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Title: Nanomechanics of cell membrane and cellular contacts in control and failing hearts
Author: Swiatlowska, Pamela
ISNI:       0000 0004 8499 6377
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2019
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In recent years, a growing number of studies have shown that mechanical properties play an important role in both structure and function of cells. Heart is an extremely dynamic organ; therefore, cardiac myocytes are constantly subjected to a mechanical stress. To date, titin protein and collagen fibers were considered to be the main regulators of tissue Young's modulus, one of the standard measures of mechanical properties. Recently, studying mechanical properties at cellular, tissue and organ level, demonstrated contribution of mechanical cues to the development of different diseases, including heart failure. During the progression of this pathology, cells undergo several changes in physiology and mechanobiology, where a significant increase in Young's modulus is observed. Work presented in this thesis examines cardiomyocyte nanomechanical properties focusing specifically on measuring transverse cortical Young's modulus by using high resolution Scanning Ion Conductance Microscopy in different mouse, rat and human disease models of heart failure. Further work investigates the role of different intracellular elements such as generic and cardiac-specific cytoskeleton, mitochondria and mechanical load that can affect cardiac mechanics. In order to determine their role RT-PCR, Western blot, Transmission Electron Microscopy and immunofluorescent staining techniques were used. To obtain a bigger picture on cardiac mechanics, co-cultures of myocytes alone and with fibroblasts were established where changes in Young's modulus at the homo- and hetero-cellular cell-cell junction were studied. Using a novel Junctional Mapper software precise quantification of intercalated disc proteins population was attainable. Scanning Ion Conductance Microscope was adapted to measure cell Young's modulus at a nanoscale resolution and used in an extensive study of cardiomyocytes mechanics. In normal myocytes, the contribution of individual cellular elements to cell mechanical properties was assessed via inhibitor analysis. Consequently, actin, microtubules and caveolae were found to have the biggest contribution to cardiomyocyte mechanics. In a rat model of heart failure (16 weeks after myocardial infarction), cardiac myocytes show a markedly increased Youngs modulus with a significantly higher value in surface crest areas than Z-grooves. This could be related to mitochondria rearrangement, actin-myosin incomplete relaxation and increased microtubular network densification. In fact, microtubule post translational modifications (acetylation and detyrosination) were found to be increased in failing cells and that will correlates with an increased Young's modulus. Moreover, a cross-talk has been revealed between these two populations of microtubules, as increased level of acetylation results in reduced detyrosination. Removal of load from both control and failing heart markedly reduces Young's modulus of myocytes; in fact, after unloading the hearts failing cardiomyocytes present a similar Young's modulus value to healthy cells. Other changes can be observed after the removal of load, for example the level of acetylated and detyrosinated microtubules and the mitochondria numbers are also reduced. Long term exposure to Angiotensin II (Ang II) is known to exert a hypertrophic effect on cardiac myocytes, whereas little is known about acute, short-term action of Ang II. This work suggests a novel, beneficial role of acute treatment with Ang II in regulating cardiomyocyte mechanics. Reduced Young's modulus is observed in Ang II treated myocytes, which is driven by changes in microtubular network, including acetylation and detyrosination modifications. More importantly, Ang II acts equally upon failing myocytes bringing Young's modulus value to the normal level. Therefore, it can be potentially used to treat diseased heart muscle cells. Overall, this thesis describes a novel technique of measuring Young's modulus in live cells. Using this method, a detailed study on changes in cardiomyocyte mechanics is presented. In line with other studies, we observe that understanding myocardial mechanobiology is imperative to fully disclose the mechanism of initiation and progression of heart failure.
Supervisor: Gorelik, Julia ; Braga, Vania ; Sanchez Alonso-Mardones, Jose Sponsor: National Heart and Lung Institute
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral