Molecular and functional remodeling of Ito by angiotensin II in the mouse left ventricle

The transient outward potassium current (I_to) in cardiac myocytes is mainly mediated by members of the Kv4 subfamily of voltage-gated potassium channels. Several in vitro studies have shown that angiotensin II (Ang II), which plays an important role in the development of cardiac hypertrophy, rapidly downregulates Kv4.3 mRNA expression. However, it is not clear whether Ang II regulates I_toin vivo and whether this regulation may depend on alterations in Kv4.3 gene expression. To address this question, we determined the effects of acute (24 h) and chronic (14 days) exogenous infusions of Ang II on I_to and the expression of its channel subunits in the mouse left ventricle. Ang II rapidly increased blood pressure and reduced Kv4.2 but not Kv4.3 mRNA levels in the absence of cardiac hypertrophy. In response to chronically elevated Ang II levels cardiac hypertrophy developed, which was associated with a downregulation of Kv4.2 and Kv4.3 mRNA levels, and an upregulation of Kv1.4 mRNA levels. In contrast, neither KChIP2 mRNA levels nor amplitude or macroscopic inactivation kinetics of I_to were affected by the acute or chronic Ang II treatments. Consistent with the unchanged I_to amplitude, Kv4.2, Kv4.3, and KChIP protein expression levels were similar after chronic Ang II and sham treatment. Our findings demonstrate that elevations of Ang II concentrations that induce hypertension and cardiac hypertrophy do not alter the amplitude of I_to in the mouse left ventricle. Furthermore, they suggest that functional expression of cardiac I_to in mice is stabilized by KChIP2.     

Celecoxib blocks cardiac Kv1.5, Kv4.3 and Kv7.1 (KCNQ1) channels: Effects on cardiac action potentials

Celecoxib is a COX-2 inhibitor that has been related to an increased cardiovascular risk and that exerts several actions on different targets. The aim of this study was to analyze the effects of this drug on human cardiac voltage-gated potassium channels (Kv) involved on cardiac repolarization Kv1.5 (I_Kur), Kv4.3 + KChIP2 (I_to1) and Kv7.1 + KCNE1 (I_Ks) and to compare with another COX-2 inhibitor, rofecoxib. Currents were recorded in transfected mammalian cells by whole-cell patch-clamp. Celecoxib blocked all the Kv channels analyzed and rofecoxib was always less potent, except on Kv4.3 + KChIP2 channels. Kv1.5 block increased in the voltage range of channel activation, decreasing at potentials positive to 0 mV. The drug modified the activation curve of the channels that became biphasic. Block was frequency-dependent, increasing at fastest frequencies. Celecoxib effects were not altered by TEA_out in R487Y mutant Kv1.5 channels but the kinetics of block were slower and the degree of block was smaller with TEA_in, indicating that celecoxib acts from the cytosolic side. We confirmed the blocking properties of celecoxib on native Kv currents from rat vascular cells, where Kv1.5 are the main contributors (IC₅₀ ≈ 7 μM). Finally, we demonstrate that celecoxib prolongs the action potential duration in mouse cardiac myocytes and shortens it in guinea pig cardiac myocytes, suggesting that Kv block induced by celecoxib may be of clinical relevance.     

Role of heteromultimers in the generation of myocardial transient outward K+ currents

