Dissociation of FKBP12.6 from ryanodine receptor type 2 is regulated by cyclic ADP-ribose but not β-adrenergic stimulation in mouse cardiomyocytes

Abstract

Aims β-Adrenergic augmentation of Ca2+ sparks and cardiac contractility has been functionally linked to phosphorylation-dependent dissociation of FK506 binding protein 12.6 (FKBP12.6) regulatory proteins from ryanodine receptors subtype 2 (RYR2). We used FKBP12.6 null mice to test the extent to which the dissociation of FKBP12.6 affects Ca2+ sparks and mediates the inotropic action of isoproterenol (ISO), and to investigate the underlying mechanisms of cyclic ADP-ribose (cADPR) regulation of Ca2+ sparks.Methods and resultsCa2+ sparks and contractility were measured in cardiomyocytes and papillary muscle segments from FKBP12.6 null mice, and western blot analysis was carried out on sarcoplasmic reticulum microsomes prepared from mouse heart. Exposure to ISO resulted in a three-and two-fold increase in Ca2+ spark frequency in wild-type (WT) and FKBP12.6 knockout (KO) myocytes, respectively, and Ca2+ spark kinetics were also significantly altered in both types of cells. The effects of ISO on Ca2+ spark properties in KO cells were inhibited by pre-treatment with thapsigargin or phospholamban inhibitory antibody, 2D12. Moreover, twitch force magnitude and the rate of force development were not significantly different in papillary muscles from WT and KO mice. Unlike β-adrenergic stimulation, cADPR stimulation increased Ca2+ spark frequency (2.8-fold) and altered spark kinetics only in WT but not in KO mice. The effect of cADPR on spark properties was not entirely blocked by pre-treatment with thapsigargin or 2D12. In voltage-clamped cells, cADPR increased the peak Ca2+ of the spark without altering the decay time. We also noticed that basal Ca2+ spark properties in KO mice were markedly altered compared with those in WT mice.ConclusionOur data demonstrate that dissociation of FKBP12.6 from the RYR2 complex does not play a significant role in β-adrenergic-stimulated Ca2+ release in heart cells, whereas this mechanism does underlie the action of cADPR.