Thrombin induces a temporal biphasic vascular response through the differential phosphorylation of endothelial nitric oxide synthase via protease-activated receptor-1 and protein kinase C.

Endothelial nitric oxide synthase (eNOS) is a critical regulatory enzyme that controls vascular tone via the production of nitric oxide. Although thrombin also modulates vascular tone predominantly via the activation of protease-activated receptors (PARs), the time course and mechanisms involved in how thrombin controls eNOS enzymatic activity are unknown. eNOS enzymatic activity is enhanced by the phosphorylation of eNOS-Ser1177 and reduced by the phosphorylation of eNOS-Thr495. In this study, we hypothesized that thrombin regulates vascular tone through the differential phosphorylation of eNOS. Using rat descending aorta, we show that thrombin modulates vascular tone in an eNOS-dependent manner via activated PAR-1. We also show that thrombin causes a temporal biphasic response. Protein kinase C (PKC) is associated with second phase of thrombin-induced response. Western blot analysis demonstrated thrombin phosphorylated eNOS-Ser1177 and eNOS-Thr495 in human umbilical vein endothelial cells. A PKC inhibitor suppressed the thrombin-induced phosphorylation of eNOS-Thr495, but not that of eNOS-Ser1177. Our results suggest that thrombin induces a temporal biphasic vascular response through the differential phosphorylation of eNOS via activated PAR-1. Thrombin causes transient vasorelaxation by the phosphorylation of eNOS-Ser1177, and subsequent attenuation of vasorelaxation by the phosphorylation of eNOS-Thr495 via PKC, leading to the modulation of vascular tone.

Novel Diagnostic Observations of Nodoventricular/Nodofascicular Pathway-Related Orthodromic Reciprocating Tachycardia Differentiating From Atrioventricular Nodal Re-Entrant Tachycardia.

OBJECTIVES: This study sought to assess the performance of current diagnostic criteria and identify additional electrophysiological features differentiating orthodromic reciprocating tachycardia (ORT) with a concealed nodoventricular/nodofascicular (NV/NF) pathway from atrioventricular nodal re-entrant tachycardia (AVNRT). BACKGROUND: Diagnosing sustained supraventricular tachycardia (SVT) despite the occurrence of ventriculoatrial block (VAB) is challenging. METHODS: We analyzed electrograms of 25 sustained SVTs (9 NV/NF-ORTs [n = 7/2] and 16 AVNRTs) with VAB and 91 AVNRTs without VAB (for reference). RESULTS: More than 1 SVT, each with a different ventriculoatrial interval, was commonly induced in AVNRT cases (75%) but not in NV/NF-ORT cases (0%; p = 0.0005). Wenckebach VAB was common in NV/NF-ORTs (78%), but VAB patterns varied in AVNRTs. The His-His interval transiently prolonged in the following beat after the VAB in most AVNRTs but rarely did in NV/NF-ORTs (79% vs. 22%; p = 0.01). NV/NF-ORT was diagnosed by His-refractory premature ventricular contractions (n = 5) and the findings during right ventricular overdrive pacing showing an uncorrected/corrected post-pacing interval (PPI)-tachycardia cycle length (TCL) ≤115/110 ms (n = 5/5), orthodromic His capture (n = 6), and V-V-A (ventricle-ventricle-atrial response) response (n = 3). A single form of induced SVT (positive predictive value [PPV]: 69%; negative predictive value [NPV]: 100%), Wenckebach VAB (PPV: 70%; NPV: 87%), stable His-His interval despite VAB (PPV: 70%; NPV: 85%), orthodromic His capture (PPV: 100%; NPV: 97%), and V-V-A response (PPV: 100%; NPV: 95%) characterized NV/NF-ORT, and a PPI-TCL of ≤125 ms (PPV: 100%; NPV: 100%) characterized NV-ORT. CONCLUSIONS: Induction of a single SVT form, Wenckebach VAB, stable His-His interval despite VAB, orthodromic His capture, and V-V-A response appeared to discriminate NV/NF-ORT from AVNRT, with a PPI-TCL of ≤125 ms discriminating NV-ORT from NF-ORT and AVNRT.

