Attenuation of I(K,slow1) and I(K,slow2) in Kv1/Kv2DN mice prolongs APD and QT intervals but does not suppress spontaneous or inducible arrhythmias

Overexpression of a truncated Kv1.1 or Kv2.1 channel polypeptide in the heart (Kv1DN or Kv2DN) resulted in mice with a prolonged action potential duration (APD) due to marked attenuation of rapidly activating, slowly inactivating K+ current (I(K,slow1)) or slowly inactivating outward K(+) current (I(K,slow2)) in ventricular myocytes. ECG monitoring, optical mapping, and programmed electrical stimulation of Kv1DN mice revealed spontaneous and inducible reentrant ventricular tachycardia due to spatial dispersion of repolarization and refractoriness. Recently, we demonstrated upregulation of I(K,slow2) in apical cardiomyocytes derived from Kv1DN mice. We therefore hypothesized that the selective upregulation of Kv2.1-encoded currents underlies the apex-to-base dispersion of repolarization and the reentrant arrhythmias. To test this hypothesis, the Kv1DN line was crossbred with the Kv2DN line to produce Kv1/Kv2DN lines. Whole cell voltage-clamp recordings from left ventricular cells of Kv1/Kv2DN confirmed that the 4-aminopyridine- and tetraethylammonium-sensitive components of IK,slow were eliminated, resulting in marked APD prolongation compared with wild-type, Kv1DN, and Kv2DN cells. Telemetric ECG recordings revealed prolongation of the corrected QT in Kv1/Kv2DN compared with Kv1DN and Kv2DN mice. However, attenuation of Kv2.1-encoded currents in Kv1DN mice did not suppress the arrhythmias. Thus, the elimination of I(K,slow2) prolongs APD and the QT intervals, but does not have an antiarrhythmic effect.     

Selective elimination of I(K,slow1) in mouse ventricular myocytes expressing a dominant negative Kv1.5alpha subunit

Although previous studies have revealed a role for the voltage-gated K+ channel alpha-subunit Kv1.5 (KCNA5) in the generation of the 4-aminopyridine (4-AP)-sensitive component of delayed rectification in mouse ventricles (IK,slow1), the phenotypic consequences of manipulating IK,slow1 expression in vivo in different (mouse) models are distinct. In these experiments, point mutations were introduced in the pore region of Kv1.5 to change the tryptophan (W) at position 461 to phenylalanine (F) to produce a nonconducting subunit, Kv1.5W461F, that is shown to function as a Kv1 subfamily-specific dominant negative (Kv1.5DN). With the use of the alpha-myosin heavy chain promoter to direct cardiac-specific expression, three lines of Kv1.5DN-expressing (C57BL6) transgenic mice were generated and characterized. Electrophysiological recordings from Kv1.5-DN-expressing left ventricular myocytes revealed that the micromolar 4-AP sensitive IK,slow1 is selectively eliminated. The attenuation of IK,slow1 is accompanied by increased ventricular action potential durations and marked QT prolongation. In contrast to previous findings in mice expressing a truncated (DN) Kv1.1 transgene; however, no electrical remodeling is evident in Kv1.5DN-expressing ventricular myocytes, and the (Kv1.5DN-induced) elimination of IK,slow1 does not result in spontaneous ventricular arrhythmias.     

Over-expression of Kv1.5 in rat cardiomyocytes extremely shortens the duration of the action potential and causes rapid excitation

BACKGROUND: Genetically abnormal action potential duration (APD) can be a cause of arrhythmias that include long and short QT interval syndrome. PURPOSE: The aim of this study was to evaluate the arrhythmogenic effect of short QT syndrome induced by the over-expression of Kv1.5 in rat. METHODS: From Sprague-Dawley rats on fetal days 18-19, cardiomyocytes were excised and cultured with and without transfection with the Kv-1.5 gene using an adenovirus vector. The expression of Kv1.5 was proven by immunohistochemistry and Western blot analysis. In the culture dish and in the whole cells, the electrical activities were recorded using the whole-cell patch-clamp technique and the effects of 4-AP and verapamil were tested. RESULTS: After transfection with Kv1.5 for 12h, immunohistochemical staining and Western blot analysis were positive for Kv1.5 while they were negative in the control transfected with only Lac-Z. In the culture dish, the myocytes showed spontaneous beating at 115beats/min (bpm) just prior to the transfection with Kv1.5 and increased to 367bpm at 24h. The control myocytes showed stable beating rates during culturing. 4-AP at 200microM slowed down the rate and verapamil abolished the beating. In the whole cells, the maximal resting membrane potential was slightly depolarized and APD was extremely abbreviated both at 50% and 90% of repolarization compared with those of the control. Rapid spontaneous activities were found in a single myocyte with Kv1.5 transfection and 4-AP slowed down the frequency of the activities with a reversal of the shortened APD. CONCLUSION: The over-expression of Kv1.5 induced short APD and triggered activities in rat cardiomyocytes. This model can be used to study the arrhythmogenic substrate of short QT syndrome.     

