Arrhythmia and sudden death associated with elevated cardiac chloride channel activity.

The identification and analysis of several cationic ion channels and their associated genes have greatly improved our understanding of the molecular and cellular mechanisms of cardiac arrhythmia. Our objective in this study was to examine the involvement of anionic ion channels in cardiac arrhythmia. We used a transgenic mouse model to overexpress the human cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a cAMP-regulated chloride channel. We used RNase protection and in situ hybridization assays to determine the level of CFTR expression, and radiotelemetry and in vivo electrophysiological study in combination with pharmacological intervention to analyse the cardiac function. Cardiac CFTR overexpression leads to stress-related sudden death in this model. In vivo intracardiac electrophysiological studies performed in anaesthetized mice showed no significant differences in baseline conduction parameters including atrial-His bundle (AH) or His bundle-ventricular (HV) conduction intervals, atrioventricular (AV) Wenckebach or 2:1 AV block cycle length and AV nodal functional refractory period. However, following isoproterenol administration, there was marked slowing of conduction parameters, including high-grade AV block in transgenic mice, with non-sustained ventricular tachycardia easily inducible using programmed stimulation or burst pacing. Our sudden death mouse model can be a valuable tool for investigation of the role of chloride channels in arrhythmogenesis and, potentially, for future evaluation of novel anti-arrhythmic therapeutic strategies and pharmacological agents.

Slow delayed rectifier K+ current block by HMR 1556 increases dispersion of repolarization and promotes Torsades de Pointes in rabbit ventricles.

BACKGROUND AND PURPOSE: The slow delayed rectifier K(+) current (I(Ks)) contributes to ventricular repolarization when the action potential (AP) is prolonged. I(Ks) block during drug-induced AP prolongation may promote Torsades de Pointes (TdP), but whether this is due to additional AP prolongation is uncertain. EXPERIMENTAL APPROACH: In bradycardic perfused rabbit ventricles, the incidence of spontaneous TdP, monophasic AP duration at 90% repolarization (MAPD(90)) and ECG interval between the peak and the end of T wave (T(peak-end)) (index of dispersion of repolarization) were measured after the administration of veratridine (125 nM, slows Na(+) channel inactivation), dofetilide (7.5 or 10 nM, a rapid delayed rectifier blocker) and HMR 1556 (HMR, 100 nM, an I(Ks) blocker), alone or in combinations (n=6 each). KEY RESULTS: HMR did not prolong MAPD(90), whereas veratridine or 7.5 nM dofetilide prolonged MAPD(90) (P<0.01) without inducing TdP. Veratridine+7.5 nM dofetilide additively prolonged MAPD(90) (P<0.05), induced 4+/-6 TdP per heart and prolonged T(peak-end) by 12+/-10 ms. Subsequent addition of HMR did not further prolonged MAPD(90), but increased the number of TdP to 22+/-18 per heart and increased T(peak-end) by 39+/-21 ms (P<0.05). Increasing dofetilide concentration from 7.5 to 10 nM (added to veratridine) produced a longer MAPD(90), but fewer TdP (5+/-5 per heart) and less T(peak-end) prolongation (17+/-8 ms) compared to the veratridine+7.5 nM dofetilide+HMR group (P<0.05). CONCLUSIONS AND IMPLICATIONS: Adding I(Ks) block markedly increases TdP incidence in hearts predisposed to TdP development by increasing the dispersion of repolarization, but without additional AP prolongation.

Impaired glucose-stimulated insulin secretion, enhanced intraperitoneal insulin tolerance, and increased beta-cell mass in mice lacking the p110gamma isoform of phosphoinositide 3-kinase.

