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Research ArticleArticle

Characterization of VU0468554, a New Selective Inhibitor of Cardiac G Protein–Gated Inwardly Rectifying K+ Channels

Allison Anderson, Baovi N. Vo, Ezequiel Marron Fernandez de Velasco, Corey R. Hopkins, C. David Weaver and Kevin Wickman
Molecular Pharmacology December 2021, 100 (6) 540-547; DOI: https://doi.org/10.1124/molpharm.121.000311

Abstract

G protein–gated inwardly rectifying K+ (GIRK) channels are critical mediators of excitability in the heart and brain. Enhanced GIRK-channel activity has been implicated in the pathogenesis of supraventricular arrhythmias, including atrial fibrillation. The lack of selective pharmacological tools has impeded efforts to investigate the therapeutic potential of cardiac GIRK–channel interventions in arrhythmias. Here, we characterize a recently identified GIRK-channel inhibitor, VU0468554. Using whole-cell electrophysiological approaches and primary cultures of sinoatrial nodal cells and hippocampal neurons, we show that VU0468554 more effectively inhibits the cardiac GIRK channel than the neuronal GIRK channel. Concentration-response experiments suggest that VU0468554 inhibits Gβγ-activated GIRK channels in noncompetitive and potentially uncompetitive fashion. In contrast, VU0468554 competitively inhibits GIRK-channel activation by ML297, a GIRK-channel activator containing the same chemical scaffold as VU0468554. In the isolated heart model, VU0468554 partially reversed carbachol-induced bradycardia in hearts from wild-type mice but not Girk4–/– mice. Collectively, these data suggest that VU0468554 represents a promising new pharmacological tool for targeting cardiac GIRK channels with therapeutic implications for relevant cardiac arrhythmias.

SIGNIFICANCE STATEMENT Although cardiac GIRK–channel inhibition shows promise for the treatment of supraventricular arrhythmias, the absence of subtype-selective channel inhibitors has hindered exploration into this therapeutic strategy. This study utilizes whole-cell patch-clamp electrophysiology to characterize the new GIRK-channel inhibitor VU0468554 in human embryonic kidney 293T cells and primary cultures. We report that VU0468554 exhibits a favorable pharmacodynamic profile for cardiac over neuronal GIRK channels and partially reverses GIRK-mediated bradycardia in the isolated mouse heart model.

Introduction

G protein–gated inwardly rectifying K+ (GIRK) channels are critical mediators of cell excitability in the heart and brain (Luscher and Slesinger, 2010; Slesinger and Wickman, 2015). In the heart, GIRK channels are heterotetrameric complexes made up of GIRK1 and GIRK4 subunits in 1:1 stoichiometry (Krapivinsky et al., 1995; Corey et al., 1998). Cardiac GIRK channels are predominantly expressed in atrial and nodal tissue, where they mediate much of the influence of the parasympathetic branch of the autonomic nervous system on cardiac physiology (Wickman et al., 1998; Bettahi et al., 2002; Mesirca et al., 2013; Lee et al., 2018). Activation of the M2 muscarinic receptor (M2R) by acetylcholine released from postganglionic parasympathetic neurons triggers the Gβγ-dependent activation of GIRK channels in sinoatrial nodal (SAN) cells and atrial myocytes (Logothetis et al., 1987; Wickman et al., 1994; Mesirca et al., 2013; Posokhova et al., 2013; Lee et al., 2018), which decreases heart rate (HR) and increases heart rate variability (Wickman et al., 1998; Lee et al., 2018). Cardiac GIRK–channel activation also shortens action potential duration (APD) and effective refractory period (Wang et al., 2013b).

Increased GIRK-channel activity in atrial and nodal tissue has been implicated in the pathophysiology of nodal and atrial rhythm disorders. A mutation in G Protein Subunit Beta 2 (Gβ2), which results in exaggerated GIRK-dependent signaling, has been identified in human patients with familial sinus node dysfunction (Stallmeyer et al., 2017; Long et al., 2020). Furthermore, increased GIRK-channel activity has been noted in patients suffering from chronic atrial fibrillation (AF) (Dobrev et al., 2005; Voigt et al., 2008). Indeed, a study using a combination of optical mapping and immunoblotting revealed that re-entry drivers for adenosine-induced AF were localized to areas with coexpression of GIRK4 and the A1 adenosine receptor in human atria (Li et al., 2016). Similar observations have been reported in mice, wherein genetic ablation of a negative regulator of GIRK-dependent signaling, regulator of G protein signaling 6, increased susceptibility to pacing-induced AF (Posokhova et al., 2010, 2013).

