Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiovascular disease that could result in sudden cardiac death. Mutations in the genes encoding sarcomeric proteins, including the thin filaments, are the most common cause of HCM. Thin filaments are an integral part of the cardiac muscle contractile unit, composed of actin, tropomyosin, and troponin (Tn) complexes which contain troponin C (TnC), troponin I (TnI) and troponin T (TnT). HCM molecular mechanisms remain unclear, partially due to the lack of a high-resolution thin filament structure and the complex molecular interactions between each component. My first goal was to investigate the effects of three TnT mutations, I79N, F110I and R287C, in human reconstituted thin filaments (RTF), using steady-state and stopped-flow fluorometry to determine Ca2+ sensitivity (Kd) and Ca2+ dissociation rates (koff), respectively. Our data showed that I79N and R278C mutations significantly decreased Kd by lowering koff, and all three mutations attenuated the functional effects of phosphomimetic TnI, suggesting an important role in impaired relaxation with HCM. My second goal was to investigate the effects of the I79N TnT mutation and the fetal cardiac R37C TnI mutation in their corresponding adult/fetal RTF. The I79N TnT mutation did not change the Ca2+ binding properties in fetal RTF but significantly decreased the Kd in adult RTF. In contrast, the R37C TnI mutation significantly increased the Kd in fetal RTF, yet its corresponding mutation, R68C TnI in adult RTF, exhibited reverse Ca2+ binding properties. My third goal was to use cryo-electron microscopy (EM) to solve the RTF structure. Optimal buffer conditions were found using negative-stain EM to ensure Tn binding on RTF with a periodicity of 38.5 nm; however, unexpected challenges arose during cryo data collection. Persistent filament aggregations obscured most of the cryo-images. Suggestions on how to address this problem are provided in the last chapter. In summary, using cardiac thin filaments as a physiologically relevant biochemical model allows us to investigate how HCM mutations alter Ca2+ binding properties. The resulting studies provide a better understanding of HCM molecular mechanism and can potentially help develop specific therapies that address the underlying causes of the disease.
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Thesis advisor: Tibbits, Glen F.
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