Protein kinase D selectively targets cardiac troponin I and regulates myofilament Ca2+ sensitivity in ventricular myocytes.
Protein kinase D (PKD) is a serine/threonine kinase with emerging myocardial functions; in skinned adult rat ventricular myocytes (ARVMs), recombinant PKD catalytic domain phosphorylates cardiac troponin I at Ser22/Ser23 and reduces myofilament Ca(2+) sensitivity. We used adenoviral gene transfer to determine the effects of full-length PKD on protein phosphorylation, sarcomere shortening and [Ca(2+)](i) transients in intact ARVMs. In myocytes transduced to express wild-type PKD, the heterologously expressed enzyme was activated by endothelin 1 (ET1) (5 nmol/L), as reflected by PKD phosphorylation at Ser744/Ser748 (PKC phosphorylation sites) and Ser916 (autophosphorylation site). The ET1-induced increase in cellular PKD activity was accompanied by increased cardiac troponin I phosphorylation at Ser22/Ser23; this measured approximately 60% of that induced by isoproterenol (10 nmol/L), which activates cAMP-dependent protein kinase (PKA) but not PKD. Phosphorylation of other PKA targets, such as phospholamban at Ser16, phospholemman at Ser68 and cardiac myosin-binding protein C at Ser282, was unaltered. Furthermore, heterologous PKD expression had no effect on isoproterenol-induced phosphorylation of these proteins, or on isoproterenol-induced increases in sarcomere shortening and relaxation rate and [Ca(2+)](i) transient amplitude. In contrast, heterologous PKD expression suppressed the positive inotropic effect of ET1 seen in control cells, without altering ET1-induced increases in relaxation rate and [Ca(2+)](i) transient amplitude. Complementary experiments in "skinned" myocytes confirmed reduced myofilament Ca(2+) sensitivity by ET1-induced activation of heterologously expressed PKD. We conclude that increased myocardial PKD activity induces cardiac troponin I phosphorylation at Ser22/Ser23 and reduces myofilament Ca(2+) sensitivity, suggesting that altered PKD activity in disease may impact on contractile function.
Cuello
Friederike
F
Cardiovascular Division, King's College London, London, UK.
Bardswell
Sonya C
SC
Haworth
Robert S
RS
Yin
Xiaoke
X
Lutz
Susanne
S
Wieland
Thomas
T
Mayr
Manuel
M
Kentish
Jonathan C
JC
Avkiran
Metin
M
D008841:Actin Cytoskeleton / Q000378:metabolism*; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002478:Cells, Cultured; D019332:Endothelin-1 / Q000494:pharmacology; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics; D006352:Heart Ventricles / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D007545:Isoproterenol / Q000494:pharmacology; D051379:Mice; D032383:Myocytes, Cardiac / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D051381:Rats; D012518:Sarcomeres / Q000187:drug effects / Q000502:physiology; D019210:Troponin I / Q000378:metabolism*
D000318:Adrenergic beta-Agonists; D019332:Endothelin-1; D019210:Troponin I; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C089212:protein kinase D; D011493:Protein Kinase C; D007545:Isoproterenol; D002118:Calcium