Previously we showed in an in situ porcine model that the thiadiazinone derivative [+]
EMD 60263, a Ca2+ 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 pCa2+/Mg(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 Ca2+ responsiveness of myofilaments during stunning. No differences were observed in the maximal activity of the Ca(2+)-stimulated Mg(2+)-
ATPase and in the pCa50 of myofibrils isolated from non-stunned and
stunned myocardium. After phosphorylation with [gamma-32P]-
ATP and excess of purified rat brain
protein kinase C, the myofibrils were separated on sodiumdodecylsulphate-
polyacrylamide gelectrophoresis and the 32P incorporation counted by the Molecular Imager. Ca2+/
phosphatidylserine/sn-
1,2 diolein-dependent 32P 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 pCa50 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(2+)-
ATPase activity. [+]
EMD 60263 (3 microM) produced a leftward shift of the myofibrillar pCa2+/Mg(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 Ca2+ responsiveness of myofilaments in
stunned myocardium. However, at this stage no differences could be found between the maximal activity of the Ca(2+)-stimulated Mg(2+)-
ATPase, the pCa50 and the degree of phosphorylation of myofibrils isolated from stunned and non-
stunned myocardium.