Takeshi Shirayama

Klotho is associated with VEGF receptor-2 and the transient receptor potential canonical-1 Ca2+ channel to maintain endothelial integrity

Klotho is a circulating protein, and Klotho deficiency disturbs endothelial integrity, but the molecular mechanism is not fully clarified. We report that vascular endothelium in Klotho-deficient mice showed hyperpermeability with increased apoptosis and down-regulation of vascular endothelial (VE)-cadherin because of an increase in VEGF-mediated internal calcium concentration ([Ca2+]i) influx and hyperactivation of Ca2+-dependent proteases. Immunohistochemical analysis, the pull-down assay using Klotho-fixed agarose, and FRET confocal imaging confirmed that Klotho protein binds directly to VEGF receptor 2 (VEGFR-2) and endothelial, transient-receptor potential canonical Ca2+ channel 1 (TRPC-1) and strengthens the association to promote their cointernalization. An in vitro mutagenesis study revealed that the second hydrolase domain of Klotho interacts with sixth and seventh Ig domains of VEGFR-2 and the third extracellular loop of TRPC-1. In Klotho-deficient endothelial cells, VEGF-mediated internalization of the VEGFR-2/TRPC-1 complex was impaired, and surface TRPC-1 expression increased 2.2-fold; these effects were reversed by supplementation of Klotho protein. VEGF-mediated elevation of [Ca2+]i was sustained at higher levels in an extracellular Ca2+-dependent manner, and normalization of TRCP-1 expression restored the abnormal [Ca2+]i handling. These findings provide evidence that Klotho protein is associated with VEGFR-2/TRPC-1 in causing cointernalization, thus regulating TRPC-1–mediated Ca2+ entry to maintain endothelial integrity.

Adenosine triphosphate-guided pulmonary vein isolation for atrial fibrillation: the UNmasking Dormant Electrical Reconduction by Adenosine TriPhosphate (UNDER-ATP) trial.

AIMS Most of recurrent atrial tachyarrhythmias after pulmonary vein isolation (PVI) for atrial fibrillation (AF) are due to reconnection of PVs. The aim of the present study was to evaluate whether elimination of adenosine triphosphate (ATP)-induced dormant PV conduction by additional energy applications during the first ablation procedure could reduce the incidence of recurrent atrial tachyarrhythmias. METHODS AND RESULTS We randomly assigned 2113 patients with paroxysmal, persistent, or long-lasting AF to either ATP-guided PVI (1112 patients) or conventional PVI (1001 patients). The primary endpoint was recurrent atrial tachyarrhythmias lasting for >30 s or those requiring repeat ablation, hospital admission, or usage of Vaughan Williams class I or III antiarrhythmic drugs at 1 year with the blanking period of 90 days post ablation. Among patients assigned to ATP-guided PVI, 0.4 mg/kg body weight of ATP provoked dormant PV conduction in 307 patients (27.6%). Additional radiofrequency energy applications successfully eliminated dormant conduction in 302 patients (98.4%). At 1 year, 68.7% of patients in the ATP-guided PVI group and 67.1% of patients in the conventional PVI group were free from the primary endpoint, with no significant difference (adjusted hazard ratio [HR] 0.89; 95% confidence interval [CI] 0.74-1.09; P = 0.25). The results were consistent across all the prespecified subgroups. Also, there was no significant difference in the 1-year event-free rates from repeat ablation for any atrial tachyarrhythmia between the groups (adjusted HR 0.83; 95% CI 0.65-1.08; P = 0.16). CONCLUSION In the catheter ablation for AF, we found no significant reduction in the 1-year incidence of recurrent atrial tachyarrhythmias by ATP-guided PVI compared with conventional PVI.

Efficacy of Antiarrhythmic Drugs Short-Term Use After Catheter Ablation for Atrial Fibrillation (EAST-AF) trial

AIMS Substantial portion of early arrhythmia recurrence after catheter ablation for atrial fibrillation (AF) is considered to be due to irritability in left atrium (LA) from the ablation procedure. We sought to evaluate whether 90-day use of antiarrhythmic drug (AAD) following AF ablation could reduce the incidence of early arrhythmia recurrence and thereby promote reverse remodelling of LA, leading to improved long-term clinical outcomes. METHODS AND RESULTS A total of 2038 patients who had undergone radiofrequency catheter ablation for paroxysmal, persistent, or long-lasting AF were randomly assigned to either 90-day use of Vaughan Williams class I or III AAD (1016 patients) or control (1022 patients) group. The primary endpoint was recurrent atrial tachyarrhythmias lasting for >30 s or those requiring repeat ablation, hospital admission, or usage of class I or III AAD at 1 year, following the treatment period of 90 days post ablation. Patients assigned to AAD were associated with significantly higher event-free rate from recurrent atrial tachyarrhythmias when compared with the control group during the treatment period of 90 days [59.0 and 52.1%, respectively; adjusted hazard ratio (HR) 0.84; 95% confidence interval (CI) 0.73-0.96; P = 0.01]. However, there was no significant difference in the 1-year event-free rates from the primary endpoint between the groups (69.5 and 67.8%, respectively; adjusted HR 0.93; 95% CI 0.79-1.09; P = 0.38). CONCLUSION Short-term use of AAD for 90 days following AF ablation reduced the incidence of recurrent atrial tachyarrhythmias during the treatment period, but it did not lead to improved clinical outcomes at the later phase.

