Rebecca A. Capel

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

Background: The Ca2+-releasing messenger nicotinic acid adenine dinucleotide phosphate (NAADP) acts via lysosomal two-pore channels (TPC2). Results: Tpcn2−/− cardiac myocytes showed reduced acute responses to β-adrenoreceptor stimulation and chronically reduced cardiac hypertrophy and arrhythmogenesis. Conclusion: Acute and chronic effects of cardiac β-adrenoreceptor stimulation depend on NAADP acting via TPC2 in lysosomes. Significance: NAADP/TPC2 signaling pathways offer new strategies for cardiac therapeutics. Ca2+-permeable type 2 two-pore channels (TPC2) are lysosomal proteins required for nicotinic acid adenine dinucleotide phosphate (NAADP)-evoked Ca2+ 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 Ca2+ responses in cardiac myocytes from Tpcn2−/− mice, unlike myocytes from wild-type (WT) mice. Ca2+/calmodulin-dependent protein kinase II inhibitors suppressed actions of NAADP in myocytes. Ca2+ transients and contractions accompanying action potentials were increased by isoproterenol in myocytes from WT mice, but these effects of β-adrenoreceptor stimulation were reduced in myocytes from Tpcn2−/− mice. Increases in amplitude of L-type Ca2+ currents evoked by isoproterenol remained unchanged in myocytes from Tpcn2−/− mice showing no loss of β-adrenoceptors or coupling mechanisms. Whole hearts from Tpcn2−/− mice also showed reduced inotropic effects of isoproterenol and a reduced tendency for arrhythmias following acute β-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 Ca2+-signaling nanodomains between lysosomes and sarcoplasmic reticulum dependent on NAADP and TPC2 comprise an important element in β-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 β-adrenergic signaling in the heart and also in stress pathways linking chronic stimulation of β-adrenoceptors to hypertrophy and associated arrhythmias.

Mapping cardiac microstructure of rabbit heart in different mechanical states by high resolution diffusion tensor imaging: A proof-of-principle study.

Myocardial microstructure and its macroscopic materialisation are fundamental to the function of the heart. Despite this importance, characterisation of cellular features at the organ level remains challenging, and a unifying description of the structure of the heart is still outstanding. Here, we optimised diffusion tensor imaging data to acquire high quality data in ex vivo rabbit hearts in slack and contractured states, approximating diastolic and systolic conditions. The data were analysed with a suite of methods that focused on different aspects of the myocardium. In the slack heart, we observed a similar transmural gradient in helix angle of the primary eigenvector of up to 23.6°/mm in the left ventricle and 24.2°/mm in the right ventricle. In the contractured heart, the same transmural gradient remained largely linear, but was offset by up to +49.9° in the left ventricle. In the right ventricle, there was an increase in the transmural gradient to 31.2°/mm and an offset of up to +39.0°. The application of tractography based on each eigenvector enabled visualisation of streamlines that depict cardiomyocyte and sheetlet organisation over large distances. We observed multiple V- and N-shaped sheetlet arrangements throughout the myocardium, and insertion of sheetlets at the intersection of the left and right ventricle. This study integrates several complementary techniques to visualise and quantify the heart’s microstructure, projecting parameter representations across different length scales. This represents a step towards a more comprehensive characterisation of myocardial microstructure at the whole organ level.

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.

High resolution structural evidence suggests the Sarcoplasmic Reticulum forms microdomains with Acidic Stores (lysosomes) in the heart

Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) stimulates calcium release from acidic stores such as lysosomes and is a highly potent calcium-mobilising second messenger. NAADP plays an important role in calcium signalling in the heart under basal conditions and following β-adrenergic stress. Nevertheless, the spatial interaction of acidic stores with other parts of the calcium signalling apparatus in cardiac myocytes is unknown. We present evidence that lysosomes are intimately associated with the sarcoplasmic reticulum (SR) in ventricular myocytes; a median separation of 20 nm in 2D electron microscopy and 3.3 nm in 3D electron tomography indicates a genuine signalling microdomain between these organelles. Fourier analysis of immunolabelled lysosomes suggests a sarcomeric pattern (dominant wavelength 1.80 μm). Furthermore, we show that lysosomes form close associations with mitochondria (median separation 6.2 nm in 3D studies) which may provide a basis for the recently-discovered role of NAADP in reperfusion-induced cell death. The trigger hypothesis for NAADP action proposes that calcium release from acidic stores subsequently acts to enhance calcium release from the SR. This work provides structural evidence in cardiac myocytes to indicate the formation of microdomains between acidic and SR calcium stores, supporting emerging interpretations of NAADP physiology and pharmacology in heart.

Cytosolic calcium ions exert a major influence on the firing rate and maintenance of pacemaker activity in guinea-pig sinus node.

