The role of monoubiquitination in endocytic degradation of human ether-a-go-go-related gene (hERG) channels under low K+ conditions

A reduction in extracellular K(+) concentration ([K(+)](o)) causes cardiac arrhythmias and triggers internalization of the cardiac rapidly activating delayed rectifier potassium channel (I(Kr)) encoded by the human ether-a-go-go-related gene (hERG). We investigated the role of ubiquitin (Ub) in endocytic degradation of hERG channels stably expressed in HEK cells. Under low K(+) conditions, UbKO, a lysine-less mutant Ub that only supports monoubiquitination, preferentially interacted and selectively enhanced degradation of the mature hERG channels. Overexpression of Vps24 protein, also known as charged multivesicular body protein 3, significantly accelerated degradation of mature hERG channels, whereas knockdown of Vps24 impeded this process. Moreover, the lysosomal inhibitor bafilomycin A1 inhibited degradation of the internalized mature hERG channels. Thus, monoubiquitination directs mature hERG channels to degrade through the multivesicular body/lysosome pathway. Interestingly, the protease inhibitor lactacystin inhibited the low K(+)-induced hERG endocytosis and concomitantly led to an accumulation of monoubiquitinated mature hERG channels, suggesting that deubiquitination is also required for the endocytic degradation. Consistently, overexpression of the endosomal deubiquitinating enzyme signal transducing adaptor molecule-binding protein significantly accelerated whereas knockdown of endogenous signal transducing adaptor molecule-binding protein impeded degradation of the mature hERG channels under low K(+) conditions. Thus, monoubiquitin dynamically mediates endocytic degradation of mature hERG channels under low K(+) conditions.     

Isolation and characterization of I(Kr) in cardiac myocytes by Cs+ permeation

Isolation of the rapidly activating delayed rectifier potassium current (I(Kr)) from other cardiac currents has been a difficult task for quantitative study of this current. The present study was designed to separate I(Kr) using Cs+ in cardiac myocytes. Cs+ have been known to block a variety of K+ channels, including many of those involved in the cardiac action potential such as inward rectifier potassium current I(K1) and the transient outward potassium current I(to). However, under isotonic Cs+ conditions (135 mM Cs+), a significant membrane current was recorded in isolated rabbit ventricular myocytes. This current displayed the voltage-dependent onset of and recovery from inactivation that are characteristic to I(Kr). Consistently, the current was selectively inhibited by the specific I(Kr) blockers. The biophysical and pharmacological properties of the Cs+-carried human ether-a-go-go-related gene (hERG) current were very similar to those of the Cs+-carried I(Kr) in ventricular myocytes. The primary sequence of the selectivity filter in hERG was in part responsible for the Cs+ permeability, which was lost when the sequence was changed from GFG to GYG, characteristic of other, Cs+-impermeable K+ channels. Thus the unique high Cs+ permeability in I(Kr) channels provides an effective way to isolate I(Kr) current. Although the biophysical and pharmacological properties of the Cs+-carried I(Kr) are different from those of the K+-carried I(Kr), such an assay enables I(Kr) current to be recorded at a level that is large enough and sufficiently robust to evaluate any I(Kr) alterations in native tissues in response to physiological or pathological changes. It is particularly useful for exploring the role of reduction of I(Kr) in arrhythmias associated with heart failure and long QT syndrome due to the reduced hERG channel membrane expression.     

Specific serine proteases selectively damage KCNH2 (hERG1) potassium channels and I(Kr)

