Phosphorylation by protein kinases A and C of myofibrillar proteins in rabbit stunned and non-stunned myocardium.
We tested the hypothesis that altered phosphorylation of myofibrillar proteins is involved in post-ischemic myocardial stunning. Myofibrillar proteins were isolated from Langendorff perfused control rabbit hearts, hearts submitted to 15 min normothermic ischemia and hearts submitted to 15 min ischemia followed by 10 min of reperfusion (stunned hearts). The in vivo level of phosphorylation of specific contractile proteins by protein kinases A and C was indirectly detected by the amount of 32P incorporated in vitro in the presence of these protein kinases and saturating concentration of [gamma-32P]-ATP (back-phosphorylation method). In control experiments the back-phosphorylation technique was able to detect PKA- or PKC-induced protein phosphorylation in hearts treated with isoproterenol and phorbol ester, respectively. In stunned hearts, contractile function was significantly suppressed compared to the period before ischemia. We found no difference in myofibrillar protein profile (on densitometry of the Coomassie-stained gels after SDS-PAGE) and in PKA mediated 32P incorporation when comparing control, ischemic and stunned myocardium. Three different PKCs were used for phosphorylation: commercial purified rat brain PKC, partially purified rat brain PKC or rabbit partially purified cardiac PKC. Cardiac PKC mainly phosphorylated troponin I, whereas brain PKC phosphorylated both troponin T and troponin I. No significant difference in 32P incorporation mediated by either brain or cardiac PKC was found between control, ischemic and ischemic/reperfused myofibrils. These data indicate that myocardial stunning does not cause changes in PKC- or PKA-mediated Pi incorporation into myofibrillar proteins detectable by the back-phosphorylation method.
Matejovicova
M
M
Centre for Experimental Surgery and Anaesthesiology, University of Leuven, Belgium.
Kaplan
P
P
Mubagwa
K
K
Raeymaekers
L
L
Pongo
E
E
Flameng
W
W
D000818:Animals; D001921:Brain / Q000201:enzymology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D006439:Hemodynamics; D009124:Muscle Proteins / Q000378:metabolism*; D017682:Myocardial Stunning / Q000201:enzymology*; D009206:Myocardium / Q000201:enzymology*; D009210:Myofibrils / Q000201:enzymology*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D011817:Rabbits; D051381:Rats
D009124:Muscle Proteins; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C