Currents In Cardiomyocytes Research Articles

Persistent human cardiac Na+ currents in stably transfected mammalian cells

Miniature persistent late Na+ currents in cardiomyocytes have been linked to arrhythmias and sudden death. The goals of this study are to establish a stable cell line expressing robust persistent cardiac Na+ currents and to test Class 1 antiarrhythmic drugs for selective action against resting and open states. After transient transfection of an inactivation-deficient human cardiac Na+ channel clone (hNav1.5-CW with L409C/A410W double mutations), transfected mammalian HEK293 cells were treated with 1 mg/ml G-418. Individual G-418-resistant colonies were isolated using glass cylinders. One colony with high expression of persistent Na+ currents was subjected to a second colony selection. Cells from this colony remained stable in expressing robust peak Na+ currents of 996 ± 173 pA/pF at +50 mV (n = 20). Persistent late Na+ currents in these cells were clearly visible during a 4-second depolarizing pulse albeit decayed slowly. This slow decay is likely due to slow inactivation of Na+ channels and could be largely eliminated by 5 μM batrachotoxin. Peak cardiac hNav1.5-CW Na+ currents were blocked by tetrodotoxin with an IC50 value of 2.27 ± 0.08 μM (n = 6). At clinic relevant concentrations, Class 1 antiarrhythmics are much more selective in blocking persistent late Na+ currents than their peak counterparts, with a selectivity ratio ranging from 80.6 (flecainide) to 3 (disopyramide). We conclude that (1) Class 1 antiarrhythmics differ widely in their resting- vs. open-channel selectivity, and (2) stably transfected HEK293 cells expressing large persistent hNav1.5-CW Na+ currents are suitable for studying as well as screening potent open-channel blockers.

ASSA13-03-46 The Effects of Verapamil On SK2 Channel In Myocardium Cells From Human Chronic Atrial Fibrillation

<h3>Objective</h3> Small conductance calcium-activated potassium channel (SK channel) plays an important role in heart. Four subtype of SK channel are classified according to their sensitive to apamin. Electrophysiological properties of SK2 channel are voltage-independent as well as solely activated by changes in intracellular calcium concentration. This study investigated the alteration of SK2 channel currents in cAF patients. Moreover, L-type calcium channel inhibitor verapamil were adopted to observe the change of SK2 channel. We plan to explore the functional relationship between L-type calcium channel and SK2 channels. <h3>Methods</h3> Single human cardiac myocyte was isolated by two-step enzyme digestion. The whole-cell patch clamp technique was used to record the SK2 current in isolated cardiomyocytes from patients with siuns rhythm (SR) or chronic atrial fibrillation (cAF). Verapamil (10 μmol/L) and apamin (100 nmol/L) were added into bath solution respectively to observe the change of current. <h3>Results</h3> The current density (pA/pF) of SK2 channel as well as the ration in the integrated inward currents were up-regulated significantly in cAF groups. At wide range of test potentials, L-type calcium channel inhibitor verapamil reduced SK2 channel currents in cardiomyocytes from patients with SR or cAF. Compared with control group, the apamin-sensitive currents density of the verapamil group showed a significant decrease in human atrial myocytes. It indicates that part of SK2 current can be inhibited by verapamil. <h3>Conclusions</h3> It suggested that the up-regulation of SK2 currents underlies the occurrence and maintenance of cAF. There may be functionally coupling between SK2 channels and L-type calcium channels in human myocardium cells. The regulation of L-type calcium channel involves the remodelling of SK2 channels in the atrial myocytes from SR and cAF patients. This work was supported by the National Natural Science Foundation of China (No. 30870903) and the Natural Science Foundation of Luzhou.

