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.     

Differential expression of Kv4 pore-forming and KChIP auxiliary subunits in rat uterus during pregnancy

Regulation of voltage-gated K(+) (K(v)) channel expression may be involved in controlling contractility of uterine smooth muscle cells during pregnancy. Functional expression of these channels is not only controlled by the levels of pore-forming subunits, but requires their association with auxiliary subunits. Specifically, rapidly inactivating K(v) current is prominent in myometrial cells and may be carried by complexes consisting of Kv4 pore-forming and KChIP auxiliary subunits. To determine the molecular identity of the channel complexes and their changes during pregnancy, we examined the expression and localization of these subunits in rat uterus. RT-PCR analysis revealed that rat uterus expressed all three Kv4 pore-forming subunits and KChIP2 and -4 auxiliary subunits. The expression of mRNAs for these subunits was dynamically and region selectively regulated during pregnancy. In the corpus, Kv4.2 mRNA level increased before parturition, whereas the expression of Kv4.1 and Kv4.3 mRNAs decreased during pregnancy. A marked increase in KChIP2 mRNA level was also seen at late gestation. In the cervix, the expression of all three pore-forming and two auxiliary subunit mRNAs increased at late gestation. Immunoprecipitation followed by immunoblot analysis indicated that Kv4.2-KChIP2 complexes were significant in uterus at late pregnancy. Kv4.2- and KChIP2-immunoreactive proteins were present in both circular and longitudinal myometrial cells. Finally, Kv4.2 and KChIP2 mRNA levels were similarly elevated in pregnant and nonpregnant corpora of one side-conceived rats. These results suggest that diffusible factors coordinate the pregnancy-associated changes in molecular compositions of myometrial Kv4-KChIP channel complexes.     

Long-Term Fish Oil Supplementation Induces Cardiac Electrical Remodeling by Changing Channel Protein Expression in the Rabbit Model

Clinical trials and epidemiological studies have suggested that dietary fish oil (FO) supplementation can provide an anti-arrhythmic benefit in some patient populations. The underlying mechanisms are not entirely clear. We wanted to understand how FO supplementation (for 4 weeks) affected the action potential configuration/duration of ventricular myocytes, and the ionic mechanism(s)/molecular basis for these effects. The experiments were conducted on adult rabbits, a widely used animal model for cardiac electrophysiology and pathophysiology. We used gas chromatography - mass spectroscopy to confirm that FO feeding produced a marked increase in the content of n-3 polyunsaturated fatty acids in the phospholipids of rabbit hearts. Left ventricular myocytes were used in current and voltage clamp experiments to monitor action potentials and ionic currents, respectively. Action potentials of myocytes from FO-fed rabbits exhibited much more positive plateau voltages and prolonged durations. These changes could be explained by an increase in the L-type Ca current (I_CaL) and a decrease in the transient outward current (I_to) in these myocytes. FO feeding did not change the delayed rectifier or inward rectifier current. Immunoblot experiments showed that the FO-feeding induced changes in I_CaL and I_to were associated with corresponding changes in the protein levels of major pore-forming subunits of these channels: increase in Cav1.2 and decrease in Kv4.2 and Kv1.4. There was no change in other channel subunits (Cav1.1, Kv4.3, KChIP2, and ERG1). We conclude that long-term fish oil supplementation can impact on cardiac electrical activity at least partially by changing channel subunit expression in cardiac myocytes.     

