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

了解控制杜氏肌营养不良症临床表现的转录网络和生理适应

• 批准号: 10223916
• 负责人: Bayardo Isidore Garay
• 金额: $ 4.98万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-27 至 2024-07-26
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10223916
• 关键词: Action Potentials; Acute; Address; Affect; Age; Animal Model; Biological Assay; Biopsy; Calcium; Cardiac Myocytes; Cell Differentiation process; Cell Line; Cell Membrane Permeability; Cell membrane; Cell physiology; Cells; Cellular biology; Chemicals; Chronic; Clinical; Data; Development; Discipline; Disease; Disease Progression; Doctor of Philosophy; Dose; Duchenne muscular dystrophy; Dystrophin; Electrophysiology (science); Evolution; Functional disorder; Gene Expression Profile; Genes; Genetic Transcription; Goals; Heart failure; Heterogeneity; Human; Impairment; In Vitro; Ion Channel; Knowledge; Laboratories; Lead; Learning; Light; Live Birth; Maintenance; Measurement; Mechanics; Microelectrodes; Modeling; Muscle; Muscle Cells; Muscle function; Muscular Dystrophies; Mutation; Myocardium; Onset of illness; Outcome; Pathologic; Patients; Pharmacology; Physicians; Physiological; Physiological Adaptation; Plasma; Proteins; Research; Research Training; Respiratory Failure; Reverse Transcription; Scientist; Severity of illness; Signal Pathway; Skeletal Muscle; Stress; Striated Muscles; Study models; System; Techniques; Teenagers; Testing; Therapeutic; Tissues; base; biological adaptation to stress; career development; clinical phenotype; disease heterogeneity; genome-wide; human disease; improved; induced pluripotent stem cell; insight; male; muscle degeneration; muscle stress; new therapeutic target; novel; paracrine; patch clamp; programs; recruit; response; skeletal; skeletal muscle differentiation; skeletal stem cell; stem; transcriptome sequencing

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.

项目概要 杜氏肌营养不良症（DMD）是一种普遍致命的疾病。 DMD 患者不表达肌营养不良蛋白 蛋白质并在 3-5 岁时出现骨骼肌 (SkM) 退化，随后出现心肌退化 (CM) 到十几岁。这些患者最终在 25-30 岁时死于呼吸或心力衰竭。这 调节 DMD 进展的潜在机制尚不清楚。使用源自患者的诱导 多能干细胞（iPSC）具有一系列突变和疾病严重程度，我们可以研究其机制 控制 SkM 和 CM 中 DMD 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 的动作电位较弱，场电位持续时间较长。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丧失会导致动态基因 网络变化会导致对因发育不当和发育不当而产生的压力反应受损 维持横纹肌的生理功能。我将在两个具体目标上检验这个中心假设。在 目标 1，我将确定转录谱以及下游电生理和机械适应 一组 DMD 患者来源的 iPSC 系中横纹肌对应激的反应。我的工作假设是 对细胞收缩的需求不断增加导致患者来源的 iPSC 中出现类似的补偿机制 SkM 和 -CM，但 CM 的反应更具保护性，因为与 不受影响的控制。在这里，我将采用电学和药理学方法来诱导收缩和 通过 RNA 测序（批量和单细胞）、电生理测量（微电极）分析效果 阵列和全细胞膜片钳）以及膜通透性测定。我们的初步研究表明，在 与对照线相比，基线、DMD iPSC-SkM 和 -CM 显示出更渗漏的质膜。瞄准 2，我将描述肌营养不良蛋白对调节发育和维持的基因网络的剂量效应 肌肉的生理功能。我的工作假设是，人类分化过程中抗肌萎缩蛋白的消耗 iPSC-SkM 和 -CM 导致可逆的转录和生理变化。使用诱导型和 未受影响的人类 iPSC 中的可逆降解系统，我们可以化学调节肌营养不良蛋白水平 在肌肉分化过程中，识别转录谱和细胞适应 不同水平的肌营养不良蛋白。总的来说，这些研究意义重大，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网络变化，这是不同临床表型和发病的基础。 进一步了解 DMD 病理生理学及其进展可能为 DMD 提供新的治疗靶点 肌营养不良症以及增进我们对正常肌肉细胞生物学和功能的理解。这 拟议的研究和培训计划为我成功完成医学博士学位提供了严格的计划 学位并将进一步促进我作为一名学术医师科学家的发展。