Rescue of lethal CaVβ2 knock‐out and identification of novel CaVβ2 variants

Disruption of the CaVβ2 gene in mice resulted in diminished L‐type Ca currents in cardiomyocytes. Thereby heart function was compromised and embryonic death occurred. Partial rescue of the Ca channel currents by Bayk8644 significantly postponed embryonic death in CaVβ2‐deficient mice. The predominant ancillary β subunit of cardiac Ca channels is the β2 subunit and three splice variants are expressed in the heart, β2aN4&gt;; β2aN1&gt;;&gt;;β2aN5. β2aN1 appears to be mainly expressed in embryonic heart and the β2aN4 in adult heart. We now rescued the lethal β2−/− phenotype by expression of a mouse β2 transgene under the control of the mouse a‐MHC promoter and generated two mouse lines: one rescued with the β2aN1 of embryonic heart, one with the β2aN4 of adult heart. Characterization of the founders and the β2−/−/tg+/‐ mice by Southern blots and Western blots show that the β2 transgenes are expressed in heart only and that the extracardial β2‐deficiency persists. Comparing β2 protein expression in adult heart and various tissues from wild‐type and transgenic mice reveals that additional and novel CaVβ2 variants are expressed in wild‐type tissues. We will characterize the novel CaVβ2 variants and the impact of β2‐deficiency in extracardial tissues. The availability of mice carrying either the N4 or the N1 β2 variant only in cardiac myocytes allows us to study the roles of either variant on L‐type Ca channels in cardiomyocytes.

Catharanthine Dilates Small Mesenteric Arteries and Decreases Heart Rate and Cardiac Contractility by Inhibition of Voltage-Operated Calcium Channels on Vascular Smooth Muscle Cells and Cardiomyocytes

Catharanthine is a constituent of anticancer vinca alkaloids. Its cardiovascular effects have not been investigated. This study compares the in vivo hemodynamic as well as in vitro effects of catharanthine on isolated blood vessels, vascular smooth muscle cells (VSMCs), and cardiomyocytes. Intravenous administration of catharanthine (0.5-20 mg/kg) to anesthetized rats induced rapid, dose-dependent decreases in blood pressure (BP), heart rate (HR), left ventricular blood pressure, cardiac contractility (dP/dt(max)), and the slope of the end-systolic pressure-volume relationship (ESPVR) curve. Catharanthine evoked concentration-dependent decreases (I(max) >98%) in endothelium-independent tonic responses of aortic rings to phenylephrine (PE) and KCl (IC(50) = 28 µM for PE and IC(50) = 34 µM for KCl) and of third-order branches of the small mesenteric artery (MA) (IC(50) = 3 µM for PE and IC(50) = 6 µM for KCl). Catharanthine also increased the inner vessel wall diameter (IC(50) = 10 µM) and reduced intracellular free Ca(2+) levels (IC(50) = 16 µM) in PE-constricted MAs. Patch-clamp studies demonstrated that catharanthine inhibited voltage-operated L-type Ca(2+) channel (VOCC) currents in cardiomyocytes and VSMCs (IC(50) = 220 µM and IC(50) = 8 µM, respectively) of MA. Catharanthine lowers BP, HR, left ventricular systolic blood pressure, and dP/dt(max) and ESPVR likely via inhibition of VOCCs in both VSMCs and cardiomyocytes. Since smaller vessels such as the third-order branches of MAs are more sensitive to VOCC blockade than conduit vessels (aorta), the primary site of action of catharanthine for lowering mean arterial pressure appears to be the resistance vasculature, whereas blockade of cardiac VOCCs may contribute to the reduction in HR and cardiac contractility seen with this agent.

Phosphodiesterase type 3A regulates basal myocardial contractility through interacting with sarcoplasmic reticulum calcium ATPase type 2a signaling complexes in mouse heart.

