New Tools for High-throughput Cardiac Muscle Experimentation

Abstract

Cardiac disease is the leading cause of death worldwide and thus there is a great need to develop a better understanding of the heart in both healthy and diseased states. Cardiac muscle cells are fundamental to the correct functioning of the heart, yet there is much to be discovered about their mechanical properties in health and disease. Cardiac trabeculae are small multicellular samples of heart muscle tissue that contain many hundreds of axially aligned myocytes, and are therefore often studied in vitro to better understand the underlying physiology of cardiac muscle. However, an order of magnitude difference between the stress production of myocytes and of cardiac trabeculae has been reported. A deeper knowledge of the properties of these tissues would assist in developing treatments for disease, as well as clarify the underlying causes of variability at different tissue levels. While both individual myocytes and cardiac trabeculae have been extensively studied to get a better understanding of cardiac mechanics, the instruments available for experimentation on these tissues often involve delicate mounting of the muscle and are limited to examining at one cell/muscle at a time. Devices and methods for high-throughput testing of cardiac tissues would provide the potential to gain deeper insight into cardiac mechanics. Experimentation under the influence of a variety of pharmacological interventions could improve the rate at which treatments for cardiac disease are developed. In this thesis, methods that are useful for high-throughput testing of both myocytes and trabeculae are explored. Robust image registration techniques are used to estimate 2D displacements within a myocyte during contraction, and to quantify internal displacement and strain fields with subpixel accuracy. Using these tracking techniques, localised sarcomere length estimation is performed using a tracked window. The internal strain field and localised sarcomere length measurements demonstrate that contraction across the cell is non-uniform. Next, these algorithms are implemented in real-time for cell length measurements, producing more robust results than currently used techniques. Methods for easy manipulation of cells and trabeculae using visible-light cured hydrogels are then developed, and experiments on cell-gel constructs are performed, using a finite element model to estimate force production from tracked displacements of the cell. Stresses reported in this thesis were similar to those found in literature. Real-time tracking is next applied to trabeculae that are secured at one end with hydrogel while the other end is manipulated via a force sensor. The muscles are stimulated while being imaged and real-time feedback control based on cantilever and trabecula movement during contraction is performed. Additionally, both ends of a trabecula are fixed and sarcomere variation along the length of the muscle is examined. Isometric contractions were performed on trabeculae based on muscle length defined using internal points in the muscle, as well as using the end tissue attachment points. The techniques developed in this thesis for experimentation of cardiac samples at both the tissue and cell level have the potential to be used in high-throughput systems to explore cardiac muscle function. Various interventions can now be applied to cardiac muscle using these techniques to determine their effects on the mechanical properties of the muscle. The devices developed in this thesis have the potential to provide new information for medical and scientific fields. The function of muscle in healthy and diseased states could be assessed, in order to develop therapies for cardiac disease.