Phosphoinositide 3-kinase (PI3 kinase) has been implicated in G protein-coupled receptor regulation of pancreatic beta-cell growth and glucose-stimulated insulin secretion. The G protein-activated p110gamma isoform of PI3 kinase was detected in insulinoma cells, mouse islets, and human islets. In 7- to 10-wk-old mice, knockout of p110gamma reduced the plasma insulin response to ip glucose injection and impaired first and second phase glucose-stimulated insulin secretion from pancreata perfused ex vivo. The p110gamma -/- mice responded to preinjection with the glucagon-like peptide-1 receptor agonist exendin 4, such that plasma glucose and insulin responses to ip glucose injection were not different from wild types. Mice lacking p110gamma were not diabetic and were only slightly glucose intolerant (ip glucose injection) compared with wild types, in part due to enhanced responsiveness to insulin as determined by an ip insulin tolerance test. Despite severely reduced insulin secretion in these animals, the p110gamma -/- mice had greater pancreatic insulin content, and an increased beta-cell mass due to beta-cell hypertrophy. These surprising results suggest that the G protein-coupled p110gamma isoform of PI3 kinase is not central to the development or maintenance of sufficient beta-cell mass but positively regulates glucose-stimulated insulin secretion.

Altered K(+) channel gene expression in diabetic rat ventricle: isoform switching between Kv4.2 and Kv1.4.

Expression of voltage-gated K(+) channels encoding the K(+) independent transient outward current in the streptozocin-induced diabetic (DM) rat ventricle was studied to determine the basis for slowed cardiac repolarization in diabetes mellitus. Although hypertrophy was not detected in diabetic rats at 12 wk after streptozocin treatment, ventricular Kv4.2 mRNA levels decreased 41% relative to nondiabetic controls. Kv1.4 mRNA levels increased 179% relative to controls, whereas Kv4.3 mRNA levels were unaffected. Immunohistochemistry and Western blot analysis of the diabetic heart showed that the density of the Kv4.2 protein decreased, whereas Kv1.4 protein increased. Thus isoform switching from Kv4.2 to Kv1.4 is most likely the mechanism underlying the slower kinetics of transient outward K(+) current observed in the diabetic ventricle. Brain Kv1.4, Kv4.2, or Kv4.3 mRNA levels were unaffected by diabetes. Myosin heavy chain (MHC) gene expression was altered with a 32% decrease in alpha-MHC mRNA and a 259% increase in beta-MHC mRNA levels in diabetic ventricle. Low-dose insulin-like growth factor-II (IGF-II) treatment during the last 6 of the 12 wk of diabetes (DM + IGF) protected against these changes in MHC mRNAs despite continued hyperglycemia and body weight loss. IGF-II treatment did not change K(+) channel mRNA levels in DM or control rat ventricles. Thus IGF treatment may prevent some, but not all, biochemical abnormalities in the diabetic heart.

Members of the Kv1 and Kv2 voltage-dependent K(+) channel families regulate insulin secretion.

In pancreatic beta-cells, voltage-dependent K(+) (Kv) channels are potential mediators of repolarization, closure of Ca(2+) channels, and limitation of insulin secretion. The specific Kv channels expressed in beta-cells and their contribution to the delayed rectifier current and regulation of insulin secretion in these cells are unclear. High-level protein expression and mRNA transcripts for Kv1.4, 1.6, and 2.1 were detected in rat islets and insulinoma cells. Inhibition of these channels with tetraethylammonium decreased I(DR) by approximately 85% and enhanced glucose-stimulated insulin secretion by 2- to 4-fold. Adenovirus-mediated expression of a C-terminal truncated Kv2.1 subunit, specifically eliminating Kv2 family currents, reduced delayed rectifier currents in these cells by 60-70% and enhanced glucose-stimulated insulin secretion from rat islets by 60%. Expression of a C-terminal truncated Kv1.4 subunit, abolishing Kv1 channel family currents, reduced delayed rectifier currents by approximately 25% and enhanced glucose-stimulated insulin secretion from rat islets by 40%. This study establishes that Kv2 and 1 channel homologs mediate the majority of repolarizing delayed rectifier current in rat beta-cells and that antagonism of Kv2.1 may prove to be a novel glucose-dependent therapeutic treatment for type 2 diabetes.

