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

doi:10.1016/j.cardiores.2007.01.019

Cardiovascular Research 2007 74(1):124-132; doi:10.1016/j.cardiores.2007.01.019

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

Axel Neulen^a^,*, Natascha Blaudeck^a, Stefan Zittrich^a, Doris Metzler^a, Gabriele Pfitzer^a^,b^,* and Robert Stehle^a^,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 myofilamentsis a major determinant in the regulation of the Ca²⁺ sensitivityof contraction. Whereas most reports have focused on effectsof phosphorylation, little is known about reverse effects ofdephosphorylation in skinned myocardium. Here we studied theeffect of the Mn²⁺-dependent catalytic subunit of protein phosphatase1 (PP1c- ${alpha}$ ) on the Ca²⁺ regulation of contraction. In particular,we tested the hypothesis that phosphorylation persists afterthe skinning procedure and thereby attenuates protein kinaseA (PKA)-induced Ca²⁺ desensitisation.

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

Results: PKA treatment significantly decreased the pCa₅₀ by0.04 pCa units. In contrast, treatment with PP1c or Mn²⁺-containingPP1c buffer significantly increased the pCa₅₀ by 0.26 unitsor 0.09 units, respectively. These Ca²⁺ sensitisations werecompletely reversed by subsequent PKA treatment. Replacementof the endogenous cardiac troponin I (cTnI) in fibres with thephospho-mimicking mutant human cTnI^S22/23D abolished the PP1c-inducedCa²⁺ sensitisation. PP1c removed ³²P which had been incorporatedinto cTnI and cardiac myosin binding protein C by PKA treatment.PKA incorporated twofold more ³²P into cTnI in fibres pre-treatedwith PP1c.

Conclusions: Mn²⁺-dependent PP1c increases the Ca²⁺ sensitivityof contraction of skinned cardiac fibres. This can be ascribedto dephosphorylation of PKA-dependent phosphorylation sites.Hence PKA-dependent phosphorylation of sarcomeric proteins persiststo 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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