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Studies of the Cardiolipin Interactome
AuthorFox, Colin Andrew
AdvisorRyan, Robert O
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Nanodisks comprised solely of the anionic glyerophospholipid cardiolipin and an apolipoprotein have been formulated and characterized. Cardiolipin nanodisks (CL ND) are composed of a planar lipid bilayer comprised solely of cardiolipin and an apolipoprotein scaffold, which circumscribes the unstable edge of the bilayer, conferring stability and complete aqueous solubility. These nanoparticles were used in a variety of experiments to better understand the interactions between cardiolipin and several members of its interactome. Calcium has previously been shown to induce a bilayer to non-bilayer transition when added to liposomes containing cardiolipin, but not in liposomes with a similar lipid composition in the absence of cardiolipin. This phenomenon was evaluated using ND technology. CL ND were found to be a homogenous population of completely aqueous soluble nanoparticles of approximately 200-300 kDa in size. CL ND undergo a bilayer to non-bilayer transformation following addition of sufficient CaCl2, similarly to liposomes and vesicles. This phenomenon is specific to divalent cations, as tested by addition of CaCl2, MgCl2, SrCl2. Addition of NaCl and KCl did not elicit a similar response. This phenomenon is also related to cardiolipin concentration as a component of the lipid bilayer, and decreasing the cardiolipin content results in increased resistance to calcium induced transformation. Ultimately, a novel molecular mechanism was put forth wherein cardiolipin molecules, upon addition of a divalent cation, reposition to allow the phosphate moieties to better bind. This repositioning increases the strain on the fatty acyl chains, forcing them to reposition and further increase the “cone” shape of the cardiolipin molecule. This interaction reduces the ability of the cardiolipin to reside stably in a planar bilayer. Once a sufficient number of molecules undergo this change, the constraining force on the nanodisk imposed by the apolipoprotein scaffold is exceeded, resulting in a transition to a non-bilayer state. CL ND were then used to investigate the interactions between cardiolipin, calcium and cytochrome c. Cytochrome c was found to stably bind to CL ND, resulting in co-elution following size exclusion chromatography. Pre-incubation of cytochrome c with CL ND increased susceptibility of CL ND to calcium induced bilayer to non-bilayer transformation, as compared to empty cardiolipin nanodisks. However, following this transformation, ~ 30% of the cytochrome c remained bound to the insoluble lipid-containing pellet. Addition of calcium insufficient to induce this transformation was found to lead to complete dissociation of cytochrome c. CL ND pre-incubated with a similar concentration of calcium no longer bind cytochrome c, suggesting competition for a specific binding site. This revelation may play an important role in furthering our understanding of cytochrome c’s release from the mitochondria during cell mediated apoptosis. Doxorubicin (DOX) is a clinically important anti-cancer drug. However, usage is restricted due to symptoms that have been termed “DOX induced cardiotoxicity”. This condition causes symptoms reminiscent, but distinct from, cardiomyopathy and often lead to heart failure. CL ND were investigated as a delivery vehicle to potentially reduce DOX induced cardiotoxicity. DOX CL ND were formulated and found to remain bound following dialysis. A novel assay was designed and tested to show that DOX, when given a choice between DNA or cardiolipin, will preferentially bind to DNA. Further studies showed that DOX CL ND retain a similar anti-cancer effect as free DOX in two different cancer cell lines while being more effective than liposomal DOX. Furthermore, it was found that DOX binding to CL ND increases cell viability as compared to free DOX in a rat cardiomyocyte cell line. Analysis of mitochondrial respiration of treated cells via XF24 Seahorse analysis suggests that binding of DOX to CL ND reduces mitochondrial dysfunction, as is evidenced by an increase in maximal respiration as compared to DOX treated samples. These data presented in this dissertation illustrates that CL ND are a useful tool to better understand mitochondrial interactions, an area that has proven difficult to study.