In the heart, endogenous nicotinic acid adenine dinucleotide phosphate (NAADP) triggers lysosomal calcium release to augment sarcoplasmic reticulum (SR) calcium sequestration, producing larger calcium transients. However, the role of lysosomal calcium signals in pacemaker activity, a distinct calcium-operated function of the sino-atrial node (SAN) or atria, a distinct calcium-operated function, has not been investigated. Pharmacological or genetic ablation of the NAADP pathway inhibits spontaneous beating rate response to β-adrenergic stimulation in intact SAN. We found intracellular signalling microdomains between lysosomes and neighbouring SR or mitochondria in mouse, rabbit, goat, and human atrial tissue. The spatial relationship between lysosomes and other calcium-handling organelles are altered in goat and human atrial fibrillation. Furthermore, we demonstrate atrial myocytes produce 3′–5′-cyclic adenosine monophosphate in response to lysosomal signalling, adding a novel trigger for cyclic nucleotide signalling. Our findings support the hypothesis that lysosomal calcium signaling directly increases cardiomyocyte cAMP and modulates pacemaker activity.

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia. Excessive stimulation of the IP3 signalling pathway has been linked to AF through abnormal calcium handling. However, little is known about the mechanisms involved in this process. We expressed the fluorescence resonance energy transfer (FRET) based cytosolic cAMP sensor EPAC-SH187 in neonatal rat atrial myocytes (NRAMs) and neonatal rat ventricular myocytes (NRVMs). In NRAMs, addition of the α-1 agonist phenylephrine (PE, 3 µM) resulted in a FRET change 21.20 ± 7.43 % and addition of membrane permeant IP3 derivative, 2,3,6-tri-O-Butyryl-myo-IP3(1,4,5)-hexakis(acetoxymethyl)ester (IP3-AM, 20 μM) resulted in a peak of 20.31 ± 6.74 %. These FRET changes imply an increase in cAMP. Prior application of IP3 receptor (IP3R) inhibitors 2-Aminoethyl diphenylborinate (2-APB, 2.5μM) or Xestospongin-C (0.3 μM) significantly inhibited the change in FRET in NRAMs in response to PE. Xestospongin-C (0.3 μM) significantly inhibited the change in FRET in NRAMs in response to IP3-AM. The FRET change in response to PE in NRVMs were not inhibited by 2-APB or Xestospongin-C. Finally, the localisation of cAMP signals was tested by expressing the FRET-based cAMP sensor, AKAP79-CUTie, which targets the intracellular surface of the plasmalemma. We found in NRAMs that PE led to FRET change corresponding to an increase in cAMP that was inhibited by 2-APB and Xestospongin C. These data support further investigation of the pro-arrhythmic nature and components of IP3 induced cAMP signalling to identify potential pharmacological targets.

Inositol trisphosphate (IP3), a calcium (Ca2+)-mobilizing second messenger, releases Ca2+ from the sarcoplasmic reticulum (SR) via IP3 receptors and modulates adenylyl cyclase (AC) activity in atrial myocytes. Lysosomes participate in Ca2+ homeostasis by mobilising Ca2+ in response to Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP). We postulate that both downstream activation of Ca2+ sensitive AC (AC1 and AC8) and lysosomal Ca2+ signalling in response to IP3R activation contribute to atrial myocyte function and pacemaking. Ectopic application of phenylephrine (PE) increased chronotropy and inotropy and this response was reduced in the presence of NAADP pathway inhibitors (BZ-194 and SAN4825) and Bafilomycin A1. PE increased cAMP activity in neonatal rat atrial myocytes (NRAMs) and this was inhibited by NAADP pathway inhibitors. This inhibition was not observed in neonatal rat ventricular myocytes (NRVMs), revealing specificity of this response to NRAMs. We investigated expression of AC1 and AC8 as a possible explanation to these observations. Genetic perturbation of AC1 and AC8 by double-knockout of Adcy1 and Adcy8 in a mouse model showed a decrease in positive chronotropic and inotropic response upon cumulative dose of PE in atrial tissue, reduced PE stimulated amplitude of Ca2+ transient in isolated atrial myocytes and presented decreased cytosolic cAMP levels in response to PE in neonatal atrial myocytes that was not inhibited by NAADP pathway inhibitors. Our data identifies a link between NAADP and α-adrenergic signalling pathways in atrial myocytes, highlighting that lysosomal Ca2+ is an important component of α-adrenergic stimulation in the cardiac atria and warrants further investigation. ### Competing Interest Statement The authors have declared no competing interest.

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.

Atrial arrhythmias, such as atrial fibrillation (AF), are a major mortality risk and a leading cause of stroke. The IP 3 signalling pathway has been proposed as an atrial specific target for AF therapy, and atrial IP 3 signalling has been linked to the activation of calcium sensitive adenylyl cyclases AC1 and AC8. Here we investigated the involvement of AC1 in the response of intact mouse atrial tissue and isolated guinea pig atrial and sinoatrial 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 14.46 % to 8.17% ( 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 = 14.20 %, ST034307 = 16.32 %; P > 0.05). Experiments were performed using isolated guinea pig atrial and SAN cells loaded with Fluo-5F-AM to record changes in calcium transient amplitude (CaT) generated by 10μM PE in the presence and absence of 1μM ST034307. ST034307 significantly reduced the beating rate of SAN cells (0.34-fold decrease; P = 0.004), but did not result in an inhibition of CaT amplitude increase in response to PE in atrial cells. The results presented here demonstrate the involvement of AC1 in the downstream response of atrial pacemaker activity to α-adrenoreceptor stimulation and IP 3 R calcium release.