Heart-on-chip: functional lipoprotein lipase (LPL) expression in co-cultures of cardiomyocytes and endothelial cells

Abstract

Introduction | Cardiovascular diseases (CVD) are the number one cause of death worldwide. Individuals with a high risk of developing CVD are nowadays mainly treated with cholesterol lowering drugs. Although disease risk can be lowered by a maximum of 40% in this way, a significant untreatable residual risk is left. Therefore, there is a big quest to find novel medicines. The enzyme lipoprotein lipase (LPL) is a key player in lipid metabolism and has recently gained a lot of attention as novel druggable target for CVD risk reduction. Although identification of LPL-activating small molecules is eagerly awaited, screening of compound libraries is nowadays impossible, as it is extremely challenging to study LPL activity in an in vitro setting due to its complex mode of action, which requires direct crosstalk between endothelial cells and metabolically active tissue. Therefore, the overall objective is to use organ-on-chip technology to develop a predictable in vitro model for functional LPL activity. Such a model is a giant leap in CVD modelling, and will open the door for discovery and development of new therapeutics for this deadly disease. Study aims | My master thesis project was the start of the overall project, and therefore mainly focused on general characterization of LPL activity in different cell types, with the aim to 1) determine the difference in fatty acid uptake and LPL activity in the medium between cardiomyocyte monocultures, cardiomyocyte endothelial cell co-cultures and endothelial cell monocultures in vitro; 2) define optimal culturing conditions for high LPL activity; and 3) design a heart-on-chip model to co-culture 3D cardiac tissue and endothelial cells such that they are in direct contact with each other. The hypothesis is that co-culturing increases functional LPL activity. Methods | Fatty acid uptake and LPL activity was compared between human pluripotent stem cell (hPSC)-derived cardiomyocyte monocultures, cardiomyocyte-endothelial cell co-cultures and endothelial cell monocultures, using tri[3H]oleate and [14C]cholesteryl oleate double radioactively labeled triglyceride-rich lipoprotein (TRL)-mimicking particles. The heart-on-chip was designed with Solidworks software. Results | LPL activity in the medium, measured as triglyceride-derived fatty acid release, was highest in co-cultures. Fatty acid uptake by the cells also seemed to be higher in co-cultures than cardiomyocyte monocultures, although differences were not statistically significant. Endothelial cells consistently showed lowest fatty acid uptake. Importantly, incubation of TRL-mimicking particles with ApoC2, which is an essential co-factor of LPL enzymatic activity, drastically increased fatty acid uptake by cardiomyocytes and co-cultures. Conclusions | The results strongly indicate that co-culturing cardiomyocytes with endothelial cells, compared to monocultures, increases fatty acid uptake by the cells and triglyceride-derived free fatty acid release in the cell culture medium, both being indicators of increased functional LPL activity. For highest LPL activity, experimental conditions should include incubation of TRL-mimicking particles with at least 0.25 µL/well ApoC2 and a particle incubation time of at least 4 hours. The future goal is to repeat the current experiments in a 3D setting using the designed heart-on-chip model.