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

Phosphorylation of proteins in cardiac myofilaments is a major determinant in the regulation of the Ca(2+) 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 Mn(2+)-dependent catalytic subunit of protein phosphatase 1 (PP1c-alpha) on the Ca(2+) regulation of contraction. In particular, we tested the hypothesis that phosphorylation persists after the skinning procedure and thereby attenuates protein kinase A (PKA)-induced Ca(2+) desensitisation.

Effects of Mn(2+) and Mn(2+)-PP1c on the Ca(2+) 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.

PKA treatment significantly decreased the pCa(50) by 0.04 pCa units. In contrast, treatment with PP1c or Mn(2+)-containing PP1c buffer significantly increased the pCa(50) by 0.26 units or 0.09 units, respectively. These Ca(2+) 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 Ca(2+) sensitisation. PP1c removed (32)P which had been incorporated into cTnI and cardiac myosin binding protein C by PKA treatment. PKA incorporated twofold more (32)P into cTnI in fibres pre-treated with PP1c.

Mn(2+)-dependent PP1c increases the Ca(2+) 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.