Valproic acid,a mood stabilizer and anticonvulsant,protects rat cerebral cortical neurons from spontaneous cell death: a role of histone deacetylase inhibition

Overexpression of a dominant-negative truncated Kv1.1 (Kv1DN) polypeptide in the mouse heart resulted in marked attenuation of a 4-aminopyridine (4-AP)-sensitive current, I_K,slow1. We used recombinant adeno-associated virus (rAAV) as a vector for direct delivery of Kv1.5 into the mouse myocardium in order to normalize the action potential duration (APD) 6 months after injection. The injection of rAAV-Kv1.5 reconstituted the 4-AP-sensitive outward potassium currents, shortened the APD, and eliminated spontaneous early afterdepolarizations. Immunoblots detected the FL-Kv1.5 polypeptides only in rAAV-Kv1.5-infected hearts. These data demonstrate long-term expression of 4-AP-sensitive potassium currents in ventricular myocytes by gene transfer using rAAV vector encodes Kv1.5.     

Trafficking defect and proteasomal degradation contribute to the phenotype of a novel KCNH2 long QT syndrome mutation

The Kv11.1 (hERG) K+ channel plays a fundamental role in cardiac repolarization. Missense mutations in KCNH2, the gene encoding Kv11.1, cause long QT syndrome (LQTS) and frequently cause channel trafficking-deficiencies. This study characterized the properties of a novel KCNH2 mutation discovered in a LQT2 patient resuscitated from a ventricular fibrillation arrest. Proband genotyping was performed by SSCP and DNA sequencing. The electrophysiological and biochemical properties of the mutant channel were investigated after expression in HEK293 cells. The proband manifested a QTc of 554 ms prior to electrolyte normalization. Mutation analysis revealed an autosomal dominant frameshift mutation at proline 1086 (P1086fs+32X; 3256InsG). Co-immunoprecipitation demonstrated that wild-type Kv11.1 and mutant channels coassemble. Western blot showed that the mutation did not produce mature complex-glycosylated Kv11.1 channels and coexpression resulted in reduced channel maturation. Electrophysiological recordings revealed mutant channel peak currents to be similar to untransfected cells. Co-expression of channels in a 1∶1 ratio demonstrated dominant negative suppression of peak Kv11.1 currents. Immunocytochemistry confirmed that mutant channels were not present at the plasma membrane. Mutant channel trafficking rescue was attempted by incubation at reduced temperature or with the pharmacological agents E-4031. These treatments did not significantly increase peak mutant currents or induce the formation of mature complex-glycosylated channels. The proteasomal inhibitor lactacystin increased the protein levels of the mutant channels demonstrating proteasomal degradation, but failed to induce mutant Kv11.1 protein trafficking. Our study demonstrates a novel dominant-negative Kv11.1 mutation, which results in degraded non-functional channels leading to a LQT2 phenotype.     

