Using Mechanical Loading to Develop Human iPSC-Derived Models of Cardiomyopathy

 描述（由申请人提供）：致心律失常性右心室心肌病（ARVC）是人类心脏病中最易致心律失常的形式，也是年轻人心脏性猝死的主要原因之一。许多研究已经建立了耐力运动和桥粒突变携带者的疾病发病率之间的联系，但运动改变疾病发病机制和疾病风险的确切机制尚不清楚。越来越多的证据表明，机械负荷可以在心脏组织重塑中发挥决定性作用，包括调节关键的细胞间蛋白，如斑珠蛋白和连接蛋白-43。对于影响锚定细胞-细胞连接的心肌病，如ARVC，这导致了机械刺激可能通过增加电生理或生物力学缺陷在疾病发病机制中发挥作用的假设。到目前为止，表征机械负荷对ARVC发病机制影响的大部分工作尚未在人类中得到验证-这一缺陷限制了机械见解，治疗选择和患者咨询。在这项研究中，我打算调查的影响，机械刺激对细胞和组织 在ARVC的3D组织模型中的疾病方面（细胞凋亡、脂肪沉积、间隙连接重构、心律失常）。特别是，我们将应用应变工程心脏切片接种诱导多能干细胞从临床证实的ARVC患者（一个杂交的无细胞组织 和器官型心脏切片技术，其重现用于细胞生长的3D微环境并实现机械相互作用）。机械刺激后，电生理测量将用于测量心脏动作电位的变化，并确定机械刺激是否会导致iPSC衍生的ARVC模型中的异位节律。接下来，我们检验了机械负荷可以概括ARVC的显性阶段表型的假设。具体而言，我们确定慢性机械负荷是否可以增加脂肪沉积，并改变关键蛋白质（即连接蛋白-43、斑珠蛋白、肌钙蛋白-T α-肌节肌动蛋白等）的表达或定位。最后，我们利用我们的平台进行药理学询问，通过确定用抗组胺剂或SB 216763（一种在ARVC的斑马鱼模型中显示具有恢复作用的化合物）治疗是否可用于预防用异丙肾上腺素刺激β-肾上腺素能的促组胺作用，以及明显疾病表型的发展。总的来说，这些研究将提供对拟生理机械刺激在ARVC进展中的作用的深入了解。

 DESCRIPTION (provided by applicant): Arrhythmogenic right ventricular cardiomyopathy (ARVC) is the most arrhythmogenic form of human heart disease, and one of the leading causes of sudden cardiac death in the young. Numerous studies have established the link between endurance exercise and disease penetrance in desmosomal mutation carriers, but the exact mechanism by which exercise alters disease pathogenesis and arrhythmic risk is unclear. There is increasing evidence that mechanical loading can play a defining role in cardiac tissue remodeling, including the regulation of key intercellular proteins such as plakoglobin and connexin-43. For cardiomyopathies affecting anchoring cell-cell junctions, such as ARVC, this lends to the hypothesis that mechanical stimulation may play a role in disease pathogenesis by augmenting electrophysiological or biomechanical defects. Thus far, much of the work characterizing the effects of mechanical loading on ARVC pathogenesis has not been verified in humans - a deficit that limits mechanistic insights, treatment options and patient counseling. In this study, I propose to investigate the influence of mechanical stimulation on cellular and tissue aspects of the disease (apoptosis, fatty deposits, gap junction remodeling, arrhythmia) in 3D tissue models of ARVC. In particular, we will apply strain to engineered heart slices seeded with induced pluripotent stem cells from clinically verified ARVC patients (a hybrid of acellular tissue and organotypic heart slice technology that recapitulates a 3D microenvironment for cell growth and enables mechanical interactions). Following mechanical stimulation, electrophysiological measurements will be used to measure changes in the cardiac action potential, and to determine if mechanical stimulation can cause ectopic rhythms in iPSC-derived model of ARVC. Next, we test the hypothesis that mechanical loading can recapitulate the overt stage phenotype of ARVC. Specifically, we determine if chronic mechanical loading can increase fat deposition, and alter the expression or localization of key proteins (i.e. connexin-43, plakoglobin, troponin-T a-sarcomeric actin, etc.). Finally, we utilize our platform for pharmacological interrogation by determining if treatment with anti-arrhythmic agents or SB216763, a compound shown to have restorative effects in a zebrafish model of ARVC, can be used to prevent the pro-arrhythmic effects of ß-adrenergic stimulation with isoproterenol, and the development of an overt-stage disease phenotype. Collectively, these studies will provide insight into the role of physio- mimetic mechanical stimulation in the progression of ARVC.