (2.5 μM) were found to reduce Ito by 44.4% and 40.4%, respectively, in addition to their known effects to augment ICa. Our findings suggest that PDE-3 inhibitors exert an ameliorative effect in the setting of ERS by producing an inward shift in the balance of current during the early phases of the epicardial AP via inhibition of Ito as well as augmentation of ICa, thus reversing the repolarization defects underlying the development of phase 2 reentry and ventricular tachycardia/ventricular fibrillation.

Effect of a novel BKCa opener on BKCa currents and contractility of the rabbit corpus cavernosum.

Large-conductance Ca(2+)-activated K(+) (BKCa) channels are thought to play a key role in the regulation of corpus cavernosum smooth muscle (CCSM) excitability. Few BKCa channel openers have been accepted for clinical development. The effect of the novel BKCa channel opener GoSlo-SR5-130 on electrical activity in isolated rabbit CCSM cells and mechanical activity in strips of rabbit CCSM was examined. Single-channel currents were observed in inside-out patches. These channels were sensitive to Ca(2+), blocked by penitrem A, and had a conductance of 291 ± 20 pS (n = 7). In the presence of GoSlo-SR5-130, the number of open BKCa channels increased. Using voltage-ramp protocols, GoSlo-SR5-130 caused currents to activate at more negative potentials in a concentration-dependent manner, shifting the half-maximal activation voltage potential to the left on the voltage axis. Therefore, BKCa channels were open within the physiological range of membrane potentials in the presence of GoSlo-SR5-130. GoSlo-SR5-130 also resulted in an increase in the activity of spontaneous transient outward currents in myocytes isolated from CCSM, and this effect was reversed by iberiotoxin. In current-clamp mode, GoSlo-SR5-130 hyperpolarized the cell membrane. Isometric tension recording of strips of rabbit corpus cavernosum showed that GoSlo-SR5-130 inhibited spontaneous contractions in a concentration-dependent manner. This effect was reversed in the presence of iberiotoxin, suggesting that GoSlo-SR5-130 exerts its effect through BKCa channels. These findings suggest that GoSlo-SR5-130 is an effective tool for the study of BKCa channels and that these channels can modulate CCSM activity and are possible targets for the treatment of erectile dysfunction.

Abstract 19562: PKG-I[[Unable to Display Character: &amp;#593;]] Oxidative Activation Regulates Small Artery Myogenic Tone Through Calcium Sparks and the BK Channel

Small resistance arteries function in a state of partial constriction generated by vascular smooth muscle cells (VSMC) in response to intraluminal pressure (‘myogenic constriction’). Collectively, the degree of myogenic constriction determines peripheral resistance and so influences systemic blood pressure. cGMP-dependent protein kinase (PKG) is widely implicated in vascular physiology, but until now has been assumed to play no role in modulation of this important vascular function on the basis that inhibitors of cGMP have no influence on pressure-induced tone. Recent identification of a novel redox mechanism by which PKG can be activated independently of the ‘classical’ NO-sGC-cGMP pathway made it important to revisit PKG’s role in these processes. To examine the relative contribution of these pathways, a transgenic mouse model was utilized, in which a single point mutation precludes activation via oxidative dimerization of PKG, whist preserving classical activation by cGMP. Diameter studies of small arteries from this ‘knock-in’ model were investigated in a pressure-myograph system, and demonstrated reduced constitutively functioning BKCa channel activity in these animals, and in consequence markedly enhanced myogenic constriction. Through imaging of calcium sparks with a spinning disc confocal microscope, the loss of BKCa channel function was shown to be due to diminished VSMC calcium sparks. Patch clamp electrophysiological studies showed that the amplitude and frequency of spontaneous transient outward currents were also reduced, consistent with these findings. Pharmacological studies using both pressure and wire myography with the application of the oxidant hydrogen peroxide provided a signal that myogenic tone initiates oxidant-induced activation of PKG. Western blot analysis of vessels fixed during relevant functional responses confirmed that generation of pressure-induced tone triggers oxidant activation of PKG. Finally, optical monitoring confirmed elevated ROS production with the induction of tone. Our results provide a conceptual framework by which vascular tone is modulated through release of ROS and oxidant-activation of PKG through calcium signalling. This may have important implications for blood pressure.

