Establishing of a cardiomyocyte-like cell culture model from equine preadipocytes in order to investigate atrial and ventricular ion channels in horses

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Atrial fibrillation is a common heart disease in both humans and horses and is often associated with limited performance, especially in race horses. This disease is treated with antiarrhythmic drugs that can modify the function of cardiac ion channels, which are responsible for spreading electrical activity and triggering cardiac contraction. Studies conducted with dogs and horses clearly indicate a species-specific expression of ion channels. The development of species-specific-models is necessary to transfer new therapeutic approaches from human to veterinary medicine. Moreover it is important to investigate whether horses also have a similar atrial-specific K+ channel, as is the case in humans, in order to understand, whether this channel could be an option for the treatment of atrial fibrillation in horses. The project aims to develop an equine-specific in vitro cardiomyocyte-like cell culture model. Our preliminary findings support the hypothesis that, as in other species (mouse, human, bovine), it is also possible to isolate multipotent stem cells from adipose tissue in the horse. The second hypothesis states that these preadipocytes can be differentiated into cardiomyocyte-like cells using appropriate transformation media. To investigate these hypotheses the preadipocytes obtained will be examined morphologically, molecular-biologically, and immunophenotypically by flow cytometry to determine their characteristics as mesenchymal stem cells. The cardiomyocyte-like cells obtained after differentiation with two different protocols (5-azacytidine vs. Activin A/ BMP-4/ DKK-1) will also be proven morphologically and the expression of cardiomyocyte-specific markers will be shown by SYBR Green RT-qPCR established in the present project. Following the differentiation of the cells, the influence of laminin-coated culture flasks on the differentiation potential of equine preadipocytes will be examined, both morphologically and on gene expression level (SYBR Green RT-qPCR). Upon successful differentiation, the electrophysiological properties of induced cardiomyocyte-like cells will be compared to those of native equine cardiomyocytes using patch-clamp technique. The resulting in vitro model will allow for the examination of the function of individual ion channels in the action potential that can be specifically tested using electrophysiological techniques. The clinical application of the model lies in the evaluation of the antiarrhythmic drug effect in atrial fibrillation of horses. By demonstrating the desired effects in action potential in vitro, drugs can be preselected before they are in clinical trials with horses. This reduces the number of experiments using live animals perspectively (3R principle).