Title

Characterization of human cardiac troponin I ARG145GLY: A mutation associated with the disease hypertrophic cardiomyopathy

Date of Award

2001

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Molecular and Cellular Pharmacology

First Committee Member

James Douglas Potter, Committee Chair

Abstract

Hypertrophic cardiomyopathy (HCM) has been associated with several mutations in the gene encoding Human Cardiac Troponin I (HCTnI). A missense mutation, which occurs in the inhibitory region of HCTnI (HCTnIR145G ), replaces an arginine residue at position 145 with a glycine. In an attempt to determine the biochemical and physiological ramifications in muscle contraction due to HCTnIR145G and its association with the disease HCM, this study focused on how the missense mutation within HCTnI affects its inhibitory function and molecular interactions within the troponin complex. Results from several different assays indicate that the inhibitory function of HCTnIR145G in actin-tropomyosin (Tm) activated myosin ATPase assays was significantly reduced as compared to the wildtype protein. In reconstituted thin filament systems, when HCTnIR145G was complexed with HCTnC and HCTnT (TnR145G) only partial inhibition of the actin-Tm activated myosin ATPase activity was observed in the absence of Ca2+ compared to wildtype Tn complex (HCTnT-HCTnI-HCTnC). There was no significant difference in the maximum level of actin-Tm activated myosin ATPase activity between wildtype Tn and TnR145G in the presence of Ca2+ . To study the effects of HCTnIR145G on steady state force development and relaxation of unregulated force, HCTnI and HCTnIR145G complexed with HCTnC were used to reconstitute HCTnT displaced skinned cardiac muscle fibers. The results indicated a significant reduction in the ability of the HCTnC/HCTnIR145G complex to inhibit force in the absence of Ca2+. In addition, skinned fibers reconstituted with the mutant complex were not able to recover maximum force development as compared to the wildtype complex. Moreover, a moderate increase in the Ca2+ dependence of force development was observed in skinned fibers reconstituted with the mutant complex. These results demonstrate impaired inhibition in reconstituted and skinned muscle systems as well as impaired force development and an increase in the Ca2+ sensitivity to force development. HCTnIR145G could be associated with contractile dysfunction in cardiac muscle resulting from abnormal interactions within the thin filament and between the thin filament and thick filament of cardiac muscle. Biacore and fluorescence measurements did not reveal a significant change in the binding affinity between HCTnIR145G and HCTnC in different metal ion buffer conditions. However, the data did reveal a trend in that the WTHCTnI had a lower affinity for HCTnC than HCTnIR145G in the absence of Ca2+. Circular dichroism measurements, to access the effect of the Arg145Gly mutation on the secondary structure of TnI, revealed that the mutation does not change the alpha helical content of the protein. Furthermore, HCTnIR145G co-sediments with the actin-tropomyosin complex in the absence and presence of Ca2+. Clearly, we observe an alteration in the inhibitory function of HCTnIR145G, that could be due to an alteration in the interaction between TnI and TnC. These results do not eliminate the possibility that this mutation has an indirect effect on other troponin subunits that indirectly affects the ability of TnI to inhibit muscle contraction.

Keywords

Biology, Molecular; Biophysics, General

Link to Full Text

http://access.library.miami.edu/login?url=http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3015565