Biomimetic electromechanical stimulation to maintain adult cardiac tissue in vitro

Abstract

Over the last few decades, progress in cardiovascular research has been significantly hindered by a lack of appropriate research models. The data collected using current in vitro approaches are often oversimplified and this makes the translation of results from the laboratory to clinical trials challenging. Additionally, adult cardiac tissue undergoes a process of rapid dedifferentiation when removed from the body, which has limited in vitro studies to acute time points. To date, there are no cardiovascular models that can be cultured without significant changes in cardiac structure and function. Myocardial slices are highly viable, ultrathin (100-400m) slices of living cardiac tissue, which retain the native multicellularity, architecture and physiology of the heart. However, we and others have previously demonstrated that myocardial slices also undergo rapid dedifferentiation in vitro.

The in vivo environment appears to be fundamental to the maturation and maintenance of the adult cardiac phenotype, particularly the cyclic dynamic mechanical load experienced by the myocardium. In this thesis, we investigated the hypothesis that biomimetic electromechanical stimulation maintains the features of the adult cardiac phenotype in vitro.

Myocardial slices were used for this project as they have a number of advantages over other cardiac in vitro models and are more amenable to culture. We developed an optimised protocol for producing myocardial slices from small and large mammalian hearts and demonstrated that they are a highly viable preparation, with only 2-3% of the total cardiomyocyte population damaged during slicing. Myocardial slices were initially cultured with a fixed preload, using bespoke myocardial slice stretchers and a novel culture system. Myocardial slices were cultured with a range of physiological preload (sarcomere length (SL) =1.8-2.4m), determined using laser diffraction. We found that the structural, functional and transcriptional properties of rat myocardial slices were optimally maintained at SL=2.2m for 24 hours. Rabbit myocardial slices cultured at SL=2.2m for 5 days did not suffer any decline in contractility and this approach was used to improve the maintenance of human donor and HF myocardial slices at 24 hours. However, the mechanical load experienced in vivo is dynamic and consists of both preload and afterload. As such, we developed two set ups capable of providing biodynamic electromechanical stimulation to myocardial slices. The first approach, using a Bose Electroforce, showed that biodynamic electromechanical stimulation may provide further benefit over fixed preload, but experiments were limited by several technical issues. As such, a second set up was developed that applied afterload using a 3-element Windkessel model. The set up was capable of developing physiological force-SL work loops and the afterload applied could be altered by modulating the individual elements of the model. However, slices cultured using this approach suffered a run down in their contractility during culture, most probably due to external sources of error.

Throughout this project, we have demonstrated that cardiac tissue is capable of significant phenotypic plasticity. We investigated the plasticity of the myocardium is response to mechanical unloading in vivo using a heterotopic abdominal rat heart transplantation model. After 48 hours, hearts underwent significant structural remodelling, including whole heart and cardiomyocyte atrophy, but this was associated with a preservation of cardiac function.

This is the first study to demonstrate the maintenance of the adult cardiac phenotype in vitro using biomimetic electromechanical stimulation. The prolonged culture of myocardial slices in vitro is likely to have significant implications for basic and translational cardiovascular research, providing a platform to test several novel therapeutic strategies.