Pharmacological inhibition and genetic ablation of GIRK channels have been shown to rescue and terminate supraventricular arrhythmias, such as AF and nodal dysfunction. The bee venom peptide tertiapin, a selective blocker of GIRK channels, terminates pharmacologically and vagally induced AF in canines (Hashimoto et al., 2006). Conversely, mice lacking cardiac GIRK channels are resistant to both pacing-induced AF and arrhythmias induced by vagus nerve stimulation (Kovoor et al., 2001; Lee et al., 2018). Additionally, genetic ablation of Girk4 rescues symptoms of sick sinus syndrome and atrioventricular block in mouse models of nodal dysfunction (Mesirca et al., 2014, 2016). Intriguingly, although loss of K+ channel activity is normally associated with an increase in ventricular arrhythmia susceptibility (Vandenberg et al., 2012; Ramalho and Freitas, 2018), ablation of GIRK channels in ventricular tissue does not increase susceptibility to ventricular arrhythmia (Anderson et al., 2018). Collectively, these studies suggest that inhibition of cardiac GIRK channels may represent a safe therapeutic intervention for the treatment of certain types of cardiac rhythm disorders.

Although the lack of pharmacological tools has impeded progress on assessing the therapeutic potential of targeting cardiac GIRK channels to treat cardiac rhythm disorders, small-molecule modulators of GIRK channels have been recently identified (Kaufmann et al., 2013; Wen et al., 2013; Xu et al., 2020; Zhao et al., 2020; Cui et al., 2021). This group includes ML297, a selective activator of GIRK1-containing GIRK channels that exhibits a modest preference for the neuronal (GIRK1/GIRK2) relative to the cardiac (GIRK1/GIRK4) GIRK–channel subtype (Wydeven et al., 2014). Using ML297 as a chemical scaffold and a fluorescence-based thallium flux assay, the structural derivative VU0468554 was identified as a novel GIRK-channel inhibitor that exhibited selectivity for the cardiac (GIRK1/GIRK4) relative to the neuronal (GIRK1/GIRK2) GIRK–channel subtype (Wen et al., 2013). Here, we probe the selectivity and mechanisms of VU0468554 inhibition of recombinant and native GIRK channels. Our work suggests that VU0468554 is a promising GIRK1/GIRK4-selective inhibitor that could serve as a lead compound for the development of new potential therapeutics for treating cardiac arrhythmias.

Materials and Methods

Animals

Animal experiments were approved by the Institutional Animal Care and Use Committee at the University of Minnesota. Both inbred and purchased (Jackson Laboratory, Bar Harbor, ME) male and female C57BL/6J mice were used for whole-cell electrophysiology and isolated heart studies. Generation of Girk1–/–, Girk2–/–, and Girk4–/– mice was described previously (Signorini et al., 1997; Wickman et al., 1998; Bettahi et al., 2002).

Human Embryonic Kidney Cell Culture and Transfection

Human embryonic kidney (HEK) 293T cells (American Type Culture Collection; Manassas, VA) were maintained at 37°C and 5% CO2 in Dulbecco’s modified Eagle’s medium containing 10% (v/v) FBS, 1% sodium pyruvate, 1% glutamax, and 1% antibiotic-antimycotic (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA). Cells were dislodged with Trypsin-EDTA solution (MilliporeSigma, Burlington, MA) at 70%–90% confluency and plated onto 8-mm glass coverslips. After at least 1 hour after plating, cells were transfected with pcDNA3-based expression constructs in the following two groups by calcium phosphate technique: group 1: M2R (50 ng/coverslip), GIRK1-AU5 (50 ng/coverslip), GIRK4-AU5 (50 ng/coverslip), and enhanced GFP (20 ng/coverslip); group 2: M2R (50 ng/coverslip), GIRK1-AU5 (50 ng/coverslip), GIRK2-myc (50 ng/coverslip), and enhanced GFP (20 ng/coverslip). Experiments were conducted 18–36 hours after transfection.

SAN Cell Culture

SAN cells were isolated from young adult (2–3 months) mice (male and female) as described (Anderson et al., 2018, 2020). In brief, mice were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg), and hearts were excised into Tyrode’s solution (in mM): 140 NaCl, 5.4 KCl, 1.2 KH2PO4, 1.0 MgCl2, 1.8 CaCl2, 5.55 glucose, and 5 HEPES (pH 7.4 with NaOH). SAN tissue was then excised and placed into a modified Tyrode’s solution containing (in mM) 140 NaCl, 5.4 KCl, 1.2 KH2PO4, 0.2 CaCl2, 50 taurine, 18.5 glucose, 5 HEPES, and 0.1% bovine serum albumin (pH 6.9 with NaOH) with elastase (0.3 mg/ml; Worthington Biochemical Corp., Lakewood, NJ) and collagenase II (0.21 mg/ml; Sigma Aldrich, St. Louis, MO) for 30 minutes at 37°C. SAN tissue was then washed in a solution containing (in mM) 100 l-glutamic acid/potassium salt, 10 l-aspartic acid/potassium salt, 25 KCl, 10 KH2PO4, 2 MgSO4, 20 taurine, 5 creatine, 0.5 EGTA, 20 glucose, 5 HEPES, and 0.1% bovine serum albumin (pH 7.2 with KOH) and triturated three times before being plated onto laminin (25 μg/ml)-coated glass coverslips. The cells were incubated at 37°C for 1 hour before recording and used within 8 hours.

