Increased Response to β2-Adrenoreceptor Stimulation Augments Inhibition of IKr in Heart Failure Ventricular Myocytes

Background Increasing evidence indicates that the rapid component of delayed rectifier potassium current (IKr) is modulated by α- and β-adrenergic stimulation. However, the role and mechanism regulating IKr through β2-adrenoreceptor (β-AR) stimulation in heart failure (HF) are unclear. Methodology/Principal Findings In the present study, we investigated the effects of fenoterol, a highly selective β2-AR agonist, on IKr in left ventricular myocytes obtained from control and guinea pigs with HF induced by descending aortic banding. IKr was measured by using whole cell patch clamp technique. In control myocytes, superfusion of fenoterol (10 µM) caused a 17% decrease in IKr. In HF myocytes, the same concentration of fenoterol produced a significantly greater decrease (33%) in IKr. These effects were not modified by the incubation of myocytes with CGP-20712A, a β1-AR antagonist, but were abolished by pretreatment of myocytes with ICI-118551, a β2-AR antagonist. An inhibitory cAMP analog, Rp-cAMPS and PKA inhibitor significantly attenuated fenoterol-induced inhibition of IKr in HF myocytes. Moreover, fenoterol markedly prolonged action potential durations at 90% (APD90) repolarization in HF ventricular myocytes. Conclusions The results indicate that inhibition of IKr induced by β2-AR stimulation is increased in HF. The inhibitory effect is likely to be mediated through a cAMP/PKA pathway in HF ventricular myocytes.


Introduction
Heart failure (HF) is associated with significant mortality, with nearly 50% of deaths occurring suddenly, primarily from ventricular tachycardia to ventricular fibrillation [1]. Sympathetic nerve activity is increased in HF [2]. It is widely accepted that the cardiac response to catecholamines is mediated primarily by badrenoreceptors (b-ARs). Arrhythmogenesis in HF is enhanced by b-adrenergic stimulation [3].
The human ether-a-go-go-related gene (hERG or KCNH2) [4] encodes the a subunit of the channel underlying I Kr [5], which is crucial for the repolarization of cardiac action potentials (AP). I Kr is modulated by catecholamines. There is increasing evidence that hERG/I Kr channels are modulated by various G protein-coupled receptors including aand b-ARs, which act through the intracellular signaling modulators cAMP, protein kinase A (PKA), and protein kinase C (PKC) [6,7,8,9]. Stimulation of b-AR by 10 mM isoprenaline decreased I Kr tail currents in guinea pig ventricular myocytes [7]. Similarly, PKA-mediated phosphorylation of expressed hERG channels significantly decreased hERG currents [10]. However, recent studies have revealed that I Kr tail currents are enhanced by 100 nM isoprenaline in canine ventricular myocytes [11]. It is well known that b 1 -AR is downregulated and b 2 -AR is relatively preserved in HF. In addition, experimental studies have demonstrated that canine ventricular response to b 2 -agonists is increased in tachypacing failure [12]. The responsiveness of cardiac L-type calcium current to b 2 -AR stimulation is increased in rats with HF induced by ligation of the coronary artery [13]. However, the role and mechanism of b 2 -AR activation in I Kr in HF have not been previously assessed. Accordingly, the present study was designed to examine the role and possible mechanisms of b 2 -AR activation in I Kr in left ventricular (LV) myocytes from HF guinea pigs using the whole cell patch clamp technique.

Validity of the Guinea Pig Model
Following 12 weeks of thoracic aortic banding, the ejection fraction (EF) and fractional shortening (FS) of the heart of guinea pigs were significantly decreased; LV end-diastolic diameter, LV end-systolic diameter, the QT interval, and corrected QT interval were significantly increased while there were no changes in heart rate (Table 1). Cell capacitance of HF LV myocytes (n = 35 cells; 20 animals) and control LV myocytes (n = 35 cells; 16 animals) was 16367 and 12163 pF, respectively (P,0.001). Cell capacitance was increased in HF LV myocytes. All guinea pigs with HF developed ascites and pleural effusion. Thus, the HF model of guinea pigs induced by pressure overload is valid.
HF Reduces I kr Tail Current Density I kr tail currents of guinea pig LV myocyte were completely abolished by the specific I kr blocker, dofetilide (1 mm) ( Figure 1A), indicating that I kr tail currents were measured free of contamination by other currents under the given experimental conditions. The amplitude of I Kr tail currents in HF myocytes was smaller than that of sham-operated controls ( Figure 1B). Figure 1C shows the current-voltage (I-V) relationship of I Kr tail current density. The I Kr tail current density in HF myocytes was significantly lower than that in control myocytes, from 0 mV to positive potentials (at +40 mV, 0.2160.01 pA/pF, n = 33, vs. 0.4360.03 pA/pF, n = 24; p,0.001).

