Cardiovascular concentration-effect relationships of amodiaquine and its metabolite desethylamodiaquine: Clinical and preclinical studies.

AIMS: Amodiaquine is a 4-aminoquinoline used extensively for the treatment and prevention of malaria. Orally administered amodiaquine is largely converted to the active metabolite desethylamodiaquine. Amodiaquine can cause bradycardia, hypotension, and electrocardiograph QT interval prolongation, but the relationship of these changes to drug concentrations is not well characterized. METHODS: We conducted a secondary analysis of a pharmacokinetic study of the cardiac safety of amodiaquine (10 mg base/kg/day over 3 days) in 54 Kenyan adults (≥18 years) with uncomplicated malaria. Nonlinear mixed effects modelling was used to assess amodiaquine and desethylamodiaquine concentration-effect relationships for vital sign (pulse rate, blood pressure) and electrocardiograph interval (QT, QRS, PR) outcomes. We also measured the spontaneous beating heart rate after cumulative dosing of amodiaquine and desethylamodiaquine in isolated mouse atrial preparations. RESULTS: Amodiaquine and desethylamodiaquine caused concentration-dependent mean decreases in pulse rate (1.9 beats/min per 100 nmol/L; 95% confidence interval: 1.5-2.4), supine systolic blood pressure (1.7 mmHg per 100 nmol/L; 1.2-2.1), erect systolic blood pressure (1.5 mmHg per 100 nmol/L; 1.0-2.0) and erect diastolic blood pressure (1.4 mmHg per 100 nmol/L; 1.0-1.7). The mean QT interval prolongation was 1.4 ms per 100 nmol/L irrespective of correction factor after adjustment for residual heart rate dependency. There was no significant effect of drug concentration on postural change in blood pressure or PR and QRS intervals. In mouse atria, the spontaneous beating rate was significantly reduced by amodiaquine (n = 6) and desethylamodiaquine (n = 8) at 3 µmol/L (amodiaquine: 10 ± 2%; desethylamodiaquine: 12 ± 3%) and 10 µmol/L (amodiaquine: 50 ± 7%; desethylamodiaquine: 46 ± 6%) concentrations with no significant difference in potency between the 2 compounds. CONCLUSION: Amodiaquine and desethylamodiaquine have concentration-dependent effects on heart rate, blood pressure, and ventricular repolarization.

Cardiac TdP risk stratification modelling of anti-infective compounds including chloroquine and hydroxychloroquine.

Hydroxychloroquine (HCQ), the hydroxyl derivative of chloroquine (CQ), is widely used in the treatment of rheumatological conditions (systemic lupus erythematosus, rheumatoid arthritis) and is being studied for the treatment and prevention of COVID-19. Here, we investigate through mathematical modelling the safety profile of HCQ, CQ and other QT-prolonging anti-infective agents to determine their risk categories for Torsade de Pointes (TdP) arrhythmia. We performed safety modelling with uncertainty quantification using a risk classifier based on the qNet torsade metric score, a measure of the net charge carried by major currents during the action potential under inhibition of multiple ion channels by a compound. Modelling results for HCQ at a maximum free therapeutic plasma concentration (free C max) of approximately 1.2 µM (malaria dosing) indicated it is most likely to be in the high-intermediate-risk category for TdP, whereas CQ at a free C max of approximately 0.7 µM was predicted to most likely lie in the intermediate-risk category. Combining HCQ with the antibacterial moxifloxacin or the anti-malarial halofantrine (HAL) increased the degree of human ventricular action potential duration prolongation at some or all concentrations investigated, and was predicted to increase risk compared to HCQ alone. The combination of HCQ/HAL was predicted to be the riskiest for the free C max values investigated, whereas azithromycin administered individually was predicted to pose the lowest risk. Our simulation approach highlights that the torsadogenic potentials of HCQ, CQ and other QT-prolonging anti-infectives used in COVID-19 prevention and treatment increase with concentration and in combination with other QT-prolonging drugs.

Emerging Evidence for cAMP-calcium Cross Talk in Heart Atrial Nanodomains Where IP3-Evoked Calcium Release Stimulates Adenylyl Cyclases.

Calcium handling is vital to normal physiological function in the heart. Human atrial arrhythmias, eg. atrial fibrillation, are a major morbidity and mortality burden, yet major gaps remain in our understanding of how calcium signaling pathways function and interact. Inositol trisphosphate (IP3) is a calcium-mobilizing second messenger and its agonist-induced effects have been observed in many tissue types. In the atria IP3 receptors (IR3Rs) residing on junctional sarcoplasmic reticulum augment cellular calcium transients and, when over-stimulated, lead to arrhythmogenesis. Recent studies have demonstrated that the predominant pathway for IP3 actions in atrial myocytes depends on stimulation of calcium-dependent forms of adenylyl cyclase (AC8 and AC1) by IP3-evoked calcium release from the sarcoplasmic reticulum. AC8 shows co-localisation with IP3Rs and AC1 appears to be nearby. These observations support crosstalk between calcium and cAMP pathways in nanodomains in atria. Similar mechanisms also appear to operate in the pacemaker region of the sinoatrial node. Here we discuss these significant advances in our understanding of atrial physiology and pathology, together with implications for the identification of potential novel targets and modulators for the treatment of atrial arrhythmias.

Optical Interrogation of Sympathetic Neuronal Effects on Macroscopic Cardiomyocyte Network Dynamics.