Hippocampal Neuron Culture

Primary cultures of hippocampal neurons were prepared as described (Vo et al., 2019). Briefly, extracted hippocampi from neonatal (P0–4) pups were placed into an ice‐cold modified Hank’s Balanced Salt Solution (MilliporeSigma) (in mM): 3 HEPES‐NaOH (pH 7.1), kynurenic acid/12 Mg2SO4, and 5.5 D-glucose. The tissue was digested for 20 minutes with papain and DNase I at 37°C with occasional inversion. Hippocampi were mechanically dissociated in growth medium containing Neural‐basal A medium, 2% B27 supplement, 0.5 mM Glutamax (MilliporeSigma), and antibiotic-antimycotic (Gibco; Thermo Fisher Scientific, Inc.) using trituration with 1-ml pipettes. Neurons were pelleted by centrifugation (150 rcf) and plated onto 8-mm glass coverslips precoated with poly(l‐lysine) in 48‐well plates. Neurons were maintained in culture in a humidified 5% CO2 incubator at 37°C, and half of the medium was replaced with fresh growth medium every 3–4 days. Neurons were kept in culture for 10–14 days before experimentation.

Electrophysiology

Electrophysiological recordings were performed as described (Vo et al., 2019). In brief, transfected HEK cells; pyramidal-shaped, hippocampal neurons (capacitances between 80 and 200 pF); or thin, striated SAN cells (capacitances between 15 and 40 pF) were transferred to a low‐K+ bath solution consisting of (in mM) 130 NaCl, 5.4 KCl, 1 CaCl2, 1 MgCl2, 5.5 D‐glucose, and 5 HEPES/NaOH (pH 7.4). Fire‐polished borosilicate patch pipettes were filled with K‐gluconate pipette solution containing (in mM) 140 K‐gluconate, 2 MgCl2, 1.1 EGTA, 5 HEPES, 2 Na2‐ATP, 0.3 Na‐GTP, and 5 Na2-phosphocreatine (pH 7.2). Upon achieving whole‐cell access, cells were held in voltage‐clamp mode at −70 mV. Currents were measured in a high‐K+ bath solution containing (in mM) 120 NaCl, 25 KCl, 1 CaCl2, 1 MgCl2, 5.5 D‐glucose, and 5 HEPES/NaOH (pH 7.4). VU0468554 and ML297 were dissolved in DMSO and then diluted with high‐K+ bath solution. Baclofen and carbachol (CCh) were dissolved in high‐K+ bath solution. Responses were measured as the difference in holding current at baseline (in high-K+ solution) and holding current after agonist, antagonist, or agonist/antagonist application. Maximal response and concentration-response experiments were performed on separate cells. Only experiments in which the access resistance was stable and low (<20 MΩ) were included in the analysis. The liquid‐junction potential predicted to be −17 mV using JPCalc software (Molecular Devices, LLC, Sunnyvale, CA) was not corrected.

Isolated Heart Recordings

Hearts from young adult (2–3 months) mice (male and female) were excised and placed into ice-cold, oxygenated Tyrode’s solution, and the aorta was quickly cannulated. Cannulated hearts were then placed into a warm (37 ± 1°C) Tyrode’s bath, and platinum recording electrodes (iWorx; Dover, NH) were placed on or near the surface of the heart. Oxygenated Tyrode’s solution was then perfused at 2–3 ml/min, and a baseline HR was recorded for 10 minutes. After the baseline HR was acquired, 1 μM CCh was perfused and allowed to stabilize for 15 minutes. While CCh was still being perfused, a bolus injection of either vehicle (1:100 DMSO) or VU0468554 (10 μM) was injected through a port in the Langendorf apparatus. The ECG signal was acquired with LabScribe v.3 software (iWorx) and filtered as appropriate. The derivative of the signal was computed to account for movements in baseline and to amplify the signal for subsequent analysis. A 15-second segment from each time point (baseline, CCh 5 minutes after, and CCh 10 minutes after) was then exported to Kubios HRV v.2 for HR analysis, utilizing artifact correction as appropriate.

