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，是指 心脏肌肉功能障碍可能导致心律失常和心力衰竭。心肌病是最常见的 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 变分是收缩函数的摄动。然而，清醒地认识到千千万万的不同 单个肌节基因变异对收缩功能的不同影响导致肥厚型心肌病和扩张型心肌病 已经实现了。此外，传统的系统已经不能有效地研究 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。诱导多能干细胞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环境中研究HCM和DCM引起的突变，但 由于技术和生物方面的障碍，这种收缩分析的模型系统一直受到限制。我的 初步数据显示，优化的生物工程平台能够产生收缩微米- 在弹性体基质上缩放二维心肌组织(简称M2D)。M2D组织展品 协调的单轴收缩、强健的肌原纤维排列和预期的收缩反应 激动剂/拮抗剂。此外，我的初步数据显示，M2D组织可以进行修改 RNA转染，使&gt；90%的突变替换收缩蛋白。我假设M2D 技术将使机械地确定失调的收缩速度和负荷关系成为可能 心肌病患者的IPSCM与对照组的比较，而且，这些分析将使 粗丝突变和细丝突变引起的收缩缺陷的细分将预测对 收缩功能的药物调节。第一个目标是测试M2D系统的能力 厚型(MYH7，MYBPC3)和薄型IPSCM患者肌肉组织收缩功能失调的鉴别 与对照组相比，总共10个患者IPSC系中的(TNNT2)细丝肌节基因变异。已修改 由于我们能够实现非常高的转染率，所以RNA转染法将被用作额外的模型 M2D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。收缩功能对钙浓度的敏感性也将在两种情况下进行评估 患者和对照肌肉组织。第二个目标将测试粗丝和细丝变体IPSCM是否具有 肌球蛋白抑制剂Myk-461对收缩失调的差异性逆转。《公约》 M2D技术将在肌节基因变异存在的情况下询问收缩功能 通过启用收缩分析，实现患者特定心肌细胞的精确分析的变革性 在工作负荷可调的生理微环境中的表型。此外，这一点的实施 新技术将是从我的K08提案到未来的R01提案的主要战略。