Protection of Rat Cardiomyocytes Against Simulated Ischemia and Reoxygenation by Treatment With Protein Kinase C Activator

From the Physiologisches Institut, Justus-Liebig-Universität, Giessen, Germany.

Correspondence to Prof H.M. Piper, Physiologisches Institut, Justus-Liebig-Universität, Aulweg 129, D-35392 Giessen, Germany. E-mail michael.piper{at}physiologie.med.uni-giessen.de

Abstract—The aim of this study was to investigate whether treatment with the protein kinase C (PKC) agonist 1,2-dioctanoyl-sn-glycerol (1,2DOG) can protect isolated adult Wistar rat cardiomyocytes against simulated ischemia and reoxygenation. Cytosolic Ca²⁺ (assessed by fura 2 fluorescence), pH_i (assessed by BCECF fluorescence), and cell length were measured during 80 minutes of simulated ischemia (anoxia, pH_o 6.4) and 20 minutes of reoxygenation (pH_o 7.4) and compared between control cells and cells treated with 20 µmol/L 1,2DOG before anoxia (10-minute treatment and 10-minute washout), before and during anoxia (two-step treatment), or only during anoxia. Treatment before anoxia attenuated rigor contracture but did not influence anoxic Ca²⁺ overload. In contrast, two-step treatment before and during anoxia accelerated rigor contracture but reduced the rate of anoxic Ca²⁺ accumulation. During reoxygenation, control cells developed irreversible hypercontracture (reduction of cell length to 43±2% of the initial cell length, n=62), which was accompanied by spontaneous oscillations of cytosolic Ca²⁺ (19.6±1.6 per minute). Two-step treatment with 1,2DOG before and during anoxia significantly reduced hypercontracture (reduction of cell length to 60±2%, P<.01 versus control, n=41) and suppressed spontaneous Ca²⁺ oscillations (2.8±0.9 per minute, P<.01 versus control). These effects could not be reproduced by treatment with 1,2DOG before anoxia or during anoxia or by a two-step treatment with the PKC-inactive 1,3-dioctanoyl-sn-glycerol and were fully abolished with 1 µmol/L bisindolylmaleimide (PKC inhibitor). We conclude that a two-step activation of PKC before and during anoxia is required for effective protection of cardiomyocytes against anoxic Ca²⁺ overload and reoxygenation-induced hypercontracture.

Key Words: protein kinase C • dioctanoyl-sn-glycerol • cardiomyocyte • ischemia • reoxygenation

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