PKA-dependent phosphorylation of cardiac myosin binding protein C in transgenic mice.
OBJECTIVE
To investigate the physiological role of cAMP-dependent protein kinase A (PKA)-mediated, cardiac myosin binding protein C (MyBP-C) phosphorylation.


METHODS
A cardiac MyBP-C cDNA lacking nine amino acids, which contained a phosphorylation site, was made, and subsequently used to generate multiple lines of transgenic mice. Upon confirming that a partial replacement of endogenous protein with transgenic protein occurred, the biochemical and physiological consequences were studied. PKA-dependent phosphorylation assays were used to estimate the phosphorylation states of major cardiac PKA substrates. Myofibril Mg-ATPase activities were also measured. Isolated working heart and whole animal exercise studies were used to measure the physiological changes.


RESULTS
Transgenic mice displayed a compensatory response, with PKA-mediated phosphorylation of both troponin I and phospholamban showing significant increases. The remaining endogenous cardiac MyBP-C also showed increased phosphorylation levels. Maximal Mg(2+)-ATPase activity was increased. Significant functional changes at both the whole organ and whole animal levels also occurred. Parameters reflecting cardiac contractility and relaxation increased about 22 and 25%, respectively, in the mutant relative to wild type mice (n=5, P<0.001). In young adults the capacity for stress exercise, quantitated using an exercise treadmill regimen, was substantially enhanced (n=6, P<0.01).


CONCLUSIONS
Cardiac MyBP-C phosphorylation plays an important physiological role and that the protein's degree of phosphorylation is coordinated with the phosphorylation levels of other proteins within the contractile apparatus.
Yang
Q
Q
Department of Pediatrics, Division of Molecular Cardiovascular Biology, The Children's Hospital Research Foundation, 333 Burnet Avenue, Cincinnati, OH 45229-3039, USA.
Hewett
T E
TE
Klevitsky
R
R
Sanbe
A
A
Wang
X
X
Robbins
J
J
D000818:Animals; D015153:Blotting, Western; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D002352:Carrier Proteins / Q000032:analysis / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D005818:Genetic Engineering; D051379:Mice; D008822:Mice, Transgenic; D009200:Myocardial Contraction / Q000502:physiology*; D009206:Myocardium / Q000737:chemistry / Q000378:metabolism*; D009210:Myofibrils / Q000201:enzymology; D010477:Perfusion; D010766:Phosphorylation; D015398:Signal Transduction / Q000502:physiology*; D000099163:Myosin Binding Protein C
D017301:Ca(2+) Mg(2+)-ATPase; D002352:Carrier Proteins; D017868:Cyclic AMP-Dependent Protein Kinases; D000099163:Myosin Binding Protein C