Drug-Induced Lysosomal Impairment Is Associated with the Release of Extracellular Vesicles Carrying Autophagy Markers

Amiodarone is a cationic amphiphilic drug used as an antiarrhythmic agent. It induces phospholipidosis, i.e., the accumulation of phospholipids within organelles of the endosomal–lysosomal system. Extracellular vesicles (EVs) are membrane-enclosed structures released by any type of cell and retrieved in every fluid of the body. EVs have been initially identified as a system to dispose cell waste, but they are also considered to be an additional manner to transmit intercellular signals. To understand the role of EVs in drug-induced phospholipidosis, we investigated EVs release in amiodarone-treated HEK-293 cells engineered to produce fluorescently labelled EVs. We observed that amiodarone induces the release of a higher number of EVs, mostly of a large/medium size. EVs released upon amiodarone treatment do not display significant morphological changes or altered size distribution, but they show a dose-dependent increase in autophagy associated markers, indicating a higher release of EVs with an autophagosome-like phenotype. Large/medium EVs also show a higher content of phospholipids. Drugs inducing lysosomal impairment such as chloroquine and bafilomycin A1 similarly prompt a higher release of EVs enriched in autophagy markers. This result suggests a mechanism associated with amiodarone-induced lysosomal impairment more than a connection with the accumulation of specific undigested substrates. Moreover, the implementation of the lysosomal function by overexpressing TFEB, a master gene regulator of lysosomal biogenesis, prevents the amiodarone-induced release of EVs, suggesting that this could be a feasible target to attenuate drug-induced abnormalities.

Amiodarone-induced thyroid dysfunction in children: insights from the THYRAMIO study

Background: Amiodarone treatment is effective against various types of arrhythmias but is associated with adverse effects affecting, among other organs, thyroid function. Amiodarone-induced thyroid dysfunction was not thoroughly evaluated in children as it was in adults, yet this affection may lead to irreversible neurodevelopmental complications. Our study aimed to define the incidence and risk factors of amiodarone-induced thyroid dysfunction in children. Methods: The study was designed as an observational study with a retrospective clinical series of 152 children treated by amiodarone in the Pediatric Cardiology Unit of our center from 1990 to 2019. All patients were divided into three groups according to their thyroid status: euthyroid, AIH (amiodarone-induced hypothyroidism) or AIT (amiodarone-induced thyrotoxicosis). Patients from these three groups were compared in terms of key clinical and therapeutic features. Results: Amiodarone-induced thyroid dysfunction was present in 23% of patients. AIT (5.3%) was three times less common than AIH (17.7%), and its occurrence increased with older age ( p < 0.05), treatment dosage ( p < 0.05), treatment duration ( p < 0.05) and the number of loading doses administered ( p < 0.05). There were no distinctive clinical features between euthyroid and AIH groups. A multivariable prediction model of AIT was built, with a yield of 66.7% as positive predictive value and 96.7% as negative predictive value. Conclusion: We observed that one in five children developed amiodarone-induced thyroid dysfunction. Special attention is required for older children with a high dosage and long-term therapy and who received a large number of loading doses, since these children are at risk to develop AIT, which is more delicate to manage than AIH.

Different effects of amiodarone and dofetilide on the dispersion of repolarization between well-coupled ventricular and Purkinje fibers1

Increased transmural dispersion of repolarization is an established contributing factor to ventricular tachyarrhythmias. In this study, we evaluated the effect of chronic amiodarone treatment and acute administration of dofetilide in canine cardiac preparations containing electrotonically coupled Purkinje fibers (PFs) and ventricular muscle (VM) and compared the effects to those in uncoupled PF and VM preparations using the conventional microelectrode technique. Dispersion between PFs and VM was inferred from the difference in the respective action potential durations (APDs). In coupled preparations, amiodarone decreased the difference in APDs between PFs and VM, thus decreasing dispersion. In the same preparations, dofetilide increased the dispersion by causing a more pronounced prolongation in PFs. This prolongation was even more emphasized in uncoupled PF preparations, while the effect in VM was the same. In uncoupled preparations, amiodarone elicited no change on the difference in APDs. In conclusion, amiodarone decreased the dispersion between PFs and VM, while dofetilide increased it. The measured difference in APD between cardiac regions may be the affected by electrotonic coupling; thus, studying PFs and VM separately may lead to an over- or underestimation of dispersion.

