Emma L. Bolton

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.

Optogenetic Control of Heart Rhythm by Selective Stimulation of Cardiomyocytes Derived from Pnmt(+) Cells in Murine Heart.

In the present study, channelrhodopsin 2 (ChR2) was specifically introduced into murine cells expressing the Phenylethanolamine n-methyltransferase (Pnmt) gene, which encodes for the enzyme responsible for conversion of noradrenaline to adrenaline. The new murine model enabled the identification of a distinctive class of Pnmt-expressing neuroendocrine cells and their descendants (i.e. Pnmt+ cell derived cells) within the heart. Here, we show that Pnmt+ cells predominantly localized to the left side of the adult heart. Remarkably, many of the Pnmt+ cells in the left atrium and ventricle appeared to be working cardiomyocytes based on their morphological appearance and functional properties. These Pnmt+ cell derived cardiomyocytes (PdCMs) are similar to conventional myocytes in morphological, electrical and contractile properties. By stimulating PdCMs selectively with blue light, we were able to control cardiac rhythm in the whole heart, isolated tissue preparations and single cardiomyocytes. Our new murine model effectively demonstrates functional dissection of cardiomyocyte subpopulations using optogenetics, and opens new frontiers of exploration into their physiological roles in normal heart function as well as their potential application for selective cardiac repair and regeneration strategies.

Synthesis of the Ca2+-mobilizing messengers NAADP and cADPR by intracellular CD38 enzyme in the mouse heart: Role in β-adrenoceptor signaling.

Nicotinic acid adenine dinucleotide phosphate (NAADP) and cyclic ADP-ribose (cADPR) are Ca2+-mobilizing messengers important for modulating cardiac excitation–contraction coupling and pathophysiology. CD38, which belongs to the ADP-ribosyl cyclase family, catalyzes synthesis of both NAADP and cADPR in vitro. However, it remains unclear whether this is the main enzyme for their production under physiological conditions. Here we show that membrane fractions from WT but not CD38−/− mouse hearts supported NAADP and cADPR synthesis. Membrane permeabilization of cardiac myocytes with saponin and/or Triton X-100 increased NAADP synthesis, indicating that intracellular CD38 contributes to NAADP production. The permeabilization also permitted immunostaining of CD38, with a striated pattern in WT myocytes, whereas CD38−/− myocytes and nonpermeabilized WT myocytes showed little or no staining, without striation. A component of β-adrenoreceptor signaling in the heart involves NAADP and lysosomes. Accordingly, in the presence of isoproterenol, Ca2+ transients and contraction amplitudes were smaller in CD38−/− myocytes than in the WT. In addition, suppressing lysosomal function with bafilomycin A1 reduced the isoproterenol-induced increase in Ca2+ transients in cardiac myocytes from WT but not CD38−/− mice. Whole hearts isolated from CD38−/− mice and exposed to isoproterenol showed reduced arrhythmias. SAN4825, an ADP-ribosyl cyclase inhibitor that reduces cADPR and NAADP synthesis in mouse membrane fractions, was shown to bind to CD38 in docking simulations and reduced the isoproterenol-induced arrhythmias in WT hearts. These observations support generation of NAADP and cADPR by intracellular CD38, which contributes to effects of β-adrenoreceptor stimulation to increase both Ca2+ transients and the tendency to disturb heart rhythm.

Possible Roles of Cannabinoid Receptors in Cardiac Myocytes

Anandamide is an endogenous ligand of G-protein coupled cannabinoid CB1 and CB2 receptors, which has been shown to have complex effects on the cardiovascular system culminating in a hypotensive response and a reduction in cardiac contractility. The aims of this study were to investigate electrophysiologically the effects of anandamide on cardiac ventricular action potential (AP) parameters and accompanying cell contractions, and to explore the involvement, if any, of CB1 and CB2 receptors.Guinea pig ventricular myocytes were stimulated to fire APs by a 2 ms depolarising current pulse applied via an intracellular microelectrode at a frequency of 1 Hz using an Axoclamp 2 (Axon Instruments) amplifier (bridge mode). Myocyte contraction was measured using an edge detection system.Anandamide (1, 3, 10 µM) caused a concentration-dependent reduction in AP duration (APD) at 90, 50 and 20 % repolarisation, which was accompanied by a reduction in the amplitude of myocyte contraction. At a concentration of 10 µM, anandamide reduced APD 90 by 38 ± 8 % (n = 7, p < 0.001). In addition, 10 µM R-(+)-methanandamide (a non-hydrolysable analogue of anandamide) similarly reduced APD and amplitude of contraction. In the presence of the CB2 receptor antagonist AM 630, but not the CB1 receptor antagonist AM 281, the magnitudes of these reductions in response to 10 µM anandamide were partially but significantly reduced.This study confirms that anandamide causes a reduction in ventricular myocyte contractility and may modulate ventricular cell Ca2+ handling. Since these effects appear only to be partially inhibited by CB2 receptor blockade, it is likely that anandamide has actions which are independent of CB1 and CB2 receptors.