Transient Outward Potassium Current Research Articles

A novel isoquinoline alkaloid HJ-69 isolated from Zanthoxylum bungeanum attenuates inflammatory pain by inhibiting voltage-gated sodium and potassium channels

Ethnopharmacology relevanceZanthoxylum bungeanum Maxim. (Z. bungeanum), a member of the Rutaceae family, has a rich history of traditional use in Asia for treating arthritis and toothache conditions. As characteristic chemical components, numerous kinds of alkaloids have been extracted from plants and their diverse biological activities have been reported. However, research on the isoquinoline alkaloid, a specific type of alkaloids, in Z. bungeanum was scarce. Aim of the studyThe study aimed to isolate a novel isoquinoline alkaloid from Z. bungeanum and explore its pharmacological activity in vitro and analgesic activity in vivo. Materials and methodsIsoquinoline alkaloid isolation and identification from Z. bungeanum were conducted using chromatographic and spectroscopic methods. The whole-cell patch-clamp technique was applied to assess its impact on neuronal excitability, and endogenous voltage-gated potassium (Kv) and sodium (Nav) currents in acutely isolated mouse small-diameter dorsal root ganglion (DRG) neurons. Its inhibitory impacts on channels were further validated with HEK293 cells stably expressing Nav1.7 and Nav1.8, and Chinese hamster ovary (CHO) cells transiently expressing Kv2.1. The formalin inflammatory pain model was utilized to evaluate the potential analgesic activity in vivo. ResultsA novel isoquinoline alkaloid named HJ-69 (N-13-(3-methoxyprop-1-yl)rutaecarpine) was isolated and identified from Z. bungeanum for the first time. HJ-69 significantly suppressed the firing frequency and amplitudes of action potentials in DRG neurons. Consistently, it state-dependently inhibited endogenous Nav currents of DRG neurons, with half maximal inhibitory concentration (IC50) values of 13.06 ± 2.06 μM and 30.19 ± 2.07 μM for the inactivated and resting states, respectively. HJ-69 significantly suppressed potassium currents in DRG neurons, which notably inhibited the delayed rectifier potassium (IK) currents (IC50 = 6.95 ± 1.29 μM) and slightly affected the transient outward potassium (IA) currents (IC50 = 523.50 ± 39.16 μM). Furtherly, HJ-69 exhibited similar potencies on heterologously expressed Nav1.7, Nav1.8, and Kv2.1 channels, which correspondingly represent the main components in neurons. Notably, intraperitoneal administration of 30 mg/kg and 100 mg/kg HJ-69 significantly alleviated pain behaviors in the mouse inflammatory pain model induced by formalin. ConclusionThe study concluded that HJ-69 is a novel and active isoquinoline alkaloid, and the inhibition of Nav and Kv channels contributes to its analgesic activity. HJ-69 may be a promising prototype for future analgesic drug discovery based on the isoquinoline alkaloid.

In vivo tebuconazole administration impairs heart electrical function and facilitates the occurrence of dobutamine-induced arrhythmias: involvement of reactive oxygen species

Tebuconazole (TEB), a widely used pesticide in agriculture to combat fungal infections, is commonly detected in global food, potable water, groundwater, and human urine samples. Despite its known in vivo toxicity, its impact on heart function remains unclear. In a 28-day study on male Wistar rats (approximately 100 g), administering 10 mg/kg/day TEB or a vehicle (control) revealed no effect on body weight gain or heart weight, but an increase in the infarct area in TEB-treated animals. Notably, TEB induced time-dependent changes in in vivo electrocardiograms, particularly prolonging the QT interval after 28 days of administration. Isolated left ventricular cardiomyocytes exposed to TEB exhibited lengthened action potentials and reduced transient outward potassium current. TEB also increased reactive oxygen species (ROS) production in these cardiomyocytes, a phenomenon reversed by N-acetylcysteine (NAC). Furthermore, TEB-treated animals, when subjected to an in vivo dobutamine (Dob) and caffeine (Caf) challenge, displayed heightened susceptibility to severe arrhythmias, a phenotype prevented by NAC. In conclusion, TEB at the no observed adverse effect level (NOAEL) dose adversely affects heart electrical function, increases arrhythmic susceptibility, partially through ROS overproduction, and this phenotype is reversible by scavenging ROS with NAC.

Inhibitory effect of aloperine on transient outward potassium currents in rat cardiac myocytes.

Aloperine (ALO) is an effective quinolizidine alkaloid. Previous research has demonstrated its antiarrhythmic effect by inhibiting voltage-gated sodium currents in rat ventricular myocytes. This study explored its effect on transient outward potassium currents (Ito) in rat atrial myocytes to identify potential targets in the context of ion channel currents. The Ito characteristics in rat atrial myocytes were recorded using a whole-cell patch-clamp technique. Molecular docking was performed to validate ligand-protein binding interactions. ALO at concentrations of 3 and 10μM significantly reduced Ito current densities. Gating kinetics analysis revealed ALO's ability to slow Ito activation, hasten inactivation, and prolong transition from inactive to resting state. Molecular docking revealed that ALO could stably bind to KCND2. ALO may inhibit Ito by slowing the activation process, accelerating inactivation, and delaying the recovery time after inactivation, potentially preventing acetylcholine-induced AF.

