Term of Award

Fall 2015

Degree Name

Master of Science in Biology (M.S.)

Document Type and Release Option

Thesis (open access)

Copyright Statement / License for Reuse

Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 License.

Department

Department of Biology

Committee Chair

Johanne M Lewis

Committee Member 1

Laura Regassa

Committee Member 2

Vinoth Sittaramane

Abstract

Approximately five million people in the United States are affected by cardiovascular related diseases yearly contributing to 300,000 annual deaths, making CVD the leading cause of mortality worldwide. It has been suggested that apoptosis (programmed cell death) contributes to the pathogenesis of cardiovascular diseases. When blood flow is reduced or cut off from the heart, usually by a thrombus, this results in oxygen deprivation (hypoxia) to the cardiomyocytes (heart cells). In response to this hypoxic stress, cardiomyocytes will undergo apoptosis. Since many species of fish can survive levels of hypoxia that would be fatal to mammals, fish are an ideal model system to study changes at the cellular and molecular level that prevent or repair hypoxia-induced damage in cardiomyocytes. For this study, the hypoxia-tolerant Nile Tilapia (Oreochromis niloticus) and the hypoxia-sensitive Striped Bass (Morone saxatilis) were subjected to gradual hypoxia exposure and their cardiac tissue was tested for key markers of apoptosis. We hypothesized that tilapia will have lower cellular markers of apoptosis due to higher anti-apoptotic and repair gene expression in their cardiomyocytes when compared to Striped Bass, which we hypothesized will have higher cellular markers of apoptosis and pro-apoptotic gene expression levels. By elucidating the cellular and molecular mechanisms present in tilapia cardiomyocytes during hypoxia exposure and reoxygenation, this study could potentially aid in the future development of therapeutic strategies for cardiovascular disease in humans. Evidence of apoptosis at the cellular level was determined by observing DNA fragmentation (late stage apoptosis) and measuring caspase-3/7 activity (early stage apoptosis) in heart tissue. No DNA fragmentation was observed in any samples across timepoints, even in our sensitive species. Additionally, there was no significant difference across timepoints and species in caspase 3/7 levels. This was surprising because we expected to see evidence of cell death in our sensitive species after hypoxia exposure and subsequent reoxygenation. To back up the results obtained at the cellular level, RT-qPCR will be used to quantify changes in the cardiomyocyte transcriptome of pro-apoptotic (bax, caspase3, and fasl), anti-apoptotic (bcl2, flip, and, p53) and repair (hsp70) genes. However, primers for hsp70 were the only ones that were successfully developed to measure gene expression. Therefore no data was collected on pro or anti-apoptotic gene expression in the hearts of Striped Bass and Nile Tilapia. Additionally, RT-qPCR efficiency for hsp70 primers were too high to collect accurate data. In conclusion, there was no cellular evidence of apoptosis in tissue samples across timepoints and between species, and no data was collected to measure pro and anti-apoptotic gene expression. The lack of cell death in Striped Bass heart samples at the cellular level could be explained by a limited capacity for hypoxia tolerance or that the fish was undergoing another mechanism in place of apoptosis such as necrosis.

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