Different Effects of Pulmonary Vein Isolation on Quality of Life Between Patients with Persistent and Paroxysmal Atrial Fibrillation.

The effect of restoring sinus rhythm by pulmonary vein isolation (PVI) on the quality of life (QOL) of patients with persistent atrial fibrillation (PerAF) has not been adequately investigated. This study was performed to compare the changes in QOL after extended PVI between patients with PerAF and paroxysmal AF (PAF).Patients with PAF (n = 38) and PerAF (n = 22) who underwent their first PVI and developed no AF recurrence 6 months after PVI were enrolled. QOL surveys were performed at baseline and 6 months post-ablation using Short Form-36 surveys.The mental component summary score (MCS) (53.4 ± 10.2 to 56.5 ± 7.1, P = 0.019) and physical component summary score (PCS) (46.1 ± 10.6 to 48.5 ± 8.3, P = 0.015) improved after PVI in the PAF group. The PCS, but not the MCS, improved after PVI in the PerAF group (45.8 ± 7.9 to 51.5 ± 6.2, P < 0.001). Changes in the PCS were greater in the PerAF group than in the PAF group (8.6 ± 6.9 versus 2.8 ± 5.2, P = 0.009). Multivariate regression analysis demonstrated that a low baseline MCS and the type of AF (PAF) were independent predictors of an increased MCS and that a low baseline PCS and the type of AF (PerAF) were independent predictors of an increased PCS.The changes in QOL differed between PAF and PerAF after PVI. Although most patients with PerAF were asymptomatic before PVI, their improvement in physical QOL was greater than that in patients with PAF. Such beneficial effects on physical QOL are likely expected in patients with PerAF with a low PCS before PVI.

Pre-ablation levels of brain natriuretic peptide are independently associated with the recurrence of atrial fibrillation after radiofrequency catheter ablation in patients with nonvalvular atrial fibrillation.

Association between pre-ablation levels of biomarkers of cardiac and endothelial dysfunctions, CHADS2, CHA2DS2-VASc, and APPLE scores and the recurrence of atrial fibrillation (AF) after radiofrequency catheter ablation has not been fully studied. A total of 254 patients with nonvalvular AF were prospectively followed for AF recurrence after a single ablation procedure. During a two-year follow-up period, AF recurred in 65 (25.6%) patients. Patients with AF recurrence had significantly greater baseline ln brain natriuretic peptide (BNP) than those without AF recurrence (P < 0.01), whereas there were no significant differences in the levels of biomarkers of endothelial dysfunction and points of scoring systems. In the Cox regression analyses, the baseline ln BNP was significantly independently associated with AF recurrence (adjusted HR =1.286, 95% CI =1.000-1.655, P < 0.05). The baseline levels of ln BNP were significantly associated with rhythm at blood collection, age, sex, and left atrial diameter, and left ventricular ejection fraction (P < 0.05).The subgroup analysis showed a significant interaction on the risk of AF recurrence between ln BNP, sex difference, and rhythm at blood collection (P for interaction < 0.05). In conclusion, the results suggest that the pre-ablation levels of ln BNP are useful to evaluate the risk of AF recurrence after ablation therapy; however, there is a need to be careful while using BNP as a biomarker for the risk of AF recurrence by taking account of the effects of rhythm status at blood collection and sex difference.

M3 Muscarinic Receptor Signaling Stabilizes a Novel Mutant Human Ether-a-Go-Go-Related Gene Channel Protein via Phosphorylation of Heat Shock Factor 1 in Transfected Cells.