PTHrP attenuates spontaneous contractions in detrusor smooth muscle of the rat bladder by activating spontaneous transient outward potassium currents.

Parathyroid hormone-related protein (PTHrP) released from detrusor smooth muscle (DSM) cells upon bladder distension attenuates spontaneous phasic contractions (SPCs) in DSM and associated afferent firing to facilitate urine storage. Here, we investigate the mechanisms underlying PTHrP-induced inhibition of SPCs, focusing on large-conductance Ca2+-activated K+ channels (BK channels) that play a central role in stabilizing DSM excitability. Perforated patch-clamp techniques were applied to DSM cells of the rat bladder dispersed using collagenase. Isometric tension changes were recorded from DSM strips, while intracellular Ca2+ dynamics were visualized using Cal520AM-loaded DSM bundles. DSM cells developed spontaneous transient outward potassium currents (STOCs) arising from the opening of BK channels. PTHrP (10nM) increased the frequency of STOCs without affecting their amplitude at a holding potential of - 30mV but not - 40mV. PTHrP enlarged depolarization-induced, BK-mediated outward currents at membrane potentials positive to + 20mV in a manner sensitive to iberiotoxin (100nM), the BK channel blocker. The PTHrP-induced increases in BK currents were also prevented by inhibitors of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) (CPA 10µM), L-type voltage-dependent Ca2+ channel (LVDCC) (nifedipine 3µM) or adenylyl cyclase (SQ22536 100µM). PTHrP had no effect on depolarization-induced LVDCC currents. PTHrP suppressed and slowed SPCs in an iberiotoxin (100nM)-sensitive manner. PTHrP also reduced the number of Ca2+ spikes during each burst of spontaneous Ca2+ transients. In conclusion, PTHrP accelerates STOCs discharge presumably by facilitating SR Ca2+ release which prematurely terminates Ca2+ transient bursts resulting in the attenuation of SPCs.

Effect of electroacupuncture on myocardial electrical remodeling in rats with acute myocardial infarction.

To investigate the mechanism of electroacupuncture (EA) at "Neiguan" (PC6) in impro-ving myocardial electrical remodeling in rats with acute myocardial infarction (AMI) by enhancing transient outward potassium current. A total of 30 male SD rats were randomly divided into control, model and EA groups, with 10 rats in each group. The AMI model was established by subcutaneous injection with isoprenaline (ISO, 85 mg/kg). EA was applied to left PC6 for 20 min, once daily for 5 days. Electrocardiogram (ECG) was recorded after treatment. TTC staining was used to observe myocardial necrosis. HE staining was used to observe the pathological morphology of myocardial tissue and measure the cross-sectional area of myocardium. Potassium ion-related genes in myocardial tissue were detected by RNA sequencing. The mRNA and protein expressions of Kchip2 and Kv4.2 in myocardial tissue were detected by real-time fluorescence quantitative PCR and Western blot, respectively. Compared with the control group, cardiomyocyte cross-sectional area in the model group was significantly increased (P<0.01), the ST segment was significantly elevated (P<0.01), and QT, QTc, QTd and QTcd were all significantly increased (P<0.05, P<0.01). After EA treatment, cardiomyocyte cross-sectional area was significantly decreased (P<0.01), the ST segment was significantly reduced (P<0.01), and the QT, QTc, QTcd and QTd were significantly decreased (P<0.01, P<0.05). RNA sequencing results showed that a total of 20 potassium ion-related genes co-expressed by the 3 groups were identified. Among them, Kchip2 expression was up-regulated most notablely in the EA group. Compared with the control group, the mRNA and protein expressions of Kchip2 and Kv4.2 in the myocardial tissue of the model group were significantly decreased (P<0.01, P<0.05), while those were increased in the EA group (P<0.01, P<0.05). EA may improve myocardial electrical remodeling in rats with myocardial infarction, which may be related to its functions in up-regulating the expressions of Kchip2 and Kv4.2.

E3 ubiquitin ligase rififylin has yin and yang effects on rabbit cardiac transient outward potassium currents (Ito) and corresponding channel proteins

