Author ORCID Identifier

https://orcid.org/0000-0002-0289-439X

Year of Publication

2017

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Medicine

Department

Physiology

First Advisor

Dr. Kenneth S. Campbell

Abstract

Heart failure is a clinical syndrome that manifests when there are structural and functional impairments to the heart that reduces the ability of the ventricles to fill or eject blood. The syndrome affects ~6 million Americans and is responsible for nearly 300,000 deaths annually. At the core of the syndrome are dysfunctional sarcomeres, the machinery that drives cardiac contraction and relaxation. By assessing the mechanical properties of human cardiac tissue, the information provided in this dissertation will provide data that demonstrates how sarcomeric dysfunction contributes to heart failure in the left and right ventricles. Additionally, these data will supply information on how probable therapeutics impact the mechanical properties of the heart and the clinical implications. Thus, the overall objective of this project is to assess the mechanical properties of failing and non-failing human myocardium while concomitantly studying the molecular mechanisms contributing to heart failure and work towards therapy.

Mechanical experiments were performed with human cardiac samples obtained from patients who were receiving heart transplants and from organ donors who did not have a history of heart failure. Cardiac samples were homogenized and chemically permeabilized (pores in the membrane). Multicellular preparations from failing and non-failing hearts were attached between a force transducer and a motor to determine the mechanical properties.

In the first study, we compared the mechanical properties of cardiac samples from the right and left ventricles of non-failing and failing hearts, as well as determined the relative phosphorylation levels of specific sarcomeric proteins. The results show that in non-failing hearts, calcium sensitivity was higher in the left ventricle, and in failing hearts, calcium sensitivity was higher in the right ventricle. The shift in the pattern of the calcium sensitivity data from non-failing samples to failing samples underpin a statistical interaction between heart failure status and the ventricles of the heart for calcium sensitivity. This interaction suggests that heart failure is altering the sensitivity of the myofilament to Ca2+ differently in the right ventricle. The mechanical data also demonstrated that heart failure significantly reduced isometric force and maximum power in both ventricles. Biochemical assays suggest that the cause of the interaction observed in the calcium sensitivity data is driven by the phosphorylation profile of sarcomeric proteins.

We then determined the effects of two small molecules (omecamtiv mecarbil and para-Nitroblebbistatin) on the mechanical properties of human myocardium. The results of those studies demonstrate that omecamtiv mecarbil increases calcium sensitivity and slows the rate of force development in a dose-dependent manner without altering maximum isometric force. Conversely, para-Nitroblebbistatin reduced isometric force, power, and calcium sensitivity without changing shortening velocity or the rate of force development.

Lastly, we measured the effects of engineered troponins on the mechanical function of failing tissue. The results show that troponin C and troponin I designed to either increase or decrease calcium sensitivity can significantly increase or decrease calcium sensitivity without altering maximum force, shortening velocity or the rate of tension development.

The findings reported in this dissertation have revealed novel mechanical data from non-failing and failing human cardiac tissue. These data present three significant results. First, the right vs. left ventricular comparison data shows that heart failure in humans reduces maximum force and power in both ventricles equally while altering myofilament calcium sensitivity of the left and right ventricles in different ways. The change in calcium sensitivity may reflect ventricle specific post-translational modifications of sarcomeric proteins. Second, the use of myosin modulators revealed that molecules like omecamtiv mecarbil and para-Nitroblebbistatin that directly target myosin function can modify calcium sensitivity and the rate of force development in human cardiac tissue. Third, the engineered troponin study showed that engineered troponins C and I can alter myofilament calcium sensitivity without affecting myosin kinetics. Clinically, the results of the small molecules and engineered protein studies suggest that small molecules and engineered proteins could potentially serve as therapy for patients suffering from heart disease.

Digital Object Identifier (DOI)

https://doi.org/10.13023/ETD.2017.411

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