I kr Inhibition Induced by b 2 -AR Stimulation in HF Ventricular Myocytes
In control myocytes, the selective b 2 -AR agonist fenoterol (10 mM) decreased I kr tail currents by 1765% (n = 6, vs. control myocytes in the absence of fenoterol, p = 0.11; Figure 2A and 2C). In contrast, fenoterol (10 mM) decreased I kr tail currents by 3367% in HF myocytes (n = 6, vs. HF myocytes in the absence of fenoterol, p,0.05; Figure 2B and 2C). The reduction of I Kr tail current occurred within 2-4 min and reached saturation about 10 min after addition of fenoterol (10 mM) into the bath. After washout of fenoterol, I kr tail current reached 83.564.5% and 67.866.7% of the control and HF value, respectively. The inhibitory effect of fenoterol was almost not reversible ( Figure 2C).

Effects of b 1 -and b 2 -AR Blockade on Fenoterol-induced Decrease in I kr
We used the selective b 2 -AR antagonist ICI118551 to examine whether the I Kr response to fenoterol was mediated through b 2 -AR in HF myocytes. When 10 mM ICI118551 was applied along with fenoterol, it almost totally prevented fenoterol-induced inhibition of I Kr (fenoterol alone, 3367% decrease, n = 6; fenoterol plus ICI118551, 863% decrease, n = 5; p,0.05; Figure 3A and 3C). However, in five HF myocytes, fenoterol decreased I Kr by 3265% in the presence of CGP20712A, a selective b 1 -AR antagonist ( Figure 3B and 3C). Under these conditions, the present results supported the role of the b 2 -AR subtype in the modulation of I Kr in HF myocytes.
b-AR mRNA and Protein Expression in HF Figure 4A and B show that b 1 -AR mRNA levels were reduced 52% in HF guinea pigs compared with controls (n = 5, p,0.05), whereas b 2 -AR mRNA levels remained unchanged. b 1 -AR protein showed a significant decline, as illustrated in Figure 4C. b 2 -AR protein remained unchanged ( Figure 4D).

I Kr Inhibition Induced by b 2 -AR Stimulation Involves the cAMP/PKA Pathway
It is known that the hERG K + channel can be modulated by cAMP and PKA. b 2 -AR is coupled to the stimulatory G s protein, which leads to the activation of adenylyl cyclase and production of cAMP. To examine whether cAMP activation mediates the I Kr response to b 2 -AR stimulation, we investigated the effect of inhibitory cAMP analog Rp-cAMPS on the stimulatory action of fenoterol. As shown in Figure 5A and 5C, with intracellular application of Rp-cAMPS (100 mM), fenoterol-induced I Kr decrease was almost fully prevented in HF myocytes (fenoterol alone, 3367% decrease, n = 6; Rp-cAMPS, 661% decrease, n = 6; p,0.05). These results suggest that I Kr inhibition induced by b 2 -AR stimulation involves cAMP activation.
We further examined the effect of PKA inhibition on the inhibitory effects of fenoterol. As illustrated in Figure 5B and 5C, fenoterol-induced I kr decrease was largely abolished by pretreatment of HF myocytes with KT5720 (fenoterol alone, 3367% decrease, n = 6; fenoterol plus KT5720, 1864% decrease, n = 5; p = 0.051), supporting an involvement of PKA activation.

Action Potential Duration (APD) is Prolonged by b 2 -AR Stimulation in HF Ventricular Myoctyes
Because action potential duration (APD) is dependent on the balance between depolarizing inward and repolarizing outward currents, an alteration in the amplitude of major repolarizing current such as I Kr would lead to substantial changes in the repolarization process. We therefore examined the effects of fenoterol on AP in LV myocytes. Figure 6A and 6B shows the superimposed traces of AP recorded before and during exposure to fenoterol (10 mM) in control and HF myocytes. A summary of corresponding data is shown in Figure 6C. APD at 90% repolarization (APD 90 ) was significantly prolonged in HF myocytes compared with control (315.168.1 ms, n = 8, vs. 274.867.3 ms, n = 9; p,0.01). Fenoterol caused significant prolongation of APD 90 in HF myocytes (9.5%, 344.666.5 vs. 315.168.1ms, n = 8; p,0.05), whereas no significant prolongation of APD 90 was observed in control myocytes (3%, 283.667.7 vs. 274.867.3 ms, n = 9). Fenoterol did not influence the AP amplitude and resting potentials in control and HF ventricular myocytes.