Regulation of Kv4 channel expression in failing rat heart by the thioredoxin system

Redox imbalance elicited by oxidative stress contributes to pathogenic remodeling of ion channels that underlies arrhythmogenesis and contractile dysfunction in the failing heart. This study examined whether the expression of K(+) channels in the remodeled ventricle is controlled by the thioredoxin system, a principal oxidoreductase network regulating redox-sensitive proteins. Ventricular dysfunction was induced in rats by coronary artery ligation, and experiments were conducted 6-8 wk postinfarction. Biochemical assays of tissue extracts from infarcted hearts showed that thioredoxin reductase activity was decreased by 32% from sham-operated controls (P < 0.05), whereas thioredoxin activity was 51% higher postinfarction (P < 0.05). These differences in activities paralleled changes in protein abundance as determined by Western blot analysis. However, whereas real-time PCR showed thioredoxin reductase mRNA levels to be significantly decreased postinfarction, thioredoxin mRNA was not altered. In voltage-clamp studies of myocytes from infarcted hearts, the characteristic downregulation of transient-outward K(+) current density was reversed by exogenous pyruvate (5 mmol/l), and this effect was blocked by the specific inhibitors of the thioredoxin system: auranofin or 13-cis-retinoic acid. Real-time PCR and Western blot analyses of myocyte suspensions from infarcted hearts showed that pyruvate increased mRNA and protein abundance of Kv4.2 and Kv4.3 channel alpha-subunits as well as the accessory protein KChIP2 when compared with time-matched, untreated cells (P < 0.05). The pyruvate-induced increase in Kv4.x expression was blocked by auranofin, but the upregulation of KChIP2 expression was not affected. These data suggest that the expression of Kv4.x channels is redox-regulated by the thioredoxin system, which may be a novel therapeutic target to reverse or limit electrical remodeling of the failing heart.     

Up‐regulation of miR‐195 contributes to cardiac hypertrophy‐induced arrhythmia by targeting calcium and potassium channels

Previous studies have confirmed that miR‐195 expression is increased in cardiac hypertrophy, and the bioinformatics website predicted by Targetscan software shows that miR‐195 can directly target CACNB1, KCNJ2 and KCND3 to regulate Cavβ1, Kir2.1 and Kv4.3 proteins expression. The purpose of this study is to confirm the role of miR‐195 in arrhythmia caused by cardiac hypertrophy. The protein levels of Cavβ1, Kir2.1 and Kv4.3 in myocardium of HF mice were decreased. After miR‐195 was overexpressed in neonatal mice cardiomyocytes, the expression of ANP, BNP and β‐MHC was up‐regulated, and miR‐195 inhibitor reversed this phenomenon. Overexpression of miR‐195 reduced the estimated cardiac function of EF% and FS% in wild‐type (WT) mice. Transmission electron microscopy showed that the ultrastructure of cardiac tissues was damaged after miR‐195 overexpression by lentivirus in mice. miR‐195 overexpression increased the likelihood of arrhythmia induction and duration of arrhythmia in WT mice. Lenti‐miR‐195 inhibitor carried by lentivirus can reverse the decreased EF% and FS%, the increased incidence of arrhythmia and prolonged duration of arrhythmia induced by TAC in mice. After miR‐195 treatment, the protein expressions of Cavβ1, Kir2.1 and Kv4.3 were decreased in mice. The results were consistent at animal and cellular levels, respectively. Luciferase assay results showed that miR‐195 may directly target CACNB1, KCNJ2 and KCND3 to regulate the expression of Cavβ1, Kir2.1 and Kv4.3 proteins. MiR‐195 is involved in arrhythmia caused by cardiac hypertrophy by inhibiting Cavβ1, Kir2.1 and Kv4.3.     

A defect in the Kv channel-interacting protein 2 (KChIP2) gene leads to a complete loss of I(to) and confers susceptibility to ventricular tachycardia.

KChIP2, a gene encoding three auxiliary subunits of Kv4.2 and Kv4.3, is preferentially expressed in the adult heart, and its expression is downregulated in cardiac hypertrophy. Mice deficient for KChIP2 exhibit normal cardiac structure and function but display a prolonged elevation in the ST segment on the electrocardiogram. The KChIP2(-/-) mice are highly susceptible to the induction of cardiac arrhythmias. Single-cell analysis revealed a substrate for arrhythmogenesis, including a complete absence of transient outward potassium current, I(to), and a marked increase in action potential duration. These studies demonstrate that a defect in KChIP2 is sufficient to confer a marked genetic susceptibility to arrhythmias, establishing a novel genetic pathway for ventricular tachycardia via a loss of the transmural gradient of I(to).     