Kinetics of frequency-dependent conduction delay by class I antiarrhythmic drugs in human atrium.

We investigated use-dependent prolongation of interatrial conduction time (IACT) by class I antiar-rhythmic drugs in 16 patients. Changes in IACT at the initiation of atrial pacing were used to evaluate the onset kinetics. We examined recovery kinetics by giving a single extra stimulus with a varying coupling interval after discontinuing train stimulation. Time constants of the onset kinetics were 1.52 ± 0.15/n(fast) and 0.087 ± 0.031/ n(slow) for mexiletine, 0.075 ± 0.015/n for aprindine, 0.078 ± 0.019/n for disopyramide, and 0.050 ± 0.006/n for pilsicainide. The recovery time constants were 203 ± 66 ms for mexiletine, 1,021 ± 162 ms for aprindine, 993 ± 101 ms for disopyramide, and 2,930 ± 569 ms for pilsicainide. Class I antiarrhythmic drugs produced use-dependent IACT prolongation in humans, with characteristic kinetics for each agent similar to that of depression of the maximum upstroke velocity of cardiac action potential (Vmax) reported in in vitro studies.

Prolonged atrial activity due to delayed conduction in the atrium of patients with paroxysmal atrial fibrillation

SummaryWe investigated the relationship between the duration of electrical atrial activity and intra-atrial conduction time to determine whether the prolonged atrial activity was due to delayed conduction in the human atrium. The study included 15 patients with paroxysmal atrial fibrillation (PAF) and 15 control patients. The duration of atrial electrical activity was measured by selecting a minimum electrographic amplitude of 50µV. In patients with PAF, the duration of atrial activity was prolonged in proportion to the delay of interatrial conduction time from the high right atrium to the coronary sinus as the coupling interval of premature extrastimuli was decreased. Both the fragmented atrial activity zone and the interatrial conduction delay zone were wider in patients with PAF than in control patients. It is concluded that assessment of the duration of atrial activity with a minimum amplitude of 50µV is useful in evaluating human atrial vulnerability since it reflects the atrial conduction delay in patients with PAF.

Nonsense-mediated mRNA decay due to a CACNA1C splicing mutation in a patient with Brugada syndrome

BACKGROUND Brugada syndrome (BrS) is an inherited cardiac arrhythmia associated with sudden death due to ventricular fibrillation. Mutations in genes related to the cardiac L-type calcium channel have been reported to be causative of BrS. Generally, the messenger RNA (mRNA) that contains a nonsense mutation is rapidly degraded via its decay pathway, which is known as nonsense-mediated mRNA decay (NMD). Previously, we reported a male patient with BrS who carried c.1896G>A (the first nucleotide of CACNA1C exon 14), which caused a synonymous mutation, p.R632R. OBJECTIVE To examine how the synonymous CACNA1C mutation p.R632R produces the phenotype of BrS, with a special emphasis on the splicing error and NMD processes. METHODS We extracted mRNA from leukocytes of the proband and his 2 children and performed reverse transcription polymerase chain reaction. Complementary DNAs were checked by using direct sequencing and quantitative analysis. RESULTS The subsequent sequence electropherogram of the complementary DNAs did not show the substitution of the nucleotide identified in the genomic DNA of the proband. In the mRNA quantification analysis, we confirmed that reduction in the CACNA1C expression level was suspected to be caused by NMD. CONCLUSIONS Mutant mRNA with a c.1896G>A substitution may be diminished by NMD, and the resultant decrease in CACNA1C message leads to a novel mechanism for inducing BrS that is distinct from that reported previously.

Two Cases of Delayed Cardiac Tamponade due to Pericarditis after Pulmonary Vein (PV) Isolation for Atrial Fibrillation

Catheter ablation is an established treatment for atrial fibrillation (AF). The incidence of major complications related to the procedure is reported to be 4.5%, and delayed cardiac tamponade (DCT) is a rare, although recently recognized, complication. However, the mechanisms underlying the development of DCT remain unclear. We herein report the cases of two men, both 49 years of age, who developed cardiac tamponade requiring pericardiocentesis a few weeks after undergoing pulmonary vein isolation for persistent AF. Physicians should explain to the patient the potential for DCT as a complication prior to performing catheter ablation and provide careful follow-up for at least a few weeks after the session.