The molecular physiology of the cardiac transient outward potassium current (I(to)) in normal and diseased myocardium.

G. Y. Oudit, Z. Kassiri, R. Sah, R. J. Ramirez, C. Zobel and P. H. Backx. The Molecular Physiology of the Cardiac Transient Outward Potassium Current (I(to)) in Normal and Diseased Myocardium. Journal of Molecular and Cellular Cardiology (2001) 33, 851-872. The Ca(2+)-independent transient outward potassium current (I(to)) plays an important role in early repolarization of the cardiac action potential. I(to)has been clearly demonstrated in myocytes from different cardiac regions and species. Two kinetic variants of cardiac I(to)have been identified: fast I(to), called I(to,f), and slow I(to), called I(to,s). Recent findings suggest that I(to,f)is formed by assembly of K(v4.2)and/or K(v4.3)alpha pore-forming voltage-gated subunits while I(to,s)is comprised of K(v1.4)and possibly K(v1.7)subunits. In addition, several regulatory subunits and pathways modulating the level and biophysical properties of cardiac I(to)have been identified. Experimental findings and data from computer modeling of cardiac action potentials have conclusively established an important physiological role of I(to)in rodents, with its role in large mammals being less well defined due to complex interplay between a multitude of cardiac ionic currents. A central and consistent electrophysiological change in cardiac disease is the reduction in I(to)density with a loss of heterogeneity of I(to)expression and associated action potential prolongation. Alterations of I(to)in rodent cardiac disease have been linked to repolarization abnormalities and alterations in intracellular Ca(2+)homeostasis, while in larger mammals the link with functional changes is far less certain. We review the current literature on the molecular basis for cardiac I(to)and the functional consequences of changes in I(to)that occur in cardiovascular disease.

Alterations in action potential profile enhance excitation-contraction coupling in rat cardiac myocytes.

Action potential (AP) prolongation typically occurs in heart disease due to reductions in transient outward potassium currents (Ito), and is associated with increased Ca2+ transients. We investigated the underlying mechanisms responsible for enhanced Ca2+ transients in normal isolated rat ventricular myocytes in response to the AP changes that occur following myocardial infarction. Normal myocytes stimulated with a train of long post-myocardial infarction (MI) APs showed a 2.2-fold elevation of the peak Ca2+ transient and a 2.7-fold augmentation of fractional cell shortening, relative to myocytes stimulated with a short control AP. The steady-state Ca2+ load of the sarcoplasmic reticulum (SR) was increased 2.0-fold when myocytes were stimulated with trains of long post-MI APs (111 +/- 21.6 micromol l(-1)) compared with short control APs (56 +/- 7.2 micromol l(-1)). Under conditions of equal SR Ca2+ load, long post-MI APs still resulted in a 1.7-fold increase in peak [Ca2+]i and a 3.8-fold increase in fractional cell shortening relative to short control APs, establishing that changes in the triggering of SR Ca2+ release are largely responsible for elevated Ca2+ transients following AP prolongation. Fractional SR Ca2+ release calculated from the measured SR Ca2+ load and the integrated SR Ca2+ fluxes was 24 +/- 3 and 11 +/- 2 % following post-MI and control APs, respectively. The fractional release (FR) of Ca2+ from the SR divided by the integrated L-type Ca2+ flux (FR/[integral]FCa,L) was increased 1.2-fold by post-MI APs compared with control APs. Similar increases in excitation-contraction (E-C) coupling gains were observed establishing enhanced E-C coupling efficiency. Our findings demonstrate that AP prolongation alone can markedly enhance E-C coupling in normal myocytes through increases in the L-type Ca2+ current (ICa,L) trigger combined with modest enhancements in Ca2+ release efficiency. We propose that such changes in AP profile in diseased myocardium may contribute significantly to alterations in E-C coupling independent of other biochemical or genetic changes.

Cytotoxic aldehyde generation in heart following acute iron-loading.

