Title

Functional and structural effects of cardiomyopathy associated mutations in alpha-tropomyosin

Date of Award

2007

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Molecular and Cellular Pharmacology

First Committee Member

James D. Potter, Committee Chair

Abstract

Familial hypertrophic and dilated cardiomyopathies (HCM and DCM, respectively) are diseases which account for significant percentages of cardiac death in the United States. The overall goals of this thesis project were to determine what, if any, are the differences between DCM and HCM associated mutations, at the level of cardiac alpha-tropomyosin (Tm) structure, and to characterize the effects of the various mutations on the function of Tm in muscle contraction.To investigate potential alterations in associations between Tm and other thin filament proteins which may account for the differential functional effects of the DCM associated mutations, E40K and E54K, and the HCM associated mutations, E62Q and L185R, structural and protein-protein interaction studies were conducted utilizing circular dichroism, actomyosin ATPase activity measurements, and actin binding assays. The results reported here in Part 1, show that each mutation investigated had distinct effects on the interactions investigated by the various methods, possibly due to the different positions of the mutations along the length of the Tm molecule and within the coiled-coil interface.To study the functional consequences of various cardiomyopathic mutations in human Tm, a method of depletion/reconstitution of native Tm and Tn complex in cardiac myofibril preparations needed to be developed. The endogenous Tm-Tn complex was selectively removed from myofibrils and replaced with recombinant wild-type or mutant proteins. Successful depletion and reconstitution steps were verified by SDS-gel electrophoresis, and by the loss and regain of Ca 2+ dependent regulation of ATPase activity. Through the use of this new depletion/reconstitution method, the functional consequences of the mutations were determined utilizing myofibrillar ATPase measurements.The results of these studies in Part 2 showed that all HCM-associated Tm mutations increased the Ca2+ sensitivity of ATPase activity and all had decreased abilities to inhibit ATPase activity. In contrast, the DCM-associated mutations both decreased the Ca2+ sensitivity of ATPase activity, and had no effect on the inhibition of ATPase activity. These findings have demonstrated that the mutations which cause HCM and DCM disrupt discrete mechanisms, which may culminate in the distinct cardiomyopathic phenotypes.

Keywords

Health Sciences, Pharmacology; Biophysics, General

Link to Full Text

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