Usage
  • 6 views
  • 5 downloads

Mutations, post-translational modifications, and small molecule binding to the cardiac troponin complex alter cardiac contractile function

  • Author / Creator
    Mahmud, Zabed
  • Ischemic heart disease is the leading cause of death worldwide. It can lead to acute myocardial infarction, commonly known as a “heart attack”, in which heart muscle dies due to disruption of its oxygen and blood supply. Even if patients survive a heart attack and receive the best medical therapy possible, many go on to develop systolic heart failure, in which the heart muscle becomes progressively weaker and more dilated, impairing its ability to pump enough blood to satisfy the needs of the body. It is important to understand how the heart regulates its pumping activity, how it becomes dysregulated in ischemic heart disease and heart failure, and how this might be corrected through novel medical therapies.
    Cardiac muscle contraction is turned on and off in a calcium dependent manner through the cardiac troponin complex, which is comprised of three subunits: troponin C (cTnC), troponin I (cTnI) and troponin T (cTnT). cTnI has a C-terminal tail (residues 135-209) that binds to the actin-tropomyosin thin filament to maintain it in a blocked state under low calcium resting conditions. During systole, calcium ions flood the cytoplasm and activate the N-terminal domain of cTnC (cNTnC), allowing it to bind to the switch region of cTnI (residues 146-158), releasing inhibition of cTnI and allowing actin-myosin cross-bridging and muscle contraction to proceed.
    cTnI is known to be cleaved in ischemia-reperfusion (I/R) injury. We demonstrate that it remains susceptible to proteolysis even while bound to actin or cTnC. Using in vitro proteolysis and mass spectrometry, we demonstrate that the switch region of cTnI is cleaved by matrix metalloproteinase-2 (MMP-2) and calpain, two intracellular proteases implicated in I/R injury. Cleavage in this essential region could account for myocardial stunning, a phenomenon whereby contraction is temporarily depressed in living and apparently structurally intact cardiac muscle.
    Physiologic regulation of human cardiac troponin activation occurs via phosphorylation of the cardiac-specific N-terminal tail of cTnI (residues 1-32). Using solution NMR spectroscopy, we demonstrate that this N-terminal tail interacts with the cNTnC to fix it to the active orientation needed to bind the switch region of cTnI. Phosphorylation of the N-terminal tail disrupts this orientation, as do some mutations in cTnC and cTnI associated with dilated cardiomyopathy. By reconstituting the cardiac troponin complex without the activating N- and C-terminal tails of cTnI, we demonstrate the presence of an alternative “dormant” orientation of the cNTnC domain that competes with its active orientation.
    Finally, we have developed RPI-194, a novel small molecule cardiac troponin activator. Using NMR and fluorescence spectroscopy, we demonstrate that it stabilizes the activated calcium-bound complex between cNTnC and cTnI. Moreover, RPI-194 acts as a calcium sensitizer in cardiac muscle, as well as in slow skeletal and fast skeletal muscle fibers. However, it appears to slow down unloaded contraction in muscle fibers as well as in individual mouse cardiomyocytes. In isolated perfused mouse heart, RPI-194 increases cardiac work.
    In summary, cardiac function is carefully regulated at the level of the cardiac troponin complex, but can be disrupted by proteolysis or cardiomyopathy-causing mutations. An exciting possibility is the development of drugs to powerfully modulate troponin activity in order to correct cardiac contractile function in disease states.

  • Subjects / Keywords
  • Graduation date
    Fall 2021
  • Type of Item
    Thesis
  • Degree
    Doctor of Philosophy
  • DOI
    https://doi.org/10.7939/r3-txvn-jb04
  • 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.