Characterization of mice with a combined suppression of I(to) and I(K,slow)

Cardiac-specific expression of a truncated Kv1.1 polypeptide (Kv1DN) attenuates the slow inactivating outward K(+) current (I(K,slow)), increases action potential duration (APD) and Q-T intervals, and induces spontaneous ventricular arrhythmias. Expression of the pore mutant of Kv4.2 (Kv4DN) eliminates the fast component of the transient outward current (I(to)) and prolongs APDs and Q-T intervals markedly; however, no arrhythmias are seen in Kv4DN mice, suggesting that APD and Q-T prolongation are not per se proarrhythmic. To test this hypothesis, the Kv1DN and Kv4DN lines were crossbred to produce animals (Kv1/Kv4DN) expressing both transgenes in an identical genetic background. Whole cell voltage-clamp recordings from left ventricular apex cells confirmed that in Kv1/Kv4DN left ventricular apex cells, both components (fast and slow) of I(to) and the 4-aminopyridine-sensitive component of I(K,slow) are eliminated, resulting in marked APD prolongation compared with wild-type, Kv1DN, or Kv4DN cells. Telemetric electrocardiogram monitoring (n = 10 mice/group) revealed a significant prolongation of Q-Tc and P-R intervals in Kv1/Kv4DN animals compared with Kv1DN or Kv4DN animals. Spontaneous arrhythmias were observed mainly in Kv1DN mice. Thus the attenuation of fast I(to) in addition to I(K,slow) in Kv1/Kv4DN mice causes significant prolongation of APD and Q-T intervals and attenuation of spontaneous arrhythmias.     

Novel characteristics of a misprocessed mutant HERG channel linked to hereditary long QT syndrome

Hereditary long QT syndrome (hLQTS) is a heterogeneous genetic disease characterized by prolonged QT interval in the electrocardiogram, recurrent syncope, and sudden cardiac death. Mutations in the cardiac potassium channel HERG (KCNH2) are the second most common form of hLQTS and reduce the delayed rectifier K(+) currents, thereby prolonging repolarization. We studied a novel COOH-terminal missense mutation, HERG R752W, which segregated with the disease in a family of 101 genotyped individuals. When the mutant cRNA was expressed in Xenopus oocytes it produced enhanced rather than reduced currents. Simulations using the Luo-Rudy model predicted minimal shortening rather than prolongation of the cardiac action potential. Consequently, a normal or shortened QT interval would be expected in contrast to the long QT observed clinically. This anomaly was resolved by our observation that the mutant protein was not delivered to the plasma membrane of mammalian cells but was retained intracellularly. We found that this trafficking defect was corrected at lower incubation temperatures and that functional channels were now delivered to the plasma membrane. However, trafficking could not be restored by chemical chaperones or E-4031, a specific blocker of HERG channels. Therefore, HERG R752W represents a new class of trafficking mutants in hLQTS. The occurrence of different classes of misprocessed channels suggests that a unified therapeutic approach for altering HERG trafficking will not be possible and that different treatment modalities will have to be matched to the different classes of trafficking mutants.     

Mutations in a dominant-negative isoform correlate with phenotype in inherited cardiac arrhythmias

The long QT syndrome is characterized by prolonged cardiac repolarization and a high risk of sudden death. Mutations in the KCNQ1 gene, which encodes the cardiac KvLQT1 potassium ion (K+) channel, cause both the autosomal dominant Romano-Ward (RW) syndrome and the recessive Jervell and Lange-Nielsen (JLN) syndrome. JLN presents with cardiac arrhythmias and congenital deafness, and heterozygous carriers of JLN mutations exhibit a very mild cardiac phenotype. Despite the phenotypic differences between heterozygotes with RW and those with JLN mutations, both classes of variant protein fail to produce K+ currents in cultured cells. We have shown that an N-terminus-truncated KvLQT1 isoform endogenously expressed in the human heart exerts strong dominant-negative effects on the full-length KvLQT1 protein. Because RW and JLN mutations concern both truncated and full-length KvLQT1 isoforms, we investigated whether RW or JLN mutations would have different impacts on the dominant-negative properties of the truncated KvLQT1 splice variant. In a mammalian expression system, we found that JLN, but not RW, mutations suppress the dominant-negative effects of the truncated KvLQT1. Thus, in JLN heterozygous carriers, the full-length KvLQT1 protein encoded by the unaffected allele should not be subject to the negative influence of the mutated truncated isoform, leaving some cardiac K+ current available for repolarization. This is the first report of a genetic disease in which the impact of a mutation on a dominant-negative isoform correlates with the phenotype.     

