Translational Studies on Anti-Atrial Fibrillatory Action of Oseltamivir by its in vivo and in vitro Electropharmacological Analyses

Oseltamivir has been shown to prolong the atrial conduction time and effective refractory period, and to suppress the onset of burst pacing-induced atrial fibrillation in vitro. To better predict its potential clinical benefit as an anti-atrial fibrillatory drug, we performed translational studies by assessing in vivo anti-atrial fibrillatory effect along with in vivo and in vitro electropharmacological analyses. Oseltamivir in intravenous doses of 3 (n = 6) and 30 mg/kg (n = 7) was administered in conscious state to the persistent atrial fibrillation model dogs to confirm its anti-atrial fibrillatory action. The model was prepared by tachypacing to the atria of chronic atrioventricular block dogs for > 6 weeks. Next, oseltamivir in doses of 0.3, 3 and 30 mg/kg was intravenously administered to the halothane-anesthetized intact dogs to analyze its in vivo electrophysiological actions (n = 4). Finally, its in vitro effects of 10–1,000 μM on IK,ACh, IKur, IKr, INa and ICaL were analyzed by using cell lines stably expressing Kir3.1/3.4, KV1.5, hERG, NaV1.5 or CaV1.2, respectively (n = 3 for IK,ACh and IKr or n = 6 for IKr, INa and ICaL). Oseltamivir in doses of 3 and 30 mg/kg terminated the atrial fibrillation in 1 out of 6 and in 6 out of 7 atrial fibrillation model dogs, respectively without inducing any lethal ventricular arrhythmia. Its 3 and 30 mg/kg delayed inter-atrial conduction in a frequency-dependent manner, whereas they prolonged atrial effective refractory period in a reverse frequency-dependent manner in the intact dogs. The current assay indicated that IC50 values for IK,ACh and IKr were 160 and 231 μM, respectively, but 1,000 µM inhibited INa, ICaL and IKur by 22, 19 and 13%, respectively. The extent of INa blockade was enhanced at faster beating rate and more depolarized resting membrane potential. Oseltamivir effectively terminated the persistent atrial fibrillation, which may be largely due to the prolongation of the atrial effective refractory period and inter-atrial conduction time induced by IK,ACh and IKr inhibitions along with INa suppression. Thus, oseltamivir can exert a powerful anti-atrial fibrillatory action through its ideal multi-channel blocking property; and oseltamivir would become a promising seed compound for developing efficacious and safe anti-atrial fibrillatory drugs.

Effects of genetic background, sex, and age on murine atrial electrophysiology

Aims Genetically altered mice are powerful models to investigate mechanisms of atrial arrhythmias, but normal ranges for murine atrial electrophysiology have not been robustly characterized. Methods and results We analyzed results from 221 electrophysiological (EP) studies in isolated, Langendorff-perfused hearts of wildtype mice (114 female, 107 male) from 2.5 to 17.7 months (mean 7 months) with different genetic backgrounds (C57BL/6, FVB/N, MF1, 129/Sv, Swiss agouti). Left atrial monophasic action potential duration (LA-APD), interatrial activation time (IA-AT), and atrial effective refractory period (ERP) were summarized at different pacing cycle lengths (PCLs). Factors influencing atrial electrophysiology including genetic background, sex, and age were determined. LA-APD70 was 18 ± 0.5 ms, atrial ERP was 27 ± 0.8 ms, and IA-AT was 17 ± 0.5 ms at 100 ms PCL. LA-APD was longer with longer PCL (+17% from 80 to 120 ms PCL for APD70), while IA-AT decreased (−7% from 80 to 120 ms PCL). Female sex was associated with longer ERP (+14% vs. males). Genetic background influenced atrial electrophysiology: LA-APD70 (−20% vs. average) and atrial ERP (−25% vs. average) were shorter in Swiss agouti background compared to others. LA-APD70 (+25% vs. average) and IA-AT (+44% vs. average) were longer in 129/Sv mice. Atrial ERP was longer in FVB/N (+34% vs. average) and in younger experimental groups below 6 months of age. Conclusion This work defines normal ranges for murine atrial EP parameters. Genetic background has a profound effect on these parameters, at least of the magnitude as those of sex and age. These results can inform the experimental design and interpretation of murine atrial electrophysiology.