Chronic fetal exposure to caffeine altered resistance vessel functions via RyRs-BKCa down-regulation in rat offspring.

Caffeine modifies vascular/cardiac contractility. Embryonic exposure to caffeine altered cardiac functions in offspring. This study determined chronic influence of prenatal caffeine on vessel functions in offspring. Pregnant Sprague-Dawley rats (5-month-old) were exposed to high dose of caffeine, their offspring (5-month-old) were tested for vascular functions in mesenteric arteries (MA) and ion channel activities in smooth muscle cells. Prenatal exposure to caffeine increased pressor responses and vasoconstrictions to phenylephrine, accompanied by enhanced membrane depolarization. Large conductance Ca2+-activated K+ (BKCa) channels in buffering phenylephrine-induced vasoconstrictions was decreased, whole cell BKCa currents and spontaneous transient outward currents (STOCs) were decreased. Single channel recordings revealed reduced voltage/Ca2+ sensitivity of BKCa channels. BKCa α-subunit expression was unchanged, BKCa β1-subunit and sensitivity of BKCa to tamoxifen were reduced in the caffeine offspring as altered biophysical properties of BKCa in the MA. Simultaneous [Ca2+]i fluorescence and vasoconstriction testing showed reduced Ca2+, leading to diminished BKCa activation via ryanodine receptor Ca2+ release channels (RyRs), causing enhanced vascular tone. Reduced RyR1 was greater than that of RyR3. The results suggest that the altered STOCs activity in the caffeine offspring could attribute to down-regulation of RyRs-BKCa, providing new information for further understanding increased risks of hypertension in developmental origins.

Cannabinoids Regulate Intestinal Motor Function and Electrophysiological Activity of Myocytes in Rodents

This study aims to investigate the effects of cannabinoid (CB)1 and CB2 receptor ligands on intestinal motor function and muscular electrophysiological activity in rodent gastrointestinal (GI) tract. Lipopolysaccharide (LPS) was used to induce intestinal hypomotility. The effect of selective CB1 and CB2 agonists and antagonists on contractility of the muscle strips from rat jejunum was measured using organ bath, and the membrane potential of the jejunal smooth muscle cells was recorded with intracellular microelectrodes. The single cell patch clamp technique was applied to record delayed rectifying potassium currents (IKV) and spontaneous transient outward currents (STOC). LPS significantly reduced contractility of the smooth muscle strips (p <0.010) and caused hyperpolarization of membrane potential of the smooth muscle cells (p<0.010). This LPS-induced effect was reversed by AM251 and AM630, selective CB1 and CB2 antagonists, respectively, which promoted contractions of smooth muscle strips and triggered cell depolarization (p <0.010). LPS-induced changes were further enhanced in the presence of CB agonists, HU210 and WIN55 (p <0.050 or p <0.010). No effect of HU210 or AM251 on IKV and STOC has been observed. This exvivo study suggests that CB1 and CB2 receptors are involved in intestinal motor function in normal and LPS-induced pathological states and the regulation of the membrane potential of smooth muscle cells is very likely one of the effective mechanisms. This is one of the first reports on neuronal regulation of intestinal motility through CB-dependent pathways with potential application in the treatment of inflammatory and functional GI disorders.

Estrogen suppresses epileptiform activity by enhancing Kv4.2-mediated transient outward potassium currents in primary hippocampal neurons.