Genome-wide association studies (GWAS) have reported a correlation between a single nucleotide polymorphism of the RING finger E3 ubiquitin protein ligase rififylin (RFFL) and QT interval variability in humans (Newton-Cheh et al., 2009). Previously, we have shown that RFFL down-regulates expression and function of the human-like ether-a-go-go-related gene (hERG) potassium channel and corresponding rapidly activating delayed rectifier potassium current (IKr) in adult rabbit ventricular cardiomyocytes (ARbCMs). Here we report that RFFL also affects the transient outward current (Ito), but in a peculiar way. RFFL overexpression in ARbCMs significantly decreases the contribution of its fast component (Ito,f) from 35% to 21%, and increases the contribution of its slow component (Ito,s) from 65% to 79%. Since Ito,f in rabbits is mainly conducted by Kv4.3, we investigated the effect of RFFL on Kv4.3 expressed in HEK293A cells. We found that RFFL overexpression reduced Kv4.3 expression and corresponding Ito,f in a RING domain-dependent manner in the presence or absence of its accessory subunit KChIP2. On the other hand, RFFL overexpression in Kv1.4-expressing HEK cells leads to an increase in both Kv1.4 expression level and Ito,s, similarly in a RING domain-dependent manner. Our physiologically detailed rabbit ventricular myocyte computational model shows that these yin and yang effects of RFFL overexpression on Ito,f, and Ito,s affect phase 1 of the action potential waveform and slightly decrease its duration in addition to suppressing IKr. Thus, RFFL modifies cardiac repolarization reserve via ubiquitination of multiple proteins that differently affect various potassium channels and cardiac action potential duration (APD).

Correction of Ito in human induced pluripotent stem Cell–derived cardiomyocyte carrying DPP6 mutation in early repolarization syndrome by CRISPR/Cas9 genome editing

Early repolarization syndrome (ERS) is defined as occurring in patients with early repolarization pattern who have survived idiopathic ventricular fibrillation with clinical evaluation unrevealing for other explanations. The pathophysiologic basis of the ERS is currently uncertain. The objective of the present study was to examine the electrophysiological mechanism of ERS utilizing induced pluripotent stem cells (iPSCs) and CRISPR/Cas9 genome editing. Whole genome sequencing was used to identify the DPP6 (c.2561T > C/p.L854P) variant in four families with sudden cardiac arrest induced by ERS. Cardiomyocytes were generated from iPSCs from a 14-year-old boy in the four families with ERS and an unrelated healthy control subject. Patch clamp recordings revealed more significant prolongation of the action potential duration (APD) and increased transient outward potassium current (Ito) (103.97 ± 18.73 pA/pF vs 44.36 ± 16.54 pA/pF at +70 mV, P < 0.05) in ERS cardiomyocytes compared with control cardiomyocytes. Of note, the selective correction of the causal variant in iPSC-derived cardiomyocytes using CRISPR/Cas9 gene editing normalized the Ito, whereas prolongation of the APD remained unchanged. ERS cardiomyocytes carrying DPP6 mutation increased Ito and lengthen APD, which maybe lay the electrophysiological foundation of ERS.

Acute antiarrhythmic effects of SGLT2 inhibitors–dapagliflozin lowers the excitability of atrial cardiomyocytes

In recent years, SGLT2 inhibitors have become an integral part of heart failure therapy, and several mechanisms contributing to cardiorenal protection have been identified. In this study, we place special emphasis on the atria and investigate acute electrophysiological effects of dapagliflozin to assess the antiarrhythmic potential of SGLT2 inhibitors. Direct electrophysiological effects of dapagliflozin were investigated in patch clamp experiments on isolated atrial cardiomyocytes. Acute treatment with elevated-dose dapagliflozin caused a significant reduction of the action potential inducibility, the amplitude and maximum upstroke velocity. The inhibitory effects were reproduced in human induced pluripotent stem cell-derived cardiomyocytes, and were more pronounced in atrial compared to ventricular cells. Hypothesizing that dapagliflozin directly affects the depolarization phase of atrial action potentials, we examined fast inward sodium currents in human atrial cardiomyocytes and found a significant decrease of peak sodium current densities by dapagliflozin, accompanied by a moderate inhibition of the transient outward potassium current. Translating these findings into a porcine large animal model, acute elevated-dose dapagliflozin treatment caused an atrial-dominant reduction of myocardial conduction velocity in vivo. This could be utilized for both, acute cardioversion of paroxysmal atrial fibrillation episodes and rhythm control of persistent atrial fibrillation. In this study, we show that dapagliflozin alters the excitability of atrial cardiomyocytes by direct inhibition of peak sodium currents. In vivo, dapagliflozin exerts antiarrhythmic effects, revealing a potential new additional role of SGLT2 inhibitors in the treatment of atrial arrhythmias.

MicroRNA-1 Deficiency Is a Primary Etiological Factor Disrupting Cardiac Contractility and Electrophysiological Homeostasis.

