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

房颤是人类和马的一种常见心脏病，经常与表现有限有关，特别是在赛马中。这种疾病是用可以改变心脏离子通道功能的抗心律失常药物治疗的，离子通道负责传播电活动并触发心脏收缩。对狗和马进行的研究清楚地表明了离子通道的物种特异性表达。为了将新的治疗方法从人类医学转移到兽医医学，特定物种模型的开发是必要的。此外，重要的是要研究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的心房颤动的一种选择。该项目旨在开发一种马特有的体外心肌细胞样细胞培养模型。我们的初步发现支持这样的假设，就像在其他物种(老鼠、人、牛)中一样，也有可能从马的脂肪组织中分离出多能干细胞。第二种假设认为，使用适当的转化介质可以将这些前脂肪细胞分化为心肌样细胞。为了验证这些假说，将通过流式细胞术对获得的前脂肪细胞进行形态、分子生物学和免疫表型的检测，以确定其作为间充质干细胞的特征。用两种不同的方法(5-氮杂胞苷和激活素A/BMP-4/DKK-1)分化后获得的心肌细胞样细胞也将得到形态学证明，并将通过本项目建立的SYBR Green RT-qPCR显示心肌细胞特异性标志的表达。在细胞分化后，将从形态和基因表达水平上检测层粘连蛋白涂层培养瓶对马前脂肪细胞分化潜能的影响(SYBR Green RT-qPCR)。在成功分化后，将利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的电生理特性进行比较。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，该动作电位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心房颤动的疗效。通过在体外展示预期的动作电位效应，药物可以在马的临床试验之前预先选择。这就减少了使用活体动物进行实验的次数(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).