THE MECHANICAL PROPERTIES OF NON-FAILING AND FAILING HUMAN MYOCARDIUM

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

Heart Failure in Humans Reduces Contractile Force in Myocardium from Both Ventricles

This study measured how heart failure affects the contractile properties of the human myocardium from the left and right ventricles. The data showed that maximum force and maximum power were reduced by approximately 30% in multicellular preparations from both ventricles, possibly because of ventricular remodeling (e.g., cellular disarray and/or excess fibrosis). Heart failure increased the calcium (Ca2+) sensitivity of contraction in both ventricles, but the effect was bigger in right ventricular samples. The changes in Ca2+ sensitivity were associated with ventricle-specific changes in the phosphorylation of troponin I, which indicated that adrenergic stimulation might induce different effects in the left and right ventricles

Omecamtiv Mecarbil Enhances the Duty Ratio of Human \u3cem\u3eβ\u3c/em\u3e-Cardiac Myosin Resulting in Increased Calcium Sensitivity and Slowed Force Development in Cardiac Muscle

The small molecule drug omecamtiv mecarbil (OM) specifically targets cardiac muscle myosin and is known to enhance cardiac muscle performance, yet its impact on human cardiac myosin motor function is unclear. We expressed and purified human β-cardiac myosin subfragment 1 (M2β-S1) containing a C-terminal Avi tag. We demonstrate that the maximum actin-activated ATPase activity of M2β-S1 is slowed more than 4-fold in the presence of OM, whereas the actin concentration required for half-maximal ATPase was reduced dramatically (30-fold). We find OM does not change the overall actin affinity. Transient kinetic experiments suggest that there are two kinetic pathways in the presence of OM. The dominant pathway results in a slow transition between actomyosin·ADP states and increases the time myosin is strongly bound to actin. However, OM also traps a population of myosin heads in a weak actin affinity state with slow product release. We demonstrate that OM can reduce the actin sliding velocity more than 100-fold in the in vitro motility assay. The ionic strength dependence of in vitro motility suggests the inhibition may be at least partially due to drag forces from weakly attached myosin heads. OM causes an increase in duty ratio examined in the motility assay. Experiments with permeabilized human myocardium demonstrate that OM increases calcium sensitivity and slows force development (ktr) in a concentration-dependent manner, whereas the maximally activated force is unchanged. We propose that OM increases the myosin duty ratio, which results in enhanced calcium sensitivity but slower force development in human myocardium

Estruturação dos objetivos de aprendizagem para ambientes de educação on-line

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro Tecnológico. Programa de Pós-Graduação em Ciência da Computação.O objetivo deste trabalho é prover o processo e a avaliação funcional dos componentes que compõem um ambiente de e-aprendizagem baseado em Objetos de aprendizagem, apresentando as definições, propriedades e aplicações dos Objetos de aprendizagem, que se referem à criação e reutilização dos objetos para desenvolver ambientes de aprendizagem. Os padrões utilizados para desenvolvimento de conteúdos estruturados para ambientes de aprendizagem para Web também serão descritos. Também é abordado o potencial da Web semântica, a qual transforma a Web em um meio em que a informação é interpretada, trocada e processada. Apresentar como a linguagem XML e a orientação a objeto se relacionam com os Objetos de aprendizagem é outro tópico abordado. O trabalho inclui, ainda, um protótipo para um ambiente de aprendizagem online utilizando os objetos de aprendizagem

Mechanobiology Assays with Applications in Cardiomyocyte Biology and Cardiotoxicity.

Cardiomyocytes are the motor units that drive the contraction and relaxation of the heart. Traditionally, testing of drugs for cardiotoxic effects has relied on primary cardiomyocytes from animal models and focused on short-term, electrophysiological, and arrhythmogenic effects. However, primary cardiomyocytes present challenges arising from their limited viability in culture, and tissue from animal models suffers from a mismatch in their physiology to that of human heart muscle. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) can address these challenges. They also offer the potential to study not only electrophysiological effects but also changes in cardiomyocyte contractile and mechanical function in response to cardiotoxic drugs. With growing recognition of the long-term cardiotoxic effects of some drugs on subcellular structure and function, there is increasing interest in using hiPSC-CMs for in vitro cardiotoxicity studies. This review provides a brief overview of techniques that can be used to quantify changes in the active force that cardiomyocytes generate and variations in their inherent stiffness in response to cardiotoxic drugs. It concludes by discussing the application of these tools in understanding how cardiotoxic drugs directly impact the mechanobiology of cardiomyocytes and how cardiomyocytes sense and respond to mechanical load at the cellular level

A Protocol for Collecting Human Cardiac Tissue for Research

This manuscript describes a protocol at the University of Kentucky that allows a translational research team to collect human myocardium that can be used for biological research. We have gained a great deal of practical experience since we started this protocol in 2008, and we hope that other groups might be able to learn from our endeavors. To date, we have procured ~4000 samples from ~230 patients. The tissue that we collect comes from organ donors and from patients who are receiving a heart transplant or a ventricular assist device because they have heart failure. We begin our manuscript by describing the importance of human samples in cardiac research. Subsequently, we describe the process for obtaining consent from patients, the cost of running the protocol, and some of the issues and practical difficulties that we have encountered. We conclude with some suggestions for other researchers who may be considering starting a similar protocol

Abnormal contractility in human heart myofibrils from patients with dilated cardiomyopathy due to mutations in TTN and contractile protein genes (vol 7, 14829, 2017)

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper