In vivo and in vitro analysis of cardiac troponin I phosphorylation.
Adrenergic stimulation induces positive changes in cardiac contractility and relaxation. Cardiac troponin I is phosphorylated at different sites by protein kinase A and protein kinase C, but the effects of these post-translational modifications on the rate and extent of contractility and relaxation during beta-adrenergic stimulation in the intact animal remain obscure. To investigate the effect(s) of complete and chronic cTnI phosphorylation on cardiac function, we generated transgenic animals in which the five possible phosphorylation sites were replaced with aspartic acid, mimicking a constant state of complete phosphorylation (cTnI-AllP). We hypothesized that chronic and complete phosphorylation of cTnI might result in increased morbidity or mortality, but complete replacement with the transgenic protein was benign with no detectable pathology. To differentiate the effects of the different phosphorylation sites, we generated another mouse model, cTnI-PP, in which only the protein kinase A phosphorylation sites (Ser(23)/Ser(24)) were mutated to aspartic acid. In contrast to the cTnIAllP, the cTnI-PP mice showed enhanced diastolic function under basal conditions. The cTnI-PP animals also showed augmented relaxation and contraction at higher heart rates compared with the nontransgenic controls. Nuclear magnetic resonance amide proton/nitrogen chemical shift analysis of cardiac troponin C showed that, in the presence of cTnI-AllP and cTnI-PP, the N terminus exhibits a more closed conformation, respectively. The data show that protein kinase C phosphorylation of cTnI plays a dominant role in depressing contractility and exerts an antithetic role on the ability of protein kinase A to increase relaxation.
Sakthivel
Sadayappan
S
Division of Molecular Cardiovascular Biology, Department of Pediatrics, The Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio 45229, USA.
Finley
Natosha L
NL
Rosevear
Paul R
PR
Lorenz
John N
JN
Gulick
James
J
Kim
Scott
S
VanBuren
Peter
P
Martin
Lisa A
LA
Robbins
Jeffrey
J
D000818:Animals; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D066298:In Vitro Techniques; D051379:Mice; D008822:Mice, Transgenic; D009200:Myocardial Contraction / Q000502:physiology; D009206:Myocardium / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D019210:Troponin I / Q000378:metabolism*
D019210:Troponin I; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C