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Publication Date

2009-08-11

Availability

UM campus only

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Molecular and Cellular Pharmacology (Medicine)

Date of Defense

2009-06-30

First Committee Member

Charles W. Luetje - Committee Chair

Second Committee Member

Danuta Szczesna-Cordary - Committee Member

Third Committee Member

Glenn W. Kerrick - Committee Member

Fourth Committee Member

James D. Potter - Mentor

Fifth Committee Member

Xupei Huang - Outside Committee Member

Abstract

Cardiac diseases associated with mutations in Tn subunits include hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM) and restrictive cardiomyopathy (RCM). Altered calcium handling in these diseases is evidenced by changes in the Ca2+ sensitivity of contraction. Mutations were generated to increase/ decrease the Ca2+ sensitivity of skinned fibers, and create the classified effects of DCM, HCM and RCM. This study mimicked the changes in Ca2+ sensitivity and relaxation properties of the muscle to determine if this was sufficient to recreate the disease. Four mutants (A23Q, S37G, V44Q, L48Q) were identified with RCM-like properties; a large increase in Ca2+ sensitivity, increased basal force and loss of ATPase inhibition. Two mutations were identified (E40A, I61Q) with DCM properties; decreased Ca2+ sensitivity in skinned fibers, decreased force recovery (%), and decreased activation of the ATPase at high Ca2+ levels (pCa 6-4). Also, the functional effects of four newly identified cTnC mutations associated with HCM were reported. Three of these HCM mutations A8V, C84Y, and D145E displayed HCM characteristics, increased Ca2+ sensitivity in skinned fibers and ATPase and A8V and D145E increased the force recovery. Only, D145E significantly increased the ATPase activation of the reconstituted thin filament. Also, Ca2+ affinity measurements using IAANS fluorescence were performed. No significant changes were found for E134D. The C84Y IAANS fluorescence measurements revealed that cTnC Ca2+ affinity of the cTn complex was unaltered. The Ca2+ affinity increased for D145E in isolated cTnC and the cTn complex, however in the regulated thin filament (RTF) with myosin subfragment-1 (S1) and rigor crossbridges the Ca2+ affinity values were similar to the fiber Ca2+ sensitivity. For A8V, the RTF significantly increased the Ca2+ affinity, and addition of S1 and rigor crossbridges caused the values to parallel the Ca2+ sensitivity values. In conclusion, direct and indirect protein-protein interactions contribute to the enhanced Ca2+ sensitivity of the HCM mutants. The cTnC mutant screen allowed selection of mutations that mimic the disease states: S37G (RCM) and, E40A (DCM); A8V (HCM) from the patient study for analysis in knock-in mice for futures studies to determine if these disease states can be recapitulated in vivo.

Keywords

Cardiomyopathy; Calcium Affinity; Hcm; Rcm; Dcm

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