Molecular Mechanisms Underlying the Enhancement of Cardiac Contraction by Modifications of Troponin

• Author / Creator

  Pineda Sanabria, Sandra E

• A fine balance between contraction and relaxation is crucial for the heart to function properly. But often the heart cannot contract with enough force to meet the demands of the body. This condition, called systolic heart failure, is the leading cause of death in the modern society. A common treatment consists in the administration of positive inotropes, which induce more forceful contractions. However, traditional inotropes carry serious adverse effects in the long term. An emerging group of therapeutics, called calcium sensitizers, also enhance contraction but without posing the risks associated with traditional inotropes. Thus, calcium sensitizers are a promising alternative in the search for better therapies for the treatment of heart failure. Calcium sensitizers act directly on the contractile proteins of the heart muscle. One of these proteins, troponin, regulates contraction in a calcium-dependent manner in cardiac and skeletal muscle. Troponin is composed of C, I, and T subunits. Troponin C and troponin I associate in the presence of calcium to form the regulatory complex that triggers contraction. Modifications to troponin, such as mutations, phosphorylation, and its interaction with small molecules, have an impact on the relaxation-contraction equilibrium of the heart. It is clear that the interaction of troponin with calcium sensitizers favors cardiac contraction. Another modification to troponin that favors contractility is the A162H mutation. However, their mechanisms of action are not fully understood. Thus, the general purpose of this thesis is to provide insight into the molecular mechanism of enhancement of cardiac contraction by these two changes on troponin. To accomplish this objective I mainly used nuclear magnetic resonance (NMR) spectroscopy to characterize the effect of the A162H substitution on cardiac troponin, and the interaction of cardiac troponin with several calcium sensitizers. I determined the biochemical and structural details of the cardiac troponin C-troponin I (A162H) interaction using NMR spectroscopy. I also investigated these details in skeletal troponin I as a means of comparison. In addition, I designed a troponin C-troponin I chimera to better study their interaction with small molecules. I screened potential drugs that target the troponin C-I interface using that chimera. I designed and synthesized a covalent calcium sensitizer (i9) that binds to troponin C. I carried out the reaction of i9 with troponin in a variety of conditions to verify the mechanism by which small molecules sensitize troponin. I also resorted to muscle fibers to study its physiologic effect. And because structure dictates function, I determined two independent NMR structures of troponin C bound to calcium sensitizers to correlate with the data already gathered. The main findings of this work indicate that the mechanism of action of the A162H substitution consists of an increase in the affinity of troponin I for troponin C, as well as a conformational change of troponin I that favors contraction in the context of the thin filament. Another major conclusion of this thesis is that effective calcium-sensitizers primarily stabilize of the open conformation of the N-domain of troponin C, having an increase in affinity of troponin I for troponin C as a secondary but desirable effect. The elucidation of these mechanisms may aid the design of effective therapies that target troponin to enhance contraction and bring balance back to the diseased heart.

• Subjects / Keywords

  • Drug discovery
  • Muscle contraction
  • NMR
  • Myocardial acidosis
  • PH senstivity
  • Myocardial ischemia
  • Cardiac troponin
  • A162H
  • Structure

• Graduation date

  Fall 2015

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R39C6S82S

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.