Mammalian cells can undergo apoptosis or programmed cell death under pathologic oxidative stress conditions, i.e. when endogenous antioxidants are overwhelmed by oxidative pressure. In heart cells, for example, oxidant-elicited apoptosis is observed in connection with disorders such as ischemia/reperfusion injury and anthracycline-induced cardiomyopathy. Apoptotic signaling often begins in mitochondria, where oxidative energy metabolism takes place. Release of respiratory chain cytochrome c (cyt c) from the mitochondrial inner membrane (IM) and movement into cytosol is known to be an early event in the intrinsic apoptotic pathway. Resting state cyt c is bound to IM via interaction with cardiolipin (CL), a phospholipid located exclusively in this compartment. Being highly unsaturated, CL can undergo oxidative modification to hydroperoxide species (CLOOHs), which lack the ability to bind cyt c and are more hydrophilic than non-oxidized CL. Based on this and our preliminary findings pertaining to CLOOH translocation and binding of the proapoptotic polypeptide tBid in a model membrane system, we present the following hypothesis: Peroxidation of CL in the IM facilitates its spontaneous or protein-mediated translocation to the outer membrane (OM), where it recruits tBid and thence oligomer-forming Bax for generation of cyt c-traversable pores. Our plan for testing this hypothesis is to study intermembrane CLOOH transfer in relation to cyt c release and tBid/Bax targeting/permeabilization in (1) liposomal IM/OM model systems with (2) measurements of association/dissociation kinetics and binding constants;(3) oxidatively modified mitoplasts and mitochondria;and (4) oxidatively stressed cardiomyocytes. Mechanistic deductions will be assisted by use of mitochondria-localizing antioxidants, viz. overexpressed MitoGPx4 and administered MitoQ. Cutting-edge analytical techniques such as Biacore-based surface plasmon resonance, high-performance liquid chromatography with electrochemical detection, and high- performance thin layer chromatography with phosphorimaging detection will be used in the project. These novel and innovative studies will provide important new mechanistic insights into early events in the intrinsic (mitochondrion-centered) pathway of oxidant-induced apoptosis. The research is also biomedically significant, considering the numerous pathological conditions associated with oxidative stress, many of which, including several cardiovascular disorders, are characterized by apoptogenic tissue damage.
