Background: Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent endogenous Ca2+ mobilizing second messenger that is known to release Ca2+ from the acidic endolysosomal vesicles. Our previous work showed that intracellular application of NAADP led to the increase in contractions of electrically stimulated guinea pig ventricular cardiomyocytes. These observations were accompanied by an increase in sarcoplasmic reticulum (SR) Ca2+ load. Previously, it has been reported that beta-adrenergic activation increases the level of NAADP in guinea pig whole heart experiments, and we therefore hypothesized that the inotropic effect of beta-adrenergic activation is partly mediated by NAADP. This study aims to test this possibility and subsequently dissect the related pathway.
Methods and results: Two-pore channel 2 (Tpc2) is the most widely discussed channel, which mediates NAADP-regulated endolysosomal calcium release. Using a genetic modification approach, we showed that intracellular application of NAADP on electrically stimulated ventricular myocytes significantly increased calcium transient amplitude in wild type but not Tpc2 knock out mice (16±5%, -6±4%; p<0.05). Strikingly, Tpc2 KO mouse ventricular cardiomyocytes also showed a reduced isoprenaline-induced increase in contraction and calcium transient amplitudes compared to wild type mice (124±23%, 57±17%; p<0.05), establishing the contribution of NAADP to the beta-adrenergic pathway-regulated inotropic effect. In order to dissect the downstream mechanism, we showed that preincubation of the Ca2+ /calmodulin-dependent protein kinase II (CaMKII) inhibitor, KN93 in electrically stimulated mouse ventricular cardiomyocytes abolished the NAADP-induced increase in calcium transient amplitude (-6±3%). This is consistent with CaMKII acting as a downstream effector of beta-adrenergic activation that amplifies SR calcium uptake. With the patch-clamp technique, we presented further data showing there is no significant difference in L-type calcium current amplitude between wild type and Tpc2KO mice upon beta-adrenergic activation, showing that an augmented calcium uptake but not calcium influx is responsible for the investigated pathway.
Conclusion: We report here that the NAADP signaling cascade is a novel downstream pathway of beta-adrenergic activation in mouse ventricular cardiomyocytes. This pathway requires functional Tpc2 channels and is partly if not entirely mediated through the activation of CaMKII by the NAADP-released Ca2+, which subsequently enhances SR calcium uptake, contributing to the inotropic effect of cardiac beta-adrenergic activation.