KCNH2 (hERG1) encodes the alpha-subunit proteins for the rapidly activating delayed rectifier K+ current (I(Kr)), a major K+ current for cardiac myocyte repolarization. In isolated myocytes I(Kr) frequently is small in amplitude or absent, yet KCNH2 channels and I(Kr) are targets for drug block or mutations to cause long QT syndrome. We hypothesized that KCNH2 channels and I(Kr) are uniquely sensitive to enzymatic damage. To test this hypothesis, we studied heterologously expressed K+, Na+, and L-type Ca2+ channels, and in ventricular myocytes I(Kr), slowly activating delayed rectifier K+ current (I(Ks)), and inward rectifier K+ current (I(K1)), by using electrophysiological and biochemical methods. 1) Specific exogenous serine proteases (protease XIV, XXIV, or proteinase K) selectively degraded KCNH2 current (I(KCNH2)) and its mature channel protein without damaging cell integrity and with minimal effects on the other channel currents; 2) immature KCNH2 channel protein remained intact; 3) smaller molecular mass KCNH2 degradation products appeared; 4) protease XXIV selectively abolished I(Kr); and 5) reculturing HEK-293 cells after protease exposure resulted in the gradual recovery of I(KCNH2) and its mature channel protein over several hours. Thus the channel protein for I(KCNH2) and I(Kr) is uniquely sensitive to proteolysis. Analysis of the degradation products suggests selective proteolysis within the S5-pore extracellular linker, which is structurally unique among Kv channels. These data provide 1) a new mechanism to account for low I(Kr) density in some isolated myocytes, 2) evidence that most complexly glycosylated KCNH2 channel protein is in the plasma membrane, and 3) new insight into the rate of biogenesis of KCNH2 channel protein within cells.     

Kv11.1 (ERG1) K+ channels localize in cholesterol and sphingolipid enriched membranes and are modulated by membrane cholesterol

The localization of ion channels to specific membrane microdomains can impact the functional properties of channels and their role in cellular physiology. We determined the membrane localization of human Kv11.1 (hERG1) alpha-subunit protein, which underlies the rapidly activating, delayed rectifier K(+) current (I(Kr)) in the heart. Immunocytochemistry and membrane fractionation using discontinuous sucrose density gradients of adult canine ventricular tissue showed that Kv11.1 channel protein localized to both the cell surface and T-tubular sarcolemma. Furthermore, density gradient membrane fractionation using detergent (Triton X-100) and non-detergent (OptiPrep) methods from canine ventricular myocytes or HEK293 cells demonstrated that Kv11.1 protein, along with MiRP1 and Kv7.1 (KCNQ1) proteins, localize in cholesterol and sphingolipid enriched membrane fractions. In HEK293 cells, Kv11.1 channels, but not long QT-associated mutant G601S-Kv11.1 channels, also localized to cholesterol and sphingolipid enriched membrane fractions. Depletion of membrane cholesterol from HEK293 cells expressing Kv11.1 channels using methyl-beta-cyclodextrin (MbetaCD) caused a positive shift of the voltage dependence of activation and an acceleration of deactivation kinetics of Kv11.1 current (I(Kv11.1)). Cholesterol loading of HEK293 cells reduced the steep voltage dependence of I(Kv11.1) activation and accelerated the inactivation kinetics of I(Kv11.1). Incubation of neonatal mouse myocytes in MbetaCD also accelerated the deactivation kinetics of I(Kr). We conclude that Kv11.1 protein localizes in cholesterol and sphingolipid enriched membranes and that membrane cholesterol can modulate I(Kv11.1) and I(Kr).     

Both EGFR kinase and Src-related tyrosine kinases regulate human ether-à-go-go-related gene potassium channels

Human ether-à-go-go-related gene (hERG or Kv11.1) encodes the rapidly activated delayed rectifier K(+) current (I(Kr)) in the human heart. Potential regulation of hERG channel by protein tyrosine kinases (PTKs) is not understood. The present study was designed to investigate whether this channel is modulated by PTKs using whole-cell patch clamp technique, and immunoprecipitation and Western blot analysis in HEK 293 cells stably expressing hERG gene. We found that the broad-spectrum PTK inhibitor genistein (30 muM), the selective EGFR (epidermal growth factor receptor) kinase inhibitor AG556 (10 muM) and the Src-family kinase inhibitor PP2 (10 muM) remarkably inhibited hERG channel current (I(hERG)), and the effects were significantly countered by the protein tyrosine phosphatase (PTP) inhibitor orthovanadate (1 mM). Immunoprecipitation and Western blot analysis demonstrated that membrane protein tyrosine phosphorylation of hERG channels was reduced by genistein, AG556, and PP2. The reduction of hERG channel phosphorylation level by genistein, AG556 or PP2 was antagonized by orthovanadate. Single point mutation(s) of Y475A and/or Y611A dramatically attenuated the inhibitory effect of I(hERG) by PP2 and/or AG556. Our results demonstrate the novel information that I(hERG) is modulated not only by Src-family kinases, but also by EGFR kinases. Y475 and/or Y611 are likely the preferred phosphorylation sites. Regulation of hERG channels by PTKs modifies the channel activity and thus likely alters electrophysiological properties including action potential duration and cell excitability in human heart and neurons.     

