Abstract 10380: Beta-Adrenergic Stimulation Enhances Ca2+ Leak From Sarcoplasmic Reticulum Through Protein Kinase A-Dependent Phosphorylation of Ryanodine Receptor under Physiological Condition
Introduction: β-adrenoceptor stimulation (β-ARS) has been considered to increase diastolic Ca2+ leak from ryanodine receptor (RyR) of sarcoplasmic reticulum (SR) via protein kinase A (PKA) and/or Ca2+/calmodulin-dependent kinase II (CaMKII)-dependent phosphorylation of RyR. The increased Ca2+ leak leads to contractile dysfunction and arrhythmia under pathophysiological condition, such as heart failure. β-ARS also shows dissociation of FK506 binding protein (FKBP) 12.6 from RyR after hyperphosphorylation by PKA under pathophysiological condition. However, the effect of β-ARS on Ca2+ leak from SR under physiological condition remains unclear. In this study, we investigated the role of β-ARS and FKBP12.6 on Ca2+ leak from SR under physiological condition.
Method: Thin trabeculae were dissected from the mouse heart and were treated with 1 μM isoproterenol (30 min), followed by permeabilization with 50 μg/ml saponin in the presence of a phosphatase inhibitor, calyculin A. Using this preparation, the amount of Ca2+ in SR was measured with fluo-3 by applying 50 mM caffeine to release Ca2+ from SR. Ca2+ leak was estimated by measuring the remaining Ca2+ in SR after leaving the preparation in the relaxing solution with EGTA to leak Ca2+ from SR for various durations (15–300 sec) following maximal Ca2+ loading (pCa 6.2, 120 sec). Western immunoblotting was carried out to estimate the amount of FKBP12.6 and the phosphorylation of RyR before and after β-ARS.
Results: The amount of Ca2+ leak from SR after maximal Ca2+ loading was significantly increased after β-ARS, which was clearly observed in the time constant of Ca2+ leak curve (control: 163.08±9.53, n=18, β-ARS: 119.53±7.06, n=18, P<0.01). The enhanced Ca2+ leak of SR was blocked by a PKA inhibitor H-89 (2 μM), but not by a CaMKII inhibitory peptide, autocamtide 2-related inhibitory peptide (100 μM). The PKA-dependent phosphorylation of RyR at Ser2808 was significantly increased, whereas CaMKII-dependent phosphorylation of RyR at Ser2814 was not altered after β-ARS. The amount of FKBP12.6 was not altered after β-ARS.
Conclusion: Under physiological condition, Ca2+ leak from SR was enhanced by PKA-dependent phosphorylation of RyR without the dissociation of FKBP12.6 after β-ARS.
- © 2010 by American Heart Association, Inc.