Statistical Analysis

The experiments included in this study were done in an exploratory manner and not done according to a preset plan. All data were analyzed using Prism v.8.2.1 software (GraphPad Software; San Diego, CA) and are presented as mean ± S.D. The level of statistical significance was set at P < 0.05. IC50 and EC50 values were calculated from data plotted as log(inhibitor)/log(agonist) versus response with nonlinear regression, variable slope models that did not assume standard slopes, with the following curve-fitting equations: Embedded Image

Specific statistical tests used for data analysis are denoted within the figure legends.

Results

We first investigated the inhibitory actions of VU0468554 (Fig. 1A) with whole-cell patch-clamp electrophysiology in HEK cells transfected with either GIRK1/GIRK4- or GIRK1/GIRK2-channel subtypes. Whole-cell GIRK-channel activity was measured in transfected HEK cells in a high-K+ bath solution at a holding potential of −70 mV. Under these conditions, application of VU0468554 (10 μM) inhibited basal GIRK–channel activity (i.e., increased holding current) in HEK cells expressing either channel subtype (Fig. 1B); the increase was slightly but not significantly larger in cells expressing GIRK1/GIRK4 channels compared with GIRK1/GIRK2-expressing cells (Fig. 1C). We also coexpressed M2R with GIRK1/GIRK4 or GIRK1/GIRK2 in HEK cells to assess the ability of VU0468554 to inhibit receptor-induced GIRK-channel activity. The nonselective muscarinic receptor agonist CCh (10 μM) evoked reliable inward currents in both GIRK1/GIRK4- and GIRK1/GIRK2-expressing HEK cells (Fig. 1B). VU0468554 blocked a significantly larger fraction of the CCh-induced current in cells expressing GIRK1/GIRK4 compared with GIRK1/GIRK2 channels (Fig. 1D), but no difference in VU0468554 potency (IC50) was detected between GIRK-channel subtypes (Fig. 1, E and F). Reversal of CCh-induced currents past baseline in GIRK1/GIRK4-expressing HEK cells likely reflects the combined inhibition of basal (Fig. 1C) and agonist-induced (Fig. 1D) GIRK-channel activity.

Fig. 1.
Fig. 1.

VU0468554 blockade of GIRK-channel subtypes expressed in HEK cells. (A) Chemical structures of VU0468554 (top) and ML297 (bottom). (B) Representative traces of holding current (Vhold = −70 mV) after application of VU0468554 (VU554, 10 μM) and CCh (10 μM) in HEK cells expressing M2R and either GIRK1/GIRK4- (top) or GIRK1/GIRK2- (bottom) channel subtypes. Scale bars: 10 seconds/500 pA. (C) Summary of the inhibition of basal GIRK–channel activity by VU0468554 (10 μM) reported as current density in HEK cells expressing either GIRK1/GIRK4- (n = 7 cells) or GIRK1/GIRK2- (n = 9 cells) channel subtypes (t14 = 1.3; P = 0.23; two-tailed unpaired t test). (D) Summary of the percent inhibition by VU0468554 (10 μM) of current evoked by CCh (10 μM) in HEK cells expressing M2R and either GIRK1/GIRK4- (n = 7 cells) or GIRK1/GIRK2- (n = 8 cells) channel subtypes (t13 = 6.7; ****P < 0.0001; two-tailed unpaired t test). (E) Concentration-response curves for VU0468554-dependent inhibition of CCh-induced currents in HEK cells expressing M2R and GIRK1/GIRK4- (n = 11 cells) or GIRK1/GIRK2- (n = 9 cells) channel subtypes. (F) Summary of the Log(IC50) extracted from concentration-response experiments examining the VU0468554-dependent inhibition of CCh-induced GIRK currents in HEK cells expressing M2R and GIRK1/GIRK4- (n = 11) or GIRK1/GIRK2- (n = 9 cells) channel subtypes (t16 = 0.9; P = 0.34; two-tailed unpaired t test).

We also examined the impact of VU0468554 on GIRK-channel activity in native cell types. Consistent with published reports (Posokhova et al., 2013; Lee et al., 2018), CCh (10 μM) elicited inward currents in SAN cells from wild-type but not Girk4–/– mice (Fig. 2A). Previously, we demonstrated that CCh-induced currents in wild-type SAN cells were reversed by application of tertiapin-Q (Anderson et al., 2020). VU0468554 reversed CCh-induced GIRK currents in SAN cells in a dose-dependent manner, inhibiting approximately 73% of the CCh-induced response at the highest dose tested (10 μM; Fig. 2, A–C). VU0468554 is a synthetic derivative of ML297, which selectively activates GIRK1-containing GIRK channels (Wydeven et al., 2014). VU0468554 (10 μM) reversibly increased the holding current of SAN cells from wild-type mice, likely because of inhibition of basal GIRK–channel activity as this effect was absent in SAN cells from Girk1–/– mice (Fig. 2, D and E). Thus, under these recording conditions, VU0468554 is selective for GIRK1-containing GIRK channels present in SAN cells.