Although the mechanism of myocardial failure following acute iron poisoning is not known, excess iron-catalyzed free radical generation is conjectured to play a role. The effects of time (0 to 360 minutes) on total iron concentrations, glutathione peroxidase activity, and cytotoxic aldehyde production in heart of mice (B6D2F1, n = 65) were first investigated following acute iron-loading (20 mg iron dextran i.p./mouse). In a subsequent experiment, the effects of dose (0 to 80 mg iron dextran i.p./mouse, n = 75) on the aforementioned parameters were investigated. Our results show that the concentrations of cytotoxic aldehydes: (1) significantly differ over-time, with corresponding increases in total concentrations of iron (r = 0.93, p < 0.001); and (2) increase parallel to the total dose of iron administered (r = 0.95, p < 0.001). Furthermore, dose-and time-dependent alterations to glutathione peroxidase activity are observed, which is most likely due to an acute up-regulation of the enzyme as an endogenous protective response to increased free radical activity in the heart subsequent to iron-loading. While no single mechanism is likely to account for the complex pathophysiology of acute iron-induced heart failure, our results shown that iron-loading can result in significant free radical generation, as quantified by cytotoxic aldehydes, in heart tissue of mice. This is the first report on the effects of time and dose on cytotoxic aldehyde generation and glutathione peroxidase activity in heart of mice following acute iron-loading.

The transgenic expression of highly inhibitory monomeric forms of phospholamban in mouse heart impairs cardiac contractility.

Transgenic mice were generated with cardiac-specific overexpression of the monomeric, dominant-acting, superinhibitory L37A and I40A mutant forms of phospholamban (PLN), and their phenotypes were compared with wild-type (wt) mice or 2-fold overexpressors of wt PLN (wtOE). The level of PLN monomer in cardiac microsomes was increased 11-13-fold, and the apparent affinity of the sarco(endo)plasmic reticulum Ca(2+)-ATPase for Ca(2+) was decreased from pCa 6.22 in wt or 6.12 in wtOE to 5.81 in L37A and 5.72 in I40A. Basal physiological parameters, measured in isolated myocytes, indicated a significant reduction in the rates of shortening (+dL/dt) and relengthening (-dL/dt). Hemodynamic measurements indicated that peak systolic pressure was unaffected but that pressure changes (+dP/dt and -dP/dt) were lowered significantly in both mutant lines, and relaxation time (tau) was also lengthened significantly. Echocardiography for both mutants showed depressed systolic function and an increase in left ventricular mass of over 1.4-fold. Significant decreases in left ventricular shortening fraction and velocity of circumferential shortening and increases in ejection time were corrected by isoproterenol. The use of antibodies specific against Ser(16)- and Thr(17)-PLN peptides showed that phosphorylation of both pentameric and monomeric PLN were increased between 1.2- and 2.4-fold in both the L37A and I40A lines but not in the wtOE line. These observations show that overexpression of superinhibitory mutant forms of PLN causes depression of contractile parameters with induction of cardiac hypertrophy, as assessed with echocardiography.

Two critical cysteine residues implicated in disulfide bond formation and proper folding of Kir2.1.

Inwardly rectifying potassium channels are important in cellular repolarization of many excitable tissues. Amino acid sequence alignment of different mammalian inward rectifier K(+) channels revealed two absolutely conserved cysteine residues in the putative extracellular face, suggesting a possible disulfide bond. Replacement of these cysteine residues in the Kir2.1 channel (i.e., C122 and C154) with either alanine or serine abolished current in Xenopus laevis oocytes although Western blotting established that the channels were fully expressed. The digestion pattern of channels treated with V8 protease combined with Western blotting under reducing and nonreducing conditions confirmed intrasubunit cross-linking of C122 and C154. Whole-cell and single channel current recordings of oocytes expressing tandem tetrameric constructs with one or two of the mutant subunits suggested that insertion of one mutant subunit is sufficient to eliminate channel function. Coexpression studies confirmed that the cysteine mutant channels eliminate wild-type Kir2.1 currents in a dominant-negative manner. Despite these results, sulfhydryl reduction did not alter the functional properties of Kir2.1 currents. Molecular modeling of Kir2.1 with the two cysteines cross-linked predicted that the extracellular loop between the first transmembrane domain and the pore helix contains a beta-hairpin structure. Distinct from the KcsA structure, the disulfide bond together with the beta-hairpin structure is expected to constrain and stabilize the P-loop and selectivity filter. Taken together, these results suggest that intramolecular disulfide bond exists between C122 and C154 of Kir2.1 channel and this cross-link might be required for proper channel folding.

