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: 10672427
• 负责人: Bayardo Isidore Garay
• 金额: $ 5.27万
• 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/10672427
• 关键词: Action Potentials; Acute; Address; Affect; Age; Animal Model; Biological Assay; Biopsy; Calcium Signaling; Cardiac; 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; Learning; Live Birth; Maintenance; Measurement; Mechanics; Microelectrodes; Modeling; Muscle; Muscle Cells; Muscle Fibers; Muscle function; Muscular Dystrophies; Mutation; Myocardium; Onset of illness; Outcome; Pathologic; Patients; Physicians; Physiological; Physiological Adaptation; Proteins; Research; Respiratory Failure; Scientist; Severity of illness; Signal Pathway; Skeletal Muscle; Stress; Striated Muscles; Study models; System; Techniques; Teenagers; Testing; Therapeutic; Tissues; Titrations; Training; biological adaptation to stress; career development; clinical phenotype; comparison control; disease heterogeneity; gene network; genome-wide; human disease; improved; induced pluripotent stem cell; insight; male; muscle degeneration; muscle stress; new therapeutic target; novel; paracrine; patch clamp; pharmacologic; programs; recruit; response; skeletal; skeletal muscle differentiation; 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.

项目摘要 Duchenne肌肉营养不良（DMD）是一种普遍致命的疾病。 DMD患者不表达肌营养不良蛋白 蛋白质并发展为3-5岁的骨骼肌肉（SKM）变性，而心脏肌肉后来变性 （厘米）到十几岁。这些患者最终在25-30岁时屈服于呼吸道或心脏衰竭。这 调节DMD进展的基本机制尚不清楚。使用患者来源的诱导 具有多种突变和疾病严重程度的多能干细胞（IPSC），我们可以研究机制 管理SKM和CM中DMD的临床表现。我们的初步数据表明DMD患者IPSC- 与对照线相比，CMS具有较弱的动作电位和较长的场电位持续时间。基于 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导致动态基因 网络变化导致对压力的反应受损，而不当发展和 维持肌肉肌肉的生理功能。我将以两个具体的目的来检验这一中心假设。在 AIM 1，我将确定转录轮廓以及下游电生理和机械适应 DMD患者衍生的IPSC系列中的压力响应压力的肌肉。我的工作假设是 对细胞收缩的需求增加导致患者衍生的IPSC-的相似补偿机制 SKM和-CMS，但由于CMS的持续募集而在CMS中受到更大的保护 未受影响的控件。在这里，我将采用电气和药物的方法来诱导收缩和 通过RNA测序（大量和单细胞），电生理测量（微电极）分析效果 阵列和全细胞斑块夹）和膜通透性评估。我们的初步研究表明，在 与对照线相比，基线，DMD IPSC -SKM和-CMS显示出叶膜膜。目标 2，我将表征肌营养不良蛋白对调节发展和维持的基因网络的剂量影响 物理肌肉功能。我的工作假设是，人类分化过程中的肌营养不良蛋白耗竭 IPSC -SKM和-CMS会导致可逆的转录和生理变化。使用诱导和 在未受影响的人IPSC中可逆降解系统，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，并确定转录曲线和细胞适应 肌营养不良蛋白的不同水平。总的来说，这些研究很重要，因为它们将阐明转录 网络因素肌肉中的肌营养不良蛋白损失而变化，这是临床表型和发作不同的基础的肌肉。 对DMD病理生理及其进展的进一步了解可能为新的治疗目标提供 肌肉营养不良，并提高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成功完成我的MD-PHD提供了严格的计划 学位，将进一步发展作为学术科学家的发展。

项目成果

Dual inhibition of MAPK and PI3K/AKT pathways enhances maturation of human iPSC-derived cardiomyocytes.

• DOI: 10.1016/j.stemcr.2022.07.003
• 发表时间: 2022-09-13
• 期刊: STEM CELL REPORTS
• 影响因子: 5.9
• 作者: Garay, Bayardo I.;Givens, Sophie;Abreu, Phablo;Liu, Man;Yucel, Dogacan;Baik, June;Stanis, Noah;Rothermel, Taylor M.;Magli, Alessandro;Abrahante, Juan E.;Goloviznina, Natalya A.;Soliman, Hossam A. N.;Dhoke, Neha R.;Kyba, Michael;Alford, Patrick W.;Dudley Jr, Samuel C.;van Berlo, Jop H.;Ogle, Brenda;Perlingeiro, Rita R. C.
• 通讯作者: Perlingeiro, Rita R. C.