MicroRNA-1 (miR1), encoded by the genes miR1-1 and miR1-2, is the most abundant microRNA in the heart and plays a critical role in heart development and physiology. Dysregulation of miR1 has been associated with various heart diseases, where a significant reduction (>75%) in miR1 expression has been observed in patient hearts with atrial fibrillation or acute myocardial infarction. However, it remains uncertain whether miR1-deficiency acts as a primary etiological factor of cardiac remodeling. miR1-1 or miR1-2 knockout mice were crossbred to produce 75%-miR1-knockdown (75%KD; miR1-1+/-:miR1-2-/- or miR1-1-/-:miR1-2+/-) mice. Cardiac pathology of 75%KD cardiomyocytes/hearts was investigated by ECG, patch clamping, optical mapping, transcriptomic, and proteomic assays. In adult 75%KD hearts, the overall miR1 expression was reduced to ≈25% of the normal wild-type level. These adult 75%KD hearts displayed decreased ejection fraction and fractional shortening, prolonged QRS and QT intervals, and high susceptibility to arrhythmias. Adult 75%KD cardiomyocytes exhibited prolonged action potentials with impaired repolarization and excitation-contraction coupling. Comparatively, 75%KD cardiomyocytes showcased reduced Na+ current and transient outward potassium current, coupled with elevated L-type Ca2+ current, as opposed to wild-type cells. RNA sequencing and proteomics assays indicated negative regulation of cardiac muscle contraction and ion channel activities, along with a positive enrichment of smooth muscle contraction genes in 75%KD cardiomyocytes/hearts. miR1 deficiency led to dysregulation of a wide gene network, with miR1's RNA interference-direct targets influencing many indirectly regulated genes. Furthermore, after 6 weeks of bi-weekly intravenous tail-vein injection of miR1 mimics, the ejection fraction and fractional shortening of 75%KD hearts showed significant improvement but remained susceptible to arrhythmias. miR1 deficiency acts as a primary etiological factor in inducing cardiac remodeling via disrupting heart regulatory homeostasis. Achieving stable and appropriate microRNA expression levels in the heart is critical for effective microRNA-based therapy in cardiovascular diseases.

Inhibition of Cardiac Kv4.3/KChIP2 Channels by Sulfonylurea Drug Gliquidone.

The Kv4.3 channel features fast N-type inactivation and also undergoes a slow C-type inactivation. The gain-of-function mutations of Kv4.3 channels cause an inherited disease called Brugada syndrome (BrS), characterized by a shortened duration of cardiac action potential repolarization and ventricular arrhythmia. The sulfonylurea drug gliquidone, an ATP-dependent K+ channel antagonist, is widely used for the treatment of type 2 diabetes. Here, we report a novel role of gliquidone in inhibiting Kv4.3 and Kv4.3/KChIP2 channels that encode the cardiac transient outward K+ currents responsible for the initial phase of action potential repolarization. Gliquidone results in concentration-dependent inhibition of both Kv4.3 and Kv4.3/KChIP2 fast or steady-state inactivation currents with an IC50 of approximately 8 μM. Gliquidone also accelerates Kv4.3 channel inactivation and shifts the steady-state activation to a more depolarizing direction. Site-directed mutagenesis and molecular docking reveal that the residues S301 in the S4 and Y312A and L321A in the S4-S5 linker are critical for gliquidone-mediated inhibition of Kv4.3 currents, as mutating those residues to alanine significantly reduces the potency for gliquidone-mediated inhibition. Furthermore, gliquidone also inhibits a gain-of-function Kv4.3 V392I mutant identified in BrS patients in voltage- and concentration-dependent manner. Taken together, our findings demonstrate that gliquidone inhibits Kv4.3 channels by acting on the residues in the S4 and the S4-S5 linker. Therefore, gliquidone may hold repurposing potential for the therapy of Brugada syndrome. SIGNIFICANCE STATEMENT: We describe a novel role of gliquidone in inhibiting cardiac Kv4.3 currents and the channel gain-of-function mutation identified from patients with Brugada syndrome, suggesting its repurposing potential for therapy for the heart disease.

Abstract 11956: Arumenamide-787 Suppresses Arrhythmogenesis Associated With the J Wave Syndrome as Well as Hypothermia

Background: The J-wave syndromes (JWS), comprised of Brugada (BrS) and early repolarization syndromes (ERS), increase the risk of life-threatening ventricular arrhythmias in patients resulting in sudden cardiac death. There are limited therapeutic strategies to treat these life-threatening conditions. Present management is via an implantable cardioverter defibrillator and/or medication, including quinidine. However, these treatments may have serious side-effects. Thus, novel therapies are needed to treat JWS. In this study, we investigate the effects of ARumenamide-787 (AR-787) in suppressing the electrocardiographic and arrhythmic manifestations of JWS and hypothermia. Methods: We studied the effects of AR-787 on sodium current (I Na ) and the delayed-rectifier potassium current (I Kr ) in HEK-293 cells stably expressing the α- and β1-subunits of the cardiac (Na V 1.5) sodium channel or the hERG channel. In addition, we studied its effect on the transient outward potassium current (I to ) and calcium current (I Ca ) from dissociated canine ventricular myocytes, along with action potentials and ECG from coronary-perfused right (RV) and left (LV) ventricular wedge preparations. The I to agonist, NS5806, I Ca blocker, verapamil, and sodium current (I Na ) blocker, ajmaline, to induce the electrocardiographic and arrhythmic manifestations of JWS (prominent J waves/ST segment elevation, phase 2 reentry, and polymorphic VT/VF) in right and left canine ventricular wedge preparations. Results: AR-787 exerted pleiotropic effects on the cardiac ion channels. The predominant effect was an inhibition of I to and enhancement of I Na , with subtle effects to inhibit the I Kr and augment I Ca . AR-787 diminished the electrocardiographic J wave and prevented and/or suppressed all arrhythmic activity in canine RV and LV experimental models of BrS, ERS and hypothermia. We observed no arrhythmogenic effects of AR-787 on I Kr . Conclusions: Our findings point to AR-787 as a promising candidate for the pharmacologic treatment of JWS and hypothermia. AR-787 suppresses the electrocardiographic and arrhythmic manifestations of JWS and hypothermia without significant prolongation of the QT interval, as with the classic BrS drug, quinidine.