The thyroid hormone analog DITPA restores I(to) in rats after myocardial infarction.

Previous studies have established that reductions in repolarizing currents occur in heart disease and can contribute to life-threatening arrhythmias in myocardium. In this study, we investigated whether the thyroid hormone analog 3, 5-diiodothyropropionic acid (DITPA) could restore repolarizing transient outward K(+) current (I(to)) density and gene expression in rat myocardium after myocardial infarction (MI). Our findings show that I(to) density was reduced after MI (14.0 +/- 1.0 vs. 10.2 +/- 0.9 pA/pF, sham vs. post-MI at +40 mV). mRNA levels of Kv4.2 and Kv4.3 genes were decreased but Kv1.4 mRNA levels were increased post-MI. Corresponding changes in Kv4.2 and Kv1.4 protein were also observed. Chronic treatment of post-MI rats with 10 mg/kg DITPA restored I(to) density (to 15.2 +/- 1.1 pA/pF at +40 mV) as well as Kv4.2 and Kv1.4 expression to levels observed in sham-operated controls. Other membrane currents (Na(+), L-type Ca(2+), sustained, and inward rectifier K(+) currents) were unaffected by DITPA treatment. Associated with the changes in I(to) expression, action potential durations (current-clamp recordings in isolated single right ventricular myocytes and monophasic action potential recordings from the right free wall in situ) were prolonged after MI and restored with DITPA treatment. Our results demonstrate that DITPA restores I(to) density in the setting of MI, which may be useful in preventing complications associated with I(to) downregulation.

Rate-dependent changes of twitch force duration in rat cardiac trabeculae: a property of the contractile system.

1. We examined the mechanisms for rate-dependent changes in twitch force duration by simultaneously measuring force and [Ca2+]i in rat cardiac trabeculae. 2. Peak force decreased when the rate of stimulation was increased from 0.2 to 0.5 Hz, whilst it increased from 1 to 2 Hz. Over the same range of frequencies, peak [Ca2+]i transients increased monotonically, whilst both force and [Ca2+]i transient duration were abbreviated. 3. Changes in peak force or peak [Ca2+]i transients were not responsible for the changes in force or [Ca2+]i transient duration. 4. The changes in twitch force and [Ca2+]i transient duration were completed roughly within one beat following an abrupt change in the rate of stimulation. 5. Rate-dependent changes resembled those observed with isoproterenol (isoprenaline) application. However, kinase inhibitors (i.e. K252-a, H-89, KN-62 and KN-93) had no effect on the rate-dependent changes of twitch force and [Ca2+]i transient kinetics, suggesting that protein kinase A (PKA), protein kinase PKG) and Ca2+-calmodulin-dependent protein kinase II (CaM/kinase II) were not responsible for these kinetic changes. 6. Despite the changes in twitch force and [Ca2+]i transient kinetics, the rate-limiting step for the rate-dependent force relaxation still resides at the level of the contractile proteins. 7. Our results suggest that rate-dependent changes in force and [Ca2+]i transients do not depend on PKA or CaM/kinase II activity but might result from intrinsic features of the contractile and/or Ca2+-handling proteins.

Targeted expression of a dominant-negative K(v)4.2 K(+) channel subunit in the mouse heart.

