Synthesis of the Ca2+-mobilizing messengers NAADP and cADPR by intracellular CD38 enzyme in the mouse heart: Role in ß-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 ß-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 ß-adrenoreceptor stimulation to increase both Ca2+ transients and the tendency to disturb heart rhythm.

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.

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.

Novel insights into mechanisms for Pak1-mediated regulation of cardiac Ca(2+) homeostasis.

A series of recent studies report novel roles for Pak1, a key member of the highly conserved family of serine-threonine protein kinases regulated by Ras-related small G-proteins, Cdc42/Rac1, in cardiac physiology and cardioprotection. Previous studies had identified Pak1 in the regulation of hypertrophic remodeling that could potentially lead to heart failure. This article provides a review of more recent findings on the roles of Pak1 in cardiac Ca(2+) homeostasis. These findings identified crucial roles for Pak1 in cardiomyocyte Ca(2+) handling and demonstrated that it functions through unique mechanisms involving regulation of the post-transcriptional activity of key Ca(2+)-handling proteins, including the expression of Ca(2+)-ATPase SERCA2a, along with the speculative possibility of an involvement in the maintenance of transverse (T)-tubular structure. They highlight important regulatory functions of Pak1 in Ca(2+) homeostasis in cardiac cells, and identify novel potential therapeutic strategies directed at manipulation of Pak1 signaling for the management of cardiac disease, particularly heart failure.