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

 描述(申请人提供)：致心律失常性右室心肌病(ARVC)是人类心脏病中最易致心律失常的形式，也是导致年轻人心脏性猝死的主要原因之一。许多研究已经确定了耐力运动与桥粒突变携带者的疾病外显性之间的联系，但运动改变疾病发病机制和心律失常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，如蛋白和连接蛋白-43。对于影响锚定细胞-细胞连接的心肌疾病，如ARVC，这有助于假设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。到目前为止，许多表征机械负荷对ARVC发病机制影响的工作尚未在人类身上得到验证--这一缺陷限制了机械洞察力、治疗选择和患者咨询。在这项研究中，我建议研究机械刺激对细胞和组织的影响 ARVC 3D组织模型中疾病的各个方面(细胞凋亡、脂肪沉积、缝隙连接重塑、心律失常)。特别是，我们将对接种了来自临床验证的ARVC患者(无细胞组织的杂交体)的诱导多能干细胞的工程化心脏切片应用应变。 以及器官型心脏切片技术，它概括了细胞生长的3D微环境并实现了机械相互作用)。在机械刺激后，电生理测量将被用来测量心脏动作电位的变化，并确定机械刺激是否能在IPSC衍生的ARVC模型中引起异位节律。接下来，我们验证了机械负荷可以概括ARVC显性阶段表型的假设。具体地说，我们确定慢性机械负荷是否会增加脂肪沉积，并改变关键蛋白(即连接蛋白-43、蛋白球蛋白、肌钙蛋白-Tα-肌节肌动蛋白等)的表达或定位。最后，我们利用我们的药理学研究平台，通过确定抗心律失常药物或SB216763(一种在ARVC斑马鱼模型中被证明具有恢复作用的化合物)的治疗是否可以用于预防异丙肾上腺素能刺激的促心律失常效应，以及是否可以防止明显的疾病表型的发展。总而言之，这些研究将深入了解模拟生理的机械刺激在ARVC进展中的作用。

 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.