The sino-atrial node (SAN) provides the electrical stimulus to initiate every heart beat. Cellular processes underlying this activity have been debated extensively, especially with regards to the role of intracellular calcium. We have used whole-cell application of 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), a rapid calcium chelator, to guinea pig isolated SAN myocytes to assess the effect of rapid reduction of intracellular calcium on SAN cell electrical activity. High-dose (10 mM) BAPTA induced rapid and complete cessation of rhythmic action potential (AP) firing (time to cessation 5.5 ± 1.7 s). Over a range of concentrations, BAPTA induced slowing of action potential firing and disruption of rhythmic activity, which was dose-dependent in its time of onset. Exposure to BAPTA was associated with stereotyped action potential changes similar to those previously reported in the presence of ryanodine, namely depolarization of the most negative diastolic potential, prolongation of action potentials and a reduction in action potential amplitude. These experiments are consistent with the view that cytosolic calcium is essential to the maintenance of rhythmic pacemaker activity.

Load-dependent effects of apelin on murine cardiomyocytes

The apelin peptide is described as one of the most potent inotropic agents, produced endogenously in a wide range of cells, including cardiomyocytes. Despite positive effects on cardiac contractility in multicellular preparations, as well as indications of cardio-protective actions in several diseases, its effects and mechanisms of action at the cellular level are incompletely understood. Here, we report apelin effects on dynamic mechanical characteristics of single ventricular cardiomyocytes, isolated from mouse models (control, apelin-deficient [Apelin-KO], apelin-receptor KO mouse [APJ-KO]), and rat. Dynamic changes in maximal velocity of cell shortening and relaxation were monitored. In addition, more traditional indicators of inotropic effects, such as maximum shortening (in mechanically unloaded cells) or peak force development (in auxotonic contracting cells, preloaded using the carbon fibre technique) were studied. The key finding is that, using Apelin-KO cardiomyocytes exposed to different preloads with the 2-carbon fibre technique, we observe a lowering of the slope of the end-diastolic stress-length relation in response to 10 nM apelin, an effect that is preload-dependent. This suggests a positive lusitropic effect of apelin, which could explain earlier counter-intuitive findings on an apelin-induced increase in contractility occurring without matching rise in oxygen consumption.

Transverse cardiac slicing and optical imaging for analysis of transmural gradients in membrane potential and Ca2+ transients in murine heart.

A robust cardiac slicing approach was developed for optical mapping of transmural gradients in transmembrane potential (Vm) and intracellular Ca2+ transient (CaT) of murine heart. Significant transmural gradients in Vm and CaT were observed in the left ventricle. Frequency‐dependent action potentials and CaT alternans were observed in all ventricular regions with rapid pacing, with significantly greater incidence in the endocardium than epicardium. The observations demonstrate the feasibility of our new approach to cardiac slicing for systematic analysis of intrinsic transmural and regional gradients in Vm and CaT.

CAN YOU TEACH AN OLD DOG NEW TRICKS? THE ANTIMALARIAL HYDROXYCHLOROQUINE SHOWS PROMISE IN CARDIAC RATE CONTROL THROUGH ACTIONS AT THE SINO-ATRIAL NODE

Hydroxychloroquine (HCQ) has been clinically prescribed since the early 1950s. Used as a prophylactic antimalarial and in chronic immune conditions, it has a good safety profile. Capitalising on a fortuitous clinical observation, we investigated the effects of HCQ on cardiac rate control. Application of HCQ to spontaneously-beating mouse atrial preparations revealed a dose-dependent rate decrease (9 ± 3% at 3 µM, 15 ± 2% at 10 µM) which did not occur in the presence of funny current (I(f)) inhibitor ZD7288 (1 µM). 1 µM HCQ significantly reduced spontaneous beating rate in isolated guinea pig sino-atrial node myocytes. Voltage clamp studies revealed a significant, dose-dependent I(f) inhibition (19 ± 2% at 3 µM, 32 ± 7% at 10 µM). Consistent with channel block, I(f) inhibition reduced maximal conductance without affecting voltage of half-activation. L-type calcium and delayed rectifier potassium currents were also significantly reduced. We performed mathematical safety modelling using methods described in. Modelling results for a maximum free therapeutic concentration of 1 µM indicated that the balance of potassium and calcium current inhibitions observed would not be expected to lengthen ventricular action potentials and placed HCQ in the ‘low risk’ category for Torsade de Pointes arrhythmia. HCQ is capable of reducing cardiac beating rate by effects on I(f). Given the excellent cardiac safety record of HCQ and safety profile modelling, it has potential as a rate-liming agent. Further work will be required in order to assess any potential cardiac benefits of HCQ in disease populations.