Angiotensin receptor type 1 forms a complex with the transient outward potassium channel Kv4.3 and regulates its gating properties and intracellular localization

We report a novel signal transduction complex of the angiotensin receptor type 1. In this complex the angiotensin receptor type 1 associates with the potassium channel alpha-subunit Kv4.3 and regulates its intracellular distribution and gating properties. Co-localization of Kv4.3 with angiotensin receptor type 1 and fluorescent resonance energy transfer between those two proteins labeled with cyan and yellow-green variants of green fluorescent protein revealed that Kv4.3 and angiotensin receptor type I are located in close proximity to each other in the cell. The angiotensin receptor type 1 also co-immunoprecipitates with Kv4.3 from canine ventricle or when co-expressed with Kv4.3 and its beta-subunit KChIP2 in human embryonic kidney 293 cells. Treatment of the cells with angiotensin II results in the internalization of Kv4.3 in a complex with the angiotensin receptor type 1. When stimulated with angiotensin II, angiotensin receptors type 1 modulate gating properties of the remaining Kv4.3 channels on the cell surface by shifting their activation voltage threshold to more positive values. We hypothesize that the angiotensin receptor type 1 provides its internalization molecular scaffold to Kv4.3 and in this way regulates the cell surface representation of the ion channel.     

KChIP2b modulates the affinity and use-dependent block of Kv4.3 by nifedipine

Rapidly activating Kv4 voltage-gated ion channels are found in heart, brain, and diverse other tissues including colon and uterus. Kv4.3 can co-assemble with KChIP ancillary subunits, which modify kinetic behavior. We examined the affinity and use dependence of nifedipine block on Kv4.3 and its modulation by KChIP2b. Nifedipine (150 microM) reduced peak Kv4.3 current approximately 50%, but Kv4.3/KChIP2b current only approximately 27%. Nifedipine produced a very rapid component of open channel block in both Kv4.3 and Kv4.3/KChIP2b. However, recovery from the blocked/inactivated state was strongly sensitive to KChIP2b. Kv4.3 Thalf,recovery was slowed significantly by nifedipine (120.0+/-12.4 ms vs. 213.1+/-18.2 ms), whereas KChIP2b eliminated nifedipine's effect on recovery: Kv4.3/KChIP2b Thalf,recovery was 45.3+/-7.2 ms (control) and 47.8+/-8.2 ms (nifedipine). Consequently, Kv4.3 exhibited use-dependent nifedipine block in response to a series of depolarizing pulses which was abolished by KChIP2b. KChIPs alter drug affinity and use dependence of Kv4.3.     

Functional Rescue of Kv4.3 Channel Tetramerization Mutants by KChIP4a

KChIP4a shows a high homology with other members of the family of Kv channel-interacting proteins (KChIPs) in the conserved C-terminal core region, but exhibits a unique modulation of Kv4 channel gating and surface expression. Unlike KChIP1, the KChIP4 splice variant KChIP4a has been shown to inhibit surface expression and function as a suppressor of channel inactivation of Kv4. In this study, we sought to determine whether the multitasking KChIP4a modulates Kv4 function in a clamping fashion similar to that shown by KChIP1. Injection of Kv4.3 T1 zinc mutants into Xenopus oocytes resulted in the nonfunctional expression of Kv4.3 channels. Coexpression of Kv4.3 zinc mutants with WT KChIP4a gave rise to the functional expression of Kv4.3 current. Oocyte surface labeling results confirm the correlation between functional rescue and enhanced surface expression of zinc mutant proteins. Chimeric mutations that replace the Kv4.3 N-terminus with N-terminal KChIP4a or N-terminal deletion of KChIP4a further demonstrate that the functional rescue of Kv4.3 channel tetramerization mutants depends on the KChIP4a core region, but not its N-terminus. Structure-guided mutation of two critical residues of core KChIP4a attenuated functional rescue and tetrameric assembly. Moreover, size exclusion chromatography combined with fast protein liquid chromatography showed that KChIP4a can drive zinc mutant monomers to assemble as tetramers. Taken together, our results show that KChIP4a can rescue the function of tetramerization-defective Kv4 monomers. Therefore, we propose that core KChIP4a functions to promote tetrameric assembly and enhance surface expression of Kv4 channels by a clamping action, whereas its N-terminus inhibits surface expression of Kv4 by a mechanism that remains elusive.     

