Download the full-sized PDF of Molecular Mechanisms Underlying the Enhancement of Cardiac Contraction by Modifications of TroponinDownload the full-sized PDF



Permanent link (DOI):


Export to: EndNote  |  Zotero  |  Mendeley


This file is in the following communities:

Graduate Studies and Research, Faculty of


This file is in the following collections:

Theses and Dissertations

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


Other title
myocardial ischemia
cardiac troponin
muscle contraction
myocardial acidosis
drug discovery
pH senstivity
Type of item
Degree grantor
University of Alberta
Author or creator
Pineda Sanabria, Sandra E
Supervisor and department
Sykes, Brian D (Biochemistry)
Examining committee member and department
Sykes, Brian D (Biochemistry)
Lemieux, Joanne (Biochemistry)
Spyracopoulos, Leonidas (Biochemistry)
Clanachan, Alexander (Pharmacology)
Shaw, Gary (Biochemistry, University of Western Ontario)
Department of Biochemistry

Date accepted
Graduation date
Doctor of Philosophy
Degree level
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.
Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.
Citation for previous publication
Pineda-Sanabria, S. E., Robertson, I. M., Li, M. X., and Sykes B. D. (2013) Cardiovascular Research 97, 481-489Robertson, I. M., Holmes, P. C., Li, M. X., Pineda-Sanabria, S. E., Baryshnikova, O. K., and Sykes, B. D. (2012) JBC 287, 4996-5007Robertson, I. M., Pineda-Sanabria, S. E., Holmes, P. C., and Sykes, B. D. (2014) Arch Biochem Biophys 552-553, 40-49Pineda-Sanabria, S. E., Robertson, I. M., Sykes, B. D. (2015) Biochemistry 54, 3583-3593Pineda-Sanabria, S. E., Julien, O., and Sykes, B. D. (2014) ACS Chem Biol 9, 2121- 2130Pineda-Sanabria, S. E., Robertson, I. M., and Sykes, B. D. (2011) Biochemistry 50, 1309-1320Robertson, I. M., Pineda-Sanabria, S. E., and Sykes, B. D. (2013). Approaches to protein-ligand structure determination by NMR spectroscopy: applications in drug binding to the cardiac regulatory protein troponin C. In Puglisi, J. D., and Margaris, M. V. (Eds.), Biophysics and structure to counter threats and challenges (pp. 121-134). Dordrecht, The Netherlands: SpringerHwang, P. M., Cai, F., Pineda-Sanabria, S. E., Corson, D. C., and Sykes, B. D. (2014) Proc Natl Acad Sci U S A 40, 14412-14417

File Details

Date Uploaded
Date Modified
Audit Status
Audits have not yet been run on this file.
File format: pdf (Portable Document Format)
Mime type: application/pdf
File size: 58986397
Last modified: 2016:06:24 17:52:07-06:00
Filename: PinedaSanabria_Sandra_E_201508_PhD.pdf
Original checksum: ecc138770e1a533a260db089ec3bfd58
Well formed: true
Valid: true
Status message: Too many fonts to report; some fonts omitted. Total fonts = 1440
File author: Sandra Pineda
Page count: 342
Activity of users you follow
User Activity Date