KCNE2 confers background current characteristics to the cardiac KCNQ1 potassium channel

Mutations in HERG and KCNQ1 (or KVLQT1) genes cause the life-threatening Long QT syndrome. These genes encode K(+) channel pore-forming subunits that associate with ancillary subunits from the KCNE family to underlie the two components, I(Kr) and I(Ks), of the human cardiac delayed rectifier current I(K). The KCNE family comprises at least three members. KCNE1 (IsK or MinK) recapitulates I(Ks) when associated with KCNQ1, whereas it augments the amplitude of an I(Kr)-like current when co-expressed with HERG. KCNE3 markedly changes KCNQ1 as well as HERG current properties. So far, KCNE2 (MirP1) has only been shown to modulate HERG current. Here we demonstrate the interaction of KCNE2 with the KCNQ1 subunit, which results in a drastic change of KCNQ1 current amplitude and gating properties. Furthermore, KCNE2 mutations also reveal their specific functional consequences on KCNQ1 currents. KCNQ1 and HERG appear to share unique interactions with KCNE1, 2 and 3 subunits. With the exception of KCNE3, mutations in all these partner subunits have been found to lead to an increased propensity for cardiac arrhythmias.     

TEA(+)-sensitive KCNQ1 constructs reveal pore-independent access to KCNE1 in assembled I(Ks) channels

I(Ks), a slowly activating delayed rectifier K(+) current through channels formed by the assembly of two subunits KCNQ1 (KvLQT1) and KCNE1 (minK), contributes to the control of the cardiac action potential duration. Coassembly of the two subunits is essential in producing the characteristic and physiologically critical kinetics of assembled channels, but it is not yet clear where or how these subunits interact. Previous investigations of external access to the KCNE1 protein in assembled I(Ks) channels relied on occlusion of the pore by extracellular application of TEA(+), despite the very low TEA(+) sensitivity (estimated EC(50) > 100 mM) of channels encoded by coassembly of wild-type KCNQ1 with the wild type (WT) or a series of cysteine-mutated KCNE1 constructs. We have engineered a high affinity TEA(+) binding site into the h-KCNQ1 channel by either a single (V319Y) or double (K318I, V319Y) mutation, and retested it for pore-delimited access to specific sites on coassembled KCNE1 subunits. Coexpression of either KCNQ1 construct with WT KCNE1 in Chinese hamster ovary cells does not alter the TEA(+) sensitivity of the homomeric channels (IC(50) approximately 0.4 mM [TEA(+)](out)), providing evidence that KCNE1 coassembly does not markedly alter the structure of the outer pore of the KCNQ1 channel. Coexpression of a cysteine-substituted KCNE1 (F54C) with V319Y significantly increases the sensitivity of channels to external Cd(2+), but neither the extent of nor the kinetics of the onset of (or the recovery from) Cd(2+) block was affected by [TEA(+)](o) at 10x the IC(50) for channel block. These data strongly suggest that access of Cd(2+) to the cysteine-mutated site on KCNE1 is independent of pore occlusion caused by TEA(+) binding to the outer region of the KCNE1/V319Y pore, and that KCNE1 does not reside within the pore region of the assembled channels.     