Fig. 2.
Fig. 2.

VU0468554 preferentially blocks cardiac over neuronal GIRK channels. (A) Representative inward currents evoked by CCh (10 μM) in wild-type SAN cells (top left) and by baclofen (100 μM) in wild-type hippocampal neurons (top right) and their reversal by VU0468554 (10 μM). CCh failed to evoke currents in SAN cells from Girk4–/– mice (bottom left), and baclofen failed to evoke responses in hippocampal neurons from Girk2–/– mice (bottom right). Scale bars: 10 seconds/500 pA. (B) Summary of the percent inhibition by VU0468554 (10 μM) of currents evoked by CCh in SAN cells (n = 9 cells) or baclofen in hippocampal neurons (n = 8 cells) (t15 = 11.8; ****P < 0.0001; two-tailed unpaired t test). (C) Summary of concentration-dependent inhibition of CCh-induced currents in SAN cells (n = 10 cells) and baclofen-induced currents in hippocampal neurons from wild-type mice (n = 7 cells). (D) Representative holding current (Vhold = −70 mV) responses to VU0468554 (10 μM) in SAN cells from wild-type (left) and Girk1−/−(right) mice. Scale bars: 5 seconds/100 pA. (E) Summary of the holding current responses evoked by VU0468554 (10 μM) in SAN cells from wild-type (n = 9) and Girk1−/− (n = 14) mice (t21 = 4.4; ***P < 0.0001; two-tailed unpaired t test).

Cultured hippocampal neurons from wild-type mice exhibit a GIRK conductance mediated by GIRK1/GIRK2 channels (Wydeven et al., 2012, 2014). Indeed, the selective GABAB receptor agonist baclofen (100 μM) elicited reliable inward currents in hippocampal neurons from C57BL/6J mice that were absent in neurons from Girk2–/– mice (Fig. 2A). VU0468554 dose-dependently inhibited baclofen-induced currents in hippocampal neurons, but the maximal inhibition observed was only 20% of the baclofen-induced response (Fig. 2, A–C), which was significantly smaller than the fractional inhibition of receptor-induced cardiac GIRK response in SAN cells (Fig. 2B). Thus, VU0468554 is a more effective inhibitor of cardiac GIRK channels than neuronal GIRK–channel subtypes, as assessed in both heterologous expression and primary culture models.

To further investigate the mechanism of inhibition exhibited by VU0468554 on the cardiac GIRK channel, we examined the impact of a maximally effective dose of VU0468554 (10 μM) on the efficacy (maximal response) and potency (EC50) of CCh (Fig. 3, A–C). VU0468554 decreased the maximal response induced by CCh, and this effect was not overcome by higher concentrations of CCh (Fig. 3C), which suggests that VU0468554 inhibits receptor-induced GIRK-channel activity in a noncompetitive manner. We also observed a slight yet significant decrease in CCh potency, as evidenced by an increase in the EC50 of CCh-induced responses (Fig. 3B). This suggests that the inhibition of CCh-induced GIRK-channel responses may be uncompetitive, in which inhibition is affected by the interaction between the GIRK channel and Gβγ, instead of solely noncompetitive.

Fig. 3.
Fig. 3.

Mechanism of VU0468554 inhibition of GIRK-channel activity in SAN cells. (A) Impact of vehicle or VU0468554 (10 μM) on the concentration-response relationship for the CCh-induced activation of GIRK channels in SAN cells. (B) Summary of the Log(EC50) extracted from concentration-response experiments examining the impact of vehicle (n = 17) or VU0468554 (10 μM; n = 11) on CCh-induced currents in wild-type SAN cells (t12.6 = 2.9; *P < 0.05; two-tailed unpaired t test, Welsh’s correction). (C) Summary of maximal CCh-induced responses (current density at 100 μM) in wild-type SAN cells in the presence (n = 10 cells/3 mice) or absence (n = 6 cells/1 mouse) of 10 μM VU0468554 (t14 = 5.4; ****P < 0.0001; two-tailed unpaired t test). (D) Summary of concentration-dependent experiments of ML297-induced currents in wild-type SAN cells in the presence (n = 12 cells/3 mice) or absence of 10 μM VU0468554 (n = 11 cells/3 mice). (E) Summary of the Log(EC50) extracted from concentration-response experiments examining the impact of vehicle (n = 11) or VU0468554 (10 μM; n = 12) on ML297-induced currents in wild-type SAN cells (t21 = 2.4; *P < 0.05; two-tailed unpaired t test). (F) Summary of maximal ML297-induced responses (current density at 100 μM) in wild-type SAN cells in the presence (n = 5 cells/1 mouse) or absence (n = 6 cells/1 mouse) of 10 μM VU0468554 (t14 = 2.2; P = 0.06; two-tailed unpaired t test).

