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

© 2015 Heart Rhythm Society. 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 (

Inhibition of adenylyl cyclase 1 by ST034307 inhibits IP₃-evoked changes in sino-atrial node beat rate.

Atrial arrhythmias, such as atrial fibrillation (AF), are a major mortality risk and a leading cause of stroke. The IP3 signalling pathway has been proposed as an atrial-specific target for AF therapy, and atrial IP3 signalling has been linked to the activation of calcium sensitive adenylyl cyclases AC1 and AC8. We investigated the involvement of AC1 in the response of intact mouse atrial tissue and isolated guinea pig atrial and sino-atrial node (SAN) cells to the α-adrenoceptor agonist phenylephrine (PE) using the selective AC1 inhibitor ST034307. The maximum rate change of spontaneously beating mouse right atrial tissue exposed to PE was reduced from 14.5% to 8.2% (p = 0.005) in the presence of 1 μM ST034307, whereas the increase in tension generated in paced left atrial tissue in the presence of PE was not inhibited by ST034307 (Control = 14.2%, ST034307 = 16.3%; p > 0.05). Experiments were performed using isolated guinea pig atrial and SAN cells loaded with Fluo-5F-AM to record changes in calcium transients (CaT) generated by 10 μM PE in the presence and absence of 1 μM ST034307. ST034307 significantly reduced the beating rate of SAN cells (0.34-fold decrease; p = 0.003) but did not inhibit changes in CaT amplitude in response to PE in atrial cells. The results presented here demonstrate pharmacologically the involvement of AC1 in the downstream response of atrial pacemaker activity to α-adrenoreceptor stimulation and IP3R calcium release

Generation of cardiomyocytes from human-induced pluripotent stem cells resembling atrial cells with ability to respond to adrenoceptor agonists

Atrial fibrillation (AF) is the most common chronic arrhythmia presenting a heavy disease burden. We report a new approach for generating cardiomyocytes (CMs) resembling atrial cells from human-induced pluripotent stem cells (hiPSCs) using a combination of Gremlin 2 and retinoic acid treatment. More than 40% of myocytes showed rod-shaped morphology, expression of CM proteins (including ryanodine receptor 2, α-actinin-2 and F-actin) and striated appearance, all of which were broadly similar to the characteristics of adult atrial myocytes (AMs). Isolated myocytes were electrically quiescent until stimulated to fire action potentials with an AM profile and an amplitude of approximately 100 mV, arising from a resting potential of approximately −70 mV. Single-cell RNA sequence analysis showed a high level of expression of several atrial-specific transcripts including NPPA, MYL7, HOXA3, SLN, KCNJ4, KCNJ5 and KCNA5. Amplitudes of calcium transients recorded from spontaneously beating cultures were increased by the stimulation of α-adrenoceptors (activated by phenylephrine and blocked by prazosin) or β-adrenoceptors (activated by isoproterenol and blocked by CGP20712A). Our new approach provides human AMs with mature characteristics from hiPSCs which will facilitate drug discovery by enabling the study of human atrial cell signalling pathways and AF. This article is part of the theme issue ‘The heartbeat: its molecular basis and physiological mechanisms’

Effects of endogenous cannabinoids and related substances on electrical activity and contraction in cardiac ventricular muscle

