Development of a high throughput microtissue model for integrative analysis of contractile function and biomechanical stress in iPSC-derived cardiomyocytes

开发高通量微组织模型，用于综合分析 iPSC 衍生心肌细胞的收缩功能和生物力学应激

• 批准号: 10312792
• 负责人: ADAM S HELMS
• 金额: $ 7.8万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-12-15 至 2022-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10312792
• 关键词: 3-Dimensional; Actins; Admixture; Affect; Agonist; Arrhythmia; Biological; Biological Models; Biomechanics; Biomedical Engineering; Biophysics; Calcium; Cardiac; Cardiac Myocytes; Cardiomyopathies; Cell Culture Techniques; Cells; Classification; Clinical Trials; Contractile Proteins; Contractile System; Contracts; Custom; Data; Defect; Dependence; Development; Dilated Cardiomyopathy; Disease; Elastomers; Engineering; Event; Exhibits; Fibroblasts; Filament; Functional disorder; Future; Gene Mutation; Generations; Genes; Genetic; Genetic Models; Genetic Variation; Genotype; Heart; Heart Contractilities; Heart failure; Human; Hydrogels; Hypertrophic Cardiomyopathy; In Vitro; Individual; Label; Laboratory Study; Lead; Link; Measures; Methodology; Methods; Microfilaments; Modeling; Mus; Muscle; Muscle Fibers; Mutation; Myocardial tissue; Myocardium; Myosin ATPase; Pathology; Patients; Pattern; Pharmacological Treatment; Pharmacology; Phenotype; Physiological; Population; RNA; Regulation; Relaxation; Reporting; Resolution; Rodent Model; Sarcomeres; Stress; Subgroup; Sudden Death; System; Techniques; Technology; Testing; Thick; Thick Filament; Thin Filament; Thinness; Time; Tissues; Traction Force Microscopy; Transfection; Variant; Workload; antagonist; biophysical model; cohort; genetic variant; improved; in vivo; in vivo Model; individual response; induced pluripotent stem cell; induced pluripotent stem cell derived cardiomyocytes; inherited cardiomyopathy; inhibitor; mutant; new technology; novel; physiologic model; precision medicine; predicting response; reconstitution; response; stem cells; tool; treatment response; two-dimensional; variant of unknown significance

ABSTRACT Cardiomyopathies, including hypertrophic (HCM) and dilated (DCM) cardiomyopathy, are conditions in which heart muscle dysfunction may lead to arrhythmias and heart failure. Cardiomyopathies are most commonly caused by variants in sarcomere genes that encode contractile proteins. The immediate effect of these genetic variants is perturbation of contractile function. However, a clear understanding of how the thousands of different variants in individual sarcomere genes differentially affect contractile function to cause HCM and DCM has not been attained. Furthermore, traditional systems have not been able to efficiently study the interaction between genetic variants affecting contractile function and varying levels of biomechanical workload that models the in vivo state. Cardiomyocytes differentiated from induced pluripotent stem cells (iPSC-CMs) are a promising model system that allow the study of HCM- and DCM-causing mutations in a human cell context, but the capacity of this model system for contractile analysis has been limited because of technical and biologic hurdles. My preliminary data shows that an optimized bioengineered platform enables generation of contracting micrometer- scale 2-dimensional heart muscle tissues (referred to as M2D) on an elastomer substrate. M2D tissues exhibit coordinated, uniaxial contraction, robust myofibrillar alignment, and expected responses to contractile agonists/antagonists. In addition, my preliminary data shows that the M2D tissues are amenable to modified RNA transfection, enabling >90% mutant replacement of contractile proteins. I hypothesize that the M2D technology will enable mechanistic determination of dysregulated contractile velocity and workload relationships in cardiomyopathy patient iPSCMs compared to controls, and, moreover, that these analyses will enable subclassification of contractile defects due to thick vs. thin filament mutations that will predict responses to pharmacologic modulation of contractile function. The first aim tests the capacity of the M2D system to discriminate contractile dysregulation in patient iPSCM muscle tissues with thick (MYH7, MYBPC3) vs thin (TNNT2) filament sarcomere gene variants in a total of 10 patient iPSC lines, as compared to controls. Modified RNA transfections will be used as additional models since we are able to achieve very high transfection efficiencies in the M2D system. Both myofibrillar alignment and contractile function will be quantified using custom analysis tools. Sensitivity of contractile function to calcium concentration will also be assessed in both patient and control muscle tissues. The second aim will test whether thick vs. thin filament variant iPSCMs have a differential reversal of contractile dysregulation with the myosin inhibitor Myk-461. The implementation of the M2D technology to interrogate contractile function in the presence of sarcomere gene variants will be transformative for precision analysis of patient-specific heart muscle cells by enabling analysis of contractile phenotypes in a physiologic microenvironment with tunable workload. In addition, the implementation of this novel technology will be a major strategy to bridge from my K08 to future R01 proposals.

摘要 心肌病，包括肥厚型（HCM）和扩张型（DCM）心肌病，是其中 心肌功能障碍可导致心律失常和心力衰竭。心肌病最常见于 由编码收缩蛋白的肌节基因变异引起。这些基因的直接影响 变量是收缩功能的扰动。但是，要清楚地认识到， 单个肌节基因的变异差异性地影响收缩功能，从而导致HCM，而DCM没有 已经实现。此外，传统的系统还不能有效地研究 影响收缩功能的遗传变异和不同水平的生物力学工作负荷， 活体状态诱导性多能干细胞（iPSC-CMs）分化为心肌细胞是一种很有前途的模型 该系统允许在人类细胞背景下研究HCM和DCM引起的突变，但其能力 这种用于收缩分析的模型系统由于技术和生物学障碍而受到限制。我 初步数据显示，优化的生物工程平台能够产生收缩微米， 在弹性体基底上缩放二维心肌组织（称为M2 D）。M2 D组织显示 协调，单轴收缩，强大的肌原纤维排列，以及对收缩的预期反应， 激动剂/拮抗剂。此外，我的初步数据显示，M2 D组织适合于修饰 RNA转染，使收缩蛋白的突变体替换率>90%。我假设M2 D 技术将能够机械地确定失调的收缩速度和工作负荷的关系 在心肌病患者iPSCM中，与对照相比，而且，这些分析将使 由于粗与细细丝突变引起的收缩缺陷的亚分类， 收缩功能的药理学调节。第一个目标测试M2 D系统的能力， 区分厚（MYH 7，MYBPC 3）与薄的患者iPSCM肌肉组织中的收缩失调 在总共10个患者iPSC细胞系中，与对照相比，图1示出了（TNNT 2）丝状肌节基因变体的基因表达。改性 RNA转染将被用作额外的模型，因为我们能够实现非常高的转染 M2 D系统的效率。肌原纤维排列和收缩功能都将使用 自定义分析工具。还将评估收缩功能对钙浓度的敏感性， 患者和对照肌肉组织。第二个目标将测试厚丝与细丝变体iPSCM是否具有 肌球蛋白抑制剂Myk-461对收缩失调的差异逆转。执行情况 M2 D技术在肌节基因变异体存在的情况下询问收缩功能， 通过分析收缩性心肌细胞， 表型在生理微环境与可调的工作量。此外，这项工作的实施 新技术将是从我的K 08方案过渡到未来R 01方案的主要策略。