We next examined the VU0468554-dependent inhibition of cardiac GIRK–channel activity evoked by ML297, which activates GIRK channels in a G protein–independent manner, presumably via direct binding (Wydeven et al., 2014) (Fig. 3D). Addition of VU0468554 (10 μM) decreased the apparent potency of ML297 (Fig. 3E), which is consistent with competitive inhibition of ML297. Although we also noted a modest VU0468554-dependent decrease in the efficacy of ML297 (Fig. 3F), the diminished amplitude of ML297-induced responses in the presence of VU0468554 likely reflects an inability to test higher concentrations of ML297 because of limited aqueous solubility of this compound.

Lastly, we investigated whether VU0468554 blocked the bradycardic effect of muscarinic receptor activation. We obtained baseline HR from isolated wild-type and Girk4–/– mouse hearts and then perfused hearts with CCh (1 μM). As expected, the decrease in HR upon perfusion of CCh was significantly blunted in hearts from Girk4–/– mice (Fig. 4B). After stabilization of the CCh-induced reduction in HR, we applied a bolus of VU0468554 (10 μM) or vehicle (1:100 DMSO). In wild-type mice, vehicle administration did not reverse the CCh-induced bradycardia (Fig. 4), as assessed at 5- and 10-minute postinjection time points. In contrast, VU0468554 partially reversed (∼20%) CCh-induced bradycardia at 5 and 10 minutes postinjection (Fig. 4, A–C). This effect was not observed on the residual CCh-induced bradycardia in isolated hearts from Girk4−/− mice, which indicates that the impact of VU0468554 is likely because of the selective inhibition of GIRK channels (Fig. 4B).

Fig. 4.
Fig. 4.

VU0468554 partially reverses CCh-induced bradycardia in the isolated heart. (A) Representative isolated heart recordings from wild-type mice showing HR at baseline (left), after perfusion of CCh (10 μM, middle), and 5 minutes postinjection of vehicle (1:100 DMSO, top) or VU0468554 (10 μM, bottom) while CCh was still being perfused; scale bar: 2 seconds. (B) Summary of the percentage of baseline beating rate (% baseline beating rate = 100 − [(beating rate at time point on x-axis/baseline beating rate) × 100)] of isolated hearts from wild-type and Girk4–/– mice. There was a significant difference in the impact of CCh on hearts from wild-type (n = 10) and Girk4–/– (n = 5) mice (t15 = 16.1, ****P < 0.0001; two-tailed unpaired t test). There was a significant difference between treatment (+/−VU554) and time point (CCh, 5 min post, and 10 min post) in wild-type hearts (F2,18 = 11.9; ***P < 0.001; two-way ANOVA), with post hoc analysis (Bonferroni) revealing a significant difference between vehicle (n = 6 hearts) and VU0468554 treatment (n = 5 hearts) 10 minutes postinjection in wild-type hearts (*P < 0.05). There was no main effect of treatment (F1,4 = 0.005; P = 0.9) or time point (F2,8 = 4.3; P = 0.1), nor was there a treatment × time point interaction (F2,8 = 0.5; P = 0.61) in Girk4–/– mouse hearts (n = 3 hearts per treatment). (C) Summary of the change in beating rate (Δ beating rate = beating rate after CCh injection – beating rate measured 5 or 10 minutes after vehicle/VU554 injection) of isolated hearts from wild-type mice. There was a significant effect of treatment (F1,9 = 31.4, ***P < 0.001; two-way ANOVA) but not time point (F1,9 = 2.5; P = 0.15), and no interaction between treatment and time point was detected (F1,9 = 0.01; P = 0.92). VU554, VU0468554.

Discussion

In this study, we characterized a novel cardiac GIRK–channel inhibitor (VU0468554) identified in a structure-activity investigation of the GIRK-channel activator ML297 (Wen et al., 2013). Our data obtained in electrophysiological assessments of recombinant and native GIRK channels show that VU0468554 preferentially inhibits the cardiac (GIRK1/GIRK4) compared with the neuronal (GIRK1/GIRK2) GIRK–channel subtype. Moreover, we show in an isolated heart model that VU0468554 can partially reverse the bradycardic influence of muscarinic receptor activation.

