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), pHi (assessed by
BCECF fluorescence), and cell length were measured during 80 minutes of simulated
ischemia (
anoxia, pHo 6.4) and 20 minutes of reoxygenation (pHo 7.4) and compared between control cells and cells treated with 20 micromol/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 micromol/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.