Previous studies have demonstrated a role for Kv4 alpha subunits in the generation of the fast transient outward K+ current, I(to,f), in the mammalian myocardium. The experiments here were undertaken to explore the role of homomeric/heteromeric assembly of Kv4.2 and Kv4.3 and of the Kv channel accessory subunit, KChIP2, in the generation of mouse ventricular I(to,f). Western blots reveal that the expression of Kv4.2 parallels the regional heterogeneity in I(to,f) density, whereas Kv4.3 and KChIP2 are uniformly expressed in adult mouse ventricles. Antisense oligodeoxynucleotides (AsODNs) targeted against Kv4.2 or Kv4.3 selectively attenuate I(to,f) in mouse ventricular cells. Adenoviral-mediated coexpression of Kv4.2 and Kv4.3 in HEK-293 cells and in mouse ventricular myocytes produces transient outward K+ currents with properties distinct from those produced on expression of Kv4.2 or Kv4.3 alone, and the gating properties of the heteromeric Kv4.2/Kv4.3 channels in ventricular cells are more similar to native I(to,f) than are the homomeric Kv4.2 or Kv4.3 channels. Biochemical studies reveal that Kv4.2, Kv4.3, and KChIP2 coimmunoprecipitate from adult mouse ventricles. In addition, most of the Kv4.2 and KChIP2 are associated with Kv4.3 in situ. Taken together, these results demonstrate that functional mouse ventricular I(to,f) channels are heteromeric, comprising Kv4.2/Kv4.3 alpha subunits and KChIP2. The results here also suggest that Kv4.2 is the primary determinant of the regional heterogeneity in I(to,f) expression in adult mouse ventricle.     

Expression of voltage-gated K<Superscript>+</Superscript> channels in human atrium

Voltage-gated K⁺ channels underlie repolarisation of the cardiac action potential and represent a potential therapeutic target in the treatment of cardiac dysrhythmias. However, very little is known about the relative expression of K⁺ channel subunits in the human myocardium. We used a semi-quantitative RT-PCR technique to examine the relative expression of mRNAs for the voltage-gated K⁺ channel subunits, Kv1.2, Kv1.4, Kv1.5, Kv2.1, Kv4.2, Kv4.3, KvLQT1, HERG and IsK in samples of human atrial appendage. Data were expressed as a percentage expression density relative to an 18S ribosomal RNA internal standard. The most abundant K⁺ channel mRNAs were Kv4.3 (80.7 ± 10.1 %), Kv1.5 (69.7 ± 11.2 %) and HERG (55.9 ± 21.5 %). Significant expression of KvLQT1 (33.5 ± 5.5 %,) and Kv1.4 (26.7 ± 9.6 %) was also detected. Levels of mRNAs for Kv1.2 and IsK were very low and neither Kv2.1 nor Kv4.2 mRNA were detected in any experiments. Whole-cell patch-clamp techniques were used to examine the outward currents of isolated human atrial myocytes at 37 °C. These recordings demonstrated the existence of transient (I_to1) and sustained (I_so) outward currents in isolated human atrial myocytes. I_to1, and not I_so, showed voltage-dependent inactivation during 100 ms pre-pulses. Both I_to1 and I_so were inhibited by high concentrations (2 mM) of the K⁺ channel blocker, 4-aminopyridine (4-AP). However, lower concentrations of 4-AP (10 μM) inhibited I_so selectively. I_to1 recovered from inactivation relatively rapidly (t ∼21 ms). These data, with published information regarding the properties of expressed K⁺ channels, suggest that Kv4.3 represents the predominant K⁺ channel subunit underlying I_to1 with little contribution of Kv1.4. The sensitivity of Iso to very low concentrations of 4-aminopyridine and the relatively low expression of mRNA for Kv1.2 and Kv2.1 is consistent with the major contribution of Kv1.5 to this current. The physiological significance of the expression of KvLQT1 and Kv1.4 mRNA in the human atrium warrants further investigation. Received: 30 August 2000, Returned for 1. revision: 21 September 2000, 1. Revision received: 21 June 2002, Returned for 2. revision: 15 July 2002, 2. Revision received: 30 July 2002, Accepted: 31 July 2002 Correspondence to: Dr. A. F. James     

Endocannabinoids and cannabinoid analogues block human cardiac Kv4.3 channels in a receptor-independent manner