Improved Survival in Hepatocellular Carcinoma Patients with Cardiac Arrhythmia by Amiodarone Treatment through Autophagy

Hepatocellular carcinoma (HCC) is a primary malignancy of the liver. In basic studies, the regulation of autophagy has offered promising results for HCC treatment. This study aimed to address the question of whether amiodarone can improve survival rates in HCC patients associated with autophagy. Using datasets from the National Health Insurance Research Database, we enrolled patients over 18 years of age that had been diagnosed with HCC between January 1997 and December 2010. Amiodarone and non-amiodarone users were matched at a 1:1 frequency, according to all variables. Additionally, HepG2 cells treated with amiodarone were evaluated by cell viability and autophagic change. Autophagic signaling was examined by immunoblotting and tissue array immunohistochemistry. Of the 10,946 patients diagnosed with HCC, each cohort included 221 patients after 1:1 propensity score matching. The median survival was 36.70 months for the amiodarone users, and 24.48 months for the non-amiodarone users. After adjusting for age, gender, comorbidities and treatment, amiodarone users had a significantly lower risk of mortality. Amiodarone users also demonstrated an improved 3-year survival rate. Furthermore, amiodarone treatment-induced autophagy in HepG2 cells was demonstrated by autophagosome formation associated with increasing LC3B-II, P62, and Beclin-1 expression. Autophagic flux also increased following amiodarone treatment with bafilomycin A1. SiRNA of LC3B knocked down endogenous LC3B formation and restored HepG2 cell viability. This study provides epidemiologic evidence that amiodarone via autophagic degradation machinery may offer survival benefits for HCC patients with a history of arrhythmia. Further randomized, blinded, and placebo-controlled trials are warranted for patients with HCC.

QT as a predictor of recurrence after atrial fibrillation ablation and the impact of amiodarone: results from the placebo-controlled AMIO-CAT trial

Aims Prolonged corrected QT interval (QTc) might be associated with arrhythmia recurrence after atrial fibrillation (AF) ablation. The effect of short-term amiodarone in this setting remains unknown. This study seeks to quantify short-term amiodarone’s impact on QTc, and to investigate QTc and amiodarone treatment as predictors of recurrence of arrhythmia after ablation. Methods and results The Short-term AMIOdarone treatment after CATheter ablation for atrial fibrillation (AMIO-CAT) trial randomized patients to 8 weeks of oral amiodarone or placebo following AF ablation. Scheduled and symptom-driven 12-lead electrocardiography and 3-day Holter-monitorings were performed. The endpoint was atrial fibrillation, atrial flutter or atrial tachycardia (AF/AT) lasting >30 s. The cut-off for prolonged QTc was 450 ms for men and 460 ms for women. A total of 212 patients were included, of which 108 were randomized to amiodarone and 104 to placebo. From baseline to 1 month QTc in the amiodarone group increased by 27 (±30) ms, while at 6 months QTc had normalized. After 3-months of blanking, new AF/AT recurrence was detected in 63% of patients with prolonged QTc vs. 41% of patients with normal QTc at baseline, and in multivariate Cox regression, prolonged QTc was associated with AF/AT recurrence [hazard ratio (HR) 2.19, P = 0.023]. Among patients with baseline QTc below median, amiodarone treatment decreased the rate of AF/AT recurrences (HR 0.43, P = 0.008). Conclusions Amiodarone increased QTc with 27 ms compared to placebo, and this effect decreased rapidly after drug discontinuation. Prolonged QTc at baseline independently predicted AF/AT recurrence, and baseline QTc identified patients who would possibly benefit from short-term amiodarone following ablation.