Altered expression of Na+ transporters at the mRNA level in rat normal and hypertrophic myocardium

Intracellular Na+ ([Na+]i) regulation plays a crucial role in the structural, mechanical, and electrical properties of myocardium. It is assumed that the [Na+]i handling system may differ not only between normal and diseased hearts but also regionally within a heart. To gain new insight concerning disease- and region-dependent differences in the [Na+]i-regulatory system, we investigated mRNA expression of Na+ transporters, the principal determinants of [Na+]i. Nonischemic pressure-overloaded hypertrophy was created by suprarenal abdominal aortic constriction of 50% for 7 weeks. mRNA abundances of Na+-Ca2+ exchanger (NCX1), Na+-H+ exchanger (NHE1), Na+-K+-2Cl− exchanger (NKCC1) and Na+, K+-ATPase multigene family(α1, α2, α3, and β1 isoforms) were measured by the real-time quantitative polymerase chain reaction method. mRNA abundance of all transporters mediating Na+ influx (NCX1, NHE1, and NKCC1) was significantly upregulated as compared to normal. In contrast, Na+-efflux-mediating transporter (Na+, K+-ATPase) mRNA expression was unaltered between normal and hypertrophic hearts. Losartan, an angiotensin II AT1 receptor antagonist, significantly attenuated upregulation of Na+-influx-mediating transporters induced by aortic constriction. The onset of Na+-influx-mediating transporter upregulation occurred within 5 days following constriction. In normal and hypertrophied hearts, mRNA of all Na+-influx-mediating transporters was expressed in order of abundance as: apex > septum ∼ free wall of left ventricles. A transmural gradient in expression was also evident in normal hearts (midcardium > endo- and epicardium), which was attenuated under hypertrophic development. Myocardial hypertrophy is associated with significant changes in the spatial distribution and expression levels of Na+ transporters. The upregulation of Na influx transporters during hypertrophy may contribute to the remodeling process, modulate contractility and promote arrhythmias.

Clinical Significance of the Atrial Fibrillation Threshold in Patients with Paroxysmal Atrial Fibrillation

INOUE, K., et al.: Clinical Significance of the Atrial Fibrillation Threshold in Patients with Paroxysmal Atrial Fibrillation. AF threshold and the other electrophysiological parameters were measured to quantify atrial vulnerability in patients with paroxysmal atrial fibrillation (PAF, n = 47), and those without AF (non‐PAF, n = 25). Stimulations were delivered at the right atrial appendage with a basic cycle length of 500 ms. The PAF group had a significantly larger percentage of maximum atrial fragmentation (%MAF, non‐PAF: mean ± SD = 149 ± 19%, PAF: 166 ± 26%, P = 0.009), fragmented atrial activity zone (FAZ, non‐PAF: median 0 ms, interquartile range 0–20 ms, PAF: 20 ms, 10–40 ms, P = 0.008). Atrial fibrillation threshold (AF threshold, non‐PAF: median 11 mA, interquartile range 6–21 mA, PAF: 5 mA, 3–6 mA, P < 0.001) was smaller in the PAF group than in the non‐PAF group. Sensitivity, specificity, and positive predictive value of electrophysiological parameters were as follows, respectively: %MAF (cut off at 150%, 78%, 52%, 76%), FAZ (cut off at 20 ms, 47%, 84%, 85%), AF threshold (cut off at 10 mA, 94%, 60%, 81%). There were no statistically significant differences between the non‐PAF and PAF groups in the other parameters (effective refractory period, interatrial conduction time, maximum conduction delay, conduction delay zone, repetitive atrial firing zone, wavelength index), that were not specific for PAF. In conclusion, the AF threshold could be a useful indicator to evaluate atrial vulnerability in patients with AF.

Effects of E-4031 on atrial fibrillation threshold in guinea pig atria: comparative study with class I antiarrhythmic drugs.

The effects of E-4031, a new class III antiarrhythmic agent, on atrial fibrillation threshold (AFT), atrial effective refractory period (ERP), and interatrial conduction time (ACT) were investigated in Langendorff-perfused guinea pig hearts; the results were then compared with those of the class I agents disopyramide, procainamide, lidocaine, and flecainide. Whole guinea pig hearts were perfused with Tyrodes solution containing acetylcholine (ACh 3 x 10(-7) M). The three indexes were measured before and after administration of the test drugs, using right atrial extrastimulus and 50-Hz continuous stimulation. Disopyramide, procainamide, and flecainide (> or = 10(-6) M) significantly increased AFT. Although E-4031 (> or = 3 x 10(-6) M) also increased AFT, this effect was less potent than that observed with the other drugs. E-4031 (> or = 10(-6) M) significantly prolonged ERP, and this prolongation was less pronounced than that observed with disopyramide but similar to that observed with procainamide or flecainide. E-4031 did not affect ACT, and the greatest prolongation of ACT was observed with flecainide. Lidocaine had no effect on any of the indexes. These findings suggest that in guinea pig hearts E-4031 exerts an antifibrillatory effect by prolonging atrial ERP alone, but this effect is less pronounced than that observed with class I drugs, because AFT measured by 50-Hz continuous stimulation is influenced by both ERP and ACT.