BACKGROUND: Long QT syndrome 2 (LQT2) is caused by mutations in the human ether-a-go-go-related gene (hERG). Most of its mutations give rise to unstable hERG proteins degraded by the proteasome. Recently, carbachol was reported to stabilize the wild-type hERG-FLAG via activation of the muscarinic type 3 receptor (M3-mAChR). Its action on mutant hERG-FLAG, however, remains uninvestigated.Methods and Results:A novel mutant hERG-FLAG carried 2 mutations: an amino acid substitution G572S and an in-frame insertion D1037_V1038insGD. When expressed in HEK293 cells, this mutant hERG-FLAG was degraded by the proteasome and failed to be transported to the cell surface. Carbachol restored stability of the mutant hERG-FLAG and facilitated cell-surface expression. Carbachol activated PKC, augmented phosphorylation of heat shock factor 1 (HSF1) and enhanced expression of heat shock proteins (hsps), hsp70 and hsp90. Both a M3-mAChR antagonist, 4-DAMP, and a PKC inhibitor, bisindolylmaleimide, abolished carbachol-induced stabilization of the mutant hERG-FLAG. CONCLUSIONS: M3-mAChR activation leads to enhancement of hsp expression via PKC-dependent phosphorylation of HSF1, thereby stabilizing the mutant hERG-FLAG protein. Thus, M3-mAChR activators may have a therapeutic value for patients with LQT2. (Circ J 2016; 80: 2443-2452).

Characterization of the novel mutant A78T-HERG from a long QT syndrome type 2 patient: Instability of the mutant protein and stabilization by heat shock factor 1.

BACKGROUND: The human ether-a-go-go-related gene (HERG) encodes the α-subunit of rapidly activating delayed-rectifier potassium channels. Mutations in this gene cause long QT syndrome type 2 (LQT2). In most cases, mutations reduce the stability of the channel protein, which can be restored by heat shock (HS). METHODS: We identified the novel mutant A78T-HERG in a patient with LQT2. The purpose of the current study was to characterize this mutant protein and test whether HS and heat shock factors (HSFs) could stabilize the mutant protein. A78T-HERG and wild-type HERG (WT-HERG) were expressed in HEK293 cells and analyzed by immunoblotting, immunoprecipitation, immunofluorescence, and whole-cell patch clamping. RESULTS: When expressed in HEK293 cells, WT-HERG gave rise to immature and mature forms of the protein at 135 and 155 kDa, respectively. A78T-HERG gave rise only to the immature form, which was heavily ubiquitinated. The proteasome inhibitor MG132 increased the expression of immature A78T-HERG and increased both the immature and mature forms of WT-HERG. WT-HERG, but not A78T-HERG, was expressed on the plasma membrane. In whole-cell patch clamping experiments, depolarizing pulses evoked E4031-sensitive HERG channel currents in cells transfected with WT-HERG, but not in cells transfected with A78T-HERG. The A78V mutant, but not A78G mutant, remained in the immature form similarly to A78T. Maturation of the A78T-HERG protein was facilitated by HS, expression of HSF-1, or exposure to geranyl geranyl acetone. CONCLUSIONS: A78T-HERG was characterized by protein instability and reduced expression on the plasma membrane. The stability of the mutant was partially restored by HSF-1, indicating that HSF-1 is a target for the treatment for LQT2 caused by the A78T mutation in HERG.

Impact of postprocedural antiarrhythmic drug therapy with bepridil on maintaining sinus rhythm after catheter ablation for persistent atrial fibrillation.

BACKGROUND: Although several studies have assessed the predictors of recurrent atrial fibrillation (AF) after catheter ablation for persistent AF, the impact of antiarrhythmic drug (AAD) therapy on maintaining sinus rhythm after catheter ablation for persistent AF has not been fully evaluated. This case-control study aimed to evaluate the effect of bepridil on maintaining sinus rhythm after catheter ablation for persistent AF. METHODS AND RESULTS: We enrolled 122 consecutive patients (87 men; mean age: 62.3 years) who underwent catheter ablation for persistent AF and were administered AAD therapy after the initial procedure. Restoration of sinus rhythm was achieved in all of the patients by catheter ablation and cardioversion after the initial procedure. After a median 12-month follow up, 51 of 122 (41.8%) patients had recurrence of AF. In Cox proportional hazard regression analysis, postprocedural AAD therapy with bepridil was a significantly correlated factor with freedom from recurrent AF after the initial ablation procedure (hazard ratio 0.446, 95% confidence interval 0.236-0.842, p=0.012). In Kaplan-Meier analysis, AF-free survival was significantly better with bepridil compared with amiodarone (AMD) and sodium channel blocker (SCB) (log-rank test, bepridil vs AMD, p=0.012; bepridil vs SCB, p=0.018). CONCLUSIONS: Bepridil reduced the recurrence of AF compared with AMD and SCB in patients who underwent catheter ablation for persistent AF.

