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

心房颤动是人类和马的一种常见心脏病，通常与表现受限有关，特别是在赛马中。这种疾病可以用抗心律失常药物治疗，这种药物可以改变心脏离子通道的功能，心脏离子通道负责传播电活动和触发心脏收缩。对狗和马进行的研究清楚地表明，离子通道具有物种特异性表达。物种特异性模型的发展对于将新的治疗方法从人类医学转移到兽医学是必要的。此外，重要的是调查马是否也有类似的心房特异性K+通道，就像人类的情况一样，以便了解该通道是否可以作为治疗马房颤的一种选择。该项目旨在开发一种马特异性体外心肌细胞样细胞培养模型。我们的初步研究结果支持这样的假设，即与其他物种（小鼠、人类、牛）一样，从马的脂肪组织中分离出多能干细胞也是可能的。第二种假设认为，这些前脂肪细胞可以通过适当的转化培养基分化为心肌细胞样细胞。为了研究这些假设，将通过流式细胞术对获得的前脂肪细胞进行形态学、分子生物学和免疫表型检查，以确定它们作为间充质干细胞的特征。两种不同方案（5-氮杂胞苷vs. Activin A/ BMP-4/ DKK-1）分化后获得的心肌细胞样细胞也将在形态学上得到证实，并通过本项目建立的SYBR Green RT-qPCR显示心肌细胞特异性标志物的表达。在细胞分化后，我们将检测层粘胶蛋白包被培养瓶对马前脂肪细胞分化潜能的影响，包括形态学和基因表达水平（SYBR Green RT-qPCR）。在成功分化后，将使用膜片钳技术将诱导的心肌细胞样细胞的电生理特性与天然马心肌细胞进行比较。由此产生的体外模型将允许检查单个离子通道在动作电位中的功能，可以使用电生理技术进行专门测试。该模型的临床应用在于评价抗心律失常药物对马房颤的疗效。通过在体外证明动作电位的预期效果，可以在马匹临床试验之前预先选择药物。这减少了活体动物实验的数量（3R原则）。

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).