Cardiac stimulation via sympathetic neurons can potentially trigger arrhythmias. We present approaches to study neuron-cardiomyocyte interactions involving optogenetic selective probing and all-optical electrophysiology to measure activity in an automated fashion. Here we demonstrate the utility of optical interrogation of sympathetic neurons and their effects on macroscopic cardiomyocyte network dynamics to address research targets such as the effects of adrenergic stimulation via the release of neurotransmitters, the effect of neuronal numbers on cardiac behavior, and the applicability of optogenetics in mechanistic in vitro studies. As arrhythmias are emergent behaviors that involve the coordinated activity of millions of cells, we image at macroscopic scales to capture complex dynamics. We show that neurons can both decrease and increase wave stability and re-entrant activity in culture depending on their induced activity-a finding that may help us understand the often conflicting results seen in experimental and clinical studies.

Two-pore Channels (TPC2s) and Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) at Lysosomal-Sarcoplasmic Reticular Junctions Contribute to Acute and Chronic ß-Adrenoceptor Signaling in the Heart.

Ca(2+)-permeable type 2 two-pore channels (TPC2) are lysosomal proteins required for nicotinic acid adenine dinucleotide phosphate (NAADP)-evoked Ca(2+) release in many diverse cell types. Here, we investigate the importance of TPC2 proteins for the physiology and pathophysiology of the heart. NAADP-AM failed to enhance Ca(2+) responses in cardiac myocytes from Tpcn2(-/-) mice, unlike myocytes from wild-type (WT) mice. Ca(2+)/calmodulin-dependent protein kinase II inhibitors suppressed actions of NAADP in myocytes. Ca(2+) transients and contractions accompanying action potentials were increased by isoproterenol in myocytes from WT mice, but these effects of ß-adrenoreceptor stimulation were reduced in myocytes from Tpcn2(-/-) mice. Increases in amplitude of L-type Ca(2+) currents evoked by isoproterenol remained unchanged in myocytes from Tpcn2(-/-) mice showing no loss of ß-adrenoceptors or coupling mechanisms. Whole hearts from Tpcn2(-/-) mice also showed reduced inotropic effects of isoproterenol and a reduced tendency for arrhythmias following acute ß-adrenoreceptor stimulation. Hearts from Tpcn2(-/-) mice chronically exposed to isoproterenol showed less cardiac hypertrophy and increased threshold for arrhythmogenesis compared with WT controls. Electron microscopy showed that lysosomes form close contacts with the sarcoplasmic reticulum (separation ∼ 25 nm). We propose that Ca(2+)-signaling nanodomains between lysosomes and sarcoplasmic reticulum dependent on NAADP and TPC2 comprise an important element in ß-adrenoreceptor signal transduction in cardiac myocytes. In summary, our observations define a role for NAADP and TPC2 at lysosomal/sarcoplasmic reticulum junctions as unexpected but major contributors in the acute actions of ß-adrenergic signaling in the heart and also in stress pathways linking chronic stimulation of ß-adrenoceptors to hypertrophy and associated arrhythmias.

Hydroxychloroquine reduces heart rate by modulating the hyperpolarization-activated current If: Novel electrophysiological insights and therapeutic potential.

BACKGROUND: Bradycardic agents are of interest for the treatment of ischemic heart disease and heart failure, as heart rate is an important determinant of myocardial oxygen consumption. OBJECTIVES: The purpose of this study was to investigate the propensity of hydroxychloroquine (HCQ) to cause bradycardia. METHODS: We assessed the effects of HCQ on (1) cardiac beating rate in vitro (mice); (2) the "funny" current (If) in isolated guinea pig sinoatrial node (SAN) myocytes (1, 3, 10 µM); (3) heart rate and blood pressure in vivo by acute bolus injection (rat, dose range 1-30 mg/kg), (4) blood pressure and ventricular function during feeding (mouse, 100 mg/kg/d for 2 wk, tail cuff plethysmography, anesthetized echocardiography). RESULTS: In mouse atria, spontaneous beating rate was significantly (P < .05) reduced (by 9% ± 3% and 15% ± 2% at 3 and 10 µM HCQ, n = 7). In guinea pig isolated SAN cells, HCQ conferred a significant reduction in spontaneous action potential firing rate (17% ± 6%, 1 µM dose) and a dose-dependent reduction in If (13% ± 3% at 1 µM; 19% ± 2% at 3 µM). Effects were also observed on L-type calcium ion current (ICaL) (12% ± 4% reduction) and rapid delayed rectifier potassium current (IKr) (35% ± 4%) at 3 µM. Intravenous HCQ decreased heart rate in anesthetized rats (14.3% ± 1.1% at 15mg/kg; n = 6) without significantly reducing mean arterial blood pressure. In vivo feeding studies in mice showed no significant change in systolic blood pressure nor left ventricular function. CONCLUSIONS: We have shown that HCQ acts as a bradycardic agent in SAN cells, in atrial preparations, and in vivo. HCQ slows the rate of spontaneous action potential firing in the SAN through multichannel inhibition, including that of If.

Macro-micro imaging of cardiac-neural circuits in co-cultures from normal and diseased hearts.

The autonomic nervous system plays an important role in the modulation of normal cardiac rhythm, but is also implicated in modulating the heart's susceptibility to re-entrant ventricular and atrial arrhythmias. The mechanisms by which the autonomic nervous system is pro-arrhythmic or anti-arrhythmic is multifaceted and varies for different types of arrhythmia and their cardiac substrates. Despite decades of research in this area, fundamental questions related to how neuron density and spatial organization modulate cardiac wave dynamics remain unanswered. These questions may be ill-posed in intact tissues where the activity of individual cells is often experimentally inaccessible. Development of simplified biological models that would allow us to better understand the influence of neural activation on cardiac activity can be beneficial. This Symposium Review summarizes the development of in vitro cardiomyocyte cell culture models of re-entrant activity, as well as challenges associated with extending these models to include the effects of neural activation.