Publication Date

2008-11-24

Availability

Open access

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Molecular and Cellular Pharmacology (Medicine)

Date of Defense

2008-08-28

First Committee Member

Nanette H. Bishopric - Committee Chair

Second Committee Member

Xupei Huang - Committee Member

Third Committee Member

Glenn W. Kerrick - Committee Member

Fourth Committee Member

Anthony H. Caswell - Committee Member

Fifth Committee Member

James D. Potter - Mentor

Abstract

Three novel mutations (G159D, L29Q and E59D/D75Y) in cardiac troponin C (CTnC) associate their clinical outcomes with a given cardiomyopathy. Current paradigms propose that sarcomeric mutations associated with dilated cardiomyopathy (DCM) decrease the myofilament calcium sensitivity while those associated with hypertrophic (HCM) cardiomyopathy increase it. Therefore, we incorporated the mutant CTnCs into skinned cardiac muscle in order to determine if their effects on the calcium regulation of tension and ATPase activity coincide with the current paradigms and phenotypic outcomes. This required the development of new calculator programs to solve complex ionic equilibria to more accurately buffer and expand the free calcium range of our test solutions. In accordance with the DCM paradigms, our result show that G159D and E59D/D75Y CTnC decrease the myofilament calcium sensitivity and force generating capabilities which would likely increase the rate of muscle relaxation and weaken the contractile force of the myocardium. Alternatively, the lack of myofilament change from L29Q CTnC (associated with HCM) may explain why the only proband is seemingly unaffected. Notably, the changes in the calcium sensitivity of tension (in fibers) do not necessarily occur in the isolated CTnC and vice versa. These counter-intuitive findings are justified through a transition in calcium affinity occurring at the level of cardiac troponin (CTn) and higher, implying that the true effects of these mutations become apparent as the hierarchal level of the myofilament increases. Despite these limitations, the regulated thin filament (RTF) retains its role as the calcium regulatory unit and best indicates a mutation's ability to sensitize (+) or desensitize (-) the muscle to calcium. Since multiple forms of cardiomyopathies exist, the identification of new drugs that sensitize (+) or desensitize (-) the calcium sensitivity could potentially reverse (+ or -) these aberrant changes in myofilament sensitivity. Therefore, we have developed an RTF mediated High Throughput Screening assay to identify compounds in libraries of molecules that can specifically modulate the calcium sensitivity of cardiac contraction. The knowledge gained from these studies will help us and others to uncover new pharmacological agents for the investigation and treatments of cardiomyopathies, hypertension and other forms of cardiovascular diseases.

Keywords

Skinned Muscle Fiber; Cardiomyopathy; Cardiac Troponin; Troponin C; Muscle Contraction; Actomyosin; Cardiac Myofibril; ATPase Activity; Fluorescence; IAANS-labeled CTnC; E59D/D75Y CTnC; Regulated Thin Filament; L29Q CTnC; CDTA; EDTA; G159D CTnC; Troponin Mutation; Myofilament; Calcium Sensitivity; Calcium Affinity; Calcium Binding

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