Abstract 16037: Vaping Associated Sudden Cardiac Arrest in a Young Healthy Patient

Introduction and Importance: While the use of vaping has increased significantly among young individuals, the effects of the various vape aerosol chemical contents on cardiac electrophysiological dynamics remain unknown. Case Presentation: A 22-year-old female with a history of energy vaping but no significant past medical history presented with cardiac arrest. Found to have no pulse, CPR was started and initial rhythm of ventricular tachycardia was obtained. Shock was administered with follow-up rhythm of ventricular fibrillation. Patient was shocked again and entered atrial fibrillation with rapid ventricular response. She later cardioverted into sinus tachycardia and was intubated. Transthoracic echocardiogram showed normal ejection fraction but was otherwise unremarkable. Patient was extubated on day four of hospitalization. Left heart catheterization and cardiac MRI were completely unremarkable. Toxicology, urine analysis, and troponins were all negative. Patient was discharged with metoprolol tartrate, and a life-vest with outpatient follow-ups for possible implantable cardioverter-defibrillator placement in the setting of negative work up for sudden cardiac arrest. Clinical Discussion: Vaping-induced sudden cardiac arrest may be attributed to a reduction in cardiac repolarization reserve. Exposure to vegetable glycerin and propylene glycol, substances present in nearly all vape products, have been found to incite arrhythmias and disrupt cardiac conduction in animals. Acrolein,a byproduct of glycerin, has also been found to induce arrhythmias due to autonomic dysfunction. Increased prevalence of ventricular arrhythmias have also been noted with exposure to aerosols containing nicotine-free propylene glycol (PG).This likely stems from the impact of PG aerosols on the transient outward potassium current, which plays a significant role in early cardiac repolarization. Conclusion: The effects of inhaling vape aerosols remain not fully understood. While there is a perceived notion that nicotine-free aerosols may not be harmful, that remains unclear.Further studies should be conducted to evaluate for pro-arrhythmogenic effects and autonomic dysfunction from the various chemical substances present in vape aerosols.

Abstract 17335: Novel Functional Genomic Studies in Scn1bb Variant Associated With Brugada Syndrome and the Ameliorative Effect of Acacetin

Background: Brugada syndrome (BrS) is a channelopathy associated with the development of life-threatening ventricular tachycardia and fibrillation (VT/VF). At present, pharmacological approaches to therapy are very limited. Objective: To evaluate the effect of an SCN1Bb variant and the effects of acacetin in experimental models of BrS. Methods: We evaluated the effect of acacetin in: 1) HEK293 cells expressing Kv4.3+ KChIP2 and Nav1.5+ SCN1B using patch clamp techniques (22 O C), 2) canine ventricular epicardial myocytes 3) coronary-perfused canine right (RV) preparations and 4) Langendorff-perfused canine whole-heart models of BrS in which the sodium channel blocker ajmaline or the calcium channel blocker verapamil, together with the transient outward current agonist NS5806 were used to provoke the arrhythmic phenotype. Results: Co-expression of SCN5A -WT+ KCND3-WT with KChIP2 + SCN1Bb -R214Q (variant associated with 3 cases of BrS and 1 case of sudden infant death (SIDS)), resulted in a 210% increase in total charge of the transient outward potassium current (I to ) as well as a 56.5% reduction in sodium channel current (I Na ) when compared to SCN1Bb-WT . In the absence of the mutation, acacetin inhibited I to with an IC 50 of 7.2±1.0 μM (n=3) in HEK cells, similar to the value obtained in native epicardial myocytes (6 μM). In the presence of the SCB1Bb mutant, IC 50 was dramatically reduced to 2.6±1 μM (n=5, p&lt;0.05) Mean ±SEM. In the wedge and whole-heart models of BrS, acacetin dose-dependently reduced the epicardial action potential notch recorded from the RV or RVOT. Acacetin (5-10 μM) totally suppressed the electrocardiographic and arrhythmic manifestation of BrS, regardless of the pharmacologic model used to induce the arrhythmic phenotype. All repolarization defects were reversed, resulting in marked diminution of J waves leading to total suppression of VT/VF. Conclusions: Our findings demonstrate the ability of an SCN1Bb variant to dramatically shift the balance of current in the early phases of the RV epicardial action potential by reducing I Na as well as augmenting I to , thus predisposing to the development of the BrS phenotype. Our observations also hold promise for acacetin as a safe and effective pharmacologic treatment for BrS and some cases of SIDS.

