TRPC6 inhibition therapy to rescue cardiac muscle dysfunction in muscular dystrophy

项目摘要 该提案测试了最近开发的，生物可利用的，选择性小- 瞬时受体电位规范6（TRPC 6）的分子抑制剂改善心脏和骨骼 Duchenne型肌营养不良症（DMD）中的肌病。这项研究实现了候选人的长期目标， 推进营养不良性心肌病的新疗法， 小鼠和工程心脏组织（EHT）研究疾病。DMD是由肌营养不良蛋白的缺失引起的， 严重进行性肌无力、脊柱畸形、纤维化、心力衰竭和早期死亡。TRPC 6是 一种在肌细胞中表达的机械敏感性非电压门控阳离子通道，在DMD中被过度激活， 介导过度机械应力诱导的力/Ca 2+反应、心律失常、心功能障碍，以及 肌肉纤维化在候选人的博士后培训期间，他领导了评估封锁影响的项目 TRPC 6基因和心脏纤维化模型中的表达。DMD的初步数据显示， 或药理学TRPC 6抑制使严重DMD模型的寿命延长2-3倍，改善纤维化 和相关病理学，以及改善心脏和骨骼肌功能。候选人首先报告了 TRPC 6药物抑制剂（BI 749327）于2019年上市，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，候选人提出了关键问题，这些问题的答案将对未来的发展产生重要影响。 DMD的翻译工作，并组织成三个目标。目的1测试慢性TRPC 6抑制的功效 通过BI 749327预防和逆转DMD骨骼和心肌功能障碍、组织病理学和TRPC 6- NFAT、促纤维化和炎症信号传导。通过单细胞RNAseq分析细胞类型表达以鉴定 表达TRPC 6的心肌细胞亚群（成纤维细胞、血管细胞和肌细胞）如何受到 用药目的2测试慢性TRPC 6抑制恢复机械激活诱导的细胞凋亡的能力。 在分离的DMD小鼠心肌细胞中的力和钙的缺陷，以及膜的抑制作用， 密封剂和其他机械敏感激活的途径。我将进一步测试TRPC 6病理生物学在一个 使用与小鼠相同的机械刺激的机械敏感性激活的新型人DMD EHT模型 心肌细胞目的3检测微小肌营养不良蛋白（μDys）基因治疗TRPC 6病理学的有效性 在最近开发的D2.mdx DMD小鼠模型中或μDys与BI 749327联合给药提供了 附加效益。该提案独特地将候选人先前的培训与其导师的专业知识相结合。 候选人具有进行这些研究所需的独特技能，结合生物医学工程，肌肉 生理学和分子信号经验。他将扩展到单细胞转录组学，干细胞衍生 EHT及其力学分析和DMD基因治疗。约翰霍普金斯的环境和专家 指导团队为候选人和项目提供成功的每一个机会。

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.