Proceedings of The Physiological Society

Future Physiology (Leeds, UK) (2017) Proc Physiol Soc 39, PC12

Poster Communications

IP3 signalling in cardiac atria and sino-atrial node; a role for calcium-sensitive adenylyl cyclases?

R. A. Capel1, T. P. Collins1, S. Rajasundaram1, D. A. Terrar1, R. A. Burton1

1. Department of Pharmacology, University of Oxford, Oxford, Oxon, United Kingdom.


Calcium handling in the heart is vital to normal physiology, yet gaps remain in our understanding of how Ca2+ signalling pathways function and interact. Inositol 1,4,5-trisphosphate (IP3) is a Ca2+-releasing second messenger acting at IP3 receptors (IP3Rs), which reside on junctional sarcoplasmic reticulum. IP3 has been implicated in acute modulation of Ca2+ in cardiac atrial1 and sino-atrial node (SAN)2, whilst atrial IP3R expression is increased in both human3 and dog models4 of atrial fibrillation. Ca2+-stimulated adenylyl cyclases (ACs), AC1 and AC8, are expressed in the cardiac atria and SAN, but negligibly within ventricular myocytes5, and have been shown to modulate L-type Ca2+ current6 and funny current5 in atrial and SAN myocytes respectively. The physiological stimuli relevant to these ACs is currently unknown. We have investigated IP3R-mediated AC modulation in the atria and SAN. Male Dunkin Hartley guinea pigs (350-400g) were culled via a Schedule 1 method and cells isolated enzymatically. For murine experiments, adult C57BL/6 wild-type (WT) or AC8 knock-out (KO, C57BL/6 background) mice were culled via a Schedule 1 method, atria isolated and mounted to a force transducer in an organ bath. All experiments were carried out at 35±1°C under oxygenated physiological solution. Data are given as mean±SEM. Phenylephrine (PE, 10µM), an α-adrenoceptor (α-AR) agonist, induced a 35±9% increase in atrial myocyte Ca2+ transient amplitude (n=8). α-ARs are Gq-coupled and would thus be expected to function via synthesis of IP3. PE responses were abolished in the presence of 2.5µM IP3R inhibitor 2-APB. Atrial cellular response to PE was significantly reduced (P<0.05, T-test) in the presence of 10µM MDL, to inhibit ACs, or 1µM H89, to inhibit PKA (to 12±4% and 13±4% increases respectively, both n=5). UV photorelease of IP3 led to a significant increase in Ca2+ transient amplitude (36±12% increase 60 s post photorelease, n=8) which was completely abolished in the presence of 3µM MDL or 1µM H89 (-9±6%, n=6, and -15±8%, n=9, at 60s). Immunocytochemistry showed type 2 IP3Rs co-localised with Ca2+-stimulated AC8, but not AC1, in a band close to the surface of isolated atrial myocytes (Pearson's R = 0.81±0.02, n=14). Spontaneously-beating atrial preparations from AC8 KO mice exhibited a significantly reduced beating rate compared to WT (P<0.05, ANOVA), but did not show a blunted response to PE. Our data suggest that IP3 in cardiac atrial myocytes acts predominantly via ACs to increase Ca2+ transient amplitude and indicates Ca2+-stimulated AC8 as the most likely effector. This is the first study to identify a physiological stimulus for cAMP production via Ca2+-stimulated ACs in the heart. The initiation of cAMP and PKA mediated signalling via Gq coupled pathways is a novel, important mechanism for future study in relation to atrial physiology and pathophysiology.

Where applicable, experiments conform with Society ethical requirements