Circulating KCNH2 Current-Activating Factor in Patients with Heart Failure and Ventricular Tachyarrhythmia

Background It is estimated that approximately half of the deaths in patients with HF are sudden and that the most likely causes of sudden death are lethal ventricular tachyarrhythmias such as ventricular tachycardia (VT) or fibrillation (VF). However, the precise mechanism of ventricular tachyarrhythmias remains unknown. The KCNH2 channel conducting the delayed rectifier K+ current (IKr) is recognized as the most susceptible channel in acquired long QT syndrome. Recent findings have revealed that not only suppression but also enhancement of IKr increase vulnerability to major arrhythmic events, as seen in short QT syndrome. Therefore, we investigated the existence of a circulating KCNH2 current-modifying factor in patients with HF. Methodology/Principal Findings We examined the effects of serum of HF patients on recombinant IKr recorded from HEK 293 cells stably expressing KCNH2 by using the whole-cell patch-clamp technique. Study subjects were 14 patients with non-ischemic HF and 6 normal controls. Seven patients had a history of documented ventricular tachyarrhythmias (VT: 7 and VF: 1). Overnight treatment with 2% serum obtained from HF patients with ventricular arrhythmia resulted in a significant enhancement in the peaks of IKr tail currents compared to the serum from normal controls and HF patients without ventricular arrhythmia. Conclusions/Significance Here we provide the first evidence for the presence of a circulating KCNH2 channel activator in patients with HF and ventricular tachyarrhythmias. This factor may be responsible for arhythmogenesis in patients with HF.


Introduction
Heart failure (HF) remains a major clinical problem all over the world. [1] It is estimated that approximately half of the deaths in these patients is sudden and that the most likely causes of sudden cardiac death (SCD) are lethal cardiac arrhythmia such as ventricular tachycardia (VT) or fibrillation (VF). [2] It has been known for a long time that the failing heart undergoes a complex electrical remodeling of ventricular myocytes and that a consequent reduction of repolarization reserve and electrical instability may predispose to an increased risk of life-threatening arrhythmia. [3,4] KCNH2, also called human ether-a-go-go-related gene (hERG) potassium channel, is responsible for the rapid components of delayed rectifier potassium currents (I Kr ). I Kr is a major contributor to the repolarization process of cardiac action potentials and is recognized as the most susceptible channel in acquired long QT syndrome. [5] Recent findings have revealed that not only suppression but also enhancement of I Kr increase vulnerability to major arrhythmic events. A gain-of-function mutation in KCNH2 has also gained recognition as a congenital disorder characterized by a higher risk for major arrhythmic events and SCD, referred to as short QT syndrome (SQT). [6,7] It has also been reported that PD-118057, an I Kr -activating agent, predisposes to cardiac arrhythmias in vitro. [8] HF also induces remodeling of I Kr , but there is no widespread consensus on suppression or enhancement of I Kr in previous studies. [9,10] Furthermore, despite the incremental progress in understanding intrinsic I Kr modulators, to our knowledge, there has been no report of naturally occurring substances with a KCNH2 current-activating effect in patients with HF. Therefore, we investigated the existence of a circulating KCNH2 current-activating factor in patients with HF.
More than half of the patients in the VT/VF (+) group took cardiotonic agents, whereas none of the patients in the VT/VF (2) group took cardiotonic agents (Table 2). However, none of the drugs, including cardiotonic agents, taken by the subjects have been reported to activate KCNH2 current.

Enhancement of KCNH2 currents by serum from HF patients with ventricular tachyarrhythmia
We investigated the effect of 2% serum of the study subjects on recombinant I Kr recorded from HEK293 cells stably expressing KCNH2. KCNH2 tail currents were similar in the control and VT/VF (2) group but were significantly increased in the VT/VF (+) group (P,0.05: VT/VF (+) vs. control and VT/VF (2)) ( Figure 1). There was no significant effect on voltage dependency of KCNH2 activation in these groups (Table 3). There was no relationship between peak tail amplitude and EF in the overall HF patients ( Figure 2).
These results indicate the presence of a circulating KCNH2 channel activator in patients with HF and ventricular tachyarrhythmias.

Efficacy of class III antiarrhythmic agents
Case No.1 was treated with amiodarone, and Case No.6 was treated with d-sotalol. Both drugs, which exert their antiarrhythmic actions essentially by blocking I Kr ,were effective in controlling VT and VF.