Pharmacogenomics of anesthetic drugs in transgenic LQT1 and LQT2 rabbits reveal genotype-specific differential effects on cardiac repolarization

Anesthetic agents prolong cardiac repolarization by blocking ion currents. However, the clinical relevance of this blockade in subjects with reduced repolarization reserve is unknown. We have generated transgenic long QT syndromes type 1 (LQT1) and type 2 (LQT2) rabbits that lack slow delayed rectifier K+ currents (IKs) or rapidly activating K+ currents (IKr) and used them as a model system to detect the channel-blocking properties of anesthetic agents. Therefore, LQT1, LQT2, and littermate control (LMC) rabbits were administered isoflurane, thiopental, midazolam, propofol, or ketamine, and surface ECGs were analyzed. Genotype-specific heart rate correction formulas were used to determine the expected QT interval at a given heart rate. The QT index (QTi) was calculated as percentage of the observed QT/expected QT. Isoflurane, a drug that blocks IKs) prolonged the QTi only in LQT2 and LMC but not in LQT1 rabbits. Midazolam, which blocks inward rectifier K+ current (IK1), prolonged the QTi in both LQT1 and LQT2 but not in LMC. Thiopental, which blocks both IKs and IK1, increased the QTi in LQT2 and LMC more than in LQT1. By contrast, ketamine, which does not block IKr, IKs, or IK1, did not alter the QTi in any group. Finally, anesthesia with isoflurane or propofol resulted in lethal polymorphic ventricular tachycardia (pVT) in three out of nine LQT2 rabbits. Transgenic LQT1 and LQT2 rabbits could serve as an in vivo model in which to examine the pharmacogenomics of drug-induced QT prolongation of anesthetic agents and their proarrhythmic potential. Transgenic LQT2 rabbits developed pVT under isoflurane and propofol, underlining the proarrhythmic risk of IKs blockers in subjects with reduced IKr.     

The congenital long QT syndrome Type 3: An update

Congenital long QT syndrome type 3 (LQT3) is the third in frequency compared to the 15 forms known currently of congenital long QT syndrome (LQTS). Cardiac events are less frequent in LQT3 when compared with LQT1 and LQT2, but more likely to be lethal; the likelihood of dying during a cardiac event is 20% in families with an LQT3 mutation and 4% with either an LQT1 or an LQT2 mutation. LQT3 is consequence of mutation of gene SCN5A which codes for the Nav1.5 Na⁺ channel α-subunit and electrocardiographically characterized by a tendency to bradycardia related to age, prolonged QT/QTc interval (mean QTc value 478 ± 52 ms), accentuated QT dispersion consequence of prolonged ST segment, late onset of T wave and frequent prominent U wave because of longer repolarization of the M cell across left ventricular wall.     

Overlapping LQT1 and LQT2 phenotype in a patient with long QT syndrome associated with loss-of-function variations in KCNQ1 and KCNH2

Long QT syndrome (LQTS) is an inherited disorder characterized by prolonged QT intervals and potentially life-threatening arrhythmias. Mutations in 12 different genes have been associated with LQTS. Here we describe a patient with LQTS who has a mutation in KCNQ1 as well as a polymorphism in KCNH2. The proband (MMRL0362), a 32-year-old female, exhibited multiple ventricular extrasystoles and one syncope. Her ECG (QT interval corrected for heart rate (QTc) = 518ms) showed an LQT2 morphology in leads V4-V6 and LQT1 morphology in leads V1-V2. Genomic DNA was isolated from lymphocytes. All exons and intron borders of 7 LQTS susceptibility genes were amplified and sequenced. Variations were detected predicting a novel missense mutation (V110I) in KCNQ1, as well as a common polymorphism in KCNH2 (K897T). We expressed wild-type (WT) or V110I Kv7.1 channels in CHO-K1 cells cotransfected with KCNE1 and performed patch-clamp analysis. In addition, WT or K897T Kv11.1 were also studied by patch clamp. Current-voltage (I-V) relations for V110I showed a significant reduction in both developing and tail current densities compared with WT at potentials >+20 mV (p < 0.05; n = 8 cells, each group), suggesting a reduction in IKs currents. K897T- Kv11.1 channels displayed a significantly reduced tail current density compared with WT-Kv11.1 at potentials >+10 mV. Interestingly, channel availability assessed using a triple-pulse protocol was slightly greater for K897T compared with WT (V0.5 = -53.1 ± 1.13 mV and -60.7 ± 1.15 mV for K897T and WT, respectively; p < 0.05). Comparison of the fully activated I-V revealed no difference in the rectification properties between WT and K897T channels. We report a patient with a loss-of-function mutation in KCNQ1 and a loss-of-function polymorphism in KCNH2. Our results suggest that a reduction of both IKr and IKs underlies the combined LQT1 and LQT2 phenotype observed in this patient.     