Canagliflozin Suppresses Atrial Remodeling in a Canine Atrial Fibrillation Model

Background Recent clinical trials have demonstrated the possible pleiotropic effects of SGLT2 (sodium–glucose cotransporter 2) inhibitors in clinical cardiovascular diseases. Atrial electrical and structural remodeling is important as an atrial fibrillation (AF) substrate. Methods and Results The present study assessed the effect of canagliflozin (CAN), an SGLT2 inhibitor, on atrial remodeling in a canine AF model. The study included 12 beagle dogs, with 10 receiving continuous rapid atrial pacing and 2 acting as the nonpacing group. The 10 dogs that received continuous rapid atrial pacing for 3 weeks were subdivided as follows: pacing control group (n=5) and pacing+CAN (3 mg/kg per day) group (n=5). The atrial effective refractory period, conduction velocity, and AF inducibility were evaluated weekly through atrial epicardial wires. After the protocol, atrial tissues were sampled for histological examination. The degree of reactive oxygen species expression was evaluated by dihydroethidium staining. The atrial effective refractory period reduction was smaller ( P =0.06) and the degree of conduction velocity decrease was smaller in the pacing+CAN group compared with the pacing control group ( P =0.009). The AF inducibility gradually increased in the pacing control group, but such an increase was suppressed in the pacing+CAN group ( P =0.011). The pacing control group exhibited interstitial fibrosis and enhanced oxidative stress, which were suppressed in the pacing+CAN group. Conclusions CAN and possibly other SGLT2 inhibitors might be useful for preventing AF and suppressing the promotion of atrial remodeling as an AF substrate.

Exploring Refractoriness as an Adjunctive Electrical Biomarker for Staging of Atrial Fibrillation

Patients diagnosed with the same subtype of atrial fibrillation according to our current classification system may differ in symptom severity, severity of the arrhythmogenic substrate, and response to antiarrhythmic therapy. Hence, there is a need for an electrical biomarker as an indicator of the arrhythmogenic substrate underlying atrial fibrillation enabling patient‐tailored therapy. The aim of this review is to investigate whether atrial refractoriness, a well‐known electrophysiological parameter that is affected by electrical remodeling, can be used as an electrical biomarker of the arrhythmogenic substrate underlying atrial fibrillation. We discuss methodologies of atrial effective refractory period assessment, identify which changes in refractoriness‐related parameters reflect different degrees of electrical remodeling, and explore whether these parameters can be used to predict clinical outcomes.

Abstract 59: Inflammasome Activation Promotes the Arrhythmogenesis of Atrial Fibrillation

Background: Inflammation is a known risk factor of atrial fibrillation (AF), the most common arrhythmia; however, the mechanistic link between the inflammatory signaling and the pathophysiology of AF has not been established. ‘NACHT, LRR and PYD domains-containing protein 3’ (NLRP3) inflammasome is a signaling platform that is responsible for the activation of caspase-1 and interleukin (IL)-1b release. The activity of NLRP3 inflammasome is enhanced in atrial tissues of paroxysmal or long-lasting persistent AF patients. Thus, we tested the hypothesis that activation of NLRP3 inflammasome promotes the development of AF. Results: To elucidate the role of NLRP3 inflammasome in cardiomyocytes (CMs) and AF development, a CM-specific knockin murine model expressing a constitutive active NLRP3 (aMHC:NLRP3 A350V/+ , CKI) was developed. At 3-month old, telemetry recordings showed that 100% of CKΙ mice (n=5) developed premature atrial contractions (PACs), whereas only 25% of control mice (n=4, P<0.05) had PACs. Rapid atrial pacing induced AF in 89% of CKI mice (n=9), a much higher incidence than control mice (20%, n=5, P<0.05). Level of the active caspase-1 was increased in atrial tissues of 3-month old CKI mice, prior to a detectable increase in the level of macrophage marker at 7-month old, suggesting that the onset of PACs and AF vulnerability is not associated with the activated macrophages. Compared to the control mice, 3-month old CKI mice exhibited atrial hypertrophy, abnormal Ca 2+ release via RyR2, and shortening of atrial effective refractory period, which were associated with the upregulation of Mef2c , Ryr2 , Kcna5 (encoding Kv1.5), Kcnj 3 (encoding Kir3.1) and Kcnj5 (encoding Kir3.4) mRNA. Lastly, inflammasome inhibitor MCC950 (i.p., 10mg/kg, 10 days) reduced the AF inducibility in CKI mice (0%, n=3, P<0.05 vs vehicle-treated CKI). Conclusion: Activation of NLRP3 inflammasome promotes structural and electrical remodeling, permissive to the AF development. In addition to its canonical function, NLRP3 inflammasome may exhibit alternative function in regulating gene transcription. Our study establishes a mechanistic link between the inflammatory signaling and the pathogenesis of AF, and the inhibition of NLRP3 may become a novel anti-AF therapy.