Endocannabinoids are amides and esters of long chain fatty acids that can modulate ion channels through both receptor-dependent and receptor-independent effects. Nowadays, their effects on cardiac K⁺ channels are unknown even when they can be synthesized within the heart. We have analyzed the direct effects of endocannabinoids, such as anandamide (AEA), 2-arachidonoylglycerol (2-AG), the endogenous lipid lysophosphatidylinositol, and cannabinoid analogues such as palmitoylethanolamide (PEA), and oleoylethanolamide, as well as the fatty acids from which they are endogenously synthesized, on human cardiac Kv4.3 channels, which generate the transient outward K⁺ current (I_to1). Currents were recorded in Chinese hamster ovary cells, which do not express cannabinoid receptors, by using the whole-cell patch-clamp. All these compounds inhibited I_Kv4.3 in a concentration-dependent manner, AEA and 2-AG being the most potent (IC₅₀ ∼ 0.3-0.4 µM), while PEA was the least potent. The potency of block increased as the complexity and the number of C atoms in the fatty acyl chain increased. The effects were not mediated by modifications in the lipid order and microviscosity of the membrane and were independent of the presence of MiRP2 or DPP6 subunits in the channel complex. Indeed, effects produced by AEA were reproduced in human atrial I_to1 recorded in isolated myocytes. Moreover, AEA effects were exclusively apparent when it was applied to the external surface of the cell membrane. These results indicate that at low micromolar concentrations the endocannabinoids AEA and 2-AG directly block human cardiac Kv4.3 channels, which represent a novel molecular target for these compounds.     

Targeted deletion of Kv4.2 eliminates I(to,f) and results in electrical and molecular remodeling, with no evidence of ventricular hypertrophy or myocardial dysfunction

Previous studies have demonstrated a role for voltage-gated K+ (Kv) channel alpha subunits of the Kv4 subfamily in the generation of rapidly inactivating/recovering cardiac transient outward K+ current, I(to,f), channels. Biochemical studies suggest that mouse ventricular I(to,f) channels reflect the heteromeric assembly of Kv4.2 and Kv4.3 with the accessory subunits, KChIP2 and Kvbeta1, and that Kv4.2 is the primary determinant of regional differences in (mouse ventricular) I(to,f) densities. Interestingly, the phenotypic consequences of manipulating I(to,f) expression in different mouse models are distinct. In the experiments here, the effects of the targeted deletion of Kv4.2 (Kv4.2(-/-)) were examined. Unexpectedly, voltage-clamp recordings from Kv4.2(-/-) ventricular myocytes revealed that I(to,f) is eliminated. In addition, the slow transient outward K+ current, I(to,s), and the Kv1.4 protein (which encodes I(to,s)) are upregulated in Kv4.2(-/-) ventricles. Although Kv4.3 mRNA/protein expression is not measurably affected, KChIP2 expression is markedly reduced in Kv4.2(-/-) ventricles. Similar to Kv4.3, expression of Kvbeta1, as well as Kv1.5 and Kv2.1, is similar in wild-type and Kv4.2(-/-) ventricles. In addition, and in marked contrast to previous findings in mice expressing a truncated Kv4.2 transgene, the elimination I(to,f) in Kv4.2(-/-) mice does not result in ventricular hypertrophy. Taken together, these findings demonstrate not only an essential role for Kv4.2 in the generation of mouse ventricular I(to,f) channels but also that the loss of I(to,f) per se does not have overt pathophysiological consequences.     

Unloaded rat hearts in vivo express a hypertrophic phenotype of cardiac repolarization

Cardiac unloading with left ventricular assist devices is increasingly used to treat patients with severe heart failure. Unloading has been shown to improve systolic and diastolic function, but its impact on the repolarization of left ventricular myocytes is not known. Unloaded hearts exhibit similar patterns of gene expression as hearts subjected to an increased hemodynamic load. We therefore hypothesized that cardiac unloading also replicates the alterations in action potential and underlying repolarizing ionic currents found in pressure-overload induced cardiac hypertrophy. Left ventricular unloading was induced by heterotopic heart transplantation in syngenic male Lewis rats. Action potentials and underlying K⁺ and Ca²⁺ currents were investigated using whole-cell patch-clamp technique. Real-time RT-PCR was used to quantify mRNA expression of Kv4.2, Kv4.3, and KChIP2. Unloading markedly prolonged cardiac action potentials and suppressed the amplitude of several repolarizing K⁺ currents, in particular of the transient outward K⁺ current I_to, in both, epicardial and endocardial myocytes. The reduction of I_to was associated with significantly lower levels of Kv4.2 and Kv4.3 mRNAs in epicardial myocytes, and of KChIP2 mRNA in endocardial myocytes. Concomitantly, the L-type Ca²⁺ current was increased in myocytes of unloaded hearts. Collectively, these results show that left ventricular unloading induces a profound remodelling of cardiac repolarization with action potential prolongation, downregulation of repolarizing K⁺ currents and upregulation of the L-type Ca²⁺ current. This indicates that unloaded rat hearts in vivo express a hypertrophic phenotype of cardiac repolarization at the cellular and the molecular level.     

