Date of Award

1-1-2016

Embargo Period

1-1-2019

Document Type

Thesis - MUSC Only

Degree Name

Master of Science (MS)

Department

Biochemistry and Molecular Biology

College

College of Graduate Studies

First Advisor

L. Ashley Cowart

Second Advisor

Michael R. Zile

Third Advisor

Dennis K. Watson

Fourth Advisor

Christina Voelkel-Johnson

Fifth Advisor

Shaun K. Olsen

Abstract

iabetic cardiomyopathy (DbCM) is defined as changes in the structure and function of the heart in the absence of coronary artery disease and hypertension. Using a mouse model with diet-induced DbCM characteristics, our laboratory found that DbCM development involved both cardiomyocyte hypertrophy and autophagy, which was dependent on de novo sphingolipid synthesis. Additional in vitro studies revealed that hypertrophy and autophagy are dependent on ceramide synthase 5 (CerS5) leading to the hypothesis that knockdown of CerS5 could protect against the development of diet-induced DbCM. Using this model of diet-induced DbCM and a CerS5 constitutive knockout mouse we determined that CerS5 knockout animals were indeed protected from diet-induced cardiac dysfunction. In additional studies of autophagy in this model we determined that CerS5 knockout also induced a block in autophagic flux and protected against diet-induced loss of mitochondrial mass. Further mechanistic cell studies focused on the signaling pathways involved in diet-induced CerS5-dependent DbCM. We identified various autophagy genes, including Atg7 that were upregulated in sphingolipid-dependent DbCM and found that many of these genes are transcriptional targets of p53, including Bnip3, Dram1, and Rb1. Cells studies revealed that sphingolipid treatment increased expression of autophagy components in a p53-dependent manner, while autophagic flux was induced by CerS5 overexpression in some, but not all studies. Recent studies from another laboratory show that Atg7 regulates p53 under stress conditions through direct binding. We therefore tested whether sphingolipid-induced autophagy is dependent on association of these proteins. Using a proximity ligation assay we observed increased association of Atg7 and p53 with sphingolipid treatment that was at least partially dependent on CerS5, but only in non-autophagy inducing conditions. Additional studies also showed evidence of p53 and Atg7 associating with ceramide under these conditions. These results support a novel model in which Atg7 and p53 associate with ceramide, and perhaps change signaling to decrease cardiomyocyte autophagic flux.

Rights

All rights reserved. Copyright is held by the author.

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