Date of Award

2021

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Cell & Molecular Biology

First Advisor

Benjamin L. Prosser

Abstract

The microtubule network of the cardiomyocyte exhibits specialized architecture, stability, and mechanical behavior that accommodates the demands of a working muscle cell. Post-translationally detyrosinated microtubules are physically coupled to the sarcomere, the contractile unit of the muscle, and resist both the contraction and relaxation of the muscle. The cumulative impact of the microtubule network on myocyte mechanics and the enzyme responsible for detyrosinating tubulin are unknown. Further, control of microtubule growth and shrinkage dynamics represents a potential intermediate in the formation of the stable, physically coupled microtubule network, yet the molecular determinates that govern dynamics have not been studied in the cardiomyocyte. I hypothesize that depolymerization of the microtubule network or knockdown of the vasohibin/small vasohibin binding protein complex, a putative tubulin carboxypeptidase in cardiomyocytes, will improve the contractile kinetics of cardiomyocytes isolated healthy or failing human hearts. Additionally, I hypothesize that desmin intermediate filaments may stabilize growing microtubules at the sarcomere Z-disk in a detyrosination-dependent manner. Using a combination of biochemical assays in tandem with direct observation of myocyte mechanics and microtubule dynamics in primary adult cardiomyocytes I find the following: 1) depolymerization of the microtubule network improves contraction and relaxation kinetics in cardiomyocytes isolated from failing human hearts; 2) knockdown of either vasohibin 1 or small vasohibin binding protein reduced levels of microtubule detyrosination resulting in improvements in contractile kinetics and a reduction in cellular stiffness; and 3) tyrosination increases renders the microtubule more dynamic while desmin intermediate filaments stabilize the growing microtubule. In summation, this dissertation establishes a mechanism for the formation of the post-translationally detyrosinated microtubule network, and further underscores the potential of detyrosination as a therapeutic target for the treatment of heart disease.

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