Cardiac-specific deletion of BRG1 ameliorates ventricular arrhythmia inmice with myocardial infarction.

Malignant ventricular arrhythmia (VA) after myocardial infarction (MI) is mainly caused by myocardial electrophysiological remodeling. Brahma-related gene 1 (BRG1) is an ATPase catalytic subunit that belongs to a family of chromatin remodeling complexes called Switch/Sucrose Non-Fermentable Chromatin (SWI/SNF). BRG1 has been reported as a molecular chaperone, interacting with various transcription factors or proteins to regulate transcription in cardiac diseases. In this study, we investigated the potential role of BRG1 in ion channel remodeling and VA after ischemic infarction. Myocardial infarction (MI) mice were established by ligating the left anterior descending (LAD) coronary artery, and electrocardiogram (ECG) was monitored. Epicardial conduction of MI mouse heart was characterized in Langendorff-perfused hearts using epicardial optical voltage mapping. Patch-clamping analysis was conducted in single ventricular cardiomyocytes isolated from the mice. We showed that BRG1 expression in the border zone was progressively increased in the first week following MI. Cardiac-specific deletion of BRG1 by tail vein injection of AAV9-BRG1-shRNA significantly ameliorated susceptibility to electrical-induced VA and shortened QTc intervals in MI mice. BRG1 knockdown significantly enhanced conduction velocity (CV) and reversed the prolonged action potential duration in MI mouse heart. Moreover, BRG1 knockdown improved the decreased densities of Na+ current (INa) and transient outward potassium current (Ito), as well as the expression of Nav1.5 and Kv4.3 in the border zone of MI mouse hearts and in hypoxia-treated neonatal mouse ventricular cardiomyocytes. We revealed that MI increased the binding among BRG1, T-cell factor 4 (TCF4) and β-catenin, forming a transcription complex, which suppressed the transcription activity of SCN5A and KCND3, thereby influencing the incidence of VA post-MI.

Phase 2 Re-Entry Without Ito: Role of Sodium Channel Kinetics in Brugada Syndrome Arrhythmias

BackgroundIn Brugada syndrome (BrS), phase 2 re-excitation/re-entry (P2R) induced by the transient outward potassium current (Ito) is a proposed arrhythmia mechanism; yet, the most common genetic defects are loss-of-function sodium channel mutations. ObjectivesThe authors used computer simulations to investigate how sodium channel dysfunction affects P2R-mediated arrhythmogenesis in the presence and absence of Ito. MethodsComputer simulations were carried out in 1-dimensional cables and 2-dimensional tissue using guinea pig and human ventricular action potential models. ResultsIn the presence of Ito sufficient to generate robust P2R, reducing sodium current (INa) peak amplitude alone only slightly potentiated P2R. When INa inactivation kinetics were also altered to simulate reported effects of BrS mutations and sodium channel blockers, however, P2R occurred even in the absence of Ito. These effects could be potentiated by delaying L-type calcium channel activation or increasing ATP-sensitive potassium current, consistent with experimental and clinical findings. INa-mediated P2R also accounted for sex-related, day and night–related, and fever-related differences in arrhythmia risk in BrS patients. ConclusionsAltered INa kinetics synergize powerfully with reduced INa amplitude to promote P2R-induced arrhythmias in BrS in the absence of Ito, establishing a robust mechanistic link between altered INa kinetics and the P2R-mediated arrhythmia mechanism.

Electrophysiological basis of cardiac arrhythmia in a mouse model of myotonic dystrophy type 1.

Introduction: Myotonic dystrophy type 1 (DM1) is a multisystemic genetic disorder caused by the increased number of CTG repeats in 3' UTR of Dystrophia Myotonia Protein Kinase (DMPK) gene. DM1 patients experience conduction abnormalities as well as atrial and ventricular arrhythmias with increased susceptibility to sudden cardiac death. The ionic basis of these electrical abnormalities is poorly understood. Methods: We evaluated the surface electrocardiogram (ECG) and key ion currents underlying the action potential (AP) in a mouse model of DM1, DMSXL, which express over 1000 CTG repeats. Sodium current (INa), L-type calcium current (ICaL), transient outward potassium current (Ito), and APs were recorded using the patch-clamp technique. Results: Arrhythmic events on the ECG including sinus bradycardia, conduction defects, and premature ventricular and atrial arrhythmias were observed in DMSXL homozygous mice but not in WT mice. PR interval shortening was observed in homozygous mice while ECG parameters such as QRS duration, and QTc did not change. Further, flecainide prolonged PR, QRS, and QTc visually in DMSXL homozygous mice. At the single ventricular myocyte level, we observed a reduced current density for Ito and ICaL with a positive shift in steady state activation of L-type calcium channels carrying ICaL in DMSXL homozygous mice compared with WT mice. INa densities and action potential duration did not change between DMSXL and WT mice. Conclusion: The reduced current densities of Ito, and ICaL and alterations in gating properties in L-type calcium channels may contribute to the ECG abnormalities in the DMSXL mouse model of DM1. These findings open new avenues for novel targeted therapeutics.