Catamenial epilepsy is a common phenomenon in female epileptic patients that is, in part, influenced by the 17-β-estradiol level during the menstrual cycle, which modulates the strength of the epileptic seizures. However, the underlying mechanism(s) for catamenial epilepsy remains unknown. In the present study, the effect of 17‑β‑estradiol on modulating epileptiform activities was investigated in cultured hippocampal neurons by focusing on the transient outward potassium current. Using the patch clamp technique, 17‑β‑estradiol was demonstrated to have a dose‑dependent U‑shape effect on epileptiform bursting activities in cultured hippocampal neurons; only the low dose (~0.1 ng/ml) of 17‑β‑estradiol had a suppressive effect on the epileptiform activities. The blockade effect of the low dose 17‑β‑estradiol could be suppressed by phrixotoxin2 (PaTx2), a selective channel blocker for voltage‑gated potassium channel type 4.2 (Kv4.2), which mediates the transient outward potassium current. Furthermore, the 17‑β‑estradiol bell‑shape‑like dose‑dependently enhanced the transient outward potassium current, which was inhibited by the estrogen receptor antagonist ICI 182,780. In conclusion, these results indicate that reduced activation of the transient outward potassium current by a high (or none) 17‑β‑estradiol level may enhance the epileptiform bursting activities in neurons, which may be one of the triggering causes for catamenial epilepsy, and therefore, maintaining a certain low 17‑β‑estradiol level may aid in the control of catamenial epilepsy.

Pro-arrhythmic action of autoantibodies against the second extracellular loop of β1-adrenoceptor and its underlying molecular mechanisms

ObjectivesThe incidence of arrhythmia is associated with autoantibodies against the second extracellular loop of β1-adrenergic receptor (β1-AAbs). The current study was designed to determine the mechanisms by which arrhythmia experimentally might be induced by β1-AAbs. MethodsBlood samples were collected from patients with varied arrhythmias or coronary heart disease (CHD) and healthy subjects. The titer of β1-AAbs was assessed. Passive immunization rat models with β1-AAbs were established to determine whether β1-AAbs induced arrhythmia. Conventional intracellular microelectrode technique and whole cell patch clamp were employed to record action potential duration (APD), resting potential (RP), L-type calcium current (ICa-L), sodium–calcium exchange current (INCX), transient outward potassium current (Ito), inward rectifier potassium current (Ik1) and delayed rectifier potassium current (Ik). ResultsHigh levels of β1-AAbs were found in the sera of heart disease patients, especially in ventricular arrhythmia (VA). Transfusion with β1-AAbs could induce arrhythmias in normal rats in vivo. β1-AAbs purified from the sera of active immunized rats induced triggered activity (TA), delayed after depolarization (DAD), and prolonged APD in the papillary muscles of rats. β1-AAbs prolonged QT interval, increased ICa-L and decreased IK1, Ito and INa–Ca in rat ventricular myocytes in vitro. All these effects can be inhibited by β1-AR blocker metoprolol. ConclusionsThese results demonstrate for the first time that β1-AAbs could directly induce ventricular arrhythmia by prolonging QT interval.

Brugada Syndrome: Clinical, Genetic, Molecular, Cellular, and Ionic Aspects

Brugada syndrome (BrS) is an inherited cardiac arrhythmia syndrome first described as a new clinical entity in 1992. Electrocardiographically characterized by distinct coved type ST segment elevation in the right-precordial leads, the syndrome is associated with a high risk for sudden cardiac death in young adults, and less frequently in infants and children. The electrocardiographic manifestations of BrS are often concealed and may be unmasked or aggravated by sodium channel blockers, a febrile state, vagotonic agents, as well as by tricyclic and tetracyclic antidepressants. An implantable cardioverter defibrillator is the most widely accepted approach to therapy. Pharmacologic therapy is designed to produce an inward shift in the balance of currents active during the early phases of the right ventricular action potential (AP) and can be used to abort electrical storms or as an adjunct or alternative to device therapy when use of an implantable cardioverter defibrillator is not possible. Isoproterenol, cilostazol, and milrinone boost calcium channel current and drugs like quinidine, bepridil, and the Chinese herb extract Wenxin Keli inhibit the transient outward current, acting to diminish the AP notch and thus to suppress the substrate and trigger for ventricular tachycardia or fibrillation. Radiofrequency ablation of the right ventricular outflow tract epicardium of patients with BrS has recently been shown to reduce arrhythmia vulnerability and the electrocardiographic manifestation of the disease, presumably by destroying the cells with more prominent AP notch. This review provides an overview of the clinical, genetic, molecular, and cellular aspects of BrS as well as the approach to therapy.

