A novel role for NAADP in the regulation of cardiac excitation-contraction coupling

S. Rakovic, M. Yamasaki, A. Macgregor, R. Parkesh, G.C. Churchill, A. Galione & D.A. Terrar. University Department of Pharmacology, Mansfield Road, Oxford OX1 3 QT, UK

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a recently described Ca2+ mobilizing intracellular messenger. We have shown previously that NAADP increases whole cell Ca2+ transients and contractions in guinea-pig ventricular myocytes when photoreleased from a caged analogue. These effects were prevented by the proton pump inhibitor bafilomycin A, indicating involvement of an acidic Ca2+ store. The aim of these experiments was to explore further the role of NAADP in cardiac muscle and to investigate the possible influence of ß-adrenoceptor stimulation on this pathway.

Male guinea-pigs (350-500 g) or rats (250 g) were killed by cervical dislocation following stunning in accordance with the Home Office Guidance On The Operation Of The Animals (Scientific Procedures) Act 1986 (H.M.S.O.) . Isolated myocytes were stimulated to fire action potentials, recorded with patch electrodes; whole-cell patch allowed introduction of membrane-impermeant drugs to the cytosol. Cell contractions were measured using an edge detection system. Ca2+ transients and sparks were imaged using a scanning laser confocal microscope (fluo-4). NAADP levels were assessed using a radio-ligand binding assay. Data are expressed as mean ± S.E.M.; statistical comparisons were made using paired or unpaired Student’s t test, with P < 0.05 taken to indicate statistical significance.

Rapid application of NAADP-AM (60 nM) increased contraction of guinea-pig ventricular myocytes (by 28 ± 5% at 20 s, n = 8, P < 0.05). This effect was inhibited by pre-treatment with bafilomycin (1 µM, 5 min) (-1 ± 1% at 20 s, n = 6, P > 0.05). In rat ventricular myocytes, application of NAADP-AM (60 nM, 20 s) increased spark frequency from 2.67 ± 1.64 to 4.52 ± 1.97 events/s/100 µM, spark amplitude by 37 ± 6 % and resting fluorescence level by 25 ± 5 % (n = 6, P < 0.05); all these actions were prevented by pre-treatment with bafilomycin. Labelling of acidic stores with lysotracker red was greatly diminished following bafilomycin, whereas labelling of the sarcopasmic reticulum (SR) with BODIPY-ryanodine or BODIPY-thapsigargin showed little or no change after addition of bafilomycin. In Langendorff perfused hearts, isoprenaline (60 nM, 5 min) increased NAADP levels from 0.220 ± 0.027 to 0.337 ± 0.043 nmol/mg protein. The enhancement of the Ca2+ transient by UV-photolysis of caged NAADP (5 µM) was smaller in the presence of 5 nM isoprenaline (18 ± 7 % increase 60 s after photolysis; n = 7, P < 0.05) than in its absence (41 ± 10 %; n = 8, P < 0.05), as might be expected if NAADP levels were elevated in the presence of a ß-adrenoceptor agonist. In contrast, cytosolic application of a high self-inactivating concentration of NAADP (1 mM, 3 min) caused a greater reduction of contraction amplitude in the presence of 2 nM isoprenaline (40 ± 6 %; n = 6, P < 0.05) than in its absence (26 ± 5 %; n = 10, P < 0.05).

These observations are consistent with an action of NAADP to increase myocyte Ca2+ transients and contractions by releasing Ca2+ from a bafilomycin-sensitive acidic store, which in turn enhances SR Ca2+ load. Furthermore, NAADP may be a novel mechanism by which ß-adrenoceptor agonists enhance contractility in the heart.