KCNE4 can co-associate with the I(Ks) (KCNQ1-KCNE1) channel complex

Voltage-gated potassium (K(V)) channels can form heteromultimeric complexes with a variety of accessory subunits, including KCNE proteins. Heterologous expression studies have demonstrated diverse functional effects of KCNE subunits on several K(V) channels, including KCNQ1 (K(V)7.1) that, together with KCNE1, generates the slow-delayed rectifier current (I(Ks)) important for cardiac repolarization. In particular, KCNE4 exerts a strong inhibitory effect on KCNQ1 and other K(V) channels, raising the possibility that this accessory subunit is an important potassium current modulator. A polyclonal KCNE4 antibody was developed to determine the human tissue expression pattern and to investigate the biochemical associations of this protein with KCNQ1. We found that KCNE4 is widely and variably expressed in several human tissues, with greatest abundance in brain, liver and testis. In heterologous expression experiments, immunoprecipitation followed by immunoblotting was used to establish that KCNE4 directly associates with KCNQ1, and can co-associate together with KCNE1 in the same KCNQ1 complex to form a 'triple subunit' complex (KCNE1-KCNQ1-KCNE4). We also used cell surface biotinylation to demonstrate that KCNE4 does not impair plasma membrane expression of either KCNQ1 or the triple subunit complex, indicating that biophysical mechanisms probably underlie the inhibitory effects of KCNE4. The observation that multiple KCNE proteins can co-associate with and modulate KCNQ1 channels to produce biochemically diverse channel complexes has important implications for understanding K(V) channel regulation in human physiology.     

Tetrahydronaphthalene-derived amino alcohols and amino ketones as potent and selective inhibitors of the delayed rectifier potassium current IKs

Class III anti-arrhythmic drugs (e.g., dofetilide) prolong cardiac action potential duration (APD) by blocking the fast component of the delayed rectifier potassium current (I(Kr)). The block of I(Kr) can result in life threatening ventricular arrhythmias (i.e., torsades de pointes). Unlike I(Kr), the role of the slow component of the delayed rectifier potassium current (I(Ks)) becomes significant only at faster heart rate. Therefore selective blockers of I(Ks) could prolong APD with a reduced propensity to cause pro-arrhythmic side effects. This report describes structure-activity relationships (SARs) of a series of I(Ks) inhibitors derived from 6-alkoxytetralones with good in vitro activity (IC(50) > or =30 nM) and up to 40-fold I(Ks)/I(Kr) selectivity.     

In silico study of action potential and QT interval shortening due to loss of inactivation of the cardiac rapid delayed rectifier potassium current

The rapid delayed rectifier K(+) current, I(Kr), plays a key role in repolarisation of cardiac ventricular action potentials (APs). In recent years, a novel clinical condition denoted the short QT syndrome (SQTS) has been identified and, very recently, gain in function mutations in the gene encoding the pore-forming sub-unit of the I(Kr) channel have been proposed to underlie SQTS in some patients. Here, computer simulations were used to investigate the effects of the selective loss of voltage-dependent inactivation of I(Kr) upon ventricular APs and on the QT interval of the electrocardiogram. I(Kr) and inactivation-deficient I(Kr) were incorporated into Luo-Rudy ventricular AP models. Inactivation-deficient I(Kr) produced AP shortening that was heterogeneous between endocardial, mid-myocardial, and epicardial ventricular cell models, irrespective of whether heterogeneity between these sub-regions was incorporated of slow delayed rectifier K(+) current (I(Ks)) alone, or of I(Ks) together with that of transient outward K(+) current. The selective loss of rectification of I(Kr) did not augment transmural dispersion of AP repolarisation, as AP shortening was greater in mid-myocardial than in endo- or epicardial cell models. Simulated conduction through a 1 D transmural ventricular strand was altered by incorporation of inactivation-deficient I(Kr) and the reconstructed QT interval was shortened. Collectively, these results substantiate the notion that selective loss of I(Kr) inactivation produces a gain in I(Kr) function that causes QT interval shortening.     

Effect of sibutramine HCl on cardiac hERG K+ channel

Common clinically used drugs block the delayed rectifier K(+) channels and prolong the cardiac action potential duration associated with long QT syndrome. Here, we investigated the mechanism of hERG K(+) channel current (I(hERG)) blockade expressed in HEK-293 cells by sibutramine HCl, a serotonin-norepinephrine reuptake inhibitor. Sibutramine HCl inhibited I (hERG) in a concentration-dependent manner with the half-maximal inhibitory concentration (IC(50)) value of 2.5 microM at -40 mV. I(hERG) inhibition by sibutramine HCl showed weak voltage dependency, but the time-dependence of I(hERG) inhibition was developed relatively rapidly on membrane depolarization. On hERG channel gating for the S6 and pore regions, the S6 residue hERG mutant Y652A and F656A largely reduced the blocking potency of I(hERG), unlike the pore-region mutants T623A and S624A. These results indicate that sibutramine HCl preferentially inhibits the hERG potassium channel through the residue Y652 and F656, in a supratherapeutic concentration should be avoided by patients with high susceptibility for cardiac arrhythmia.     