Epinephrine reversed high-concentration bupivacaine-induced inhibition of calcium channels and transient outward potassium current channels, but not on sodium channel in ventricular myocytes of rats.

BackgroundEpinephrine is a first-line drug for cardiopulmonary resuscitation, but its efficacy in the treatment of bupivacaine-induced cardiac toxicity is still in question. We hypothesized that epinephrine can reverse cardiac inhibition of bupivacaine by modulating ion flows through the ventricular myocyte membrane channels of rats. The aim of this study was to observe and report the effects of epinephrine on high-concentration bupivacaine-induced inhibition of sodium (INa), L-type calcium (ICa-L), and transient outward potassium (Ito) currents in the ventricular myocytes of rats.MethodsThe ventricular myocytes were isolated from Sprague-Dawley rats (250-300 g) by acute enzymatic dissociation. The whole-cell patch clamp technique was used to record the ion channel currents in single ventricular myocytes both before and after administration of medications.ResultAdministration of bupivacaine 100 μmol/L significantly reduced INa, (P < 0.05). However, administration of bupivacaine 100 μmol/L in conjunction with epinephrine 0.15 μg/ml had no effect in restoring INa to its previous state. Similarly, a sharp decline of ICa-L and Ito was observed after administration of bupivacaine 100 μmol/L (P < 0.05). In contrast to INa, ICa-L and Ito were significantly improved after the administration of the aforementioned combination of bupivacaine and epinephrine (P < 0.05).ConclusionEpinephrine can reverse high-concentration bupivacaine induced inhibition of ICa-L and Ito, but not INa. Thus, epinephrine’s effectiveness in reversal of bupivacaine-induced cardiac toxicity secondary to sodium channel inhibition may be limited.

High Activity of Large‐conductance Ca 2+ ‐activated K + channels Underlies Weak Myogenic Tone of Rat Mesenteric Arteries in Comparison of Cerebral Artery

Myogenic tone (MT) of resistance artery, vasoconstriction in response to increasing luminal pressure (Plum), stabilizes regional blood flow albeit the fluctuation of perfusion pressure. The levels of MT differ between vascular beds. Mechanosensitive depolarization of membrane potential and activation of voltage-gated Ca2+ channels (VOCCs) are cellular mechanisms for MT in smooth muscle cells (SMCs). Large-conductance Ca2+-activated K+ channel (BKCa) in SMCs may antagonize the contractile signals in MT. Using video-analysis of pressurized (60 mmHg) artery, we found that MT in mesenteric artery (MA) is significantly smaller than that of cerebral artery (CA). Iberiotoxin, a selective BKCa inhibitor, augmented the MT of MA by larger extent than CA. Patch clamp analysis showed that not only the BKCa current density but also unitary slope conductance of MASMC are higher than CASMCs. In addition, the frequency of spontaneous transient outward currents (STOCs) reflecting the BKCa activity in responses to Ca2+ sparks was higher in MASMCs. Voltage-gated K+ channel (Kv) activity was also higher in MASMCs. Although Ba2+-sensitive inwardly rectifying K+ current is larger in CASMCs, pretreatment with Ba2+did not induce significant augmentation of iberiotoxin sensitivity of MT in CA. Taken together, it is suggested that more frequent STOCs and larger average outward current via BKCa could explain the weak MT in MA.

Involvement of large-conductance Ca2+-activated K+ channels in chloroquine-induced force alterations in pre-contracted airway smooth muscle.

