This Is AuburnElectronic Theses and Dissertations

Protection from Oxidative Stress in the Cardiac H9c2-Cell Line by the Transcription Factor Nrf2




Gray-Edwards, Heather

Type of Degree



Biomedical Sciences


Cardiovascular disease (CVD) is the leading cause of death in the United States, and its prevalence is increasing in all populations and age groups worldwide (71). In 2002 alone, more than 927,000 Americans died from heart disease-related conditions. Oxidative stress, or the generation of reactive oxygen species (ROS), is a contributing factor to the progression of many cardiovascular diseases. One approach to combat the detrimental effects of oxidative stress in cardiac disease is to use the cell’s inherent ability to increase the expression of various stress-related proteins to eliminate the oxidative stress. NF-E2 related factor 2 (Nrf2) is the transcriptional activator of the antioxidant response element (ARE) found in the promoter region of antioxidant and phase II detoxifying genes. In this study, we examined the role of Nrf2 in protecting the H9c2 cardiac-like cell line against oxidative stress. Nrf2 was induced using the known Nrf2 activator tert-butyl hydroquinone (tBHQ). We measured heme oxygenase-1 (HO1) and NAD(P)H:quinone oxidoreductase 1 (NQO1) gene and protein expression as markers of ARE-driven gene activation in response to tBHQ. We also determined ROS generation using the fluorescent probe carboxy-H2DCFDA, and cell viability in response to the prooxidants tert-butyl hydroperoxide (tBHP) and H2O2 in the presence and absence of activated Nrf2. To directly evaluate the roles of Nrf2 and its inhibitory protein Keap1, we utilized Nrf2 and Keap1 siRNAs to knockdown Nrf2 and Keap1 expression. H9c2 cells overexpressing Nrf2 tagged with either green fluorescent protein (GFP) were also generated to examine Nrf2 localization using fluorescent microscopy and gene expression of ARE-containing genes. In H9c2 cells tBHQ activated Nrf2 resulting in the translocation of Nrf2 into the nucleus and increased the transcription and translation of the ARE-driven genes HO1 and NQO1. The prooxidant, tBHP increased ROS generation and cell death in H9c2 cells and pretreatment with tBHQ abrogated both these effects. Nrf2 knockdown experiments resulted in a blunted induction of tBHQ-induced HO1 and NQO1 expression and a loss in the protective effect of tBHQ on tBHP-induced ROS generation. Cell death in response to tBHP and H2O2 was augmented in cells treated with Nrf2 siRNAs, but rescued by Keap1 knockdown. H9c2 cells overexpressing Nrf2-GFP displayed fluorescence that was sequestered in the cytoplasm and translocated to the nucleus upon treatment with tBHQ. Along similar lines, HO1 and NQO1 gene expression remained at basal levels, despite Nrf2 overexpression and cytoplasmic localization. HO1 and NQO1 gene expression was enhanced in Nrf2 overexpressing cells after tBHQ treatment. From these studies, we conclude that Nrf2 protects the cardiac-like H9c2 cells from oxidative stress.