The role of oxidized cytochrome c in regulating mitochondrial reactive oxygen species production and its perturbation in ischaemia
Résumé
Oxidized cytochrome c (Cyt-c) is a powerful superoxide scavenger within the mitochondrial intermembrane space, the importance of this role in situ has not been well explored. Here we investigate this with particular emphasis on whether loss of Cyt-c from mitochondria during heart ischaemia may mediate the increased production of reactive oxygen species during subsequent reperfusion that induces mitochondrial permeability transition pore (mPTP) opening. Mitochondrial Cyt-c depletion was induced in vitro with digitonin, or by 30 minutes ischaemia of the perfused rat heart. Control and Cyt-c-deficient mitochondria were incubated with mixed respiratory substrates and an ADP-regenerating system (State 3.5) to mimic physiological conditions. This contrasts with most published studies performed with a single substrate and without significant ATP turnover. Cyt-c-deficient mitochondria produced more H2O2 than control mitochondria, and exogenous Cyt-c addition reversed this increase. In the presence of increasing [KCN] rates of H2O2 production by both pre-ischaemic and end-ischaemic mitochondria correlated with the oxidized Cyt-c content but not with rates of respiration or NAD(P)H autofluorescence. Cyt-c loss during ischaemia was not mediated by mPTP opening (cyclosporine-A insensitive); nor was it associated with changes in mitochondrial Bax, Bad, Bak or Bid. However, bound hexokinase 2 and Bcl-xl were decreased in end-ischaemic mitochondria. We conclude that Cyt-c loss during ischaemia, caused by outer membrane permeabilisation, is a major determinant of H2O2 production by mitochondria under pathophysiological conditions. We further suggest that in hypoxia production of H2O2 to activate signaling pathways may be also mediated by decreased oxidized Cyt-c and less superoxide scavenging.
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