PHD, Kent State University, 2014, College of Arts and Sciences / School of Biomedical Sciences

Ischemic postconditioning has been shown to reduce injury in response to ischemia/reperfusion. Because of limitations on the clinical use of ischemic postconditioning protocols, pharmacological agents that elicit a postconditioning (PostC) effect are highly desired. Previous studies have shown that stimulation of alpha1-adrenergic receptors (α1-ARs) is cardioprotective, thus, the first aim of this dissertation was to examine the effects of post-ischemic stimulation of α1-ARs on cardiac myocyte cell death. Adult rat ventricular myocytes and HL-1 cardiac myocytes were subjected to simulated ischemia-reperfusion injury. Cell membrane permeability, evaluated by measuring released lactate dehydrogenase (LDH) or propidium iodide uptake (PI), was used as an estimate of cell death. Lower amounts of LDH and PI uptake were detected when α1-ARs were stimulated at the onset of reperfusion. Further, lower levels of apoptosis were measured using TUNEL and DNA laddering to evaluate DNA cleavage and Annexin V staining to evaluate outer membrane phosphatidylserine. Prior studies suggest that increased autophagy following ischemia is protective. The second aim of this dissertation was to determine whether post-ischemic α1-AR stimulation inhibits cardiac myocyte death through modulation of autophagy. Alpha1-AR-mediated reductions in cell death were reversed in the presence of ATG inhibitor, 3-Methyladenine. Western blot for autophagosomal marker, LC3-II indicated modulation of autophagy, and two methods were used to measure autophagic flux. LC3-II turnover examined with and without autophagosome-lysosome fusion inhibitor chloroquine revealed an increase in autophagic flux or induction. HL-1 cells transfected with plasmid to express a tandem fluorescent-tagged LC3 molecule also indicated an increase in autophagic flux or induction. Finally, the third aim of this dissertation was to examine the molecular pathways stimulated by α1-ARs that lead to decreased cell death. PI fluorescence as (open full item for complete abstract)

Committee: June Yun Ph.D. (Advisor); J. Gary Meszaros Ph.D. (Committee Member); Angelo DeLucia Ph.D. (Committee Member); Werner Geldenhuys Ph.D. (Committee Member); Joel Hughes Ph.D. (Committee Member) Subjects: Biology; Biomedical Research

Doctor of Philosophy (PhD), University of Toledo, 2014, College of Medicine

Acute myocardial infarction, the clinical manifestation of ischemia-reperfusion (IR) injury, is a leading cause of death worldwide. Although percutaneous coronary interventions and thrombolytic therapies are effective in limiting the duration of ischemia, the re-introduction of blood flow to previously ischemic area causes additional damage, collectively known as reperfusion injury. One of the most effective ways to reduce reperfusion injury is ischemic preconditioning (IPC), which is induced by several cycles of brief ischemia and reperfusion bouts prior to the prolonged ischemia. IPC was found to be mediated through signaling pathways (including activation of Src, PI3K-IB, and PKCe), and mimicked by a number of pharmacological or mechanical interventions. However, 25 years after the first report of IPC, preconditioning research has not translated into clinical application against cardiac reperfusion injury. Contributing to this somewhat surprising and disappointing failure to translate preconditioning into the clinic, the applicability and efficacy of preconditioning treatments in the setting of acute myocardial infarction (MI) have not always been carefully considered in the research setting. In particular, the impact of comorbidities on cardioprotective signaling or the unpredictable nature of MI has limited the impact of IPC. Against this background, the overall objective of this work was to investigate the potential benefit of using cardiac glycosides (CG) drugs to trigger cardioprotection in conditions relevant to acute myocardial infarction. Indeed, treatment with low doses of the CG ouabain before ischemia has been shown to induce cardioprotective effects against IR injury through a mechanism known as ouabain preconditioning (OPC). Rather than the classic specific inhibition of Na/K-ATPase-mediated ion transport, the mechanism underlying OPC is the activation of the more recently recognized signaling function of Na/K-ATPase, which includes Src-PKCe, ROS a (open full item for complete abstract)

Committee: Sandrine Pierre Ph.D (Advisor); Sandrine Pierre Ph.D (Committee Chair); Zi-Jian Xie Ph.D (Committee Member); Jiang Tian Ph.D (Committee Member); Lijun Liu M.S., M.D. (Committee Member); Guillermo Vazquez Ph.D (Committee Member); Andrew Beavis Ph.D (Committee Member) Subjects: Biomedical Research