Hypertrophic Cardiomyopathy (HCM) is the leading cause of sudden cardiac death in young adults under the age of 35; a devastating disease that is not yet well understood. To date, greater than 1000 HCM-associated mutations have been found in genes that encode mostly sarcomeric proteins. Familial Hypertrophic Cardiomyopathy (FHC) is the heritable form of HCM. The overlying phenotype of FHC is thought to be derived from an increase in calcium (Ca2+) sensitivity of contraction and impaired relaxation of the myocardium. Dilated Cardiomyopathy (DCM) associated mutations are thought to have the opposite functional effect. This study focuses on cardiac troponin C (cTnC) a component of the cardiac troponin complex where binding of Ca2+ acts as the regulatory switch, leading to a series of conformational changes that culminate in muscle contraction. This project explores Ca2+ binding by focusing on the proximal-most unit of the contractile apparatus. The interaction of Ca2+ with the regulatory domain of cTnC is studied through isothermal titration calorimetry in conjunction with Molecular Dynamics simulations to understand structural and functional changes in the N-terminal region of cTnC. Initially, we established a workflow by exploring the functional consequences of sequence variations in coordinating Ca2+ binding and the genetic control of paralog expression in response to environmental temperature change in zebrafish. We then focused on a series of FHC-associated mutations (A8V, L29Q, A31S, and C84Y), as well as an engineered Ca2+ sensitizing mutation (L48Q), and a DCM-associated mutation (Q50R). The effects of temperature in modulating the Ca2+-cTnC interaction was also studied in these mutants. We further explored the role of cellularly abundant magnesium (Mg2+) which also interacts with cTnC and may modulate the Ca2+ coordinating capabilities of this contractile protein. Lastly, the role of Mg2+ binding to the mutants of interest, under normal cellular condition and in energy depleted states was explored to better understand the etiology of FHC and provide biomedical and physiological insight into potential treatments for this disease.
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Thesis advisor: Tibbits, Glen
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