cAMP is an important regulator of myocardial function, and regulation of cAMP hydrolysis by cyclic nucleotide phosphodiesterases (PDEs) is a critical determinant of the amplitude, duration, and compartmentation of cAMP-mediated signaling. The role of different PDE isozymes, particularly PDE3A vs PDE3B, in the regulation of heart function remains unclear. To determine the relative contribution of PDE3A vs PDE3B isozymes in the regulation of heart function and to dissect the molecular basis for this regulation. Compared with wild-type littermates, cardiac contractility and relaxation were enhanced in isolated hearts from PDE3A(-/-), but not PDE3B(-/-), mice. Furthermore, PDE3 inhibition had no effect on PDE3A(-/-) hearts but increased contractility in wild-type (as expected) and PDE3B(-/-) hearts to levels indistinguishable from PDE3A(-/-). The enhanced contractility in PDE3A(-/-) hearts was associated with cAMP-dependent elevations in Ca(2+) transient amplitudes and increased sarcoplasmic reticulum (SR) Ca(2+) content, without changes in L-type Ca(2+) currents of cardiomyocytes, as well as with increased SR Ca(2+)-ATPase type 2a activity, SR Ca(2+) uptake rates, and phospholamban phosphorylation in SR fractions. Consistent with these observations, PDE3 activity was reduced ≈8-fold in SR fractions from PDE3A(-/-) hearts. Coimmunoprecipitation experiments further revealed that PDE3A associates with both SR calcium ATPase type 2a and phospholamban in a complex that also contains A-kinase anchoring protein-18, protein kinase type A-RII, and protein phosphatase type 2A. Our data support the conclusion that PDE3A is the primary PDE3 isozyme modulating basal contractility and SR Ca(2+) content by regulating cAMP in microdomains containing macromolecular complexes of SR calcium ATPase type 2a-phospholamban-PDE3A.

Selective inhibition of Cx43 hemichannels by Gap19 and its impact on myocardial ischemia/reperfusion injury

Connexin-43 (Cx43), a predominant cardiac connexin, forms gap junctions (GJs) that facilitate electrical cell-cell coupling and unapposed/nonjunctional hemichannels that provide a pathway for the exchange of ions and metabolites between cytoplasm and extracellular milieu. Uncontrolled opening of hemichannels in the plasma membrane may be deleterious for the myocardium and blocking hemichannels may confer cardioprotection by preventing ionic imbalance, cell swelling and loss of critical metabolites. Currently, all known hemichannel inhibitors also block GJ channels, thereby disturbing electrical cell-cell communication. Here we aimed to characterize a nonapeptide, called Gap19, derived from the cytoplasmic loop (CL) of Cx43 as a hemichannel blocker and examined its effect on hemichannel currents in cardiomyocytes and its influence in cardiac outcome after ischemia/reperfusion. We report that Gap 19 inhibits Cx43 hemichannels without blocking GJ channels or Cx40/pannexin-1 hemichannels. Hemichannel inhibition is due to the binding of Gap19 to the C-terminus (CT) thereby preventing intramolecular CT-CL interactions. The peptide inhibited Cx43 hemichannel unitary currents in both HeLa cells exogenously expressing Cx43 and acutely isolated pig ventricular cardiomyocytes. Treatment with Gap19 prevented metabolic inhibition-enhanced hemichannel openings, protected cardiomyocytes against volume overload and cell death following ischemia/reperfusion in vitro and modestly decreased the infarct size after myocardial ischemia/reperfusion in mice in vivo. We conclude that preventing Cx43 hemichannel opening with Gap19 confers limited protective effects against myocardial ischemia/reperfusion injury.

Modulation of excitability, membrane currents and survival of cardiac myocytes by N-acylethanolamines

N-Acylethanolamines (NAE) are endogenously produced lipids playing important roles in a diverse range of physiological and pathological conditions. In the present study, using whole-cell patch clamp technique, we have for the first time investigated the effects of the most abundantly produced NAEs, N-stearoylethanolamine (SEA) and N‐oleoylethanolamine (OEA), on electric excitability and membrane currents in cardiomyocytes isolated from endocardial, epicardial, and atrial regions of neonatal rat heart. SEA and OEA (1–10μM) attenuated electrical activity of the myocytes from all regions of the cardiac muscle by hyperpolarizing resting potential, reducing amplitude, and shortening the duration of the action potential. However, the magnitudes of these effects varied significantly depending on the type of cardiac myocyte (i.e., endocardial, epicardial, atrial) with OEA being generally more potent. OEA and to a lesser extent SEA suppressed in a concentration-dependent manner currents through voltage-gated Na+ (VGSC) and L-type Ca2+ (VGCC) channels, but induced variable cardiac myocyte type-dependent effects on background K+ and Cl− conductance. The mechanisms of inhibitory action of OEA on cardiac VGSCs and VGCCs involved influence on channels' activation/inactivation gating and partial blockade of ion permeation. OEA also enhanced the viability of cardiac myocytes by reducing necrosis without a significant effect on apoptosis. We conclude that SEA and OEA attenuate the excitability of cardiac myocytes mainly through inhibition of VGSCs and VGCC-mediated Ca2+ entry. Since NAEs are known to increase during tissue ischemia and infarction, these effects of NAEs may mediate some of their cardioprotective actions during these pathological conditions.