Discussion
The present study demonstrates, for the first time, that increased response to b 2 -AR stimulation produces an inhibitory effect on I Kr in ventricular myocytes of guinea pigs with descending aortic banding-induced heart failure, and this effect is induced by the activation of the cAMP/PKA pathway.    The hERG channel (Kv11.1) underlies the rapid component of delayed rectifier potassium current (I kr ) and is critical for the kinetics of cardiac AP repolarization [5,14]. There is increasing evidence that hERG/I Kr channels are modulated by various G protein-coupled receptors including aand b-ARs, acting through the intracellular signaling modulators cAMP, PKA, and PKC [6,7,8,9,11]. It has been increasingly recognized that b 1 -and b 2 -AR coexist in the heart. In large mammal hearts, b 2 -AR may account for approximately 40% of b-AR in total. The reported ratio of b 1 -/b 2 -AR was 85/15 in guinea pig hearts [15]. Consistent with previous observations [16], in our HF guinea pigs, b 1 -AR was downregulated; however, b 2 -AR was unaltered. The increase of b 2 -AR density has been demonstrated in the LV of dogs with pacing-induced HF [17]. It has been demonstrated that in diseased ventricles, b 2 -AR stimulation exhibits a heightened response [12,13]. Our results demonstrate, for the first time, that the responsiveness of I Kr to b 2 -AR stimulation is enhanced in HF guinea pigs ventricular myocytes. The effect of selective b 2 -AR agonist fenoterol (10 mM) on I Kr was far less effective in control myocytes; whereas, I Kr was significantly inhibited by 10 mM fenoterol in HF myocytes. The reduction of I Kr tail current occurred within 2-4 min and reached saturation about 10 min after addition of fenoterol into the bath. After washout of fenoterol, the inhibitory effect on I Kr tail current was almost irreversible. In order to identify the b 2 -AR specific effect, we used salbutamol (10 mM), another highly selective b 2 -AR agonist, and achieved similar results (data not shown). Moreover, the inhibitory effect of fenoterol was almost completely abolished by b 2 -AR antagonist, but not by the b 1 -AR antagonist. Therefore, our findings indicate that the I Kr inhibition induced by fenoterol is due to b 2 -AR activation.
Heart failure induced by pressure overload is a non-ischemic heart failure, which exhibits clinical and pathological features typical of hypertension-induced heart failure. Consistent with previous observations, myocytes from these hearts were enlarged. This model of heart failure is widely applied in the study of cardiac cellular electrophysiology. Sympathetic nervous system is activated in response to exercise and emotional stress, which results in increased levels of local and circulating catecholamines. Plasma norepinephrine levels of , 3 mM and peak exercise norepinephrine levels of , 15 mM have been detected in chronic HF in humans [18], giving our present findings utilizing 10 mM fenoterol a clinical relevance. Stimulation of different G protein-coupled receptors, including a 1 -and b-ARs, regulates I Kr through intracellular messengers and provides a link between autonomic stimulation and cardiac repolarization [19]. Stimulation of b 2 -AR in the heart has classically been known to result in G s -mediated stimulation of adenylyl cyclase (AC), which leads to increased cellular cAMP, thus activating PKA [20]. It has been demonstrated in the Xenopus laevis oocyte expression system that PKA can reduce hERG currents via direct phosphorylation of all four putative PKA consensus sites [10]. In the present study, we demonstrated that fenoterol-induced inhibition of I Kr was fully prevented by intracellular application of Rp-cAMPS, an inhibitory cAMP analog, suggesting that this effect may be mediated through a cAMP-dependent mechanism in HF ventricular myocytes. Furthermore, fenoterol-induced inhibition of I Kr was partly attenuated by PKA inhibitor, implicating the involvement of PKA activation. The dual regulation of I Kr by cAMP and PKA phosphorylation has been previously demonstrated [21]. hERG current may be decreased by cAMP-dependent activation of PKA; however, a putative direct binding of cAMP to the channel causes opposite effects. The net effect of dual hERG current regulation by PKA and cAMP is current reduction. A recent study has shown that b 2 -AR redistribution in HF can change cAMP compartmentation [22]. Our results do not exclude the possibility of direct binding of cAMP to the channel.
Cardiac b 2 -AR can couple with both G s and G i proteins. The complexity of the b 2 -AR signaling pathway, including functional compartmentalization of signaling mediated by G i , phosphatidylinositol 3-kinase, and mitogen-activated protein kinases (MAPKs), has been documented [20,23]. Recently, Li et al. has reported that one or more A-kinase anchoring proteins (AKAPs) targets PKA to HERG channels and may contribute to the acute regulation of I Kr by cAMP [24]. AKAPs are a structurally diverse group of proteins that lack primary structure sequence homology but share the function of localizing PKA to subcellular structures, substrates, and oftentimes with other members of the signaling pathway [25]. b-adrenergic signaling is maintained by the localization of PKA and phosphodiesterases to subcellular microdomains. In addition, in the rat brain, b 2 -ARs are found to be associated directly with Cav1.2 in a macromolecular signaling complex [26]. Indeed, the precise mechanism for b 2 -adrenergic stimulation-induced inhibition of I Kr for HF myocytes remains to be further elucidated. For example, it remains unclear whether G i pathway or a b 2 -ARmacromolecular signaling complex mediates the inhibition of I Kr via b 2 -AR in HF ventricular myocytes.

