Phosphorylation by protein kinase C and the responsiveness of Mg2+-ATPase to Ca2+ of myofibrils isolated from stunned and non-stunned porcine myocardium

Previously we showed in an in situ porcine model that the thiadiazinone derivative [+]EMD 60263, a Ca^sup 2+^ sensitizer without phosphodiesterase III inhibitory properties, increased contractility more profoundly in stunned than in non-stunned myocardium. This finding was consistent with the observed leftward shifts of the pCa^sup 2+^/Mg^sup 2+^-ATPase curves of isolated myofibrils induced by [+]EMD 60263. The aim of the present investigation was to study the possible involvement of protein kinase C in the mechanism of reduced Ca^sup 2+^ responsiveness of myofilaments during stunning. No differences were observed in the maximal activity of the Ca^sup 2+^-stimulated Mg^sup 2+^-ATPase and in the pCa^sub 50^ of myofibrils isolated from non-stunned and stunned myocardium. After phosphorylation with [gamma-^sup 32^P]-ATP and excess of purified rat brain protein kinase C, the myofibrils were separated on sodiumdodecylsulphate-polyacrylamide gelectrophoresis and the^sup 32^ P incorporation counted by the Molecular Imager. Ca^sup 2+^/phosphatidylserine/sn-1,2 diolein-dependent^sup 32^ P incorporation catalyzed by excess of purified rat brain protein kinase C in C-protein, TnT and TnI subunits did not show any differences between myofibrils from non-stunned and stunned myocardium. However, protein kinase C-induced phosphorylation of myofibrils isolated from ventricular myocardium of sham-operated pigs resulted in a marked leftward shift of the pCa^sub 50^ from 6.03 ± 0.04 to 6.44 ± 0.06 (p < 0.05), while porcine heart cyclic AMP-dependent protein kinase-induced phosphorylation resulted in an expected small rightward shift to 5.97, although statistical significance was not reached. Protein kinase C-induced phosphorylation also stimulated (80%) the maximal myofibrillar Mg^sup 2+^-ATPase activity. [+]EMD 60263 (3 µM) produced a leftward shift of the myofibrillar pCa^sup 2+^/Mg^sup 2+^-ATPase curve which was unaffected by prior protein kinase C-induced phosphorylation. In conclusion, the findings with isolated myofibrils from myocardium of anaesthetized open-chest pigs indicate that protein kinase C might be involved in the mechanism of reduced Ca^sup 2+^ responsiveness of myofilaments in stunned myocardium. However, at this stage no differences could be found between the maximal activity of the Ca^sup 2+^-stimulated Mg^sup 2+^-ATPase, the pCa^sub 50^ and the degree of phosphorylation of myofibrils isolated from stunned and non-stunned myocardium.[PUBLICATION ABSTRACT