Dissociation of FKBP12.6 from ryanodine receptor type 2 is regulated by cyclic ADP-ribose but not beta-adrenergic stimulation in mouse cardiomyocytes.
OBJECTIVE
Beta-adrenergic augmentation of Ca(2+) 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 Ca(2+) sparks and mediates the inotropic action of isoproterenol (ISO), and to investigate the underlying mechanisms of cyclic ADP-ribose (cADPR) regulation of Ca(2+) sparks.


RESULTS
Ca(2+) 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 Ca(2+) spark frequency in wild-type (WT) and FKBP12.6 knockout (KO) myocytes, respectively, and Ca(2+) spark kinetics were also significantly altered in both types of cells. The effects of ISO on Ca(2+) 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 beta-adrenergic stimulation, cADPR stimulation increased Ca(2+) 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 Ca(2+) of the spark without altering the decay time. We also noticed that basal Ca(2+) spark properties in KO mice were markedly altered compared with those in WT mice.


CONCLUSIONS
Our data demonstrate that dissociation of FKBP12.6 from the RYR2 complex does not play a significant role in beta-adrenergic-stimulated Ca(2+) release in heart cells, whereas this mechanism does underlie the action of cADPR.
Zhang
Xu
X
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101, China.
Tallini
Yvonne N
YN
Chen
Zheng
Z
Gan
Lu
L
Wei
Bin
B
Doran
Robert
R
Miao
Lin
L
Xin
Hong-Bo
HB
Kotlikoff
Michael I
MI
Ji
Guangju
G
D000200:Action Potentials; D000318:Adrenergic beta-Agonists / Q000494:pharmacology*; D000818:Animals; D020013:Calcium Signaling / Q000187:drug effects*; D002135:Calcium-Binding Proteins / Q000378:metabolism; D036563:Cyclic ADP-Ribose / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D007545:Isoproterenol / Q000494:pharmacology*; D007700:Kinetics; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D009200:Myocardial Contraction / Q000187:drug effects*; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000378:metabolism; D010210:Papillary Muscles / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation; D011943:Receptors, Adrenergic, beta / Q000187:drug effects* / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism; D022021:Tacrolimus Binding Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D019284:Thapsigargin / Q000494:pharmacology; D000099211:Phospholamban
D000318:Adrenergic beta-Agonists; D002135:Calcium-Binding Proteins; D036563:Cyclic ADP-Ribose; D017868:Cyclic AMP-Dependent Protein Kinases; D004791:Enzyme Inhibitors; D007545:Isoproterenol; D011943:Receptors, Adrenergic, beta; D019837:Ryanodine Receptor Calcium Release Channel; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D022021:Tacrolimus Binding Proteins; D019284:Thapsigargin; D000099211:Phospholamban; C509259:FKBP1B protein, mouse