Hydrogen Sulfide Inhibits L-Type Calcium Currents Depending upon the Protein Sulfhydryl State in Rat Cardiomyocytes

Hydrogen sulfide (H2S) is a novel gasotransmitter that inhibits L-type calcium currents (I Ca, L). However, the underlying molecular mechanisms are unclear. In particular, the targeting site in the L-type calcium channel where H2S functions remains unknown. The study was designed to investigate if the sulfhydryl group could be the possible targeting site in the L-type calcium channel in rat cardiomyocytes. Cardiac function was measured in isolated perfused rat hearts. The L-type calcium currents were recorded by using a whole cell voltage clamp technique on the isolated cardiomyocytes. The L-type calcium channel containing free sulfhydryl groups in H9C2 cells were measured by using Western blot. The results showed that sodium hydrosulfide (NaHS, an H2S donor) produced a negative inotropic effect on cardiac function, which could be partly inhibited by the oxidant sulfhydryl modifier diamide (DM). H2S donor inhibited the peak amplitude of I Ca, L in a concentration-dependent manner. However, dithiothreitol (DTT), a reducing sulfhydryl modifier markedly reversed the H2S donor-induced inhibition of I Ca, L in cardiomyocytes. In contrast, in the presence of DM, H2S donor could not alter cardiac function and L type calcium currents. After the isolated rat heart or the cardiomyocytes were treated with DTT, NaHS could markedly alter cardiac function and L-type calcium currents in cardiomyocytes. Furthermore, NaHS could decrease the functional free sulfhydryl group in the L-type Ca2+ channel, which could be reversed by thiol reductant, either DTT or reduced glutathione. Therefore, our results suggest that H2S might inhibit L-type calcium currents depending on the sulfhydryl group in rat cardiomyocytes.

The Use of Ranolazine to Facilitate Electrical Cardioversion in Cardioversion‐Resistant Patients: A Case Series

Occasionally atrial fibrillation (AF) is resistant to electrical cardioversion (EC). Ranolazine (RZ) is an antianginal agent, which inhibits abnormal late Na(+) channel currents in cardiomyocytes and decreases Na(+) /Ca(++) overload. RZ is a potent inhibitor of after-depolarizations and triggered activity and prolongs atrial refractory periods. We postulated RZ could facilitate EC in patients resistant to EC. Over a 3-year period, we identified 25 EC-resistant patients who had been administered oral RZ shortly after failing attempted EC. The anterior-posterior cardioversion approach was used and each patient had failed to be restored to sinus rhythm despite using up to the maximum output of a biphasic cardioversion device. Repeat EC was performed 3.5-4 hours after administration of 2 g of oral RZ using the same device, sedation, and lead placement. Sinus rhythm was successfully restored in 19 (76%) of 25 EC-resistant patients. Three patients spontaneously converted before the second attempt at EC within 4 hours of the RZ dose. Of the 22 patients undergoing another attempt at EC, 16 were successfully converted to sinus rhythm. Five of the six patients who were refractory to repeat EC despite RZ had AF of unknown duration and each is now in permanent AF. No adverse effects were noted. RZ shows promise as a safe and convenient agent to facilitate EC in EC-resistant patients. It appears to be most effective in patients whose AF duration is known to be less than 3 months.

