Browsing by Subject "Congenital, Hereditary, and Neonatal Diseases and Abnormalities"
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Item Effect of Phosphorylation on Muscle Physiology and Biophysical Characterization of Mutations Responsible for Familial Hypertrophic Cardiomyopathy(2016-08-01) Duggal, Divya; Borejdo, Julian; Gryczynski, Ignacy; Clark, Abbot F.Familial hypertrophic cardiomyopathy (FHC) is the most common cause of sudden cardiac death in young individuals. Molecular mechanisms underlying this disorder are largely unknown; this study aims at revealing how disruptions in actin-myosin interactions can play a role in the pathogenesis of this disorder. Cross-bridge (XB) kinetics and the degree of order were examined in contracting myofibrils from the ex vivo ventricles of transgenic (Tg) mice expressing FHC regulatory light chain (RLC) mutation K104E and Troponin I mutation, R21C. Because the degree of order and the kinetics are best studied when an individual XB makes a significant contribution to the overall signal, the number of observed XBs in an ex vivo ventricle was minimized to 20. Autofluorescence and photobleaching were minimized by using a relatively long-lived red-emitting dye. In case of K104E, mutated XBs were significantly better ordered during steady-state contraction and during rigor, but the mutation had no effect on the degree of order in relaxed myofibrils. The K104E mutation increased the rate of XB binding to thin filaments and the rate of execution of the power stroke. In case of R21C, differences were investigated in the left (LV) and right ventricle (RV) mutant where it was found that the mutation imposed significant difference in the distribution of angles that actin makes with thin filament axis: during contraction, actin angles from LV were more tightly distributed compared to actin angles from RV. Collectively, the data indicates that the mutation-induced changes in the interaction of myosin with actin during the contraction- relaxation cycle may contribute to altered contractility and the development of FHC. Phenotypic differences of the R21C mutation in the left versus right mouse ventricles, even though both ventricles express the same isoform of the cardiac highlights the importance of functional differences between the two ventricles of cardiac disease.Item North Texas Health & Science - 2011, Issue 2(University of North Texas Health Science Center at Fort Worth, 2011-01-01)Item Study of Cross Bridge Kinetics in Hypertrophic Ventricular Muscle(2009-05-01) Muthu, Priya; Borejdo, JulianCardiovascular diseases are the leading cause of mortality worldwide; with heart failure being highly prevalent in most affluent parts of the world. There is a need for a better understanding of the mechanism underlying these diseases. Familial hypertrophic cardiomyopathy (FHC), one such disease, is a genetic disorder of the heart characterized by increased growth or hypertrophy in the thickness of the wall of the left ventricle, the largest of the four chambers of the heart. This research project is focused on one kind of FHC, the D166V mutation in the regulatory light chain in myosin, which is associated with a particularly malignant form of the disease. The overall goal of this project was to study cross bridge kinetics (contraction and ATP utilization) in cardiac muscle from transgenic mice and to develop assays to apply this to human samples. The real time orientation changes of myosin and actin during a single cross bridge cycle beginning in a state of rigor was studied by Fluorescence anisotropy. Rabbit psoas fibers were isolated and used to achieve imaging of a few fluorophores or cross bridges. This technique was then applied to study cardiac myofibrils from transgenic mice, carrying the mutation causing the disease (FHC). Methods to achieve single molecule detection to aid studying human samples suffering from this disease were developed using silver island films, monolayers of nanoparticles and surface plasmon coupled emission. The conclusions of this dissertation were that a mutation in a light chain in myosin cause changes in the cross bridge kinetics. Myofibrils from the mutated mice displayed a significant slower rate of detachment during contraction as well as increased ATPase activity, which if severe enough could cause the heart to compensate by increasing wall thickness (hypertrophy). Despite significant clinical advances in the treatment of various cardiovascular diseases, mortality rates remain high. No therapy currently exists to treat or delay progression from hypertrophy to heart failure. This proposal help answer an important question regarding the molecular basis of FHC-mediated pathology in the heart. Also, achieving imaging of a single fluorophore has numerous implications in the biological field, like studying ligand-receptor interactions in live cells, involvement of protein molecules in internalization of bacteria by cells, monitoring the conformational fluctuations of DNA, diagnosis of prion diseases and also in detection of viruses at an early phase of infection.