Involvement of Caveolin in Low K+-induced Endocytic Degradation of Cell-surface Human Ether-a-go-go-related Gene (hERG) Channels

Reduction in the rapidly activating delayed rectifier K⁺ channel current (I_Kr) due to either mutations in the human ether-a-go-go-related gene (hERG) or drug block causes inherited or drug-induced long QT syndrome. A reduction in extracellular K⁺ concentration ([K⁺]_o) exacerbates long QT syndrome. Recently, we demonstrated that lowering [K⁺]_o promotes degradation of I_Kr in rabbit ventricular myocytes and of the hERG channel stably expressed in HEK 293 cells. In this study, we investigated the degradation pathways of hERG channels under low K⁺ conditions. We demonstrate that under low K⁺ conditions, mature hERG channels and caveolin-1 (Cav1) displayed a parallel time-dependent reduction. Mature hERG channels coprecipitated with Cav1 in co-immunoprecipitation analysis, and internalized hERG channels colocalized with Cav1 in immunocytochemistry analysis. Overexpression of Cav1 accelerated internalization of mature hERG channels in 0 mm K⁺_o, whereas knockdown of Cav1 impeded this process. In addition, knockdown of dynamin 2 using siRNA transfection significantly impeded hERG internalization and degradation under low K⁺_o conditions. In cultured neonatal rat ventricular myocytes, knockdown of caveolin-3 significantly impeded low K⁺_o-induced reduction of I_Kr. Our data indicate that a caveolin-dependent endocytic route is involved in low K⁺_o-induced degradation of mature hERG channels.     

Quantitative comparison of cardiac ventricular myocyte electrophysiology and response to drugs in human and nonhuman species

Explanations for arrhythmia mechanisms at the cellular level are usually based on experiments in nonhuman myocytes. However, subtle electrophysiological differences between species may lead to different rhythmic or arrhythmic cellular behaviors and drug response given the nonlinear and highly interactive cellular system. Using detailed and quantitatively accurate mathematical models for human, dog, and guinea pig ventricular action potentials (APs), we simulated and compared cell electrophysiology mechanisms and response to drugs. Under basal conditions (absence of β-adrenergic stimulation), Na(+)/K(+)-ATPase changes secondary to Na(+) accumulation determined AP rate dependence for human and dog but not for guinea pig where slow delayed rectifier current (I(Ks)) was the major rate-dependent current. AP prolongation with reduction of rapid delayed rectifier current (I(Kr)) and I(Ks) (due to mutations or drugs) showed strong species dependence in simulations, as in experiments. For humans, AP prolongation was 80% following I(Kr) block. It was 30% for dog and 20% for guinea pig. Under basal conditions, I(Ks) block was of no consequence for human and dog, but for guinea pig, AP prolongation after I(Ks) block was severe. However, with β-adrenergic stimulation, I(Ks) played an important role in all species, particularly in AP shortening at fast rate. Quantitative comparison of AP repolarization, rate-dependence mechanisms, and drug response in human, dog, and guinea pig revealed major species differences (e.g., susceptibility to arrhythmogenic early afterdepolarizations). Extrapolation from animal to human electrophysiology and drug response requires great caution.     

The Serum- and Glucocorticoid-inducible Kinases SGK1 and SGK3 Regulate hERG Channel Expression via Ubiquitin Ligase Nedd4-2 and GTPase Rab11

