Impact of cellular pathways of fatty acid metabolism and cholesterol synthesis on cardiomyocyte cell death

Fatty acids have long been implicated in increasing the severity and the extent of myocardial infarction. The common fatty acid palmitate, a 16C saturated fatty acid, was investigated for its role in myocardial cell death. Palmitate was observed to induce significant cardiomyocyte death, both apoptosis and necrosis/oncosis in a specific manner. Palmitate induced an increase in nuclear size, a decrease in intramitochondrial respiration, and a loss of mitochondrial potential. Palmitate was observed to induce an increase in mitochondrial permeability, evidenced by the release of cytochrome C from the mitochondria into the cytoplasm which was inhibitable by cyclosporin A, a drug that closes mitochondrial pores. Enhanced mitochondrial metabolism ofpalmitate with carnitine, a factor necessary for palmitate transportation into the mitochondria via the carnitine-palmitoyl transferase 1 (CPT-1) enzyme, also increased apoptosis. The CPT-1 inhibitor oxfenicine blocked this enhanced carnitinepalmitate death. Shifting palmitate metabolism from mitochondria to peroxisomes with long term treatment with the peroxisome proliferators fenofibrate and WY14643 resulted in a decrease in palmitate-induced apoptosis, suggesting that the location of palmitate oxidation is also responsible for apoptosis. Fenofibrate pretreatment also increased the expression ofperoxisome proliferator activated receptors (PPARs): nuclear receptors known to be involved in the regulation of transcription of B-oxidation enzymes. As palmitate can be incorporated into the apoptotic inducer ceramide, the effects of ceramide were investigated. The ceramide synthase inhibitor fumonisin Bl was ineffective at inhibiting palmitate-induced apoptosis by 24 h suggesting that de novo ceramide synthesis is involved in palmitate's action. Like palmitate, exogenous addition of ceramide induced apoptosis, a decrease in mitochondrial potential, and an increase in mitochondrial permeability. However, unlike palmitate, ceramide-induced an increase in intramitochondrial enzyme activity that was unaffected by cyclosporin A. Ceramide also induced a parallel decrease in ERK activation and an increase of SAPK activation. As well, phosphorylated p38 MAPK was observed to locate in the mitochondrial fraction and to be partially responsible for ceramide-induced apoptosis and loss of mitochondrial potential. Another mechanism hypothesized to explain palmitate-induced death involves acetyl CoA, a byproduct of mitochondrial fatty acid oxidation, which eventually forms the starting substrate for cholesterol biosynthesis. Lovastatin, a HMG-CoA reductase inhibitor, was used in combination with palmitate to inhibit cholesterol biosynthesis during palmitate metabolism. The combination of palmitate and lovastatin resulted in apoptotic death greater than either palmitate or lovastatin alone. This observation suggests that palmitate's death action is not through increased cholesterol biosynthesis as lovastatin did not reduce palmitate-induced death: Further investigation showed that lovastatin co-treated with mevalonic acid, a product downstream of HMG-CoA reductase, blocked lovastatin-induced apoptosis suggesting that if allowed to continue, cholesterol biosynthesis is beneficial to the cardiomyocyte. Lovastatin activated caspase-2 and caspase-3 to a lesser extent and also induced dramatic changes in morphology. The morphological changes were distinct from those produced by palmitate. A physical association was observed to link changes in morphology and the apoptotic process, specifically the association of the small G-protein RhoB and caspase-2. This association appears constitutive and was unaffected by inhibition of caspase-2 activity. In summary, this thesis unraveled the mechanism of action ofpalmitate-induced cell death in cardiomyocytes and indicated that palmitate acts via a mitochondrial mechanism involving palmitate metabolism and mitochondrial permeability. Palmitate does not induce apoptosis via ceramide as ceramide induces a separate distinct mechanism of cell death.Medicine, Faculty ofMedicine, Department ofExperimental Medicine, Division ofGraduat