Pathways of cardiac myocyte apoptosis induced by hypoxia-acidosis and hypoxia-reoxygenation

Date of Award




Degree Name

Doctor of Philosophy (Ph.D.)



First Committee Member

Keith Webster - Committee Chair


Hypoxia-acidosis and hypoxia-reoxygenation were investigated as two models of ischemic heart disease which induce apoptosis in cultured cardiac myocytes. The first research objective was to characterize Bnip3 stabilization and subsequent induction of death by hypoxia-acidosis in cardiac myocytes. Experiments were designed based on the hypothesis that an acidic pH induces a conformational change in Bnip3 which mediates its integration into the mitochondrial membrane thereby protecting it from degradation and facilitating its ability to induce apoptosis. Bnip3 was found to be preferentially cleaved to four products in acidosis and this cleavage was found to strongly correlate with the induction of apoptosis. Bnip3 species were demonstrated to have a greater half-life in acidic conditions, but were selectively degraded by the proteasome in neutral conditions. Results showed that Bnip3 species associate more stringently with the mitochondrial membrane, possess greater hydrophobicity, and have increased resistance to protease digestion in acidic conditions and suggested that this increased membrane association protects Bnip3 from proteasome targeting and facilitates apoptotic induction. Bnip3 proteolytic processing and subsequent induction of apoptosis was also determined to be contingent on calpain activity, the calcium transient, and membrane association as well as an increase in H+ concentration. The second research objective was to elucidate the upstream components of hypoxia-reoxygenation induced JNK activation in cardiac myocytes. Experiments were designed based on the hypothesis that calcium is an additional component of reoxygenation induced stress which, along with ROI, leads to the sequentially activation of PKC, Pyk2, and JNK that ultimately influences cell fate. Results demonstrated that calcium is an essential upstream mediator of reoxygenation induced JNK signaling cascade along with oxidative stress. Pyk2 was also found to serve as a calcium dependent kinase upstream of JNK activation and was determined to be cardioprotective under physiological glucose conditions as has been previously reported for JNK. PKCalpha and PKCepsilon were found to be additional mediators of JNK activation upon reoxygenation. Lastly, JNK activation by calcium and Pyk2 was demonstrated to be unique to the reoxygenation pathway as inhibition of these molecules did not effect the activation of JNK by other stimuli.


Biology, Molecular; Biology, Cell; Health Sciences, Pharmacology

Link to Full Text


Link to Full Text