The initial characterization of VU0468554 was conducted in transfected HEK cells and used a fluorescence-based thallium flux assay (Wen et al., 2013). In this assay, VU0468554 suppressed the basal activity of GIRK1/GIRK2- and GIRK1/GIRK4-channel subtypes and exhibited a 3-fold increased potency as an inhibitor of GIRK1/GIRK4 (IC50: 0.85 μM) relative to GIRK1/GIRK2 (IC50: 2.6 μM) channels. Using whole-cell electrophysiological assessments, we did not detect a significant channel subtype–dependent difference in VU0468554 potency in the inhibition of M2R-activated responses. We did, however, detect a difference in the magnitude of inhibition of maximal CCh-induced responses between GIRK-channel subtypes expressed in HEK cells. Indeed, VU0468554 blocked nearly all of the M2R-induced response in HEK cells expressing GIRK1/GIRK4 channels but only about 50% of the response in GIRK1/GIRK2-expressing cells. Similar observations were made in primary cultures of SAN cells and hippocampal neurons, with VU0468554 exhibiting a significantly stronger blockade of receptor-induced GIRK currents in SAN cells (∼70%) as compared with neurons (20%). The apparent discrepancies in relative VU0468554 potency for GIRK1/GIRK2- and GIRK1/GIRK4-channel subtypes measured using thallium flux and whole-cell recording assays could be attributable to differences between inhibition of basal activity versus G protein–coupled receptor–activated GIRK-channel activity.

VU0468554 is structurally related to ML297, a direct Gβγ-independent activator of GIRK1-containing GIRK channels (Kaufmann et al., 2013; Wydeven et al., 2014). The initial structure-activity investigation involving VU0468554 and related compounds showed that they did not act on GIRK1-lacking GIRK channels (Wen et al., 2013). Site-directed mutagenesis revealed two GIRK1 residues, one in the pore helix and the other in the second membrane-spanning domain, that are necessary for the ML297-induced activation of GIRK1-containing GIRK channels (Wydeven et al., 2014). Here, we showed that VU0468554 did not impact holding current in recordings in SAN cells from Girk1–/– mice (Fig. 2, D and E). Although this finding supports the contention that VU0468554 is a selective inhibitor of GIRK1-lacking GIRK channels, further investigation of the impact of VU0468554 on this subcategory of GIRK channels is warranted.

VU0468544 provoked a rightward shift in the ML297 concentration-response relationship for GIRK-channel activation in SAN cells, as expected for a competitive inhibitor. We also observed a significant reduction in the amplitude of CCh-induced responses, as expected for a noncompetitive inhibitor of Gβγ-dependent GIRK-channel activation. Surprisingly, however, we also observed a significant reduction in CCh potency in the presence of VU0468554, suggesting that VU0468554 exhibits uncompetitive inhibition, in which inhibition is dependent on the formation of a substrate-bound complex (Ring et al., 2014). This phenomenon could be explained if the binding of the GIRK channel to Gβγ is required for its inhibition by VU0468554. Indeed, some of the basal GIRK–channel activity measured in heterologous systems and native cell types is Gβγ-dependent (Rishal et al., 2005; Rubinstein et al., 2007). Therefore, it is possible that VU0468554 inhibition of basal GIRK–channel activity by VU0468554 may depend on the formation of a GIRK:Gβγ complex.

Previous work has explored the potential of GIRK-channel inhibitors for the treatment of cardiac arrhythmias. Tertiapin, a bee venom peptide that is relatively selective for GIRK channels (Jin et al., 1999; Jin and Lu, 1999), completely reversed CCh-induced currents in SAN cells from wild-type mice (Anderson et al., 2020), and it suppressed atrial tachyarrhythmias in canines after pacing-induced cardiac remodeling (Cha et al., 2006). NIP-142, which inhibits GIRK channels at low concentrations (EC50 = 0.64 μM) and hERG channels at substantially higher concentrations (EC50 = 44 μM), reversed CCh- and adenosine-induced shortening of APD (Matsuda et al., 2006) and was shown to terminate and prevent reinitiation of AF and atrial flutter in atrial canine preparations (Nagasawa et al., 2002). Here, we show that although VU0468554 strongly inhibited CCh-induced GIRK-channel activity in isolated SAN cells, it only partially reversed (15%–20%) CCh-induced bradycardia in the isolated mouse heart model. This apparent discrepancy could be due to the fact that although GIRK-channel activation contributes to the bradycardic effect of muscarinic receptor activation in the heart (Mesirca et al., 2013; Posokhova et al., 2013), other effectors, including the cAMP-dependent "funny" current (If) and voltage-gated Ca2+ channels, also play a role (DiFrancesco et al., 1989; Mangoni et al., 2003; Kozasa et al., 2018). The significantly blunted response in the Girk4–/– mouse, however, indicates that the contribution of other effectors under the recording conditions is small. The relatively modest inhibition of GIRK-channel activity by VU0468554 in the isolated heart model likely also reflects the poor aqueous solubility of the compound and the consequent inability to test higher concentrations in this model. Indeed, we were unable to fully resuspend the compound in aqueous solutions at concentrations higher than 10 μM.