Increasing I(Ks) corrects abnormal repolarization in rabbit models of acquired LQT2 and ventricular hypertrophy

Excessive action potential (AP) prolongation and early afterdepolarizations (EAD) are triggers of malignant ventricular arrhythmias. A slowly activating delayed rectifier K+ current (I(Ks)) is important for repolarization of ventricular AP. We examined the effects of I(Ks) activation by a new benzodiazepine (L3) on the AP of control, dofetilide-treated, and hypertrophied rabbit ventricular myocytes. In both control and hypertrophied myocytes, L3 activated I(Ks) via a negative shift in the voltage dependence of activation and a slowing of deactivation. L3 had no effect on L-type Ca(2+) current or other cardiac K+ currents tested. L3 shortened AP of control, dofetilide-treated, and hypertrophied myocytes more at 0.5 than 2 Hz. Selective activation of I(Ks) by L3 attenuates prolonged AP and eliminated EAD induced by rapidly activating delayed rectifier K+ current inhibition in control myocytes at 0.5 Hz and spontaneous EAD in hypertrophied myocytes at 0.2 Hz. Pharmacological activation of I(Ks) is a promising new strategy to suppress arrhythmias resulting from excessive AP prolongation in patients with certain forms of long QT syndrome or cardiac hypertrophy and failure.     

Quantitative relationship between plasma potassium levels and QT interval in beagle dogs

The aim of the current study was to establish the quantitative relationship between plasma potassium concentrations and the QT interval of the electrocardiogram in dogs. Furosemide, a potent diuretic, was given at increasing doses (5-60 mg/kg) to five male and five female beagle dogs. Electrocardiogram (ECG) was recorded three times each day, simultaneous to blood sampling for measurement of plasma potassium. Furosemide treatment produced a clear hypokalaemia, which was associated with an increase in QT and corrected QT intervals (QTc) duration. On average, the slopes of the negative linear correlation between potassium plasma levels and QT or QTc were steeper in females than in males. These results show that a decrease in potassium plasma level may explain a concomitant increase in QT duration in a toxicity study in dogs, in particular if potassium values are decreased below 3.3 mmol/L. Correction of QT interval for K+ plasma level has, therefore, been established separately for males and females. A global formula correcting QT for K+ and heart rate simultaneously was established. Hypokalaemia was also associated with changes in the morphology of the T wave recorded in CV5RL, in particular, with a flattening and/or a notching of the wave (appearance of a second peak), biphasic aspect or inversion of polarity. These changes are probably related to an increased heterogeneity of repolarization between different populations of cardiomyocytes. In conclusion, hypokalaemia is quantitatively associated with an increase in QT and QTc duration in dogs. The relationship is apparently stronger for females than for males. A formula may be used to correct QT for potassium plasma level.     

Long QT syndrome-associated mutations in the Per-Arnt-Sim (PAS) domain of HERG potassium channels accelerate channel deactivation

Mutations in the human ether-a-go-go-related gene (HERG) cause long QT syndrome, an inherited disorder of cardiac repolarization that predisposes affected individuals to life-threatening arrhythmias. HERG encodes the cardiac rapid delayed rectifier potassium channel that mediates repolarization of ventricular action potentials. In this study, we used the oocyte expression system and voltage clamp techniques to determine the functional consequences of eight long QT syndrome-associated mutations located in the amino-terminal region of HERG (F29L, N33T, G53R, R56Q, C66G, H70R, A78P, and L86R). Mutant subunits formed functional channels with altered gating properties when expressed alone in oocytes. Deactivation was accelerated by all mutations. Some mutants shifted the voltage dependence of channel availability to more positive potentials. Voltage ramps indicated that fast deactivation of mutant channels would reduce outward current during the repolarization phase of the cardiac action potential and cause prolongation of the corrected QT interval, QTc. The amino-terminal region of HERG was recently crystallized and shown to possess a Per-Arnt-Sim (PAS) domain. The location of these mutations suggests they may disrupt the PAS domain and interfere with its interaction with the S4-S5 linker of the HERG channel.     