Discussion
The major new finding of this work is that the factor causing activation of the KCNH2 current is present in serum of patients with HF accompanied by cardiac arrhythmia. Although there are several reports on suppression of I Kr by circulating hormones or auto-antibodies, this is the first report providing evidence for a naturally occurring I Kr activator. [5,11,12] I Kr conducted by KCNH2 channels is the major repolarizing outward current of ventricular action potential, and it has been shown to have a strong association with life-threatening arrhythmia in many pathological circumstances. Our finding indicates that enhancement of I Kr can play a key role in arrhythmogenesis in the setting of HF.
The majority of research efforts have focused classically on reduction of I Kr as an approach for understanding arrhtymogenesis because it is well recognized that reduction of I kr means decrease in net repolarizing current that results in prolongation of action potential duration (APD) and QT interval in the ECG and development of early afterdepolarization-induced triggered activity following torsade de pointes (TdP). [13] On the other hand, much attention has been paid to upregulation of KCNH2 in recent years because of its causal relationship to cardiac arrhythmias. SQT is a recently recognized clinical concept characterized by short QT intervals in the ECG, and it is associated with major cardiac events leading to SCD without organic heart disease. [7] The first identified form of SQT resulted from gain-of-function mutation in KCNH2. Mutation N588K in KCNH2 causes an increase in net repolarizing current. [6] In addition, recent studies have revealed a significant association between early repolarization and SQT in clinical phenotype. [14,15] Because of the high prevalence of early repolarization detected in SQT, it is thought that enhancement of repolarization is a common mechanism underlying arryhthmogenicity in early repolarization and SQT. [14] Since QT interval is affected by other ion channels and cardiac enlargement, QT interval of HF patients with ventricular tachyarrhythmia in this study was not short. However, the circulating KCNH2 channel activator in this study is potentially responsible for electrical instability. Effects of KCNH2-activating agents have also been evaluated. It has been reported that PD-118057, established as a selective KCNH2 current enhancer without affecting activation, confers inducibility of both ventricular tachyarrhythmia and atrial fibrillation. [8,16] The functional similarity between this agent and circulating KCNH2-activating factor in having no significant effect on the voltage dependence of activation suggests a causal relationship of this intrinsic enhancer with arrhythmogenesis in HF. In addition, this concept is endorsed by the efficacy of class III antiarrhythmic agents for treatment of ventricular tachyarrhythmia in the study subjects. Since the dominant action of class III antiarrhythmic agents is to lengthen the APD by a reduction of I Kr , it is thought that the therapeutic effect of the agents against arrhythmia in the study subjects is manifested by offsetting oversupplied KCNH2 currents.
On the other hand, there are only a few reports on the antiarrhythmic potential of an I Kr activator in specialized pathologic settings. PD-118057 prevented the early after-depolarization induced by class III antiarrhythmic agents, and NS3623 decreased the frequency of bradycardia-induced extrasystoles in vitro. [17,18] However, these data were obtained only in constrained experimental conditions and couldn't refer clinical benefit for arrhythmogenic disorders such as long QT syndrome.
The mechanism by which exposure of ion channels to this factor activates the KCNH2 current remains to be elucidated. It is also uncertain whether the KCNH2-activating effect is the cause or consequence of HF-induced remodeling of action potential. However, exertion of their functional effects may be involved in arrhythmogenesis in HF. Further studies are needed to clarify these points.
In conclusion, our data show that a novel KCNH2 activator exists in serum of HF patients with ventricular tachyarrhythmia and is potentially responsible for electrical instability.

Methods
All of the studies were approved by the Ethics Committee of Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, and written informed consent was obtained from all patients before the procedure. The investigation also conforms to the principles outlined in the Declaration of Helsinki.

Study population
Serum samples were obtained from 14 HF patients referred to Okayama University Hospital, Okayama, Japan between December 2007 and December 2008 who consented to undergo evaluation of the pathogenesis of HF. HF was defined as the presence of current or previous symptoms of exercise intolerance and EF of 50% and less with no other cause of exercise intolerance. Exclusion criteria included coronary heart disease, primary valvular heart disease, severe systemic disease, or severe pulmonary disease. The mean age of the patients was 52610 years, and 8 patients (57%) were male. Control sera were obtained from 6 subjects without cardiovascular disease or abnormal ECG findings. Mean age of the control subjects was 43615 years, and 4 subjects (67%) were male.

Patch-clamp recordings
KCNH2 channel currents were recorded at room temperature (2261uC) by using the whole-cell patch-clamp technique as previously described. [11] The control bath solution contained (mM): 132 NaCl, 4.8 KCl, 1.2 MgCl 2 , 2 CaCl 2 , 5 glucose, 10 Hepes, pH 7.4. Pipettes (2-4 MV resistances) were filled with a pipette solution containing (mM): 110 potassium aspartate, 5 K2-ATP, 11 EGTA, 5 Hepes, 1 CaCl 2 , and 1 MgCl 2 , pH 7.3. Currents were normalized to cell capacitance to give the measure of current density. There was no difference in membrane capacitance between the groups (14.162.0 pF, 14.262.8 pF, and 14.761.6 pF for the control, VT/VF (2) and VT/VF (+) groups, respectively; P = 0.88). KCNH2 channel tail-current amplitude was monitored at 0.1 Hz by analysis of peak deactivating tail current recorded at 240 mV after 2-s depolarizing test pulses to +20 mV from a holding potential (V H ) of 280 mV. Current zero level (no activation) was determined by applying 25-ms pulses to 240 mV preceding the test pulses. HEK293 cells stably expressing KCNH2 were cultured for 1 day   in a medium to which 2% serum obtained from the study subjects had been added. The KCNH2 current activation was studied by analysis of deactivating tail currents recorded at 240 mV after a series of 2-s test pulses from 250 mV to +70 mV (10 mV increments; V H , 280 mV). Pulse frequency was 0.1 Hz. Peaks of tail currents were plotted as function of the KCNH2 activation. Activation curves were analyzed with a fit of each data to the Boltzmann equation, I/I max = G/G max = {1+exp[2(V m 2V 1/2 )/ k]} 21 . G/G max is normalized chord conductance at V m to the maximum chord conductance. V 1/2 is the potential where the conductance is half-maximally activated, and k is the slope factor.

ECG measurements
Twelve-lead ECGs were recorded to measure the parameters of repolarization. The QT interval was measured from the start of the QRS complex to the end of the T wave, defined as the return to the iso-electric baseline. They were corrected to heart rate using Bazett's formula: QTc (QT/!RR).

Data Analysis
All values are presented as means6.D. Statistical significance was assessed with ANOVA followed by post hoc Scheffe's method. A value of P,.05 was considered statistically significant. pCLAMP 9.2 software (Axon Instruments) was used to both acquire and analyze data for the patch-clamp experiments. Graphical analyses were carried out using Origin 7.0 J software (Microcal). Statistical analysis was performed with StatView 5.0 (Abacus Concepts, Inc., Berkeley, CA, U.S.A.).