Stimulation of cardiac β-adrenoceptors is the primary mechanism by which cardiac output is increased to meet metabolic demands of the body. Recently, nicotinic acid adenine dinucleotide phosphate (NAADP), has been implicated as a novel component of the β-adrenoceptor signalling pathway. NAADP is thought to mobilise Ca2+ from acidic endolysosomal stores which then supplements sarcoplasmic reticulum Ca2+ load, leading to a positive inotropic effect. Recent progress in the field has been made with the identification of two-pore channel 2 (TPC2) as a candidate NAADP receptor. Isolated ventricular myocytes from mice lacking TPC2 proteins (Tpcn2-/-) displayed blunted maximal responses to the β-adrenoceptor agonist isoprenaline. This blunted response was also evident in Langendorff-perfused Tpcn2-/- hearts. Furthermore, a blunted response was observed in guinea pig ventricular myocytes which had been treated with bafilomycin A1, which disrupts the integrity of acidic endolysosomal stores. These data add to the body of evidence that NAADP signalling forms an important additional component of the β-adrenoceptor signalling pathway. Chronic activation of the β-adrenoceptor pathway is associated with certain disease states including heart failure and arrhythmias. Anandamide is an endogenous cannabinoid, ('endocannabinoid'), with similar properties to δ9-tetrahydrocannabinol (δ9-THC), the primary active constituent of Cannabis sativa. These compounds have widespread physiological effects through actions at cannabinoid CB1 and CB2 receptors, which are negatively coupled to adenylyl cyclases and are expressed in cardiac muscle. Exposure of guinea pig ventricular myocytes to anandamide resulted in a reduction in the amplitude of contractions and Ca2+ transients. This was associated with a reduction in action potential duration and amplitude of ICaL. These effects of anandamide could not be prevented by cannabinoid receptor antagonists, and could not be mimicked by cannabinoid receptor agonists. An inhibition of IKs was also observed. Given the reported Gi-protein coupling of cannabinoid receptors, it may be expected that additional negative inotropic actions of anandamide might be observed when adenylyl cyclases are stimulated. However, the effects of anandamide to reduce amplitude of contraction and ICaL were no greater in the presence of isoprenaline. Furthermore, the effects of anandamide were not prevented by pre-treatment of myocytes with pertussis toxin (PTX). This is in contrast to the actions of adenosine, which displayed clear PTX sensitive actions in the presence of isoprenaline. These data suggest that cannabinoid receptors are not involved in mediating the negative inotropic actions of anandamide. Another endocannabinoid, 2-arachidonoylglycerol, was without significant effect on action potentials or contractions in the absence or presence of isoprenaline. δ9-THC shared many of the actions of anandamide. It appears that anandamide and δ9-THC exert significant effects on cardiac muscle through direct modulation of ion channel function, although additional actions, for example, on the sarcoplasmic reticulum or myofilaments, cannot be ruled out.This thesis is not currently available in ORA

Actions of NAADP and other agents in cardiac myocytes

Modulation of cardiac rate and contraction through calcium-dependent and independent means are of central import to the ability of an organism to adapt to its environment. Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent calcium-releasing second messenger across a broad range of tissues and organisms. In cardiac myocytes, NAADP is thought to stimulate calcium release from acidic stores which then bolsters filling and release during CICR. Questions remain: as to the potential need for amplification to generate the size of responses observed and the physiological role of the NAADP pathway. In contractile myocytes, photorelease of NAADP caused significant increase in calcium transient amplitude and velocity of transient upstroke and decay. Effects were absent during NAADP photorelease in the presence of Ned-19 or CaMKII inhibitors. Cellular calcium transient responses to β-adrenergic stimulation were significantly reduced in the presence of inhibitors of the NAADP pathway. These data support the hypothesis that NAADP-induced calcium release is relevant during adrenergic stimulation and requires amplification through CaMKII. Rate modulation at the sino-atrial node can occur through the hyperpolarisation-activated current I(f). Basal cardiac rate is a major determinant in cardiac mortality and compounds which specifically affect rate have clinical utility. A compound currently used to treat inflammatory conditions was found to have a significant rate-reducing effect in sino-atrial node preparations mediated by inhibition of I(f). Apelin, an endogenous peptide, has been reported to potently generate improved contractility without development of hypertrophy. Study of its effects in single cells have provided conflicting information, at least in part because of the difficulty in working with the compound. A method for the consistent observation of apelin-mediated contractile responses is presented, focusing on the timecourse of cell contraction. These observations suggest a role for apelin in both inotropy and lusitropy and will enable further research.This thesis is not currently available in ORA