Andrea Deranek's Abstracts

Andrea Deranek's Abstracts

     Andrea Deranek
     Ph.D. Student
     Biomedical Engineering GIDP

     Conference Summary
     Biophysical Society 61st Annual Meeting
     New Orleans, Louisiana

Lay Audience Abstract

Hypertrophic cardiomyopathies (HCM), one type of heart disease, affects 1 in 500 Americans. HCM leads to thickening of the heart, causing improper function of the heart, specifically improper relaxation. Inherited HCM is due to a mutation found within a number of cardiac muscle proteins. One such protein, tropomyosin (Tm), helps regulate the contraction of the heart muscle. My research focuses on 4 mutations within Tm, Ala22Thr (A22T), Asp175Asn (D175N), Ser215Leu (S215L), and Met281Thr (M281T), that have all been clinically linked to HCM. These mutations, spread along the entire length of the Tm protein, which will be useful to provide insight into how the location of the mutation might affect its progression to disease. To examine the structural and functional effects of these mutations, I will utilize differential scanning calorimetry (DSC), calcium dissociation kinetics, and in-vitro motility (IVM). DSC will allow me to determine how the mutated tropomyosin interacts with the other regulatory proteins of the heart. For example, we can determine whether there is a structural change that leads to stiffening in any of the protein interactions, thus hindering the activation of contraction within the heart. I have collected preliminary results for the Tm A22T, D175N, and M281T mutations, all of which cause destabilization of the tropomyosin molecule alone. For S215L I have found a differential effect of stabilization within the molecule, such that one terminal end of tropomyosin is stabilized while the other terminal end is destabilized. To better understand these results in the context of activation of contraction, I will repeat these DSC experiments with the other regulatory proteins and determine if the effects described above are maintained. Calcium dissociation kinetics will show if there is a change in the rate of calcium release from the mutated thin filament. If a decreased dissociation rate is measured, that indicates an inability of the heart to properly relax and fill with blood for the subsequent beat, thus compromising function. IVM will measure whether there are any functional effects of the mutations, specifically alterations in muscle activation. This assay can provide insight into whether the mutation causes a change in the tendency of the muscle to be activated as well as the amount of force that is generated with contraction. Once all the experimental data is collected, I hope to develop a model that will allow for prediction of how different mutations can lead to complex HCM to improve clinical management of patients.

Investigating the Structural and Functional Effects of 4 HCM Linked Tropomyosin Mutations

One of the most common causes for cardiomyopathies are mutations in the sarcomeric proteins. Several mutations have been found in the sarcomeric protein, tropomyosin (Tm), a regulatory protein of cardiac contraction. The canonical, alpha-helical coiled coil structure of the tropomyosin dimer is defined by seven positions (A-G) that form a helical wheel in the dimerized molecules and seven periods (P1-P7) spatially defined by the binding of Tm to actin within the sarcomere. We looked at 4 tropomyosin mutations, A22T, D175N, S215L, and M281T, that have been clinically linked to hypertrophic cardiomyopathy (HCM). These mutations are found throughout the helical wheel positions (A22T and M281T in A position, D175N in G, and S215L in E) and spread along the seven periods (A22T in P1, D175N in P5, S215L in P6, and M281T in P7). These differences led us to wanting to investigate how mutations located throughout Tm could all lead to HCM. To analyze the properties of these mutations, each mutated Tm protein was bacterially expressed and purified. Differential scanning calorimetry (DSC) will provide insight into the thermal stability of the protein. Preliminary results of mutated Tm alone have shown that A22T, D175N, and M281T cause overall destabilization of the protein, with M281T having the most pronounced effect, while S215L causes stabilization of the C terminus and destabilization of the N terminus. These experiments will be repeated with fully reconstituted thin filaments to determine if these results hold in a more biologically complex system. Future studies will be conducted with these mutants, including calcium dissociation kinetics and regulated in vitro motility to determine the functional effects of the mutants. With this data, we hope to develop a model that will allow for prediction of how different mutations can lead to complex HCM to improve clinical management of patients.