APD Prolongation by b 2 -AR Stimulation
One of the most characteristic electrophysiological remodeling in failing heart is APD prolongation, which is believed to mainly result from the downregulation of repolarizing outward potassium currents, including I to (transient outward K + current ), I Ks and I Kr in heart failure [27,28]. In the present study, we found that QT interval was increased in failing guinea pigs and APD was prolonged in failing ventricular myocytes. Fenoterol caused significant prolongation of APD 90 in failing ventricular myocytes, whereas no significant prolongation of APD 90 was observed in control myocytes. The present study provides evidence that fenoterol-induced inhibition of I Kr may result in delay in cardiac repolarization via stimulation of b 2 -AR in failing ventricular myocytes. Because the I Kr is crucial for the repolarization of cardiac AP, inhibition of I Kr induced by stimulation of b 2 -AR in failing ventricular myocytes may in part contribute to the delay in cardiac repolarization.Ventricular myocytes from the guinea pig heart possess a time-and voltage-independent Cl 2 current induced by b-adrenoceptor activation [29,30]. Activation of this current will result in outward current during the plateau phase of AP, shortening APD [31]. One limitation of the present study is that the AP experiments were performed without considering cAMP modulated Cl 2 current. Therefore, the effect of fenoterol on cardiac repolarization should be studied without ignoring other currents in the future.
In conclusion, the inhibitory effect of I Kr induced by b 2 -AR stimulation is increased in HF. The effect is likely to be mediated through a cAMP/PKA pathway mechanism in HF ventricular myocytes. This regulatory pathway of the b 2 -adrenergic system on I kr current provides a possible relationship between stress and lifethreatening arrhythmias in HF.

Materials and Methods
All experiments were performed in accordance with animal care protocols approved by the Nanjing Medical University Institutional Animal Care and Use Committee.

Heart Failure Model in Guinea Pigs
Heart failure was induced in adult male guinea pigs (250 to 300 g) by subtotal descending thoracic aortic banding. After sodium pentobarbital anesthesia (25 mg/kg, i.p.), the descending thoracic aorta was exposed sterilely through an incision in the left third intercostal space. A uniform degree of constriction around the descending thoracic aorta was produced by tightening a 2-0 surgical silk ligature around an 18-gauge needle. The needle was then withdrawn from the ligature, and the chest incision was closed. Sham animals underwent the same operation, with the exception that the aorta was not banded. Sham-operated animals were used as control. Aortic-banded animals and sham animals were housed and fed under identical conditions and were used 12 weeks after surgery. To monitor the progress of HF during the observation period, two-dimensional echocardiography (Sonos 5500, Hewlett Packard, Andover, MA, USA) was carried out periodically. ECGs and two-dimensional echocardiography were obtained under anesthesia (ketamine hydrochloride; 30 mg/kg, i.p.). The QTc was calculated by Bazett's formula where QTc = QT/!RR. Following 12 weeks of aortic banding, guinea pigs showed signs of HF, including ventricular dilation, decreased ejection fraction (EF) and fractional shortening (FS), ascites, and pleural effusions.