Cardioprotection by Klotho through downregulation of TRPC6 channels in the mouse heart

Klotho is a membrane protein predominantly produced in the kidney that exerts some anti-ageing effects. Ageing is associated with an increased risk of heart failure; whether Klotho is cardioprotective is unknown. Here we show that Klotho-deficient mice have no baseline cardiac abnormalities but develop exaggerated pathological cardiac hypertrophy and remodeling in response to stress. Cardioprotection by Klotho in normal mice is mediated by downregulation of TRPC6 channels in the heart. We demonstrate that deletion of Trpc6 prevents stress-induced exaggerated cardiac remodeling in Klotho-deficient mice. Furthermore, mice with heart-specific overexpression of TRPC6 develop spontaneous cardiac hypertrophy and remodeling. Klotho overexpression ameliorates cardiac pathologies in these mice and improves their long-term survival. Soluble Klotho present in the systemic circulation inhibits TRPC6 currents in cardiomyocytes by blocking phosphoinositide-3-kinase-dependent exocytosis of TRPC6 channels. These results provide a new perspective on the pathogenesis of cardiomyopathies and open new avenues for treatment of the disease.

Effects of ginkgo biloba extract and bilobalide, a main constituent, on the ionic currents in guinea pig ventricular cardiomyocytes.

Effects of Ginkgo biloba extract (GBE, 0.01 to 1 mg/ml) and a main constituent, bilobalide (0.1 to 1 mumol/l), on the action potentials and the underlying ionic currents in guinea pig ventricular cardiomyocytes were investigated using a patch-clamp technique. Both GBE and bilobalide at high concentrations caused depressant actions on the action potential configuration. GBE (0.3 mg/ml) decreased the Vmax by 17.1 +/- 2.1% (n = 6, p < 0.05), and bilobalide (1 mumol/l) by 14.7 +/- 2.2% (n = 8, p < 0.05). GBE prolonged the action potential duration at 90% repolarization (APD90), by 70.6 +/- 2.8% (n = 6, p < 0.001) at 1 mg/ml; in contrast, bilobalide shortened APD, by 11.1 +/- 2.0% (n = 8, p < 0.05) at 3 mumol/l. In voltage-clamp experiments, GBE (1 mg/ml) markedly inhibited the Ca2+ current (ICa) at 10 mV by 90.1 +/- 3.0% (n = 6, p < 0.001), the delayed rectifier K+ current (IK) at 60 mV by 63.7 +/- 3.0% (n = 6, p < 0.01), and the inwardly rectifying K+ current (IK1) at -120 mV by 47.8 +/- 2.6% (n = 6, p < 0.01). On the other hand, bilobalide at 1 mumol/l enhanced the ICa by 40.0 +/- 2.3% (n = 6, p < 0.05), and the IK by 14.0 +/- 2.3% (n = 6, p < 0.05), concentration-dependently. The IK1 was unaffected. These responses were reversible (to approximately 50-80%) after 10- to 20-min washout. These results indicate that even after acute administrations, GBE and bilobalide produced active actions on the APD and the ionic currents in cardiomyocytes. Although each chemical exhibited the responses in opposite directions, GBE acts totally as a mixture.

Sustained CaMKII activity mediates transient oxidative stress-induced long-term facilitation of L-type Ca2+ current in cardiomyocytes

Oxidative stress remodels Ca2+ signaling in cardiomyocytes, which promotes altered heart function in various heart diseases. Ca2+/calmodulin-dependent protein kinase II (CaMKII) was shown to be activated by oxidation, but whether and how CaMKII links oxidative stress to pathophysiological long-term changes in Ca2+ signaling remain unknown. Here, we present evidence demonstrating the role of CaMKII in transient oxidative stress-induced long-term facilitation (LTF) of L-type Ca2+ current (ICa,L) in rat cardiomyocytes. A 5-min exposure of 1mM H2O2 induced an increase in ICa,L, and this increase was sustained for ~1h. The CaMKII inhibitor KN-93 fully reversed H2O2-induced LTF of ICa,L, indicating that sustained CaMKII activity underlies this oxidative stress-induced memory. Simultaneous inhibition of oxidation and autophosphorylation of CaMKII prevented the maintenance of LTF, suggesting that both mechanisms contribute to sustained CaMKII activity. We further found that sarcoplasmic reticulum Ca2+ release and mitochondrial ROS generation have critical roles in sustaining CaMKII activity via autophosphorylation- and oxidation-dependent mechanisms. Finally, we show that long-term remodeling of the cardiac action potential is induced by H2O2 via CaMKII. In conclusion, CaMKII and mitochondria confer oxidative stress-induced pathological cellular memory that leads to cardiac arrhythmia.

