Date of Award

2001

Embargo Period

8-1-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular and Cellular Biology and Pathobiology

College

College of Graduate Studies

First Advisor

George Cooper

Second Advisor

Donald Menick

Third Advisor

Perry V. Halushka

Fourth Advisor

Barry Ledford

Fifth Advisor

Paul J. McDermott

Abstract

This work focused on the regulation of the different isotypes of beta-tubulin during cardiac hypertrophy, with the specific hypothesis that increased levels of the beta I isotype leads to the increase in microtubule density seen during hypertrophy. Partial cDNAs for the three beta-tubulin isotypes expressed in the heart (I, II, and IV) have been cloned and used to show that there is a differential upregulation of the beta I and beta II isotypes during cardiac hypertrophy. Messenger RNA half-life studies of cells isolated from hypertrophied myocardium have shown that this upregulation is due to increased transcription of the genes encoding those isotypes. This work has also shown the presence in terminally differentiated cells of the co-translational autoregulatory mechanism by which increased free tubulin protein levels leads to decreased mRNA levels. The beta-tubulin isotypes that are expressed in the heart have been cloned into tetracycline-regulated expression vectors and adenoviral vectors. Overexpression in CHO cells has shown that, contrary to some previous reports, overexpression of beta I, beta II, and beta IV does not affect microtubule stability as measured by taxol resistance. In addition, overexpression of beta I in adult cardiocytes does not affect microtubule stability. However, overexpression of the predominant heart microtubule associated protein, MAP 4, is alone enough to cause increased microtubule stability in vitro. Because there is an increase in MAP 4 protein levels during cardiac hypertrophy in vivo, we believe that this is the primary mechanism of increased microtubule stability in the hypertrophied myocardium. This work also focused on development of a mouse model in which to study the effects of microtubule stability on the development of heart failure in vivo. Mutation of the beta-tubulin protein in a string of leucine residues destabilized the microtubules in CHO cells. Constructs encoding these mutant tubulin proteins have been inserted into a heart-specific vector and cloned into transgenic mice. Several lines have been initially characterized for levels of expression, and one line has shown heart-specific expression of a mutated beta-tubulin protein. These lines will be used for future studies of the effect of destabilized microtubules on the development of heart failure.

Rights

All rights reserved. Copyright is held by the author.

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