Stabilization of Kv1.5 channel protein by the inotropic agent olprinone.

Olprinone is an inotropic agent that inhibits phosphodiesterase (PDE) III and causes vasodilation. Olprinone has been shown to be less proarrhythmic and possibly affect expression of functional Kv1.5 channels that confer the ultra-rapid delayed-rectifier K+ channel current (IKur) responsible for action potential repolarization. To reveal involvement of Kv1.5 channels in the less arrhythmic effect of olprinone, we examined effects of the agent on the stability of Kv1.5 channel proteins expressed in COS7 cells. Olprinone at 30-1000 nM increased the protein level of Kv1.5 channels in a concentration-dependent manner. Chase experiments showed that olprinone delayed degradation of Kv1.5 channels. Olprinone increased the immunofluorescent signal of Kv1.5 channels in the endoplasmic reticulum (ER) and Golgi apparatus as well as on the cell surface. Kv1.5-mediated membrane currents, measured as 4-aminopyridine-sensitive currents, were increased by olprinone without changes in their activation kinetics. A protein transporter inhibitor, colchicine, abolished the olprinone-induced increase of Kv.1.5-mediated currents. The action of olprinone was inhibited by 4-aminopyridine, and was not mimicked by the application of 8-Bromo-cAMP. Taken together, we conclude that olprinone stabilizes Kv1.5 proteins at the ER through an action as a chemical chaperone, and thereby increases the density of Kv1.5 channels on the cell membrane. The enhancement of Kv1.5 currents could underlie less arrhythmogenicity of olprinone.

Right- and left-sided carcinoid heart disease without intracardiac shunting.

We present a case of a 54-year-old man with carcinoid heart disease and mitral valve involvement. He had hepatic carcinoid with an extremely elevated urinary excretion of 5-hydroxyindole acetic acid and was referred to our division for shortness of breath and leg edema. Transthoracic echocardiography showed the thickened and retracted tricuspid valve leaflets and severe tricuspid regurgitation. Moderate mitral regurgitation was observed, with the involvement of mitral valve leaflets. A patent foramen ovale was not detected on transesophageal echocardiography. The extremely high concentration of circulating serotonin and/or other vasoactive substances may have contributed to left- as well as right-sided carcinoid heart disease.

Enhancing effects of salicylate on quinidine-induced block of human wild type and LQT3 related mutant cardiac Na+ channels.

It is unknown whether salicylate enhances the action of antiarrhythmic agents on human Na+ channels with state dependency and tissue specificity. We therefore investigated effects of salicylate on quinidine-induced block of human cardiac and skeletal muscle Na+ channels. Human cardiac wild-type (hH1), LQT3-related mutant (ΔKPQ), and skeletal muscle (hSkM1) Na+ channel α subunits were expressed in COS7 cells. Effects of salicylate on quinidine-induced tonic and use-dependent block of Na+ channel currents were examined by the whole-cell patch-clamp technique. Salicylate enhanced the quinidine-induced tonic and use-dependent block of both hH1 and hSkM1 currents at a holding potential (HP) of -100 mV but not at -140 mV. Salicylate decreased the IC50 value for the quinidine-induced tonic block of hH1 at an HP of -100 mV, and produced a negative shift in the steady-state inactivation curve of hH1 in the presence of quinidine. According to the modulated receptor theory, it is probable that salicylate decreases the dissociation constant for quinidine binding to inactivated-state channels. Furthermore, salicylate significantly enhanced the quinidine-induced tonic and use-dependent block of the peak and steady-state ΔKPQ channel currents. The results suggest that salicylate enhances quinidine-induced block of Na+ channels via increasing the affinity of quinidine to inactivated state channels.

Atrial defibrillation threshold as a novel predictor of clinical outcome of catheter ablation for persistent atrial fibrillation.