Myocardial KChIP2 Expression in Guinea Pig Resolves an Expanded Electrophysiologic Role

Cardiac ion channels and their respective accessory subunits are critical in maintaining proper electrical activity of the heart. Studies have indicated that the K⁺ channel interacting protein 2 (KChIP2), originally identified as an auxiliary subunit for the channel Kv4, a component of the transient outward K⁺ channel (I_to), is a Ca²⁺ binding protein whose regulatory function does not appear restricted to Kv4 modulation. Indeed, the guinea pig myocardium does not express Kv4, yet we show that it still maintains expression of KChIP2, suggesting roles for KChIP2 beyond this canonical auxiliary interaction with Kv4 to modulate I_to. In this study, we capitalize on the guinea pig as a system for investigating how KChIP2 influences the cardiac action potential, independent of effects otherwise attributed to I_to, given the endogenous absence of the current in this species. By performing whole cell patch clamp recordings on isolated adult guinea pig myocytes, we observe that knock down of KChIP2 significantly prolongs the cardiac action potential. This prolongation was not attributed to compromised repolarizing currents, as I_Kr and I_Ks were unchanged, but was the result of enhanced L-type Ca²⁺ current due to an increase in Cav1.2 protein. In addition, cells with reduced KChIP2 also displayed lowered I_Na from reduced Nav1.5 protein. Historically, rodent models have been used to investigate the role of KChIP2, where dramatic changes to the primary repolarizing current I_to may mask more subtle effects of KChIP2. Evaluation in the guinea pig where I_to is absent, has unveiled additional functions for KChIP2 beyond its canonical regulation of I_to, which defines KChIP2 as a master regulator of cardiac repolarization and depolarization.     

Loss of the normal epicardial to endocardial gradient of cftr mRNA expression in the hypertrophied rabbit left ventricle

The electrical instability of hypertrophied and failing hearts is caused by delayed repolarisation, which is thought to be due in part to altered levels and/or patterns of expression of ion channel genes. The aim of this study was to investigate changes in the levels and pattern of cystic fibrosis transmembrane conductance regulator (cftr) mRNA expression in a combined pressure and volume overload model of heart failure in the rabbit, using in situ mRNA hybridisation. There was a decrease in cftr mRNA expression, primarily due to a decrease in epicardial expression and, hence, loss of the normal epicardial to endocardial gradient of cftr mRNA expression in the rabbit left ventricle. In contrast there was an increase in atrial natriuretic factor (anf) mRNA expression in the hypertrophied hearts with preferential reexpression in subendocardial regions. The patterns of both cftr and anf mRNA expression in the hypertrophied hearts were similar to those seen in embryonic hearts. This suggests that the reversion to an embryonic pattern of gene expression in cardiac hypertrophy applies to ion channel genes. The loss of the normal transmural gradient of repolarising ion channels is likely to contribute to instability of repolarisation in the hypertrophied heart and hence increased risk of cardiac arrhythmias in patients with heart failure.     

