Length and protein kinase A modulations of myocytes in cardiac myosin binding protein C-deficient mice.
OBJECTIVE
Beta-adrenergic stimulation modulates cardiac contractility through protein kinase A (PKA), which phosphorylates proteins such as troponin I (cTnI) and C-protein (cMyBP-C). The relative contributions of cTnI and cMyBP-C to the regulation of myofilament Ca(2+) sensitivity are still controversial because of difficulty in targeting specific protein phosphorylation. Recently, impaired relaxation was found in cMyBP-C-deficient mice (KO) in vivo under basal conditions and after beta-adrenergic stimulation. The goal of this study was to analyse the length-dependent and PKA-dependent modulations of the cardiac contractile machinery in a mouse model lacking cMyBP-C.


METHODS
In the present work, we studied the PKA effect on myofilament Ca(2+) sensitivity of left ventricular skinned myocytes isolated from 5-week- and 55-week-old wild-type (WT) and cMyBP-C knockout (KO) mice at 1.9 and 2.3 mum sarcomere lengths (SL). The cTnI content and phosphorylation status were examined by Western blot analysis.


RESULTS
Without PKA stimulation and at the shorter SL, Ca(2+) sensitivity was higher in KO compared to WT. The difference disappeared at the longer SL. No difference in passive tension or maximal active tension was observed. PKA stimulation induced a desensitization of WT myofilaments at both SL but had almost no effect in KO myofilaments despite similar levels of cTnI phosphorylation. We also observed expression of slow skeletal TnI in KO animals that was not correlated with the PKA effects.


CONCLUSIONS
The results suggest that cMyBP-C contributes to the regulation of cardiac contraction at short sarcomere length and that myofilament desensitization induced by PKA requires the presence of cMyBP-C and does not depend only upon TnI phosphorylation.
Cazorla
Olivier
O
INSERM U-637, CHU Arnaud de Villeneuve, F-34295 Montpellier, France. cazorla@montp.inserm.fr
Szilagyi
Szabolcs
S
Vignier
Nicolas
N
Salazar
Guillermo
G
Krämer
Elisabeth
E
Vassort
Guy
G
Carrier
Lucie
L
Lacampagne
Alain
A
D008841:Actin Cytoskeleton / Q000378:metabolism / Q000648:ultrastructure; D000818:Animals; D015153:Blotting, Western / Q000379:methods; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D005260:Female; D051379:Mice; D018345:Mice, Knockout; D032383:Myocytes, Cardiac / Q000378:metabolism* / Q000648:ultrastructure; D010766:Phosphorylation; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012518:Sarcomeres / Q000378:metabolism / Q000648:ultrastructure; D019210:Troponin I / Q000378:metabolism; D000099163:Myosin Binding Protein C
D002118:Calcium; D002352:Carrier Proteins; D017868:Cyclic AMP-Dependent Protein Kinases; D019210:Troponin I; D000099163:Myosin Binding Protein C