Targeting of protein phosphatases PP2A and PP2B to the C-terminus of the L-type calcium channel Ca v1.2.

The L-type Ca(2+) channel Ca(v)1.2 forms macromolecular signaling complexes that comprise the β(2) adrenergic receptor, trimeric G(s) protein, adenylyl cyclase, and cAMP-dependent protein kinase (PKA) for efficient signaling in heart and brain. The protein phosphatases PP2A and PP2B are part of this complex. PP2A counteracts increase in Ca(v)1.2 channel activity by PKA and other protein kinases, whereas PP2B can either augment or decrease Ca(v)1.2 currents in cardiomyocytes depending on the precise experimental conditions. We found that PP2A binds to two regions in the C-terminus of the central, pore-forming α(1) subunit of Ca(v)1.2: one region spans residues 1795-1818 and the other residues 1965-1971. PP2B binds immediately downstream of residue 1971. Injection of a peptide that contained residues 1965-1971 and displaced PP2A but not PP2B from endogenous Ca(v)1.2 increased basal and isoproterenol-stimulated L-type Ca(2+) currents in acutely isolated cardiomyocytes. Together with our biochemical data, these physiological results indicate that anchoring of PP2A at this site of Ca(v)1.2 in the heart negatively regulates cardiac L-type currents, likely by counterbalancing basal and stimulated phosphorylation that is mediated by PKA and possibly other kinases.

Glycine inhibits the LPS-induced increase in cytosolic Ca2+ concentration and TNFα production in cardiomyocytes by activating a glycine receptor

Previous studies have demonstrated that glycine (GLY) markedly reduces lipopolysaccharide (LPS)-induced myocardial injury.However, the mechanism of this effect is still unclear. The present study investigated the effect of GLY on cytosolic calcium concentration([Ca2+]c) and tumor necrosis factor-alpha (TNFalpha) production in cardiomyocytes exposed to LPS, as well as whether the glycine-gated chloride channel is involved in this process. Neonatal rat cardiomyocytes were isolated, and the [Ca2+]c and TNFalpha levels were determined by using Fura-2 and a Quantikine enzyme-linked immunosorbent assay, respectively. The distribution of the GLY receptor and GLY-induced currents in cardiomyocytes were also investigated using immunocytochemistry and the whole-cell patch-clamp technique, respectively. LPS at concentrations ranging from 10 ng/mL to 100 microg/mL significantly stimulated TNFalpha production. GLY did not inhibit TNFalpha production induced by LPS at concentrations below 10 ng/mL but did significantly decrease TNFalpha release stimulated by 100 microg/mL LPS and prevented an LPS-induced increase in [Ca2+]c, which was reversed by strychnine, a glycine receptor antagonist. GLY did not block the isoproterenol-induced increase in [Ca2+]c, but did prevent the potassium chloride-induced increase in [Ca2+]c in cardiomyocytes.Strychnine reversed the inhibition of the KCl-stimulated elevation in [Ca2+]c by GLY. In chloride-free buffer, GLY had no effect on the dipotassium hydrogen phosphate-induced increase in [Ca2+]c. Furthermore, GLY receptor alpha1 and beta subunit-immunoreactive spots were observed in cardiomyocytes, and GLY-evoked currents were blocked by strychnine. Cardiomyocytes possess the glycine-gated chloride channel, through which GLY prevents the increase in [Ca2+]c and inhibits the TNFalpha production induced by LPS at high doses in neonatal rat cardiomyocytes.

Effect of antipsychotic drug perphenazine on fast sodium current and transient outward potassium current in rat ventricular myocytes

Antipsychotic drug perphenazine belongs to the phenothiazine group commonly reported to induce ECG changes and tachyarrhythmias. Data about its effect on ionic membrane currents in cardiomyocytes are missing. We analyzed the effect of perphenazine (0.1-100 microM) on fast sodium current I (Na) and transient outward potassium current I (to) in enzymatically isolated rat right ventricular myocytes by the whole-cell patch-clamp technique at room temperature. Perphenazine reversibly blocked I (Na) (reducing its amplitude; IC(50) = 1.24 +/- 0.10 microM) and I (to) (accelerating its apparent inactivation with a slight decrease of its amplitude; IC(50) = 38.2 +/- 3.5 microM, evaluated from changes of the time integral). The fast time constant of I (to) inactivation was significantly decreased in a concentration-dependent manner (IC(50) = 30.0 +/- 6.6 microM). Both blocks were use and frequency dependent at 3.3 Hz. We conclude that perphenazine causes concentration-, use-, and frequency-dependent block of I (Na) and I (to) . Computer simulations suggest that perphenazine interacts preferentially with I (Na) channels in inactivated states and with I (to) channels in both open and open-inactivated states.