The hERG (human ether-a-go-go-related gene) encodes the α subunit of the rapidly activating delayed rectifier potassium channel (I_Kr). Dysfunction of hERG channels due to mutations or certain medications causes long QT syndrome, which can lead to fatal ventricular arrhythmias or sudden death. Although the abundance of hERG in the plasma membrane is a key determinant of hERG functionality, the mechanisms underlying its regulation are not well understood. In the present study, we demonstrated that overexpression of the stress-responsive serum- and glucocorticoid-inducible kinase (SGK) isoforms SGK1 and SGK3 increased the current and expression level of the membrane-localized mature proteins of hERG channels stably expressed in HEK 293 (hERG-HEK) cells. Furthermore, the synthetic glucocorticoid, dexamethasone, increased the current and abundance of mature ERG proteins in both hERG-HEK cells and neonatal cardiac myocytes through the enhancement of SGK1 but not SGK3 expression. We have previously shown that mature hERG channels are degraded by ubiquitin ligase Nedd4-2 via enhanced channel ubiquitination. Here, we showed that SGK1 or SGK3 overexpression increased Nedd4-2 phosphorylation, which is known to inhibit Nedd4-2 activity. Nonetheless, disruption of the Nedd4-2 binding site in hERG channels did not eliminate the SGK-induced increase in hERG expression. Additional disruption of Rab11 proteins led to a complete elimination of SGK-mediated increase in hERG expression. These results show that SGK enhances the expression level of mature hERG channels by inhibiting Nedd4-2 as well as by promoting Rab11-mediated hERG recycling.     

The hERG K(+) channel S4 domain L532P mutation: characterization at 37°C

hERG (human Ether-à-go-go Related Gene) is responsible for ion channels mediating rapid delayed rectifier potassium current, I(Kr), which is key to cardiac action potential repolarization. Gain-of-function hERG mutations give rise to the SQT1 variant of the Short QT Syndrome (SQTS). Reggae mutant zebrafish, with a S4 zERG mutation (Leucine499Proline; L499P), display arrhythmic features analogous to those seen in the SQTS. The affected S4 domain ERG residue is highly conserved. This study was executed to determine how the homologous hERG mutation (L532P) influences channel function at 37°C. Whole-cell measurements of current (I(hERG)) were made from HEK 293 cells expressing WT or L532P hERG. The half maximal activation voltage (V(0.5)) of L532P I(hERG) was positively shifted by ~+36mV compared to WT I(hERG); however at negative voltages a pronounced L532P I(hERG) was observed. Both activation and deactivation time-courses were accelerated for L532P I(hERG). The inactivation V(0.5) for L532P I(hERG) was shifted by ~+32mV. Under action potential (AP) voltage-clamp, L532P I(hERG) exhibited a dome-shaped current peaking at ~+16mV, compared to ~-31mV for WT-I(hERG). The L532P mutation produced an ~5-fold increase in the IC(50) for dronedarone inhibition of I(hERG). Homology modeling indicated that the L532 residue within the S4 helix lies closely apposed to the S5 region of an adjacent hERG subunit. Alterations to the S4 domain structure and, potentially, to interactions between adjacent hERG subunits are likely to account for the functional effects of this mutation.     

Mechanisms of I(Ks) suppression in LQT1 mutants

Mutations in the cardiac potassium ion channel gene KCNQ1 (voltage-gated K(+) channel subtype KvLQT1) cause LQT1, the most common type of hereditary long Q-T syndrome. KvLQT1 mutations prolong Q-T by reducing the repolarizing cardiac current [slow delayed rectifier K(+) current (I(Ks) )], but, for reasons that are not well understood, the clinical phenotypes may vary considerably even for carriers of the same mutation, perhaps explaining the mode of inheritance. At present, only currents expressed by LQT1 mutants have been studied, and it is unknown whether abnormal subunits are transported to the cell surface. Here, we have examined for the first time trafficking of KvLQT1 mutations and correlated the results with the I(Ks) currents that were expressed. Two missense mutations, S225L and A300T, produced abnormal currents, and two others, Y281C and Y315C, produced no currents. However, all four KvLQT1 mutations were detected at the cell surface. S225L, Y281C, and Y315C produced dominant negative effects on wild-type I(Ks) current, whereas the mutant with the mildest dysfunction, A300T, did not. We examined trafficking of a severe insertion deletion mutant Delta544 and detected this protein at the cell surface as well. We compared the cellular and clinical phenotypes and found a poor correlation for the severely dysfunctional mutations.     