Enhanced Trafficking of Tetrameric Kv4.3 Channels by KChIP1 Clamping

The cytoplamsic auxiliary KChIPs modulate surface expression and gating properties of Kv4 channels. Recent co-crystal structure of Kv4.3 N-terminus and KChIP1 reveals a clamping action of the complex in which a single KChIP1 molecule laterally binds two neighboring Kv4.3 N-termini at different locations, thus forming two contact interfaces involved in the protein–protein interaction. In the second interface, it functions to stabilize the tetrameric assembly, but the role it plays in channel trafficking remains elusive. In this study, we examined the effects of KChIP1 on Kv4 protein trafficking in COS-7 cells expressing EGFP-tagged Kv4.3 channels using confocal microscopy. Mutations either in KChIP1 (KChIP1 L39E-Y57A-K61A) or Kv4.3 (Kv4.3 E70A-F73E) that disrupt the protein–protein interaction within the second interface can reduce surface expression of Kv4 channel proteins. Kv4.3 C110A, the Zn²⁺ binding site mutation in T1 domain, that disrupts the tetrameric assembly of the channels can be rescued by WT KChIP1, but not the KChIP1 triple mutant. These results were further confirmed by whole cell current recordings in oocytes. Our findings show that key residues of second interface involved in stabilizing tetrameric assembly can regulate the channel trafficking, indicating an intrinsic link between tetrameric assembly and channel trafficking. The results also suggest that formation of octameric Kv4 and KChIP complex by KChIPs clamping takes place before their trafficking to final destination on the cell surface. Special issue article in honor of Dr. Ji-Sheng Han.     

Growth factor-induced mobilization of cardiac progenitor cells reduces the risk of arrhythmias, in a rat model of chronic myocardial infarction

Heart repair by stem cell treatment may involve life-threatening arrhythmias. Cardiac progenitor cells (CPCs) appear best suited for reconstituting lost myocardium without posing arrhythmic risks, being commissioned towards cardiac phenotype. In this study we tested the hypothesis that mobilization of CPCs through locally delivered Hepatocyte Growth Factor and Insulin-Like Growth Factor-1 to heal chronic myocardial infarction (MI), lowers the proneness to arrhythmias. We used 133 adult male Wistar rats either with one-month old MI and treated with growth factors (GFs, n = 60) or vehicle (V, n = 55), or sham operated (n = 18). In selected groups of animals, prior to and two weeks after GF/V delivery, we evaluated stress-induced ventricular arrhythmias by telemetry-ECG, cardiac mechanics by echocardiography, and ventricular excitability, conduction velocity and refractoriness by epicardial multiple-lead recording. Invasive hemodynamic measurements were performed before sacrifice and eventually the hearts were subjected to anatomical, morphometric, immunohistochemical, and molecular biology analyses. When compared with untreated MI, GFs decreased stress-induced arrhythmias and concurrently prolonged the effective refractory period (ERP) without affecting neither the duration of ventricular repolarization, as suggested by measurements of QTc interval and mRNA levels for K-channel α-subunits Kv4.2 and Kv4.3, nor the dispersion of refractoriness. Further, markers of cardiomyocyte reactive hypertrophy, including mRNA levels for K-channel α-subunit Kv1.4 and β-subunit KChIP2, interstitial fibrosis and negative structural remodeling were significantly reduced in peri-infarcted/remote ventricular myocardium. Finally, analyses of BrdU incorporation and distribution of connexin43 and N-cadherin indicated that cytokines generated new vessels and electromechanically-connected myocytes and abolished the correlation of infarct size with deterioration of mechanical function. In conclusion, local injection of GFs ameliorates electromechanical competence in chronic MI. Reduced arrhythmogenesis is attributable to prolongation of ERP resulting from improved intercellular coupling via increased expression of connexin43, and attenuation of unfavorable remodeling.     

Kinetic properties of Kv4.3 and their modulation by KChIP2b

KChIPs are a family of Kv4 K(+) channel ancillary subunits whose effects usually include slowing of inactivation, speeding of recovery from inactivation, and increasing channel surface expression. We compared the effects of the 270 amino acid KChIP2b on Kv4.3 and a Kv4.3 inner pore mutant [V(399, 401)I]. Kv4.3 showed fast inactivation with a bi-exponential time course in which the fast time constant predominated. KChIP2b expressed with wild-type Kv4.3 slowed the fast time constant of inactivation; however, the overall rate of inactivation was faster due to reduction of the contribution of the slow inactivation phase. Introduction of [V(399, 401)I] slowed both time constants of inactivation less than 2-fold. Inactivation was incomplete after 20s pulse durations. Co-expression of KChIP2b with Kv4.3 [V(399, 401)I] slowed inactivation dramatically. KChIP2b increased the rate of recovery from inactivation 7.6-fold in the wild-type channel and 5.7-fold in Kv4.3 [V(399,401)I]. These data suggest that inner pore structure is an important factor in the modulatory effects of KChIP2b on Kv4.3 K(+) channels.     

