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Abstract

Previously we showed in an in situ porcine model that the thiadiazinone derivative [+]EMD 60263, a Ca 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 2+ /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 Ca 2+ responsiveness of myofilaments during stunning. No differences were observed in the maximal activity of the Ca 2+ -stimulated Mg 2+ -ATPase and in the pCa 50 of myofibrils isolated from non-stunned and stunned myocardium. After phosphorylation with [gamma-32 P]-ATP and excess of purified rat brain protein kinase C, the myofibrils were separated on sodiumdodecylsulphate-polyacrylamide gelectrophoresis and the 32 P incorporation counted by the Molecular Imager. Ca 2+ / phosphatidylserine/sn-1,2 diolein-dependent 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 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 2+ -ATPase activity.[+]EMD 60263 (3 µM) produced a leftward shift of the myofibrillar pCa 2+ /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 Ca 2+ 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 pCa 50 and the degree of phosphorylation of myofibrils isolated from stunned and non-stunned myocardium. (Mol Cell Biochem 176: 211-218, 1997) Key words: cardiac myofibrils, cardiac sarcoplasmic reticulum, human, pig, Ca 2+ stimulated Mg 2+ -ATPase, thiadiazinone derivatives, stunningAbbreviations: ATPase -Adenosine-5′-triphosphatase; EGTA -ethylene glycol bis(β-aminoethylether) N,N,N′,N′-tetraacetic acid; DTT -dithiothreitol; MOPS -4-morpholino-propane sulfonic acid; P i -inorganic phosphate; PS -phosphatidylserine; DG -sn-1,2 diolein; SDS-PAAGE -sodiumdodecylsulphate-polyacrylamide gelectrophoresis; PMSF -phenylmethanesulfonylfluoride; PDE III -phosphodiesterase III; LADCA -left anterior descending coronary artery; LCXCA -left circumflex coronary artery; Tn...

Cardiac Disorders and Pathophysiology of Sarcomeric Proteins

“…cTnI proteolysis may thus explain part of the dysfunction observed in the stunned myocardium. In subsequent years, proteomic analyses of stunned myocardium and calpain I-incubated cardiac tissue revealed a multitude of protein changes: degradation of multiple structural and sarcomeric proteins (5,72,335,443,470,471,478) and altered posttranslational modifications of sarcomeric proteins (33,68,491). Based on these studies, it can be concluded that reperfusion injury reduces contractility of myofilaments via a myriad of sarcomeric protein changes and contributes to systolic dysfunction of the stunned heart.…”