Contribution of L-type Ca2+ channels to early afterdepolarizations induced by I Kr and I Ks channel suppression in guinea pig ventricular myocytes

Early afterdepolarizations (EADs) induced by suppression of cardiac delayed rectifier I (Kr) and/or I (Ks) channels cause fatal ventricular tachyarrhythmias. In guinea pig ventricular myocytes, partial block of one of the channels with complete block of the other reproducibly induced EADs. Complete block of both I (Kr) and I (Ks) channels depolarized the take-off potential and reduced the amplitude of EADs, which in some cases were not clearly separated from the preceding action potentials. A selective L-type Ca(2+) (I (Ca,L)) channel blocker, nifedipine, effectively suppressed EADs at submicromolar concentrations. As examined with the action potential-clamp method, I (Ca,L) channels mediated inward currents with a spike and dome shape during action potentials. I (Ca,L) currents decayed mainly due to inactivation in phase 2 and deactivation in phase 3 repolarization. When EADs were induced by complete block of I (Kr) channels with partial block of I (Ks) channels, repolarization of the action potential prior to EAD take-off failed to increase I (K1) currents and thus failed to completely deactivate I (Ca,L) channels, which reactivated and mediated inward currents during EADs. When both I (Kr) and I (Ks) channels were completely blocked, I (Ca,L) channels were not deactivated and mediated sustained inward currents until the end of EADs. Under this condition, the recovery and reactivation of I (Ca,L) channels were absent before EADs. Therefore, an essential mechanism underlying EADs caused by suppression of the delayed rectifiers is the failure to completely deactivate I (Ca,L) channels.     

Transmural action potential and ionic current remodeling in ventricles of failing canine hearts

Heart failure (HF) produces important alterations in currents underlying cardiac repolarization, but the transmural distribution of such changes is unknown. We therefore recorded action potentials and ionic currents in cells isolated from the endocardium, midmyocardium, and epicardium of the left ventricle from dogs with and without tachypacing-induced HF. HF greatly increased action potential duration (APD) but attenuated APD heterogeneity in the three regions. Early afterdepolarizations (EADs) were observed in all cell types of failing hearts but not in controls. Inward rectifier K(+) current (I(K1)) was homogeneously reduced by approximately 41% (at -60 mV) in the three cell types. Transient outward K(+) current (I(to1)) was decreased by 43-45% at +30 mV, and the slow component of the delayed rectifier K(+) current (I(Ks)) was significantly downregulated by 57%, 49%, and 58%, respectively, in epicardial, midmyocardial, and endocardial cells, whereas the rapid component of the delayed rectifier K(+) current was not altered. The results indicate that HF remodels electrophysiology in all layers of the left ventricle, and the downregulation of I(K1), I(to1), and I(Ks) increases APD and favors occurrence of EADs.     

IKr and IKs remodeling differentially affects QT interval prolongation and dynamic adaptation to heart rate acceleration in bradycardic rabbits

Bradycardic ventricular electrical remodeling predisposes to lethal tachyarrhythmias. We investigated the early temporal sequence and reversibility of electrical remodeling in a rabbit complete heart block model subjected to bradycardic ventricular pacing for either 2 or 8 days, with a third group of animals undergoing 8 days of bradycardic pacing followed by 8 days of physiological-rate pacing. At specified time points after complete heart block induction and pacing initiation, steady-state QT interval measurements and variability as well as dynamic QT interval adaptation to abrupt heart rate acceleration were assessed in the absence and presence of isoproterenol. Rapidly (I(Kr)) and slowly (I(Ks)) activating delayed rectifier repolarizing K(+) tail current densities were evaluated using whole cell patch clamp in isolated right ventricular myocytes. Steady-state QT interval prolongation at both 2 and 8 days was associated with moderate I(Kr) reduction. I(Ks) downregulation was apparent by day 2 but more profound at day 8. Dynamic QT interval adaptation was impaired under baseline conditions at day 8 but only during isoproterenol administration at day 2. Both in vivo and cellular manifestations of remodeling reverted toward control values after 8 days of physiological-rate pacing. In conclusion, in this bradycardic model, I(Ks) downregulation 1) proceeds more gradually but more extensively than that of I(Kr) and 2) is most prominently associated with impaired dynamic QT interval adaptation to heart rate acceleration. Isoproterenol blunts the dynamic QT interval response in animals with partially downregulated I(Ks), consistent with stress-related phenomena in known I(Ks)-impaired states. Relative early sparing of I(Ks) could explain the delay in the onset of lethal tachyarrhythmia predisposition in bradycardic electrical remodeling. Reversibility of remodeling supports the potential utility of preventive pacing intervention soon after bradycardia onset.