Advanced electromechanical methods to culture adult myocardium in vitro

Abstract

The adult heart is a terminally differentiated organ that responds to changes in its environment by phenotypic adaptions. This process involves electrical, mechanical, structural, and molecular changes and it is termed cardiac remodelling or plasticity. Understanding the mechanisms behind remodelling is crucial in understanding cardiac physiology and pathology and the transition between the two. A canonical driver of remodelling is mechanical load. One method to examine plasticity in response to load is to subject cardiac tissue to different loads during in vitro culture and examine its temporal response and the terminal remodelled phenotype. There are multiple cardiac preparations used for in vitro culture, including engineered heart tissues, isolated cardiomyocytes, trabeculae, and papillary muscles. Throughout this thesis, we used a novel cardiac preparation known as living myocardial slices (LMS). These are 300 μm thick slices prepared from the explanted hearts of animals or humans. LMS are organotypic, meaning that they maintain the cellular stoichiometry, functional, structural, metabolic, and molecular profile of the tissue from which they are prepared. LMS were used as they offer several advantages compared to other models. To date, mechanical load has been studied in vitro using either auxotonic or isometric loading protocols. These have enabled the progression of the field and our understanding of the effects of load on the myocardium. However, they are oversimplified as they fail to recapitulate the mechanical events of the in vivo cardiac cycle, which broadly include isometric contraction followed by ejection, isometric relaxation, and diastolic refilling. In this thesis, we aimed to develop a culture platform to recreate the fine sequence of mechanical events that occur during the in vivo cardiac cycle and apply them in vitro on LMS. We did this by using a Three-Element Windkessel model to describe afterload and sarcomere length to describe preload. As both preload and afterload were parametrised in our platform, we were able to culture LMS under a range of pathophysiological loads. In Chapter 3, we cultured LMS for 3-days under physiological load (normal preload, normal afterload), pressure-overload (high afterload, normal preload), or volume-overload (high preload, normal afterload). Our results show that LMS demonstrate distinct functional, structural, and molecular profiles with activation of both shared and unique gene networks as a function of mechanical load profile applied to them. To our knowledge, this was the first time that cardiac tissue has been cultured and studied in vitro under a mechanical load protocol that recreates the in vivo cardiac cycle In Chapter 4, we show the development of MyoLoop, a patented bioreactor designed to culture LMS in vitro under the advanced electromechanical stimulation protocol developed in Chapter 3, which enables recreation of the in vivo cardiac work loop. We describe the novelties, advantages, and limitations of MyoLoop relatively to other culture set-ups and commercially available systems. Finally in Chapter 5, we shift our attention to acute physiological studies that examine transmural mechanical heterogeneity in the adult rat LV wall. To do that, we leverage the unique method of preparation of LMS, which involves the sequential generation of intact slices from the subendocardium to the subepicardium. We show that different layers of the left ventricular wall have different mechanical properties and provide a physiological conceptual framework for the presence of these.