Modeling myosin mechanobiology towards understanding the mechanisms of hypertrophic cardiomyopathy

模拟肌球蛋白力学生物学以了解肥厚型心肌病的机制

• 批准号: 10747039
• 负责人: Alison Schroer Vander Roest
• 金额: $ 24.9万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-01 至 2026-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10747039
• 关键词: ATP phosphohydrolase; Adhesions; Advisory Committees; Affect; Alleles; Area; Automobile Driving; Award; Biological Models; Biomechanics; Biomedical Engineering; CRISPR/Cas technology; Cardiac; Cardiac Myocytes; Categories; Cell Adhesion; Cell-Cell Adhesion; Cells; Cellular biology; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Computer Models; Contracts; Cues; Development; Disease; Disease Progression; Elements; Engineering; Environment; Event; Extracellular Matrix; Fibroblasts; Fibrosis; Fluorescence Resonance Energy Transfer; Functional disorder; Gene Expression; Generations; Genes; Genotype; Goals; Health; Heart; Heart Diseases; Heart failure; Heritability; Heterogeneity; Human; Hypertrophic Cardiomyopathy; Hypertrophy; ITGB3 gene; In Vitro; Induced Mutation; Integrins; Intercellular Junctions; Kinetics; Knowledge; Lead; Link; MAP Kinase Gene; Measurement; Measures; Mechanics; Mediator; Mentors; Mentorship; Modeling; Molecular; Molecular Biology; Mutation; Myofibrils; Myofibroblast; Myosin ATPase; Myosin Heavy Chains; Organ; Pathologic; Pathway interactions; Patients; Pattern; Pharmacotherapy; Phase; Phenotype; Point Mutation; Positioning Attribute; Production; Proteins; Regulation; Relaxation; Research; Research Support; Role; Sarcomeres; Severity of illness; Signal Pathway; Signal Transduction; System; Technical Expertise; Techniques; Testing; Therapeutic; Tissues; Traction Force Microscopy; Training; Universities; Validation; Variant; Vinculin; beta-Myosin; career; career development; disease phenotype; disease-causing mutation; experience; extracellular; improved; in silico; in vivo; individualized medicine; induced pluripotent stem cell; induced pluripotent stem cell derived cardiomyocytes; innovation; insight; molecular phenotype; mutant; new therapeutic target; novel; research and development; response; sensor; single molecule; skill acquisition; skills; species difference; tool; transcriptome; transcriptome sequencing; transcriptomics

PROJECT SUMMARY: This proposed project focuses on understanding how mutations in beta cardiac myosin with diverse and often opposing effects on myosin molecular function contribute to similar disease phenotypes of hypertrophic cardiomyopathy (HCM): cardiomyocyte (CM) hypertrophy, hypercontractility, and tissue fibrosis. Understanding disease mechanisms and the heterogeneity of phenotypes across multiple scales (molecular, cellular, and clinical) will enable the development of better and more individualized therapies for patients with HCM. The first aim of this proposal will clarify the mechanisms by which altered intracellular forces affect intracellular signaling and CM hypertrophy. The second aim will use computational modeling to examine how alterations to interrelated parameters of myosin function change force production temporally and spatially. The third aim will define how altered extracellular mechanics affect CM and cardiac fibroblast phenotypes. The proposal uses several innovative molecular, cellular, bioengineering, and computational modeling tools to examine and contextualize the role of altered cellular mechanics in driving changes in cardiac cell phenotypes. CRISPR/Cas9 gene editing in human induced pluripotent stem cells provides a model system to investigate the effects of specific mutations in a dynamic cellular context. Micropatterned engineered platforms will be used to improve myofibril alignment and allow direct measurement of intracellular force production by traction force microscopy. Molecular biology and transcriptomic techniques will be used to measure changes in intracellular signaling and gene expression in response to mechanical perturbation. Another innovation is the novel application of a vinculin tension sensor FRET probe to directly measure intracellular forces at sarcomere Z disks and at cellular adhesions. Together these platforms will allow direct validation of temporal and spatial cellular forces predicted using computational modeling (molecular myosin models and cell-specific finite element models). Finally, investigating the effect of altered extracellular mechanics on CM function and cardiac fibroblast activation in engineered environments will give insights into the effects of fibrotic remodeling. The research and career development training plans will enable the transition of Dr. Vander Roest to an independent career. During the mentored (K99 phase) of this proposal, Dr. Vander Roest will develop technical skills in molecular biomechanics, FRET measurements, and transcriptome analysis, and expand her skills in computational modeling in the context of cardiac disease. This training will take place at Stanford University, under the mentorship of Drs. Bernstein and Spudich, as well as an expert trans-disciplinary advisory committee, including 2 experts in computational modeling (Drs. Regnier and Campbell). The development of these skills will support the research plan for the independent phase of this project, combining new skills and experience gained during this award with Dr. Vander Roest’s past experience in myofibroblast mechanobiology to facilitate a successful transition to independence.

项目摘要：这项拟议的项目侧重于了解β-心肌肌球蛋白的突变 对肌球蛋白分子功能的不同和经常相反的影响导致相似的疾病表型 肥厚型心肌病(HCM)：心肌细胞(CM)肥大、收缩过度和组织纤维化。 了解疾病机制和表型在多尺度上的异质性 (分子、细胞和临床)将使更好和更个性化的治疗方法的发展成为可能 适用于肥厚型心肌炎患者。这项提议的第一个目标将阐明细胞内改变的机制 作用力影响细胞内信号转导和心肌肥大。第二个目标将使用计算建模来 研究肌球蛋白功能相关参数的改变如何改变时间和压力产生 在空间上。第三个目标将定义改变的细胞外机制如何影响心肌细胞和心脏成纤维细胞 表型。该提案使用了几种创新的分子、细胞、生物工程和计算 用于检查和研究改变的细胞力学在驱动心脏改变中的作用的建模工具 细胞表型。人诱导多能干细胞中CRISPR/Cas9基因编辑提供了一个模型系统 研究特定突变在动态细胞环境中的影响。微图案化工程平台 将用于改善肌原纤维的排列，并允许通过以下方式直接测量细胞内力产生 牵引力显微镜。分子生物学和转录技术将被用来测量 细胞内信号和基因表达对机械扰动的响应。另一项创新是 纽蛋白张力传感器FRET探头直接测量肌节细胞内力的新应用 Z盘和细胞粘连。这些平台结合在一起将允许直接验证时间和空间 使用计算模型(分子肌球蛋白模型和特定于细胞的有限元)预测细胞力 模特)。最后，研究改变的细胞外力对心肌功能和心肌成纤维细胞的影响。 工程环境中的激活将使人们对纤维化重塑的影响有更深入的了解。该研究和 职业发展培训计划将使范德·罗斯特博士能够过渡到独立的 职业生涯。在此建议书的指导阶段(K99阶段)，Vander Roest博士将在 分子生物力学、FRET测量和转录组分析，并扩展她在 心脏疾病背景下的计算建模。这项培训将在斯坦福大学进行， 在伯恩斯坦和斯普迪奇博士的指导下，以及一个跨学科的专家咨询委员会， 包括2名计算建模专家(雷尼耶博士和坎贝尔博士)。这些技能的发展将 支持本项目独立阶段的研究计划，结合所获得的新技能和经验 在这个奖项中，Vander Roest博士过去在肌成纤维细胞机械生物学方面的经验促进了 成功地过渡到独立。