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(Circulation Research. 2007;100:864.)
© 2007 American Heart Association, Inc.

Cellular Biology

Protein Kinase D Selectively Targets Cardiac Troponin I and Regulates Myofilament Ca²⁺ Sensitivity in Ventricular Myocytes

Friederike Cuello^*, Sonya C. Bardswell^*, Robert S. Haworth, Xiaoke Yin, Susanne Lutz, Thomas Wieland, Manuel Mayr, Jonathan C. Kentish, Metin Avkiran

From the Cardiovascular Division (F.C., S.C.B., R.S.H., X.Y., M.M., J.C.K., M.A.), King’s College London, UK; and Institut für Pharmakologie und Toxikologie (S.L., T.W.), Universität Heidelberg, Mannheim, Germany.

Correspondence to Prof Metin Avkiran, Cardiovascular Division, King’s College London, The Rayne Institute, St. Thomas’ Hospital, London SE1 7EH, UK. E-mail metin.avkiran{at}kcl.ac.uk

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 Ca²⁺ sensitivity. We used adenoviral gene transfer to determine the effects of full-length PKD on protein phosphorylation, sarcomere shortening and [Ca²⁺]_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 [Ca²⁺]_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 [Ca²⁺]_i transient amplitude. Complementary experiments in "skinned" myocytes confirmed reduced myofilament Ca²⁺ 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 Ca²⁺ sensitivity, suggesting that altered PKD activity in disease may impact on contractile function.

Key Words: protein kinase D • cardiac troponin I • protein phosphorylation • contractile function • calcium sensitivity

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