AIMS: Catheter ablation for persistent atrial fibrillation (AF) is currently performed with different procedural endpoints. When AF did not terminate during ablation procedure, electrical cardioversion was performed at different defibrillation threshold (DFT) according to AF characteristics and atrial electrophysiologic substrates. We sought to evaluate the impact of atrial DFT after catheter ablation for persistent AF on clinical outcome. METHODS AND RESULTS: We studied 128 patients with persistent AF (age 63±9 years, 106 men). After completion of circumferential pulmonary vein isolation, the left atrial substrate ablation was performed until AF terminated or all identified complex fractionated electrograms were eliminated. If AF did not terminate during ablation, an internal cardioversion protocol was started at 5J and was increased incrementally in 5 J steps until successful cardioversion was accomplished. Procedural AF termination was achieved in 50 patients (Group A). Atrial fibrillation was terminated by cardioversion with DFT≤10 J in 47 patients (Group B) and with DFT>10 J in 31 patients (Group C). At 14±7 follow-up months after 1.3±0.5 sessions, 47 (94%) Group A patients, 42 (89%) Group B patients, and 14 (45%) Group C patients remained in sinus rhythm. In multivariate analysis of Group B and Group C, DFT (hazard ratio 5.54, P<0.001) and AF duration (hazard ratio 3.74, P=0.011) were independent predictors of recurrent arrhythmia. CONCLUSION: When AF does not terminate after the completion of predetermined stepwise ablation, further extensive ablation to terminate AF might be unnecessary if the AF can be successfully terminated by electrical cardioversion at low DFT.

Exact location of the branching bundle in the living heart.

AIMS: The His bundle electrogram is believed to reflect the exact location of the His bundle. However, the distinction between distal His bundle potential and proximal right bundle branch potential is challenging. The aim of this study was to pinpoint the location of the branching point of the His bundle, and to compare that site with the site of recording of the largest His bundle electrogram (LH) during sinus rhythm. METHODS: We hypothesized that the site of earliest His activation (EH) during retrograde conduction via the left bundle branch is the branching point. We studied 15 nonconsecutive patients (mean age = 40 +/- 22 years; eight men). We performed a programmed stimulation from right ventricular apex until retrograde right bundle branch block appeared. At that point we measured (1) the distance between antegrade LH site and retrograde EH site and (2) the atrial-to-ventricular amplitude ratio (A/V ratio) at both sites. RESULTS: EH was recorded at the proximal electrode of the His bundle catheter in all patients. Mean distance between EH and LH was 9.8 +/- 2.5 mm. The mean A/V ratios at the EH site and the LH site were 1.01 +/- 0.42 and 0.08 +/- 0.06, respectively. DISCUSSION: This study showed that the EH site is located approximately 10-mm proximal to the LH site. The mean A/V ratio at the EH site during sinus rhythm is approximately 1.0. These observations suggest that the majority of His potentials reflect proximal right bundle activation. Before delivering radiofrequency energy in the para-Hisian area, attention should be paid to the presence of a His potential and to the A/V ratio, rather to the amplitude of the His electrogram.

Left atrial branches of coronary arteries; clinical implications related to linear catheter ablation for atrial fibrillation.

BACKGROUND: Coronary artery damage has been reported during catheter ablation procedures. Recently, linear ablation of thin left atrial tissue has been performed for atrial fibrillation. OBJECTIVE AND METHODS: Because we have little information about the arteries in the left atrium, this study was performed to evaluate the anatomy of these arteries, and to compare them with previously reported ablation lines. Coronary angiography was performed in 262 patients. Atrial coronary arteries between the left atrial appendage and the left superior pulmonary vein (LAA-LSPV region), as well as between the left inferior pulmonary vein and the mitral annulus ("mitral isthmus" region) were examined. RESULTS: Atrial coronary arteries extending to the LAA-LSPV region were found in 92 subjects (35%), while arteries crossing the mitral isthmus region were found in 119 subjects (46%). Atrial coronary arteries crossed the ablation line in about 69% of subjects overall. CONCLUSION: These results might suggest a risk of acute complications due to left atrial ablation. Alternatively, recurrence of atrial fibrillation might be caused by protected myocardium around the atrial arteries. We should note that atrial coronary arteries cross the ablation line in many patients.

QRS complex widening due to loss of left bundle branch capture: pitfall of para-Hisian pacing.