Molecular correlates of altered expression of potassium currents in failing rabbit myocardium

Action potential (AP) prolongation is a hallmark of failing myocardium. Functional downregulation of K currents is a prominent feature of cells isolated from failing ventricles. The detailed changes in K current expression differ depending on the species, the region of the heart, and the mechanism of induction of heart failure. We used complementary approaches to study K current downregulation in pacing tachycardia-induced heart failure in the rabbit. The AP duration (APD) at 90% repolarization was significantly longer in cells isolated from failing hearts compared with controls (539 +/- 162 failing vs. 394 +/- 114 control, P < 0.05). The major K currents in the rabbit heart, inward rectifier potassium current (I(K1)), transient outward (I(to)), and delayed rectifier current (I(K)) were functionally downregulated in cells isolated from failing ventricles. The mRNA levels of Kv4.2, Kv1.4, KChIP2, and Kir2.1 were significantly downregulated, whereas the Kv4.3, Erg, KvLQT1, and minK were unaltered in the failing ventricles compared with the control left ventricles. Significant downregulation in the long splice variant of Kv4.3, but not in the total Kv4.3, Kv4.2, and KChIP2 immunoreactive protein, was observed in cells isolated from the failing ventricle with no change in Kv1.4, KvLQT1, and in Kir2.1 immunoreactive protein levels. Multiple cellular and molecular mechanisms underlie the downregulation of K currents in the failing rabbit ventricle.     

Evidence against functional heteromultimerization of the KATP channel subunits Kir6.1 and Kir6.2

K(ATP) channels consist of pore-forming potassium inward rectifier (Kir6.x) subunits and sulfonylurea receptors (SURs). Although Kir6.1 or Kir6.2 coassemble with different SUR isoforms to form heteromultimeric functional K(ATP) channels, it is not known whether Kir6.1 and Kir6.2 coassemble with each other. To define the molecular identity of K(ATP) channels, we used adenoviral gene transfer to express wild-type and dominant-negative constructs of Kir6.1 and Kir6.2 in a heterologous expression system (A549 cells) and in native cells (rabbit ventricular myocytes). Dominant-negative (DN) Kir6.2 gene transfer suppressed current through heterologously expressed SUR2A + Kir6.2 channels. Conversely, DN Kir6.1 suppressed SUR2B + Kir6.1 current but had no effect on coexpressed SUR2A + Kir6. 2. We next probed the ability of Kir6.1 and Kir6.2 to affect endogenous K(ATP) channels in adult rabbit ventricular myocytes, using adenoviral vectors to achieve efficient gene transfer. Infection with the DN Kir6.2 virus for 72 h suppressed pinacidil-inducible K(ATP) current density measured by whole-cell patch clamp. However, there was no effect of infection with the DN Kir6.1 on the K(ATP) current. Based on these functional assays, we conclude that Kir6.1 and Kir6.2 do not heteromultimerize with each other and that Kir6.2 is the sole K(ATP) pore-forming subunit in the surface membrane of heart cells.     

Phospholipid lysophosphatidylcholine as a metabolic trigger and HERG as an ionic pathway for extracellular K accumulation and "short QT syndrome" in acute myocardial ischemia.