Phenotypic differences in transient outward K+ current of human and canine ventricular myocytes: insights into molecular composition of ventricular Ito

The Ca(2+)-independent transient outward K(+) current (I(to)) plays an important electrophysiological role in normal and diseased hearts. However, its contribution to ventricular repolarization remains controversial because of differences in its phenotypic expression and function across species. The dog, a frequently used model of human cardiac disease, exhibits altered functional expression of I(to). To better understand the relevance of electrical remodeling in dogs to humans, we studied the phenotypic differences in ventricular I(to) of both species with electrophysiological, pharmacological, and protein-chemical techniques. Several notable distinctions were elucidated, including slower current decay, more rapid recovery from inactivation, and a depolarizing shift of steady-state inactivation in human vs. canine I(to). Whereas recovery from inactivation of human I(to) followed a monoexponential time course, canine I(to) recovered with biexponential kinetics. Pharmacological sensitivity to flecainide was markedly greater in human than canine I(to), and exposure to oxidative stress did not alter the inactivation kinetics of I(to) in either species. Western blot analysis revealed immunoreactive bands specific for Kv4.3, Kv1.4, and Kv channel-interacting protein (KChIP)2 in dog and human, but with notable differences in band sizes across species. We report for the first time major variations in phenotypic properties of human and canine ventricular I(to) despite the presence of the same subunit proteins in both species. These data suggest that differences in electrophysiological and pharmacological properties of I(to) between humans and dogs are not caused by differential expression of the K channel subunit genes thought to encode I(to), but rather may arise from differences in molecular structure and/or posttranslational modification of these subunits.     

Multistep ion channel remodeling and lethal arrhythmia precede heart failure in a mouse model of inherited dilated cardiomyopathy

Background

Patients with inherited dilated cardiomyopathy (DCM) frequently die with severe heart failure (HF) or die suddenly with arrhythmias, although these symptoms are not always observed at birth. It remains unclear how and when HF and arrhythmogenic changes develop in these DCM mutation carriers. In order to address this issue, properties of the myocardium and underlying gene expressions were studied using a knock-in mouse model of human inherited DCM caused by a deletion mutation ΔK210 in cardiac troponinT.

Methodology/Principal Findings

By 1 month, DCM mice had already enlarged hearts, but showed no symptoms of HF and a much lower mortality than at 2 months or later. At around 2 months, some would die suddenly with no clear symptoms of HF, whereas at 3 months, many of the survivors showed evident symptoms of HF. In isolated left ventricular myocardium (LV) from 2 month-mice, spontaneous activity frequently occurred and action potential duration (APD) was prolonged. Transient outward (I_to) and ultrarapid delayed rectifier K⁺ (I_Kur) currents were significantly reduced in DCM myocytes. Correspondingly, down-regulation of Kv4.2, Kv1.5 and KChIP2 was evident in mRNA and protein levels. In LVs at 3-months, more frequent spontaneous activity, greater prolongation of APD and further down-regulation in above K⁺ channels were observed. At 1 month, in contrast, infrequent spontaneous activity and down-regulation of Kv4.2, but not Kv1.5 or KChIP2, were observed.

Conclusions/Significance

Our results suggest that at least three steps of electrical remodeling occur in the hearts of DCM model mice, and that the combined down-regulation of Kv4.2, Kv1.5 and KChIP2 prior to the onset of HF may play an important role in the premature sudden death in this DCM model. DCM mice at 1 month or before, on the contrary, are associated with low risk of death in spite of inborn disorder and enlarged heart.