TRPC6 inhibition therapy to rescue cardiac muscle dysfunction in muscular dystrophy

项目摘要 这项建议测试了一种新近开发的、生物可用的、选择性的小剂量... 瞬时受体电位经典6(TRPC6)分子抑制剂改善心脏和骨骼 Duchenne肌营养不良症(DMD)的肌病。这项研究实现了候选人的长期目标 营养不良性心肌病新疗法的发展和机械敏感信号分析的应用 小鼠和工程心脏组织(EHT)来研究疾病。DMD是由于Dstrophin的缺失而引起的 严重的进行性肌肉无力、脊柱畸形、纤维化、心力衰竭和早期死亡。TRPC6是 一种在肌肉细胞中表达的机械敏感的非电压门控阳离子通道，在DMD中过度激活， 介导过度机械应激诱导的力/钙反应、心律失常、心功能不全和 肌肉纤维化。在博士后培训期间，他领导了评估封锁影响的项目 TRPC6基因和药理学在心肌纤维化模型中的作用。DMD的初步数据显示基因 或药物TRPC6抑制延长严重DMD模型的寿命2-3倍，改善纤维化 和相关的病理，以及改善心脏和骨骼肌功能。这位候选人首先报道了 2019年推出的TRPC6药物抑制剂(BI 749327)及其临床衍生物目前正在进行肺和肾的人体试验 疾病。在这份提案中，候选人解决了关键问题，这些问题的答案将对未来产生重要影响 DMD的翻译努力，并被组织为三个目标。目的1检测慢性抑制TRPC6的疗效 由BI 749327预防和逆转DMD骨骼肌和心肌功能障碍、组织病理学和TRPC6- NFAT、促肝纤维化和炎症信号。通过单细胞RNAseq分析细胞类型的表达 如何影响表达TRPC6(成纤维细胞、血管和肌细胞)的心肌细胞亚群 治疗。目的2检测慢性抑制TRPC6恢复机械激活的能力 DMD小鼠心肌细胞内力和钙离子的缺陷及膜的作用 密封剂和其他机械敏感激活途径。我将进一步测试TRPC6病理生物学在 使用与小鼠相同的机械刺激建立新的人DMD EHT机械敏感激活模型 心肌细胞。目的3检测微营养不良蛋白(μDys)基因治疗TRPC6病理生物学的疗效 在最近开发的D2.mdx DMD小鼠模型或μDys与BI 749327的组合中提供了 附加福利。这项提议独一无二地将候选人之前接受的培训与他导师的专业知识结合在一起。 候选人拥有进行这些研究所需的独特技能，结合了生物医学工程、肌肉 生理学和分子信号传递经验。他将扩展到单细胞转录学，干细胞来源 EHT及其力学分析，DMD基因治疗。约翰·霍普金斯大学的环境与专家 指导团队为候选人和项目提供了一切成功的机会。

Project Summary This proposal tests the efficacy and mechanisms by which a recently developed, bioavailable, selective small- molecule inhibitor of Transient Receptor Potential Canonical 6 (TRPC6) ameliorates cardiac and skeletal myopathy in Duchenne muscular dystrophy (DMD). This research fulfills the candidate’s long-term goals of advancing novel therapies for dystrophic cardiomyopathy and applying mechanosensitive signaling assays in mice and engineered heart tissues (EHT) to study disease. DMD results from a loss of dystrophin, inducing profound progressive muscle weakness, spinal deformities, fibrosis, heart failure, and early mortality. TRPC6 is a mechanosensitive, non-voltage gated cation channel expressed in muscle cells that is hyper-activated in DMD, mediating excessive mechanical stress-induced force/Ca2+ responses, arrhythmias, cardiac dysfunction, and muscle fibrosis. During the candidate’s postdoctoral training, he led projects assessing the impact of blocking TRPC6 genetically and pharmacologically in models of cardiac fibrosis. Preliminary data in DMD show genetic or pharmacological TRPC6 inhibition prolongs lifespan in severe DMD models by 2-3 fold, ameliorating fibrosis and associated pathology, and improving heart and skeletal muscle function. The candidate first reported on the TRPC6 drug inhibitor (BI 749327) in 2019, and its clinical derivatives are now in human trials for lung and renal disease. In this proposal, the candidate addresses key questions whose answers will importantly inform future DMD translational efforts, and is organized into three aims. Aim 1 tests the efficacy of chronic TRPC6 inhibition by BI 749327 to prevent and reverse DMD skeletal and cardiac muscle dysfunction, histopathology, and TRPC6- NFAT, pro-fibrotic, and inflammatory signaling. Cell-type expression is analyzed by single-cell RNAseq to identify how subpopulations of cardiac cells that express TRPC6 (fibroblasts, vascular, and myocytes) are impacted by the treatment. Aim 2 tests the capacity of chronic TRPC6 suppression to restore mechanical activation-induced defects in force and calcium in isolated DMD mouse cardiomyocytes, and to obviate effects of membrane sealants and other mechanosensitive-activated pathways. I will further test the role of TRPC6 pathobiology in a novel human DMD EHT model of mechanosensitive activation using the same mechanical stimuli as in mouse cardiomyocytes. Aim 3 tests the efficacy of micro-dystrophin (μDys) gene therapy to treat TRPC6 pathobiology in the recently-developed D2.mdx DMD mouse model or the combination of μDys with BI 749327 provides additive benefits. The proposal uniquely combines the candidate’s prior training with expertise from his mentors. The candidate has the unique skills needed to conduct these studies, combining biomedical engineering, muscle physiology, and molecular signaling experience. He will expand into single-cell transcriptomics, stem cell derived EHT and their mechanical analysis, and DMD gene therapy. The environment at Johns Hopkins and expert mentoring team provides every opportunity for success for the candidate and project.