High-mobility group box 1 (HMGB1) downregulates cardiac transient outward potassium current (Ito) through downregulation of Kv4.2 and Kv4.3 channel transcripts and proteins

Transient outward potassium currents (I_to) are major early repolarization currents in shaping cardiac action potential (AP). Downregulation of I_to contributes to AP configuration alteration in myocardial infarction (MI) and numerous other heart diseases. High-mobility group box 1 (HMGB1), a proinflammatory cytokine, has been reported to increase dramatically in the serum of patients with MI, participating in ischemia-reperfusion injury and recovery of post-infarction failing heart. This study investigated the possible role of HMGB1 in regulating cardiac I_to and electrical stability. HMGB1 treatment for 24 h significantly inhibited the current densities of heterologously expressed Kv4.3 and Kv4.2 in COS-7 cells and native I_to in neonatal rat ventricular myocytes (NRVMs) in a dose-dependent manner. HMGB1 decreased the mRNA and protein levels of the I_to α subunits Kv4.2 and Kv4.3 channels, but not the β subunit KChIP2 and KCNE2 in NRVMs. The receptor binding domain (150-186 amino acid residues) responsible for receptor of advanced glycation end product (RAGE) binding similarly inhibited I_to, while treatment with soluble RAGE that blocks binding of ligands to cell-surface RAGE partially restored I_to current density and Kv4 protein expressions. Box A which possesses no proinflammatory activity of HMGB1 still remained part of the I_to suppression effect. In addition to downregulating I_to, HMGB1 modestly inhibited L-type Ca²⁺ current, but not I_K1. The AP duration (APD) was slightly prolonged by HMGB1 treatment. These results collectively establish HMGB1 as a novel pathological factor downregulating I_to partially through HMGB1-RAGE interaction, providing new insights into the potential molecular mechanisms underlying the electrical remodeling in MI.     

Molecular determinants of cardiac transient outward potassium current (Ito) expression and regulation

Rapidly activating and inactivating cardiac transient outward K⁺ currents, I_to, are expressed in most mammalian cardiomyocytes, and contribute importantly to the early phase of action potential repolarization and to plateau potentials. The rapidly recovering (I_to,f) and slowly recovering (I_to,s) components are differentially expressed in the myocardium, contributing to regional heterogeneities in action potential waveforms. Consistent with the marked differences in biophysical properties, distinct pore-forming (α) subunits underlie the two I_to components: Kv4.3/Kv4.2 subunits encode I_to,f, whereas Kv1.4 encodes I_to,s, channels. It has also become increasingly clear that cardiac I_to channels function as components of macromolecular protein complexes, comprising (four) Kvα subunits and a variety of accessory subunits and regulatory proteins that influence channel expression, biophysical properties and interactions with the actin cytoskeleton, and contribute to the generation of normal cardiac rhythms. Derangements in the expression or the regulation of I_to channels in inherited or acquired cardiac diseases would be expected to increase the risk of potentially life-threatening cardiac arrhythmias. Indeed, a recently identified Brugada syndrome mutation in KCNE3 (MiRP2) has been suggested to result in increased I_to,f densities. Continued focus in this area seems certain to provide new and fundamentally important insights into the molecular determinants of functional I_to channels and into the molecular mechanisms involved in the dynamic regulation of I_to channel functioning in the normal and diseased myocardium.     