Action potential duration is prolonged in many forms of heart disease, often as a result of reductions in Ca(2+)-independent transient outward K(+) currents (ie, I(to)). To examine the effects of a primary reduction in I(to) current in the heart, transgenic mice were generated that express a dominant-negative N-terminal fragment of the K(v)4.2 pore-forming potassium channel subunit under the control of the mouse alpha-myosin heavy chain promoter. Two of 6 founders died suddenly, and only 1 mouse successfully transmitted the transgene in mendelian fashion. Electrophysiological analysis at 2 to 4 weeks of age demonstrated that I(to) density was specifically reduced and action potential durations were prolonged in a subset of transgenic myocytes. The heterogeneous reduction in I(to) was accompanied by significant prolongation of monophasic action potentials. In vivo hemodynamic studies at this age revealed significant elevations in the mean arterial pressure, peak systolic ventricular pressures, and +/-dP/dt, indicative of enhanced contractility. Surprisingly, by 10 to 12 weeks of age, transgenic mice developed clinical and hemodynamic evidence of congestive heart failure. Failing transgenic hearts displayed molecular and cellular remodeling, with evidence of hypertrophy, chamber dilatation, and interstitial fibrosis, and individual myocytes showed sharp reductions in I(to) and I(K1) densities, action potential duration prolongation, and increased cell capacitance. Our results confirm that K(v)4.2 subunits contribute to I(to) in the mouse and demonstrate that manipulation of cardiac excitability may secondarily influence contractile performance.

Local anesthetic anchoring to cardiac sodium channels. Implications into tissue-selective drug targeting.

Local anesthetics inhibit Na+ channels in a variety of tissues, leading to potentially serious side effects when used clinically. We have created a series of novel local anesthetics by connecting benzocaine (BZ) to the sulfhydryl-reactive group methanethiosulfonate (MTS) via variable-length polyethylether linkers (L) (MTS-LX-BZ [X represents 0, 3, 6, or 9]). The application of MTS-LX-BZ agents modified native rat cardiac as well as heterologously expressed human heart (hH1) and rat skeletal muscle (rSkM1) Na+ channels in a manner resembling that of free BZ. Like BZ, the effects of MTS-LX-BZ on rSkM1 channels were completely reversible. In contrast, MTS-LX-BZ modification of heart and mutant rSkM1 channels, containing a pore cysteine at the equivalent location as cardiac Na+ channels (ie, Y401C), persisted after drug washout unless treated with DTT, which suggests anchoring to the pore via a disulfide bond. Anchored MTS-LX-BZ competitively reduced the affinity of cardiac Na+ channels for lidocaine but had minimal effects on mutant channels with disrupted local anesthetic modification properties. These results establish that anchored MTS-LX-BZ compounds interact with the local anesthetic binding site (LABS). Variation in the linker length altered the potency of channel modification by the anchored drugs, thus providing information on the spatial relationship between the anchoring site and the LABS. Our observations demonstrate that local anesthetics can be anchored to the extracellular pore cysteine in cardiac Na+ channels and dynamically interact with the intracellular LABS. These results suggest that nonselective agents, such as local anesthetics, might be made more selective by linking these agents to target-specific anchors.

Effect of Cd2+ on Kv4.2 and Kv1.4 expressed in Xenopus oocytes and on the transient outward currents in rat and rabbit ventricular myocytes.