Myocyte Isolation and Electrophysiological Recordings
Single left ventricular (LV) myocytes were enzymatically dissociated from the heart of guinea-pigs (550-600 g) as described previously [6] with minor modification. Cardiomyocytes were transferred to a recording chamber that was continuously perfused with a bath solution. Pipettes had resistances of 3-6 MV after filling with the pipette solution. Following seal formation, access developed within 2-5 min and series resistance was compensated up to 80%. Whole-cell patch-clamp recordings were performed with an EPC-9 amplifier (HEKA, Germany). Flow rate through the chamber was maintained at 2-3 ml/min. I kr was determined using the following test pulse protocol: after a holding potential of 240 mV, test pulses were applied at various voltages from 240 to +40 mV (step width 20 mV, step duration 200 ms) prior to returning to 240 mV for tail current recording [6,7]. I Kr current was recorded at 3760.5uC. Substance effects were investigated on tail currents after the test pulse was applied at +40 mV. Measurements were repeated every two min.

Solutions and Drugs
Preparation of the solution was performed as described previously by Wang et al. [6]. Calcium currents were blocked by the addition of 10 mM nifedipine in the bath solution, and 10 mM chromanol was used to block I ks . Na 2 -ATP, EGTA, creatine phosphate, L-glutamic acid, HEPES, taurine, bovine serum albumin (BSA), nifedipine, isoproterenol, xamoterol, fenoterol, CGP20712A, ICI118551, and Rp-cAMPS were purchased from Sigma (St. Louis, MO), collagenase II from Invitrogen (Carlsbad, CA), and KT5720 from Merck KGaA (Darmstadt, Germany). Dofetilide, a specific blocker of I kr or hERG, was kindly provided by Pfizer Inc (NY, USA). All other reagents were obtained from Amresco (OH, USA).
For stock solutions, chromanol and KT5720 were dissolved in dimethyl sulfoxide (DMSO) at a final concentration of 1 mM and stored at 220uC until use. Final concentration of DMSO was less than 0.5% in the bath, which exerted no effect on the currents measured.

Real Time RT-PCR Analysis
RNA was extracted from 100 mg of LV tissue using the Trizol reagent (Invitrogen) according to the manufacturer's protocol. RNA concentration was determined spectrophotometrically. Reverse transcription of 2 mg RNA was performed in a 20-ml reaction mixture using an RT kit (Takara Biotech). The GAPDH housekeeping gene was used as a reference for loading control. The primers for genes examined are shown in Table 2. Optimized PCR cycle parameters included 2 min at 95uC, 10 sec at 95uC followed by 10 sec at 95uC, 10 sec at 57uC or 55uC, and 45 sec at 72uC for 40 cycles. Standard curves were determined for PCR efficiency. Quantitative real-time (RT) polymerase chain reaction PCR) was performed using the Applied Biosystems 7500 Real-time PCR System (Applied Biosystems, Foster City, CA, USA) with SYBR Green I (Invitrogen). Relative mRNA expression was determined using the 2 2gCt method.

Western Blot Analysis
Membrane fractions were prepared as previously described [32]. Protein samples were separated by 10% SDS-PAGE. The separated proteins were transferred by electrophoresis to PVDF membranes. Membranes were blocked for two h with Tris-Tween buffered saline (TTBS) solution containing 5% nonfat dried milk and incubated overnight with primary antibody against b 1 -AR and b 2 -AR (both from Santa Cruz Biotechnology, Santa Cruz, CA). After washing, blots were incubated for one h with appropriate peroxidase-conjugated goat anti-rabbit IgG secondary antibody (Santa Cruz Biotechnology). Staining was determined with chemiluminescence and quantified using image acquisition and analysis software. All data were expressed relative to GAPDH staining for the same samples on the same gels.

Data Analysis
The data were acquired using Pulse +Pulsefit V 8.53 and were expressed as mean 6 SEM. Western blot band intensities were expressed after background subtraction as optical density units (ODUs) normalized against GAPDH signal intensity for the same sample. Data comparisons were made using Student's t-test (paired or unpaired). Differences were considered statistically significant if p,0.05.