Chronic treatment with anabolic steroids induces ventricular repolarization disturbances: Cellular, ionic and molecular mechanism

The illicit use of supraphysiological doses of androgenic steroids (AAS) has been suggested as a cause of arrhythmia in athletes. The objectives of the present study were to investigate the time-course and the cellular, ionic and molecular processes underlying ventricular repolarization in rats chronically treated with AAS. Male Wistar rats were treated weekly for 8 weeks with 10 mg/kg of nandrolone decanoate (DECA n = 21) or vehicle (control n = 20). ECG was recorded weekly. Action potential (AP) and transient outward potassium current (I_to) were recorded in rat hearts. Expression of KChIP2, Kv1.4, Kv4.2, and Kv4.3 was assessed by real-time PCR. Hematoxylin/eosin and Picrosirius red staining were used for histological analysis. QTc was greater in the DECA group. After DECA treatment the left, but not right, ventricle showed a longer AP duration than did the control. I_to current densities were 47.5% lower in the left but not in the right ventricle after DECA. In the right ventricle the I_to inactivation time-course was slower than in the control group. After DECA the left ventricle showed lower KChIP2 (∼ 26%), Kv1.4 (∼ 23%) and 4.3 (∼ 70%) expression while the Kv 4.2 increased in 4 (∼ 250%) and diminished in 3 (∼ 30%) animals of this group. In the right ventricle the expression of I_to subunits was similar between the treatment and control groups. DECA-treated hearts had 25% fewer nuclei and greater nuclei diameters in both ventricles. Our results strongly suggest that supraphysiological doses of AAS induce morphological remodeling in both ventricles. However, the electrical remodeling was mainly observed in the left ventricle.     

Effects of atorvastatin and simvastatin on atrial plateau currents

Recent evidence has shown that the inhibitors of the 3-hydroxy-3-methylglutaryl coenzyme A reductase (statins) might exert antiarrhythmic effects both in experimental models and in humans. In this study we analyzed the effects of atorvastatin and simvastatin acid (SVA) on the currents responsible for the duration of the plateau of human atrial action potentials: hKv1.5, Kv4.3, and L-type Ca(2+) (I(Ca,L)). hKv1.5 and Kv4.3 currents were recorded in transfected Ltk(-) and Chinese hamster ovary cells, respectively, and I(Ca,L) in mouse ventricular myocytes, using whole-cell patch-clamp. Atorvastatin and SVA produced a concentration-dependent block of hKv1.5 channels (IC(50)=4.5+/-1.7 microM and 5.7+/-0.03 microM, respectively) and shifted the midpoint of the activation and inactivation curves to more negative potentials. Importantly, atorvastatin- and SVA-induced block was added to that produced by quinidine, a drug that blocks hKv1.5 channels by binding to their pore cavity. Atorvastatin and SVA blocked Kv4.3 channels in a concentration-dependent manner (IC(50)=13.9+/-3.6 nM and 7.0+/-0.8 microM, respectively). Both drugs accelerated the inactivation kinetics and shifted the inactivation curve to more negative potentials. SVA (10 nM), but not atorvastatin, also blocked I(Ca,L) producing a frequency-dependent block that, at 2 Hz, reached a 50.2+/-1.5%. As a consequence of these effects, at nanomolar concentrations, atorvastatin lengthened, whereas SVA shortened, the duration of mouse atrial action potentials. The results suggest that atorvastatin and SVA alter Kv1.5 and Kv4.3 channel activity following a complex mechanism that does not imply the binding of the drug to the channel pore.     

Molecular composition and functional properties of f-channels in murine embryonic stem cell-derived pacemaker cells