Mechanisms of cardiac arrhythmias and sudden death in transgenic rabbits with long QT syndrome

Long QT syndrome (LQTS) is a heritable disease associated with ECG QT interval prolongation, ventricular tachycardia, and sudden cardiac death in young patients. Among genotyped individuals, mutations in genes encoding repolarizing K+ channels (LQT1:KCNQ1; LQT2:KCNH2) are present in approximately 90% of affected individuals. Expression of pore mutants of the human genes KCNQ1 (KvLQT1-Y315S) and KCNH2 (HERG-G628S) in the rabbit heart produced transgenic rabbits with a long QT phenotype. Prolongations of QT intervals and action potential durations were due to the elimination of IKs and IKr currents in cardiomyocytes. LQT2 rabbits showed a high incidence of spontaneous sudden cardiac death (>50% at 1 year) due to polymorphic ventricular tachycardia. Optical mapping revealed increased spatial dispersion of repolarization underlying the arrhythmias. Both transgenes caused downregulation of the remaining complementary IKr and IKs without affecting the steady state levels of the native polypeptides. Thus, the elimination of 1 repolarizing current was associated with downregulation of the reciprocal repolarizing current rather than with the compensatory upregulation observed previously in LQTS mouse models. This suggests that mutant KvLQT1 and HERG interacted with the reciprocal wild-type alpha subunits of rabbit ERG and KvLQT1, respectively. These results have implications for understanding the nature and heterogeneity of cardiac arrhythmias and sudden cardiac death.

Antiarrhythmic properties of a rapid delayed-rectifier current activator in rabbit models of acquired long QT syndrome

Impaired repolarization in cardiac myocytes can lead to long QT syndrome (LQTS), with delayed repolarization and increased susceptibility to Torsades de Pointes (TdP) arrhythmias. Current pharmacological treatment of LQTS is often inadequate. This study sought to evaluate the antiarrhythmic effect of a novel compound (NS1643) that activates the rapid delayed-rectifier K+ current, I(Kr), in two rabbit models of acquired LQTS. We used two clinically relevant in vivo rabbit models of TdP in which we infused NS1643 or vehicle: (i) three-week atrioventricular block with ventricular bradypacing; (ii) dofetilide-induced I(Kr) inhibition in methoxamine-sensitized rabbits. In addition, we studied effects on ionic currents in cardiomyocytes with I(Kr) suppressed by bradycardia remodelling or dofetilide exposure. Bradypaced rabbits developed QT interval prolongation, spontaneous ventricular ectopy, and TdP. Infusion of NS1643 completely suppressed arrhythmic activity and shortened the QT interval; vehicle had no effect. NS1643 also suppressed ventricular tachyarrhythmias caused by infusion of dofetilide to methoxamine-sensitized rabbits, and reversed dofetilide-induced QT prolongation. NS1643 increased I(Kr) in cardiomyocytes isolated from normal and bradycardia-remodelled rabbits by approximately 75% and 50%, respectively (P < 0.001 for each). Similarly, NS1643 restored I(Kr) suppressed by 5 nmol/L dofetilide (tail current 0.28 +/- 0.03 pA/pF pre-dofetilide, 0.20 +/- 0.01 pA/pF in the presence of dofetilide, 0.27 +/- 0.02 pA/pF after adding NS1643 to dofetilide-containing solution, P < 0.01). Pharmacological activation of I(Kr) reverses acquired LQTS and TdP caused by bradycardic remodelling and I(Kr)-blocking drugs. I(Kr)-activating drug therapy could be a potentially interesting treatment approach for LQTS.