The aim of the present study was to compare the biophysical properties and Cd2+ sensitivity of Kv4.2 and Kv1.4 in Xenopus oocytes with those of native transient outward potassium currents in rat and rabbit ventricular myocytes. In Xenopus oocytes, Kv4.2 inactivated at hyperpolarized voltages (V(1/2)inact = -58.4 +/- 0.96 mV, n = 12) and recovered from inactivation rapidly (time constant = 224 +/- 23 ms, n = 3). Cd2+ induced large (approx. 30 mV with 500 microM Cd2+), concentration-dependent rightward shifts in Kv4.2 steady-state activation and inactivation. Kv1.4 inactivated over more depolarized voltages than Kv4.2 (V(1/2)inact = -49.3 +/- 1.4 mV, n = 12). Recovery from inactivation of Kv1.4 was dominated by a large slow component (time constant = 9,038 +/- 1,178 ms, n = 4). Cd2+ exerted only modest effects on Kv1.4 gating, with 500 microM Cd2+ shifting the voltage dependence of steady-state activation and inactivation by approximately 12 mV. We show that the biophysical properties and Cd2+ sensitivity of rat ventricular Ito resemble those of heterologously expressed Kv4.2. These findings support previous suggestions that Kv4.2 is an important molecular component of Ito in adult rat heart. In addition, our findings show that Ito in rabbit ventricular myocytes and Kv1.4-based currents in Xenopus oocytes share similar biophysical properties and sensitivity to Cd2+, suggesting that Kv1.4 may underlie Ito in rabbit ventricle. However, a number of discrepancies exist between the properties of native currents and their putative molecular counterparts, suggesting that additional proteins and/or modulatory factors may also play a role in determining the biophysical and pharmacological properties of these native currents.

Modulation of iron uptake in heart by L-type Ca2+ channel modifiers: possible implications in iron overload.

Heart failure is the leading cause of mortality in patients with transfusional iron (Fe) overload in which myocardial iron uptake ensues via a transferrin-independent process. We examined the ability of L-type Ca2+ channel modifiers to alter Fe2+ uptake by isolated rat hearts and ventricular myocytes. Perfusion of rat hearts with 100 nmol/L 59Fe2+ and 5 mmol/L ascorbate resulted in specific 59Fe2+ uptake of 20.4+/-1.9 ng of Fe per gram dry wt. Abolishing myocardial electrical excitability with 20 mmol/L KCl reduced specific 59Fe2+ uptake by 60+/-7% (P<0.01), which suggested that a component of myocardial Fe2+ uptake depends on membrane voltage. Accordingly, 59Fe2+ uptake was inhibited by 10 micromol/L nifedipine (45+/-12%, P<0.02) and 100 micromol/L Cd2+ (86+/-3%; P<0. 001) while being augmented by 100 nmol/L Bay K 8644 (61+/-18%, P<0. 01) or 100 nmol/L isoproterenol (40+/-12%, P<0.05). By contrast, uptake of 100 nmol/L ferric iron (59Fe3+) was significantly lower (1. 4+/-0.3 ng Fe per gram dry wt; P<0.001) compared with divalent iron. These data suggest that a component of Fe2+ uptake into heart occurs via the L-type Ca2+ channel in myocytes. To investigate this further, the effects of Fe2+ on cardiac myocyte L-type Ca2+ currents were measured. In the absence of Ca2+, noninactivating nitrendipine-sensitive Fe2+ currents were recorded with 15 mmol/L [Fe2+]o. Low concentrations of Fe2+ enhanced Ca2+ current amplitude and slowed inactivation rates, which was consistent with Fe2+ entry into the cell, whereas higher Fe2+ levels caused dose-dependent decreases in peak current. Fe3+ had no effect on current amplitude or decay. Combined, our data suggest that myocardial Fe2+ uptake occurs via L-type Ca2+ channels and that blockade of these channels might be useful in the treatment of patients with excessive serum iron levels.

Antibodies against Tityus discrepans venom do not abolish the effect of Tityus serrulatus venom on the rat sodium and potassium channels.