GIRK channels are also expressed at relatively low levels in ventricular tissue (Dobrzynski et al., 2001, 2002; Yang et al., 2010; Anderson et al., 2018), and there is evidence that they mediate, in part, muscarinic and adenosine receptor shortening of APD and effective refractory period (Liang et al., 2014; Anderson et al., 2018). Notably, a GIRK4/KCNJ5 loss-of-function mutation was identified in human LQT13, a ventricular repolarization disorder (Yang et al., 2010; Wang et al., 2013a). Recent work, however, has indicated that a loss of GIRK-channel function does not appear to increase ventricular arrhythmia susceptibility (Anderson et al., 2018). Therefore, therapeutic interventions aimed at suppressing GIRK-channel activity for the treatment of atrial arrhythmias could represent a safer alternative to therapies that predispose patients to ventricular dysfunction.

Given the medical significance of AF and other supraventricular arrhythmias and the limitations of existing therapeutic approaches (Mankad and Kalahasty, 2019), it is critical to pursue multiple therapeutic avenues. VU0468554 represents a new structural class of cardiac GIRK–channel inhibitor with a promising pharmacodynamic profile. Indeed, the relative selectivity of VU0468554 for cardiac GIRK channels is important because inhibition of neuronal GIRK channels could provoke seizures or have undesirable neurologic effects (Signorini et al., 1997; Lujan et al., 2014). Of relevance to the potential neurologic effects of GIRK-channel inhibitors, the GIRK-channel inhibitor BMS914392 (NTC-801) was tested in human patients with AF and did not significantly reduce AF burden. This apparent lack of therapeutic efficacy, however, was likely due to the fact that the dose employed was lowered substantially to avoid neurologic side effects noted previously for this compound (Machida et al., 2011; Yamamoto et al., 2014; Podd et al., 2016).

In conclusion, VU0468554 represents a new structural class of GIRK-channel inhibitor that holds promise as a lead compound for development of selective cardiac GIRK–channel inhibitors. Next-generation agents with improved solubility could prove useful not only as being a new pharmacological tool for studying GIRK channels but also having therapeutic implications in relevant cardiac arrhythmia settings.

Acknowledgments

The authors would like to thank Courtney Wright for taking exceptional care of the mouse colony.

Authorship Contributions

Participated in research design: Anderson, Vo, de Velasco, Wickman.

Conducted experiments: Anderson, Vo.

Contributed new reagents or analytic tools: Hopkins, Weaver.

Performed data analysis: Anderson, Vo.

Wrote or contributed to the writing of the manuscript: Anderson, Vo, de Velasco, Hopkins, Weaver, Wickman.

Footnotes

    • Received May 3, 2021.
    • Accepted September 1, 2021.

  • This work was supported by National Institutes of Health (NIH) National Heart, Lung, and Blood Institute [Grant RO1 HL105550], National Institute on Drug Abuse [Grant R01 DA034696], and National Institute on Alcohol Abuse and Alcoholism [Grant R01 AA027544] (to K.W.); NIH National Heart, Lung, and Blood Institute [Grant F31 HL139090] (to A.A.); and NIH National Institute of Mental Health [Grant R01 MH107399] (to K.W., C.R.H., and C.D.W.).

  • The authors declare no conflicts of interest.

  • Part of this work was presented as follows: Anderson A (2020) Physiological contribution and molecular details of GIRK-dependent signaling in the heart. Department of Pharmacology, University of Minnesota, Minneapolis, MN.

  • https://doi.org/10.1124/molpharm.121.000311.

Abbreviations

AF
atrial fibrillation
APD
action potential duration
CCh
carbachol
GIRK
G protein–gated inwardly rectifying K+
HEK
human embryonic kidney
HR
heart rate
M2R
M2 muscarinic receptor
SAN
sinoatrial node

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A New Inhibitor of Cardiac GIRK Channels

Allison Anderson, Baovi N. Vo, Ezequiel Marron Fernandez de Velasco, Corey R. Hopkins, C. David Weaver and Kevin Wickman
Molecular Pharmacology December 1, 2021, 100 (6) 540-547; DOI: https://doi.org/10.1124/molpharm.121.000311
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