Role of protein kinase C in mediating effects of hydrogen peroxide in guinea-pig ventricular myocytes

Abstract

The present study examined the effects of hydrogen peroxide (H 2O 2) on intracellular calcium transients and unloaded cell shortening in the presence of the protein kinase C (PKC) inhibitors 1-(5-isoquinolinesulfonyl-2-methylpiperazine (H7) or chelerythrine chloride (CHC) or the PKC activator phorbol 12-myristate 13-acetate (PMA). Calcium transient amplitudes and cell shortening were measured simultaneously in single, enzymatically dissociated ventricular myocytes loaded with fura2-AM. Exposure of myocytes to H 2O 2, 25 μ m or 75 μ m, for 15 min caused a time- and concentration-dependent increase in calcium transient amplitude, cell shortening and the diastolic 340 380 fluorescence ratio. Significant increases in calcium transient amplitude were observed from 7 to 15 min of superfusion with 25 μ m H 2O 2 and the transient amplitude remained elevated throughout the 10 min washout period. In the presence of 75 μ m H 2O 2, transient amplitude was elevated following 2 min and remained elevated for the remainder of the experiment. Significant increases in cell shortening were also observed from 7 to 15 min in the presence of either 25 or 75 μ m H 2O 2. This effect was reversed upon washout of the lower concentration of H 2O 2 but persisted during the initial 5 min of washout at the higher concentration. The diastolic 340 380 fluorescence ratio was unaltered in the presence of 25 μ m of H 2O 2, however this parameter was significantly increased from 7 to 15 min following exposure to 75 μ m H 2O 2 and remained elevated throughout the washout period. The H 2O 2-induced increases in calcium transient amplitude and cell shortening were significantly attenuated in myocytes which were pretreated with either H7 or CHC. In addition. H7 pretreatment abolished the increase in the diastolic ratio following exposure to the higher concentration of the oxidant. Addition of 10 −12 M PMA in the presence of 25 μ m H 2O 2 did not result in any further increase in either the calcium transient amplitude or in cell shortening, however PMA plus H 2O 2 did cause a significant increase in the diastolic ratio, an effect which had not been observed in the presence of this concentration of H 2O 2 alone. Pretreatment of cells with H7 prior to exposure to the combination of H 2O 2 and PMA significantly attenuated all changes seen in the absence of inhibitor. The results of this study suggest that the increase in the calcium transient amplitude and cell shortening following exposure of isolated myocytes to low micromolar concentrations of H 2O 2 are mediated through direct activation of PKC.