Anti-(Tityus serrulatus + Tityus bahiensis) and anti-Tityus discrepans venom polyclonal antisera were used to investigate whether antigenic differences exist between the venoms of the Brazilian T. serrulatus and the Venezuelan T. discrepans scorpions. Both antisera recognised the toxin-containing electrophoretic fractions of their cognate venoms and also those from Tityus zulianus and Tityus trinitatis venoms on Western blots. The anti-T. discrepans antiserum reacted only weakly with T. serrulatus toxic polypeptides. The effect of T. serrulatus alpha- or beta-toxins on rat skeletal muscle Na+ channels expressed in Xenopus laevis oocytes was abolished by pre-incubating the venom with anti-(T. serrulatus + T. bahiensis) serum but not with anti-T. discrepans serum. Nor did the Brazilian or the Venezuelan sera prevent the reduction in K+ currents by T. serrulatus venom in X. laevis oocytes expressing the rat brain delayed rectifying Shaker K+ channel (Kv1.2). These results indicate that toxins from T. serrulatus and T. discrepans venoms, which primarily target mammalian Na+ channels, are antigenically distinct, although they probably share common epitopes. Our results also suggest that Na+ channel-active toxins are the immunodominant antigens of the T. serrulatus venom.

Relationship between K+ channel down-regulation and [Ca2+]i in rat ventricular myocytes following myocardial infarction.

1. Cardiac hypertrophy and prolongation of the cardiac action potential are hallmark features of heart disease. We examined the molecular mechanisms and the functional consequences of this action potential prolongation on calcium handling in right ventricular myocytes obtained from rats 8 weeks following ligation of the left anterior descending coronary artery (post-myocardial infarction (MI) myocytes). 2. Compared with myocytes from sham-operated rats (sham myocytes), post-MI myocytes showed significant reductions in transient outward K+ current (Ito) density (sham 19.7 +/- 1.1 pA pF-1 versus post-MI 11.0 +/- 1.3 pA pF-1; means +/- s.e.m.), inward rectifier K+ current density (sham -13.7 +/- 0.6 pA pF-1 versus post-MI -10.3 +/- 0.9 pA pF-1) and resting membrane potential (sham -84.4 +/- 1.3 mV versus post-MI -74.1 +/- 2.6 mV). Depressed Ito amplitude correlated with significant reductions in Kv4.2 and Kv4.3 mRNA and Kv4.2 protein levels. Kv1.4 mRNA and protein levels were increased and coincided with the appearance of a slow component of recovery from inactivation for Ito. 3. In current-clamp recordings, post-MI myocytes showed a significant increase in [Ca2+]i transient amplitude compared with sham myocytes. Using voltage-clamp depolarizations, no intrinsic differences in Ca2+ handling by the sarcoplasmic reticulum or in L-type Ca2+ channel density (ICa,L) were detected between the groups. 4. Stimulation of post-MI myocytes with an action potential derived from a sham myocyte reduced the [Ca2+] transient amplitude to the sham level and vice versa. 5. The net Ca2+ influx per beat via ICa,L was increased about 2-fold in myocytes stimulated with post-MI action potentials compared with sham action potentials. 6. Our findings demonstrate that reductions in K+ channel expression in post-MI myocytes prolong action potential duration resulting in elevated Ca2+ influx and [Ca2+]i transients.

Regional contributions of Kv1.4, Kv4.2, and Kv4.3 to transient outward K+ current in rat ventricle.

The aim of the present study was to assess differences in transient outward potassium current (Ito) between the right ventricular free wall and the interventricular septum of the adult rat ventricle and to evaluate the relative contributions of Kv4.2, Kv4.3, and Kv1.4 to Ito in these regions. The results show that Ito is composed of both rapidly and slowly recovering components in the right wall and septum. The fast component had a significantly higher density in the right free wall than in the septum, whereas the slow component did not differ between the two sites. Kv4.2 mRNA and protein levels were also highest in the right wall and correlated with Ito density, whereas Kv4.3 was expressed uniformly in these regions. The kinetics of the rapidly recovering component of Ito in myocytes was similar to that recorded in tsa-201 cells expressing Kv4.2 and Kv4.3 channels. Kv1.4 mRNA and protein expression correlated well with the density of the slowly recovering Ito, whereas the recovery kinetics of the slow component were identical to Kv1.4 expressed in tsa-201 cells. In conclusion, expression of Kv1.4, Kv4.2, and Kv4.3 differs between regions in rat hearts. Regionally specific differences in the genetic composition of Ito can account for the region-specific properties of this current.