Angiotensin-(1–7) attenuates atrial tachycardia-induced sympathetic nerve remodeling

Introduction: The effect of Angiotensin-(1–7) (Ang-(1–7)) on atrial autonomic remodeling is still unknown. We hypothesized that Ang-(1–7) could inhibit sympathetic nerve remodeling in a canine model of chronic atrial tachycardia. Materials and methods: Eighteen dogs were randomly assigned to sham group, pacing group and Ang-(1–7) group. Rapid atrial pacing was maintained for 14 days in the pacing and Ang-(1–7) groups. Ang-(1–7) was administered intravenously in the Ang-(1–7) group. The atrial effective refractory period and atrial fibrillation inducibility level were measured at baseline and under sympathetic nerve stimulation after 14 days of measurement. The atrial sympathetic nerves labeled with tyrosine hydroxylase were detected using immunohistochemistry and Western blotting, and tyrosine hydroxylase and nerve growth factor mRNA levels were measured by reverse transcription polymerase chain reaction. Results: Pacing shortened the atrial effective refractory period and increased the atrial fibrillation inducibility level at baseline and under sympathetic nerve stimulation. Ang-(1–7) treatment attenuated the shortening of the atrial effective refractory period and the increase in the atrial fibrillation inducibility level. Immunohistochemistry and Western blotting showed sympathetic nerve hyperinnervation in the pacing group, while Ang-(1–7) attenuated sympathetic nerve proliferation. Ang-(1–7) alleviated the pacing-induced increases in tyrosine hydroxylase and nerve growth factor mRNA expression levels. Conclusion: Ang-(1–7) can attenuate pacing-induced atrial sympathetic hyperinnervation.

Simvastatin attenuates sympathetic hyperinnervation to prevent atrial fibrillation during the postmyocardial infarction remodeling process

Statin, as a 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitor, has been shown to prevent atrial fibrillation (AF) due to its anti-inflammatory and antioxidant effects. However, it is still not known whether statin can improve autonomic remodeling to prevent AF. In the present study, using an in vivo rat myocardial infarction (MI) model, we aimed to test whether simvastatin can attenuate nerve sprouting and sympathetic hyperinnervation to prevent AF during the post-MI remodeling process. Our data demonstrate that simvastatin, delivered 3 days after MI for 4 wk, can result in significant decreases in plasma levels of both TNF-α (239 ± 23 pg/ml) and IL-1β (123 ± 11 pg/ml) compared with MI rats without therapy (TNF-α, 728 ± 57 pg/ml; IL-1β, 213 ± 21 pg/ml; P < 0.05), which, however, were still higher than sham-operated rats (TNF-α, 194 ± 20 pg/ml; IL-1β, 75 ± 8 pg/ml; P < 0.05). The similar pattern of changes in inflammation responses was also observed in TNF-α and IL-1β protein expression in the left atrium free wall. The suppressed inflammation responses were associated with reduced superoxide and malondialdehyde generation in the atrium. These changes account for decreases in neural growth factor expression at levels of both mRNA (1.2 ± 0.09 AU vs. MI group, 1.78 ± 0.16 AU) and protein (1.57 ± 0.17 AU vs. MI group, 2.24 ± 0.19 AU; P < 0.05), thus resulting in reduced nerve sprouting and sympathetic hyperinnervation. Accordingly, the rate adaptation of the atrial effective refractory period also recovered, leading to the decreased inducibility of AF. These data suggest that simvastatin administration after MI can prevent AF through reduced sympathetic hyperinnervation.