The most profound abnormalities during acute myocardial ischemia are extracellular K(+) accumulation ([K(+)](o)- upward arrow) and shortening of action potential duration or QT interval (APD- downward arrow or QT- downward arrow), which are pivotal in the genesis of ischemic arrhythmias and sudden cardiac death. The ionic mechanisms however remained obscured. We performed studies in a rabbit model of acute global myocardial ischemia in order to explore ionic and metabolic mechanisms for ischemic [K(+)](o)- upward arrow and QT- downward arrow. Exogenous 1-palmitoyl-lysophosphatidylcholine (LPC-16) mimicked the low-perfusion ischemia to produce significant [K(+)](o)- upward arrow and QT- downward arrow. The [K(+)](o)- upward arrow and QT- downward arrow induced by either LPC-16 or ischemia were prevented by dofetilide, a blocker of rapid delayed rectifier K(+) current (I(Kr)), but not by blockers for other K(+) channels. Consistently, dofetilide efficiently abolished the ventricular tachy-arrhythmias induced by ischemia or LPC-16. LPC-16 remarkably shortened APD and enhanced the function of I(Kr) and HERG (the pore-forming subunit of I(Kr)). The effects of LPC-16 manifested with shorter APD (faster repolarization rate) and at more negative potential (membrane repolarization). Dofetilide abolished the I(Kr)/HERG enhancing and APD shortening effects of LPC-16. Our results suggest that LPC-16 accumulation/HERG enhancement may be a link between metabolic trigger and ionic pathway for ischemic [K(+)](o)- upward arrow and QTc- downward arrow. This represents the first documentation of I(Kr)/HERG as the ionic mechanism in ischemic [K(+)](o)- upward arrow and QTc- downward arrow. Inhibition of LPC-16 production and accumulation and/or of I(Kr)/HERG may be a promising therapeutic strategy to attenuate the incidence of lethal arrhythmias associated with ischemic heart disease.     

Bcl-2 decreases voltage-gated K+ channel activity and enhances survival in vascular smooth muscle cells

Cell shrinkage is an incipient hallmark of apoptosis in a variety of cell types. The apoptotic volume decrease has been demonstrated to attribute, in part, to K+ efflux; blockade of plasmalemmal K+ channels inhibits the apoptotic volume decrease and attenuates apoptosis. Using combined approaches of gene transfection, single-cell PCR, patch clamp, and fluorescence microscopy, we examined whether overexpression of Bcl-2, an anti-apoptotic oncoprotein, inhibits apoptosis in pulmonary artery smooth muscle cells (PASMC) by diminishing the activity of voltage-gated K+ (Kv) channels. A human bcl-2 gene was infected into primary cultured rat PASMC using an adenoviral vector. Overexpression of Bcl-2 significantly decreased the amplitude and current density of Kv currents (I(Kv)). In contrast, the apoptosis inducer staurosporine (ST) enhanced I(Kv). In bcl-2-infected cells, however, the ST-induced increase in I(Kv) was completely abolished, and the ST-induced apoptosis was significantly inhibited compared with cells infected with an empty adenovirus (-bcl-2). Blockade of Kv channels in control cells (-bcl-2) by 4-aminopyridine also inhibited the ST-induced increase in I(Kv) and apoptosis. Furthermore, overexpression of Bcl-2 accelerated the inactivation of I(Kv) and downregulated the mRNA expression of the pore-forming Kv channel alpha-subunits (Kv1.1, Kv1.5, and Kv2.1). These results suggest that inhibition of Kv channel activity may serve as an additional mechanism involved in the Bcl-2-mediated anti-apoptotic effect on vascular smooth muscle cells.     

Electrical remodeling of cardiac myocytes from mice with heart failure due to the overexpression of tumor necrosis factor-alpha

Mice that overexpress the inflammatory cytokine tumor necrosis factor-alpha in the heart (TNF mice) develop heart failure characterized by atrial and ventricular dilatation, decreased ejection fraction, atrial and ventricular arrhythmias, and increased mortality (males > females). Abnormalities in Ca2+ handling, prolonged action potential duration (APD), calcium alternans, and reentrant atrial and ventricular arrhythmias were previously observed with the use of optical mapping of perfused hearts from TNF mice. We therefore tested whether altered voltage-gated outward K+ and/or inward Ca2+ currents contribute to the altered action potential characteristics and the increased vulnerability to arrhythmias. Whole cell voltage-clamp recordings of K+ currents from left ventricular myocytes of TNF mice revealed an approximately 50% decrease in the rapidly activating, rapidly inactivating transient outward K+ current Ito and in the rapidly activating, slowly inactivating delayed rectifier current IK,slow1, an approximately 25% decrease in the rapidly activating, slowly inactivating delayed rectifier current IK,slow2, and no significant change in the steady-state current Iss compared with controls. Peak amplitudes and inactivation kinetics of the L-type Ca2+ current ICa,L were not altered. Western blot analyses revealed a reduction in the proteins underlying Kv4.2, Kv4.3, and Kv1.5. Thus decreased K+ channel expression is largely responsible for the prolonged APD in the TNF mice and may, along with abnormalities in Ca2+ handling, contribute to arrhythmias.