The participation of large-conductance Ca2+ activated K+ channels (BKs) in chloroquine (chloro)-induced relaxation of precontracted airway smooth muscle (ASM) is currently undefined. In this study we found that iberiotoxin (IbTx, a selective inhibitor of BKs) and chloro both completely blocked spontaneous transient outward currents (STOCs) in single mouse tracheal smooth muscle cells, which suggests that chloro might block BKs. We further found that chloro inhibited Ca2+ sparks and caffeine-induced global Ca2+ increases. Moreover, chloro can directly block single BK currents completely from the intracellular side and partially from the extracellular side. All these data indicate that the chloro-induced inhibition of STOCs is due to the blockade of chloro on both BKs and ryanodine receptors (RyRs). We also found that low concentrations of chloro resulted in additional contractions in tracheal rings that were precontracted by acetylcholine (ACH). Increases in chloro concentration reversed the contractile actions to relaxations. In the presence of IbTx or paxilline (pax), BK blockers, chloro-induced contractions were inhibited, although the high concentrations of chloro-induced relaxations were not affected. Taken together, our results indicate that chloro blocks BKs and RyRs, resulting in abolishment of STOCs and occurrence of contraction, the latter will counteract the relaxations induced by high concentrations of chloro.

Cold-Inducible RNA-Binding Protein Regulates Cardiac Repolarization by Targeting Transient Outward Potassium Channels

Cold-inducible RNA-binding protein (CIRP) is constitutively expressed at low levels across various tissues. It is rapidly upregulated by multiple stresses, underlying a general role for CIRP in organic adaptations to pathophysiological conditions. However, the role of CIRP in the heart remains unclear. To examine the biofunctions of CIRP in the mammalian heart. Rats with targeted disruption of Cirp were generated using the TALEN (transcription activator-like effector nucleases)-based genome editing technique. The Cirp-knockout rats had structurally and functionally normal hearts. Resting ECG recordings revealed a short rate-corrected QT (QTc) interval in Cirp-null rats without any abnormalities in PR interval, RR interval or QRS waves as compared to wild-type animals. The shortened QTc interval from Cirp ablation was tightly linked to an abbreviated action potential duration in cardiac myocytes, which was attributable to increased transient outward potassium current (Ito). Furthermore, our findings uncovered that CIRP protein selectively bonded to KCND2 and KCND3 mRNAs encoding the functional α-subunits of Ito channel proteins. CIRP deficiency did not change the transcriptional activity of KCND2 or KCND3, but it facilitated their translation. Cirp knockout had no effect on the functional expression of ion channels other than Ito channels. CIRP modulates cardiac repolarization by negatively adjusting the expression and function of Ito channels. Our study may open a window to decipher the potential function of RNA-binding proteins in bioelectric activity.

Functional properties of a Brazilian derived mouse embryonic stem cell line.

Pluripotent mouse embryonic stem cells (mESC) are cell lines derived from the inner cell mass of blastocyst-stage early mammalian embryos. Since ion channel modulation has been reported to interfere with both growth and differentiation process in mouse and human ESC it is important to characterize the electrophysiological properties of newly generated mESC and compare them to other lines. In this work, we studied the intercellular communication by way of gap junctions in a Brazilian derived mESC (USP-1, generated by Dr. Lygia Pereira's group) and characterized its electrophysiological properties. We used immunofluorescence and RT-PCR to reveal the presence of connexin 43 (Cx43), pluripotency markers and ion channels. Using a co-culture of neonatal mouse cardiomyocytes with mESC, where the heart cells expressed the enhanced Green Fluorescent Protein, we performed dye injections to assess functional coupling between the two cell types observing dye diffusion. The patch-clamp study showed outward currents identified as two types of potassium currents, transient outward potassium current (Ito) and delayed rectifier outward potassium current (Iks), by use of specific drug blockage. Calcium or sodium currents in undifferentiated mESC were not identified. We conclude that USP-1 mESC has functional Cx43 channels establishing intercellular communication among themselves and with cardiomyocytes and has a similar electrophysiological profile compared to other mESC cell lines.