Mouse embryonic stem cells (mESCs) differentiate into all cardiac phenotypes, and thus represent an important potential source for cardiac regenerative therapies. Here we characterize the molecular composition and functional properties of “funny” (f-) channels in mESC-derived pacemaker cells. Following differentiation, a fraction of mESC-derived myocytes exhibited action potentials characterized by a slow diastolic depolarization and expressed the I_f current. I_f plays an important role in the pacemaking mechanism of these cells since ivabradine (3 μM), a specific f-channel inhibitor, inhibited I_f by about 50% and slowed rate by about 25%. Analysis of I_f kinetics revealed the presence of two populations of cells, one expressing a fast- and one a slow-activating I_f; the two components are present both at early and late stages of differentiation and had also distinct activation curves. Immunofluorescence analysis revealed that HCN1 and HCN4 are the only isoforms of the pacemaker channel expressed in these cells. Rhythmic cells responded to β-adrenergic and muscarinic agonists: isoproterenol (1 μM) accelerated and acetylcholine (0.1 μM) slowed spontaneous rate by about 50 and 12%, respectively. The same agonists caused quantitatively different effects on I_f: isoproterenol shifted activation curves by about 5.9 and 2.7 mV and acetylcholine by − 4.0 and − 2.0 mV in slow and fast I_f-activating cells, respectively. Accordingly, β1- and β2-adrenergic, and M2-muscarinic receptors were detected in mESC-derived myocytes. Our data show that mESC-derived pacemaker cells functionally express proteins which underlie generation and modulation of heart rhythm, and can therefore represent a potential cell substrate for the generation of biological pacemakers.     

KChIP2 attenuates cardiac hypertrophy through regulation of Ito and intracellular calcium signaling

Recent evidence shows that the auxiliary subunit KChIP2, which assembles with pore-forming Kv4-subunits, represents a new potential regulator of the cardiac calcium-independent transient outward potassium current (I_to) density. In hypertrophy and heart failure, KChIP2 expression has been found to be significantly decreased. Our aim was to examine the role of KChIP2 in cardiac hypertrophy and the effect of restoring its expression on electrical remodeling and cardiac mechanical function using a combination of molecular, biochemical and gene targeting approaches. KChIP2 overexpression through gene transfer of Ad.KChIP2 in neonatal cardiomyocytes resulted in a significant increase in I_to-channel forming Kv4.2 and Kv4.3 protein levels. In vivo gene transfer of KChIP2 in aortic banded adult rats showed that, compared to sham-operated or Ad.β-gal-transduced hearts, KChIP2 significantly attenuated the developed left ventricular hypertrophy, robustly increased I_to densities, shortened action potential duration, and significantly altered myocyte mechanics by shortening contraction amplitudes and maximal rates of contraction and relaxation velocities and decreasing Ca²⁺ transients. Interestingly, blocking I_to with 4-aminopyridine in KChIP2-overexpressing adult cardiomyocytes significantly increased the Ca²⁺ transients to control levels. One-day-old rat pups intracardially transduced with KChIP2 for two months then subjected to aortic banding for 6–8 weeks (to induce hypertrophy) showed similar echocardiographic, electrical and mechanical remodeling parameters. In addition, in cultured adult cardiomyocytes, KChIP2 overexpression increased the expression of Ca²⁺-ATPase (SERCA2a) and sodium calcium exchanger but had no effect on ryanodine receptor 2 or phospholamban expression. In neonatal myocytes, KChIP2 notably reversed Ang II-induced hypertrophic changes in protein synthesis and MAP-kinase activation. It also significantly decreased calcineurin expression, NFATc1 expression and nuclear translocation and its downstream target, MCiP1.4. Altogether, these data show that KChIP2 can attenuate cardiac hypertrophy possibly through modulation of intracellular calcium concentration and calcineurin/NFAT pathway.     

Specificities of atrial electrophysiology: Clues to a better understanding of cardiac function and the mechanisms of arrhythmias

The electrical properties of the atria and ventricles differ in several aspects reflecting the distinct role of the atria in cardiac physiology. The study of atrial electrophysiology had greatly contributed to the understanding of the mechanisms of atrial fibrillation (AF). Only the atrial L-type calcium current is regulated by serotonine or, under basal condition, by phosphodiesterases. These distinct regulations can contribute to I_Ca down-regulation observed during AF, which is an important determinant of action potential refractory period shortening. The voltage-gated potassium current, I_Kur, has a prominent role in the repolarization of the atrial but not ventricular AP. In many species, this current is based on the functional expression of K_V1.5 channels, which might represent a specific therapeutic target for AF. Mechanisms regulating the trafficking of K_V1.5 channels to the plasma membrane are being actively investigated. The resting potential of atrial myocytes is maintained by various inward rectifier currents which differ with ventricle currents by a reduced density of I_K1, the presence of a constitutively active I_KACh and distinct regulation of I_KATP. Stretch-sensitive or mechanosensitive ion channels are particularly active in atrial myocytes and are involved in the secretion of the natriuretic peptide. Integration of knowledge on electrical properties of atrial myocytes in comprehensive schemas is now necessary for a better understanding of the physiology of atria and the mechanisms of AF.     

