Understanding the Transcriptional Networks and Physiologic Adaptations Governing the Clinical Manifestations of Duchenne Muscular Dystrophy

项目总结 Duchenne肌营养不良症(DMD)是一种常见的致命性疾病。DMD患者不表达抗肌营养不良蛋白 蛋白质和骨骼肌(SKM)在3-5岁之前变性，随后在心肌中变性 (厘米)十几岁左右。这些患者最终在25-30岁时死于呼吸或心力衰竭。这个 调节DMD进展的潜在机制尚不清楚。使用患者派生的诱导 多能干细胞(IPSCs)具有一系列突变和疾病严重性，我们可以研究其机制 总结DMD在SKM和CM中的临床表现。我们的初步数据显示，DMD患者IPSC- 与对照相比，细胞质雄性不育系具有较弱的动作电位和较长的场电位持续期。基于 这些初步结果和动物模型研究，我假设Dystrophin的丢失导致了动态基因 网络变化，导致对来自不适当发展和压力的反应受损 维持横纹肌的生理功能。我将在两个具体目标上检验这一中心假设。在……里面 目标1，我将确定转录谱和下游的电生理和机械适应 在一组DMD患者衍生的IPSC系中，横纹肌对压力的反应。我的工作假设是 不断增加的细胞收缩需求导致患者来源的IPSC产生类似的代偿机制- SKM和-CMS，但与CMS相比，CMS的反应更具保护性，因为它们不断招募 未受影响的对照。在这里，我将使用电学和药理学的方法来诱导宫缩和 通过RNA测序(散装和单细胞)、电生理测量(微电极)分析影响 阵列和全细胞膜片钳)和膜通透性分析。我们的初步研究表明，在 与对照品系相比，基线、DMD、IPSC-SKM和-CMS表现出更多的质膜渗漏。在AIM 2，我将描述dystrophin对调节发育和维持的基因网络的剂量效应 生理肌肉功能。我的工作假设是在人类分化过程中肌营养不良蛋白的耗竭 IPSC-SKM和-CMS导致可逆的转录和生理变化。使用诱导式和 在未受影响的人ipscs中的可逆降解系统中，我们可以化学调节dystrophin蛋白的水平 在肌肉分化和，确定转录图谱和细胞适应反应 不同程度的肌营养不良蛋白。总的来说，这些研究具有重要意义，因为它们将揭示转录 由于横纹肌中肌营养不良蛋白的丢失而导致的网络改变，这是不同的临床表型和发病的基础。 进一步了解DMD的病理生理机制及其进展可能为DMD提供新的治疗靶点 肌肉营养不良以及促进我们对正常肌肉细胞生物学和功能的理解。这个 建议的研究和培训计划为我顺利完成医学博士学位提供了一个严格的计划 学位，并将推动我作为一名学术内科科学家的发展。

PROJECT SUMMARY Duchenne muscular dystrophy (DMD) is a universally fatal disease. DMD patients do not express dystrophin protein and develop skeletal muscle (SkM) degeneration by age 3-5 with later degeneration in cardiac muscle (CM) by mid-teens. These patients ultimately succumb to respiratory or cardiac failure by age 25-30. The underlying mechanisms that regulate DMD progression are not well understood. Using patient-derived induced pluripotent stem cells (iPSCs) with a spectrum of mutations and disease severity, we can study the mechanisms governing the clinical manifestations of DMD in SkM and CM. Our preliminary data show that DMD patient iPSC- CMs have weaker action potentials and longer field potential duration when compared to control lines. Based on these preliminary results and animal model studies, I hypothesize that loss of dystrophin results in dynamic gene network changes that cause impaired responses to stress stemming from improper development and maintenance of striated muscle’s physiologic functions. I will test this central hypothesis in two specific aims. In Aim 1, I will identify the transcriptional profile and downstream electrophysiological and mechanical adaptations of striated muscle in response to stress in a panel of DMD patient-derived iPSC lines. My working hypothesis is that increasing demand for cell contraction leads to similar compensatory mechanisms in patient-derived iPSC- SkM and -CMs, but the response is more protective in CMs due to their constant recruitment when compared to unaffected controls. Here, I will employ electrical- and pharmacological approaches to induce contractions and analyze the effects via RNA sequencing (bulk and single-cell), electrophysiologic measurements (microelectrode array and whole-cell patch clamp), and membrane permeability assays. Our preliminary studies reveal that, at baseline, DMD iPSC-SkM and -CMs show a leakier plasma membrane when compared to control lines. In Aim 2, I will characterize dose effects of dystrophin on gene networks that regulate the development and maintenance of physiologic muscle function. My working hypothesis is that dystrophin depletion during differentiation of human iPSC-SkM and -CMs results in reversible transcriptional and physiologic changes. Using an inducible and reversible degradation system in unaffected human iPSCs, we can chemically modulate dystrophin protein levels during muscle differentiation and, identify the transcriptional profiles and cellular adaptations in response to varying levels of dystrophin. Collectively, these studies are significant in that they will shed light on transcriptional network changes due to loss of dystrophin in striated muscle that underlie varying clinical phenotype and onset. Further understanding of DMD pathophysiology and its progression may offer new therapeutic targets for muscular dystrophies as well as advance our understanding of normal muscle cell biology and function. The proposed research and training plans provide a rigorous program for successful completion of my MD-PhD degrees and will further my development as an academic physician-scientist.