| Dietary copper deficiency leads to cardiac hypertrophy, which is characterized by mitochondria proliferation and numerous morphological defects. In this dissertation, the impact of copper deficiency upon mitochondria biology was studied. Cardiac mitochondrial respiration, ATP synthase activity, and membrane potential and intactness were evaluated in copper deficient rats. In the presence of NADH, both copper deficient and copper adequate mitochondria had low oxygen consumption rates, indicating membrane intactness. Copper-deficient mitochondria had lower membrane potential, lower state 3 oxygen consumption rates and were less sensitive to inhibition by oligomycin, an ATP synthase inhibitor, than copper-adequate mitochondria. These studies suggested that cardiac mitochondrial respiration is compromised in copper deficient rats, and may be related to an altered ATP synthase complex and/or a decreased mitochondrial membrane potential.;The question as to whether the changes in the in vivo model was due to copper deficiency per se or to the disease state, was pursued using C 2C12 cells treated with the copper chelator tetraethylenepentamine (TEPA) to induce a copper-deficiency. Consistent with animal studies, the proteins levels of the nuclear encoded subunits of cytochrome c oxidase decreased, but the mitochondrial encoded subunits remained unchanged. Since the first study indicated decreased mitochondrial membrane potential, and early indicator of apoptosis, programmed cell death was studied. Decreased mitochondrial membrane potential was indicated in TEPA treated cells, but further investigation of the potential induction of apoptosis by measuring caspase-3 activity, Bcl-2 and Bax protein levels, DNA fragmentation suggested that apoptosis was not induced in TEPA treated C2C12 cells. Unexpectedly, higher caspace-3 activity and increased Bcl-2 protein levels were observed in TEPA treated cells, which could indicate greater oxidative stress in the TEPA treated cells. When the cells were exposed to hydrogen peroxide as a stressor, the TEPA treated cells had a more dramatic drop in membrane potential. These data suggest that copper deficiency per se does not induce apoptosis, but TEPA treated cells become more susceptible to oxidative stress. Decreased mitochondrial membrane potential could explain the decreased levels of cytochrome c oxidase nuclear encoded subunits, but cells with decreased mitochondrial membrane potential were not destined for apoptosis. |