Naringin exerts antiarrhythmic effects by inhibiting channel currents in mouse cardiomyocytes.

Naringin, a flavonoid extracted from citrus plants, has a variety of biological effects. Studies have shown that increasing the consumption of flavonoid-rich foods can reduce the incidence of cardiac arrhythmia. Naringin has been reported to have beneficial cardiovascular effects and thus can be used to prevent cardiovascular diseases, but the electrophysiological mechanism through which it prevents arrhythmias has not been elucidated. This study was conducted to investigate the effect of naringin on the transmembrane ion channel currents in mouse ventricular myocytes and the antiarrhythmic effect of this compound on Langendorff-perfused mouse hearts. Action potentials (APs) and ionic currents were recorded in isolated ventricular myocytes using the whole-cell patch-clamp technique. Anemone toxin II (ATX II) and CaCl2 were used to induce early afterdepolarizations (EADs) and delayed afterdepolarizations (DADs), respectively. Electrocardiogram (ECG) recordings were conducted in Langendorff-perfused mouse hearts with a BL-420F biological signal acquisition and analysis system. At the cellular level, naringin shortened the action potential duration (APD) of ventricular myocytes and decreased the maximum depolarization velocity (Vmax) of APs.Naringin inhibited the L-type calcium current (ICa.L) and ATX II enhanced the late sodium current (INa.L) in a concentration-dependent manner with IC50 values of 508.5μmol/L (n=9) and 311.6μmol/L (n=10), respectively. In addition, naringin also inhibited the peak sodium current (INa·P) and delayed the rectifier potassium current (IK) and the transient outward potassium current (Ito). Moreover, naringin reduced ATX II-induced APD prolongation and EADs and had a significant inhibitory effect on CaCl2-induced DADs as well. At the organ level, naringin reduced the incidence of ventricular tachycardia (VT) and ventricular fibrillation (VF) induced by ATX II and shortened the duration of both in isolated hearts. Naringin can inhibit the occurrence of EADs and DADs at the cellular level; furthermore, it can inhibit INa.L, ICa.L, INa·P, IK, and Ito in ventricular myocytes. Naringin also inhibits arrhythmias induced by ATX II in hearts. By investigating naringin with this electrophysiological method for the first time, we determined that this flavonoid may be a multichannel blocker with antiarrhythmic effects.

Effect of Zhenwu Decoction on electrical remodeling of cardiomyocytes in heart failure via I_(to)/Kv channels

This study aimed to investigate the underlying mechanism of Zhenwu Decoction in the treatment of heart failure by regulating electrical remodeling through the transient outward potassium current(I_(to))/voltage-gated potassium(Kv) channels. Five normal SD rats were intragastrically administered with Zhenwu Decoction granules to prepare drug-containing serum, and another seven normal SD rats received an equal amount of distilled water to prepare blank serum. H9c2 cardiomyocytes underwent conventional passage and were treated with angiotensin Ⅱ(AngⅡ) for 24 h. Subsequently, 2%, 4%, and 8% drug-containing serum, simvastatin(SIM), and BaCl_2 were used to interfere in H9c2 cardiomyocytes for 24 h. The cells were divided into a control group [N, 10% blank serum + 90% high-glucose DMEM(DMEM-H)], a model group(M, AngⅡ + 10% blank serum + 90% DMEM-H), a low-dose Zhenwu Decoction-containing serum group(Z1, AngⅡ + 2% drug-containing serum of Zhenwu Decoction + 8% blank serum + 90% DMEM-H), a medium-dose Zhenwu Decoction-containing serum group(Z2, AngⅡ + 4% drug-containing serum of Zhenwu Decoc-tion + 6% blank serum + 90% DMEM-H), a high-dose Zhenwu Decoction-containing serum group(Z3, AngⅡ + 8% drug-containing serum of Zhenwu Decoction + 2% blank serum + 90% DMEM-H), an inducer group(YD, AngⅡ + SIM + 10% blank serum + 90% DMEM-H), and an inhibitor group(YZ, AngⅡ + BaCl_2 + 10% blank serum + 90% DMEM-H). The content of ANP in cell extracts of each group was detected by ELISA. The relative mRNA expression levels of ANP, Kv1.4, Kv4.2, Kv4.3, DPP6, and KChIP2 were detected by real-time quantitative PCR. The protein expression of Kv1.4, Kv4.2, Kv4.3, DPP6, and KChIP2 was detected by Western blot. I_(to) was detected by the whole cell patch-clamp technique. The results showed that Zhenwu Decoction at low, medium, and high doses could effectively reduce the surface area of cardiomyocytes. Compared with the M group, the Z1, Z2, Z3, and YD groups showed decreased ANP content and mRNA level, increased protein and mRNA expression of Kv4.2, Kv4.3, DPP6, and KChIP2, and decreased protein and mRNA expression of Kv1.4, and the aforementioned changes were the most notable in the Z3 group. Compared with the N group, the Z1, Z2, and Z3 groups showed significantly increased peak current and current density of I_(to). The results indicate that Zhenwu Decoction can regulate myocardial remodeling and electrical remodeling by improving the expression trend of Kv1.4, Kv4.2, Kv4.3, KChIP2, and DPP6 proteins and inducing I_(to) to regulate Kv channels, which may be one of the mechanisms of Zhenwu Decoction in treating heart failure and related arrhythmias.

