Nitroprusside induces cardiomyocyte death: interaction with hydrogen peroxide.

Abstract

We examined the hypothesis that sodium nitroprusside (SNP) produces cell death in cardiomyocytes through generation of H(2)O(2). Embryonic chick cardiomyocytes in culture were treated with SNP, and cell viability was assessed by trypan blue, MTT assay, and fluorescent activated cell sorting (FACS) analysis. SNP for 24 h induced a significant (P < 0.001) dose-dependent loss of cell viability. On MTT assay, the half-maximal effective concentration was 0.53 mM (confidence interval 0.45-0.59 mM). SNP-treated cardiomyocytes displayed characteristic microscopic features of apoptosis: reduced cell size, nuclear disintegration, and membrane bleb formation. FACS analysis demonstrated SNP-induced apoptosis as well as cell changes consistent with necrosis. The proportion of cells with nuclear changes of apoptosis, identified by propidium iodide (PI) staining of permeabilized cells, increased significantly (P < 0.05) after 0.5 mM SNP for 24 h. The proportion of apoptotic cells, characterized by dual staining of intact cardiomyocytes with fluorescein diacetate and PI, was significantly (P < 0.05) increased after treatment with 0.5 mM SNP for 24 h. SNP metabolism and NO production was suggested by the significant (P < 0.05) increase in nitrite generation in the media with 0.5 mM SNP compared with control. SNP-mediated H(2)O(2) production was implicated in the mechanism of SNP-induced cell death. First, SNP produced a significant (P < 0.05) increase in H(2)O(2) detected in the media after 6 or 24 h of SNP treatment. Second, catalase completely blocked the reduction of cell viability induced by 0.1 mM SNP and significantly (P < 0.05) blunted the effect of 0.5 mM SNP. In contrast, the iron chelator deferoxamine did not alter SNP-induced loss of cell viability. FACS analysis showed that the combination of low concentrations of H(2)O(2) (10(-8) M) that did not alter cell viability augmented SNP-induced apoptosis. In contrast, the amount of necrotic cell death was unchanged by the combination of H(2)O(2) and SNP. H(2)O(2) plus SNP produced a dramatic alteration in cell structure with greater membrane bleb formation, shrunken cells, and more intense cytosolic acridine orange staining and nuclear fragmentation than either agent alone. These data indicate the vulnerability of cardiomyocytes to SNP and suggest the involvement of H(2)O(2) in the pathogenesis of SNP-induced cardiomyocyte cell death. Establishing apoptosis as a component of the type of cell death induced by SNP permitted the recognition that SNP-induced apoptosis was increased by chronic treatment with low (subtoxic) concentrations of H(2)O(2).