Date of Award

5-2009

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Field of Study

Biochemistry and Molecular Biology

Department

Graduate School of Biomedical Sciences

First Advisor

Dr. Julian Borejdo

Abstract

Cardiovascular diseases are the leading cause of mortality worldwide; with heart failure being highly prevalent in most affluent parts of the world. There is a need for a better understanding of the mechanism underlying these diseases. Familial hypertrophic cardiomyopathy (FHC), one such disease, is a genetic disorder of the heart characterized by increased growth or hypertrophy in the thickness of the wall of the left ventricle, the largest of the four chambers of the heart. This research project is focused on one kind of FHC, the D166V mutation in the regulatory light chain in myosin, which is associated with a particularly malignant form of the disease. The overall goal of this project was to study cross bridge kinetics (contraction and ATP utilization) in cardiac muscle from transgenic mice and to develop assays to apply this to human samples.

The real time orientation changes of myosin and actin during a single cross bridge cycle beginning in a state of rigor was studied by Fluorescence anisotropy. Rabbit psoas fibers were isolated and used to achieve imaging of a few fluorophores or cross bridges. This technique was then applied to study cardiac myofibrils from transgenic mice, carrying the mutation causing the disease (FHC). Methods to achieve single molecule detection to aid studying human samples suffering from this disease were developed using silver island films, monolayers of nanoparticles and surface plasmon coupled emission.

The conclusions of this dissertation were that a mutation in a light chain in myosin cause changes in the cross bridge kinetics. Myofibrils from the mutated mice displayed a significant slower rate of detachment during contraction as well as increased ATPase activity, which if severe enough could cause the heart to compensate by increasing wall thickness (hypertrophy).

Despite significant clinical advances in the treatment of various cardiovascular diseases, mortality rates remain high. No therapy currently exists to treat or delay progression from hypertrophy to heart failure. This proposal help answer an important question regarding the molecular basis of FHC-mediated pathology in the heart. Also, achieving imaging of a single fluorophore has numerous implications in the biological field, like studying ligand-receptor interactions in live cells, involvement of protein molecules in internalization of bacteria by cells, monitoring the conformational fluctuations of DNA, diagnosis of prion diseases and also in detection of viruses at an early phase of infection.

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