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

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 I (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 (pCa(50)) 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 pCa(50) by 0.04 pCa units. In contrast, treatment with PP1c or Mn2+-containing PP1c buffer significantly increased the pCa(50) 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 cTnI(S22/23D) abolished the PP1c-induced Ca2+ sensitisation. PP1c removed P-32 which had been incorporated into cTnI and cardiac myosin binding protein C by PKA treatment. PKA incorporated twofold more P-32 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. (c) 2007 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.