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Cardiovascular Research 2007 74(1):124-132; doi:10.1016/j.cardiores.2007.01.019
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Copyright © 2007, European Society of Cardiology

Mn2+-dependent protein phosphatase 1 enhances protein kinase A-induced Ca2+ desensitisation in skinned murine myocardium

Axel Neulena,*, Natascha Blaudecka, Stefan Zittricha, Doris Metzlera, Gabriele Pfitzera,b,* and Robert Stehlea,b

aInstitute of Vegetative Physiology, University of Cologne, Robert-Koch-Strasse 39, D-50931 Köln, Germany
bCenter of Molecular Medicine, University of Cologne, Robert-Koch-Strasse 39, D-50931 Köln, Germany

* Corresponding authors. Institut für Vegetative Physiologie, Universität zu Köln, Robert-Koch-Strasse 39, D-50931 Köln, Germany. Tel.: +49 221 4786950; fax: +49 221 4786965. Email address: axel.neulen{at}uni-koeln.de gabriele.pfitzer{at}uni-koeln.de

Objective: Phosphorylation of proteins in cardiac myofilaments is a major determinant in the regulation of the Ca2+ sensitivity of contraction. Whereas most reports have focused on effects of phosphorylation, little is known about reverse effects of dephosphorylation in skinned myocardium. Here we studied the effect of the Mn2+-dependent catalytic subunit of protein phosphatase 1 (PP1c-{alpha}) on the Ca2+ regulation of contraction. In particular, we tested the hypothesis that phosphorylation persists after the skinning procedure and thereby attenuates protein kinase A (PKA)-induced Ca2+ desensitisation.

Methods: Effects of Mn2+ and Mn2+-PP1c on the Ca2+ sensitivity of contraction (pCa50) were investigated in triton-skinned cardiac fibres from mice and compared with those of PKA treatment. Phosphorylation of proteins was monitored by autoradiography.

Results: PKA treatment significantly decreased the pCa50 by 0.04 pCa units. In contrast, treatment with PP1c or Mn2+-containing PP1c buffer significantly increased the pCa50 by 0.26 units or 0.09 units, respectively. These Ca2+ sensitisations were completely reversed by subsequent PKA treatment. Replacement of the endogenous cardiac troponin I (cTnI) in fibres with the phospho-mimicking mutant human cTnIS22/23D abolished the PP1c-induced Ca2+ sensitisation. PP1c removed 32P which had been incorporated into cTnI and cardiac myosin binding protein C by PKA treatment. PKA incorporated twofold more 32P into cTnI in fibres pre-treated with PP1c.

Conclusions: Mn2+-dependent PP1c increases the Ca2+ sensitivity of contraction of skinned cardiac fibres. This can be ascribed to dephosphorylation of PKA-dependent phosphorylation sites. Hence PKA-dependent phosphorylation of sarcomeric proteins persists to a functionally relevant degree after the skinning procedure.

KEYWORDS Contractile function; Protein phosphatases; Protein phosphorylation; Protein kinase A

Time for primary review 17 days


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