Functional role of CLC-2 chloride inward rectifier channels in cardiac sinoatrial nodal pacemaker cells

A novel Cl⁻ inward rectifier channel (Cl,ir) encoded by ClC-2, a member of the ClC voltage-gated Cl⁻ channel gene superfamily, has been recently discovered in cardiac myocytes of several species. However, the physiological role of Cl,ir channels in the heart remains unknown. In this study we tested the hypothesis that Cl,ir channels may play an important role in cardiac pacemaker activity. In isolated guinea-pig sinoatrial node (SAN) cells, Cl,ir current was activated by hyperpolarization and hypotonic cell swelling. RT-PCR and immunohistological analyses confirmed the molecular expression of ClC-2 in guinea-pig SAN cells. Hypotonic stress increased the diastolic depolarization slope and decreased the maximum diastolic potential, action potential amplitude, APD₅₀, APD₉₀, and the cycle-length of the SAN cells. These effects were largely reversed by intracellular dialysis of anti-ClC-2 antibody, which significantly inhibited Cl,ir current but not other pacemaker currents, including the hyperpolarization-activated non-selective cationic “funny” current (I_f), the L-type Ca²⁺ currents (I_Ca,L), the slowly-activating delayed rectifier I_Ks and the volume-regulated outwardly-rectifying Cl⁻ current (I_Cl,vol). Telemetry electrocardiograph studies in conscious ClC-2 knockout (Clcn2^−/−) mice revealed a decreased chronotropic response to acute exercise stress when compared to their age-matched Clcn2^+/+ and Clcn2^+/− littermates. Targeted inactivation of ClC-2 does not alter intrinsic heart rate but prevented the positive chronotropic effect of acute exercise stress through a sympathetic regulation of ClC-2 channels. These results provide compelling evidence that ClC-2-encoded endogenous Cl,ir channels may play an important role in the regulation of cardiac pacemaker activity, which may become more prominent under stressed or pathological conditions.     

Contribution of KChIP2 to the developmental increase in transient outward current of rat cardiomyocytes

The Ca(2+)-independent, voltage-gated transient outward current (I(to)) displays a marked increase during development of cardiomyocytes. However, the molecular mechanism remained unclear. In rat adult ventricular myocytes, I(to) can be divided into a fast (I(to,f)) and a slow (I(to,s)) component by recovery process from inactivation. Voltage-gated K(+) channel-interacting proteins 2 (KChIP2) has recently been shown to modify membrane expressions and current densities of I(to,f). Here we examined the developmental change of I(to) and the putative molecular correlates of I(to,f) (Kv4.2 and Kv4.3) and KChIP2 in rat ventricular myocytes. Even in rat embryonic day 12 (E12) myocytes, we detected I(to). However, I(to) in E12 was solely composed of I(to,s). In postnatal day 10 (P10), we recorded much increased I(to) composed of two components (I(to,f) and I(to,s)), and I(to,f) was dominant. Thus, the developmental increase of I(to) from E12 to P10 can be explained by the dramatic appearance of I(to,f). Real-time RT-PCR revealed that Kv4.2 and Kv4.3 mRNA levels were slightly changed. By contrast, KChIP2 mRNA level increased from E12 to P10 by 731-fold. Therefore, the huge increase of KChIP2 expression was likely to be the cause of the great increase of I(to,f). In order to confirm that KChIP2 is crucial to induce I(to,f), we used adenoviral gene transfer technique. When KChIP2 was over-expressed in E12 myocytes, a great amplitude of I(to,f) appeared. Immunocytochemical experiments also demonstrated that KChIP2 enhanced the trafficking of Kv4.2 channels to cell surface. These results indicate that KChIP2 plays an important role in the generation of functional I(to,f) channels during development.