During para-Hisian pacing, widening of the paced QRS complex usually indicates loss of His bundle capture. We describe a patient without any accessory pathways in whom widening of the paced QRS complex occurred due to loss of left bundle branch capture during para-Hisian pacing. After initial widening of the QRS complex, further widening was observed due to loss of His bundle capture. With the initial QRS widening, the stimulus-atrial interval and retrograde atrial activation sequence were almost unchanged, so the findings mimicked retrograde conduction over an accessory pathway. This may be a pitfall of the para-Hisian pacing technique.

Long-term reliability of AAI mode pacing in patients with sinus node dysfunction and low Wenckebach block rate.

AIMS: To compare the risk of atrioventricular (AV) conduction disturbance between patients with sinus node dysfunction on AAI pacing who had a low or high Wenckebach block rate (WBR). METHODS AND RESULTS: Patients with sinus node dysfunction and normal AV conduction those underwent an electrophysiological study were studied. The patients were classified into two groups: Group L was with the patients with a WBR of 100 to 129 per minute and Group H was with the patients with a WBR > or = 130 per minute. All patients followed up every 3-6 months after an AAI pacemaker implantation. A total of 102 patients, including 35 Group L and 67 Group H, were followed for 90 +/- 44 months. Six patients died from non-cardiac cause and five patients required a new atrial lead implantation due to lead failure during follow-up. Symptomatic bradycardia requiring a new ventricular lead implantation developed in four patients (annual incidence 0.5%). In Group L, two patients developed AV block (annual incidence 0.7%). In Group H, two patients developed bradycardic atrial fibrillation (annual incidence 0.4%). Kaplan-Meier analysis revealed no significant difference between the two groups (P = 0.2983). CONCLUSION: These results suggest that a long-term risk of developing AV conduction disturbance is low even in patients with a WBR of 100 to 129 per minute.

Macroreentrant atrial tachycardia with an isolated pathway mimicking focal activation on three-dimensional electroanatomical mapping.

A 76-year-old man with two different sustained atrial arrhythmias that occurred after coronary artery bypass grafting underwent electrophysiological studies. Macroreentrant atrial tachycardias were detected with an isolated slow pathway mimicking focal activation on three-dimensional electroanatomical mapping. The slow conduction pathway in the right atrial free wall was assumed to represent tissue damaged by right atrial cannulation during previous coronary artery bypass grafting.

Inhibition of beta-adrenergic signaling by intracellular AMP is independent of cell-surface adenosine receptors in rat cardiac cells.

We report a novel action of intracellular adenosine monophosphate (AMP) to inhibit beta-adrenergic signaling in isolated rat ventricular myocytes. Extracellular application of adenosine or AMP suppressed isoproterenol (Iso)-induced prolongation of action potential duration (APD). This effect was completely abolished by an A(1)-receptor antagonist, DPCPX. Intracellular application of AMP, but not adenosine, attenuated Iso-induced APD prolongation. Iso-induced increases in the L-type Ca(2+) current (I(Ca,L)) were also inhibited by intracellular AMP. These inhibitory effects were not affected by either DPCPX or glibenclamide. In vitro, AMP directly inhibited PKA activity via binding to its regulatory subunit. These results suggest that intracellular AMP attenuates beta-adrenergic signaling by directly inhibiting PKA activity, independently of A(1)-purinergic receptor.

Radiofrequency catheter ablation for atrial tachycardia originating from the left atrial appendage.

A 36-year-old woman presented with drug-refractory atrial tachycardia. During the tachycardia episodes, P waves were positive in leads II, III, aVF, and V1, while they were negative in leads I and aVL. It was hard to determine whether the origin was the left atrial appendage or left superior pulmonary vein on the surface electrocardiogram. Electrophysiologic evaluation revealed that the earliest endocardial activation occurred at the base of the left atrial appendage, preceding the onset of P waves by 38 ms. On initiation of the tachycardia, a warm-up phenomenon was observed. There was a fixed relation between the coupling interval of a single extrastimulus and the return cycle length during the tachycardia. These findings suggested that the mechanism of the tachycardia was automaticity. Application of radiofrequency energy at the left atrial appendage terminated the tachycardia and it was not inducible after ablation.