Altered cardiac repolarization associated with cellular electrophysiological remodeling following chronic testosterone undecanoate administration in a canine model

Abstract Funding Acknowledgements Type of funding sources: None. Background The influence of testosterone on ventricular ion channels is widely investigated. Despite the legally stipulated availability of different androgen anabolic steroids (AAS), these agents are very popular among young adults, however, the direct effects of supra-physiological testosterone level are still unclear. Supposedly, the chronic use of AASs may result in structural and functional remodeling of the heart. Purpose The aim of our study was to investigate the potential electrophysiological effects of chronic administration of testosterone-undecanoate in a canine model in in vivo and in vitro studies. Methods Eight male beagle dogs were randomized into control (’Cont’) and treated (’Tr’) groups (n = 4; n = 4). The latter group received 15 mg/kg of long-lasting testosterone-undecanoate intramuscular injections weekly for 3 months. Blood samples were taken for monitoring testosterone levels. To investigate the altered repolarization in conscious dogs electrocardiography studies were performed. Ventricular myocytes were enzymatically dissociated via retrograde perfusion. The transmembrane ionic currents were recorded using the whole-cell configuration of the patch-clamp technique and the action potential duration (APD) was measured by the perforated patch-clamp technique. Results Testosterone level was significantly higher in the ‘Tr’ group compared to the ‘Cont’ group (47.02 nm/L vs. 15.23 nm/L; p=0.0002). The chronic treatment did not affect the heart rate between the examined groups (93.7±23.5 vs.108.8±21.4 beats/min), however, it resulted in significantly shortened QT (226±49 vs. 244.9±26.6 ms; p&amp;lt;0.05) and QTc (26.06±2.5 vs. 29.6±3.01 ms, p&amp;lt;0.05) intervals. ECG recordings also presented prolonged PQ (112.1±15.5 vs. 61.65±12.7ms; p&amp;lt;0.05), QRS (72.37±15.4 vs. 61.65±12.7 ms; p&amp;lt;0.05), and Tp-Te (32.95±7.45 vs. 53.46±16.6 ms; p&amp;lt;0.05) intervals in the ‘Tr’ group. Additionally, the APD of isolated left ventricular myocytes significantly shortened in the ‘Tr’ group compared to the ‘Con’ group (235.2±26.7 vs. 283.6±28.5 ms; p&amp;lt;0.05). Patch-clamp experiments revealed increased magnitude of transient outward potassium current (Ito), the inward rectifier potassium current (Ik1), and the slowed delayed rectifier potassium current (Iks) in the ‘Tr’ group. Conclusion The repolarization of the canine ventricular myocardium was significantly modified by constantly high level of testosterone. Many beneficial effects are attributed to physiological testosterone levels; however, the supra-physiological testosterone level may lead to potentially harmful alternations in the cardiac repolarization that may promote arrhythmogenesis, especially in the presence of various heart diseases. Presumably, the constantly high testosterone level induced electrophysiological changes may contribute to the development of life-threatening arrhythmias under certain circumstances.

Amyloid-β Protein Precursor Regulates Electrophysiological Properties in the Hippocampus via Altered Kv1.4 Expression and Function in Mice.

Amyloid-β protein precursor (AβPP) is enriched in neurons. However, the mechanism underlying AβPP regulation of neuronal activity is poorly understood. Potassium channels are critically involved in neuronal excitability. In hippocampus, A-type potassium channels are highly expressed and involved in determining neuronal spiking. We explored hippocampal local field potential (LFP) and spiking in the presence and absence of AβPP, and the potential involvement of an A-type potassium channel. We used in vivo extracellular recording and whole-cell patch-clamp recording to determine neuronal activity, current density of A-type potassium currents, and western blot to detect changes in related protein levels. Abnormal LFP was observed in AβPP-/- mice, including reduced beta and gamma power, and increased epsilon and ripple power. The firing rate of glutamatergic neurons reduced significantly, in line with an increased action potential rheobase. Given that A-type potassium channels regulate neuronal firing, we measured the protein levels and function of two major A-type potassium channels and found that the post-transcriptional level of Kv1.4, but not Kv4.2, was significantly increased in the AβPP-/- mice. This resulted in a marked increase in the peak time of A-type transient outward potassium currents in both glutamatergic and gamma-aminobutyric acid-ergic (GABAergic) neurons. Furthermore, a mechanistic experiment using human embryonic kidney 293 (HEK293) cells revealed that the AβPP deficiency-induced increase in Kv1.4 may not involve protein-protein interaction between AβPP and Kv1.4. This study suggests that AβPP modulates neuronal firing and oscillatory activity in the hippocampus, and Kv1.4 may be involved in mediating the modulation.