Functional Expression of the TRPM4 Cationic Current in Ventricular Cardiomyocytes From Spontaneously Hypertensive Rats

Cardiac hypertrophy is associated with electrophysiological modifications, including modification of action potential shape that can give rise to arrhythmias. We report here a higher detection of a calcium-activated nonselective cation current in cardiomyocytes of spontaneously hypertensive rats (SHRs), a model of hypertension and heart hypertrophy when compared with Wistar-Kyoto (WKY) rat, its normotensive equivalent. Freshly isolated cells from the left ventricles of 3- to 6-month-old WKY rats or SHRs were used for patch-clamp recordings. In inside-out patches, the channel presented a linear conductance of 25+/-0.5 pS, did not discriminate Na(+) over K(+), and was not permeable to Ca(2+). Open probability was increased by depolarization and a rise in [Ca(2+)](i) (dissociation constant=10+/-5.4 micromol/L) but reduced by 0.5 mmol/L [ATP](i), 10 micromol/L glibenclamide, or flufenamic acid (IC(50)=5.5+/-1.7 micromol/L). Thus, it owns the fingerprint of the TRPM4 current. Although rarely detected in WKY cardiomyocytes, the current was present in >50% of patches from SHR cardiomyocytes. Moreover, by performing RT-PCR from ventricular samples, we observed that TRPM4 mRNA detection was higher in SHRs than in WKY rats. We propose that a TRPM4 current is expressed in ventricular cardiomyocytes from SHRs. According to its properties, this channel may contribute to the transient inward current implicated in delayed-after-depolarizations observed during [Ca(2+)] overload of cardiomyocytes.

Effect of haloperidol on transient outward potassium current in rat ventricular myocytes

Although sigma ligand haloperidol is known to affect repolarization in heart, its effect on potassium currents in cardiomyocytes has not yet been studied. We analyzed the effect of 1 μmol/l haloperidol on transient outward K + current ( I to ) in enzymatically isolated rat right ventricular cardiomyocytes using the whole-cell patch-clamp technique at room temperature. Haloperidol induced a decrease of amplitude and an acceleration of apparent inactivation of I to , both in a voltage-independent manner. The averaged inhibition of I to , evaluated as a change of its time integral, was 23.0 ± 3.2% at stimulation frequency of 0.1 Hz. As a consequence of slow recovery of I to from the haloperidol-induced block (time constant 1482 ±783 ms), a cumulation of the block up to about 40% appeared at 3.3 Hz. We conclude that haloperidol causes a voltage-independent block of I to that cumulates at higher stimulation frequencies. Based on the computer reconstruction of experimental data, a block of I to -channels in both open and open-inactivated states appears to be likely mechanism of haloperidol-induced inhibition of I to .

Reduced Cardiac L-Type Ca 2+ Current in Ca v β 2 −/− Embryos Impairs Cardiac Development and Contraction With Secondary Defects in Vascular Maturation

Cardiac myocyte contraction depends on transmembrane L-type Ca2+ currents and the ensuing release of Ca2+ from the sarcoplasmic reticulum. Here we show that these L-type Ca2+ currents are essential for cardiac pump function in the mouse at developmental stages where the functional significance of the heart becomes imperative to blood flow and to the continuing growth and survival of the embryo. Disruption of the Ca(V)beta2 gene, which encodes for the predominant ancillary beta subunit of cardiac Ca2+ channels, resulted in diminished L-type Ca2+ currents in cardiomyocytes of embryonic day 9.5 (E9.5). This led to a functionally compromised heart, causing defective remodeling of intra- and extraembryonic blood vessels and embryonic death following E10.5. The defects in vascular remodeling were also observed when the Ca(V)beta2 gene was selectively targeted in cardiomyocytes, demonstrating that they are secondary to cardiac failure rather than a result of the lack of Ca(V)beta2 proteins in the vasculature. Partial rescue of the Ca2+ channel currents by a Ca2+ channel agonist significantly postponed embryonic death in Ca(V)beta2-/- mice. Taken together, these data strongly support the essential role of L-type Ca2+ channel activity in cardiomyocytes for normal heart development and function and that this is a prerequisite for proper maturation of the vasculature.