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

项目摘要 杜氏肌营养不良症（DMD）是一种常见的致死性疾病。DMD患者不表达肌营养不良蛋白 蛋白质和骨骼肌（SkM）在3-5岁时退化，随后在心肌中退化 (CM)十几岁的时候这些患者最终在25-30岁时死于呼吸衰竭或心力衰竭。的 调节DMD进展的潜在机制还不清楚。使用患者源性诱导 多能干细胞（iPSC）与一系列的突变和疾病的严重程度，我们可以研究的机制 控制DMD在SkM和CM中的临床表现。我们的初步数据显示DMD患者iPSC- 与对照线相比，CM具有较弱的动作电位和较长的场电位持续时间。基于 根据这些初步结果和动物模型研究，我假设肌营养不良蛋白的缺失导致动态基因表达， 网络变化导致对源于不适当发展的压力的反应受损， 维持横纹肌的生理功能。我将在两个具体目标中检验这一核心假设。在 目的1，我将确定转录谱和下游的电生理和机械适应 在一组DMD患者来源的iPSC系中，横纹肌对应激的反应。我的假设是 细胞收缩需求的增加导致患者源性iPSC中出现类似的补偿机制- SkM和-CM，但由于它们的不断招募，与 未受影响的对照组。在这里，我将采用电和药理学方法来诱导收缩， 通过RNA测序（整体和单细胞）、电生理测量（微电极） 阵列和全细胞膜片钳）和膜渗透性测定。我们的初步研究表明，在 基线时，DMD iPSC-SkM和-CM显示出与对照系相比更渗漏的质膜。在Aim中 2，我将描述dystrophin对调控发育和维持的基因网络的剂量效应， 生理性肌肉功能。我的工作假设是，在人类分化过程中， iPSC-SkM和-CM导致可逆的转录和生理变化。使用诱导型和 在未受影响的人iPSC中，我们可以通过化学方法调节抗肌萎缩蛋白的蛋白水平， 在肌肉分化过程中，确定转录谱和细胞适应， 不同程度的抗肌萎缩蛋白总的来说，这些研究是重要的，因为它们将阐明转录 由于横纹肌中肌营养不良蛋白的丢失而导致的网络变化，其是不同临床表型和发病的基础。 对DMD病理生理学及其进展的进一步了解可能为DMD的治疗提供新的靶点。 肌营养不良症以及推进我们对正常肌肉细胞生物学和功能的理解。的 拟议的研究和培训计划提供了一个严格的计划，成功完成我的MD-PhD 学位，并将进一步发展我作为一个学术物理学家，科学家。

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.