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.

抽象的 心肌病，包括肥厚（HCM）和扩张（DCM）心肌病，是 心肌功能障碍可能导致心律不齐和心力衰竭。心肌病最常见 由编码收缩蛋白的肌节基因中的变异引起。这些遗传的直接作用 变体是收缩功能的扰动。但是，清楚地了解了成千上万的不同 单个肌节基因的变体会差异地影响收缩功能会导致HCM和DCM 已达到。此外，传统系统无法有效研究 影响收缩功能和不同水平的生物力学工作负载的遗传变异，以建模 体内状态。与诱导的多能干细胞（IPSC-CM）区分的心肌细胞是一个有前途的模型 允许在人类细胞环境中研究HCM和DCM引起的突变的系统，但能力 由于技术和生物障碍，这种用于收缩分析的模型系统受到限制。我的 初步数据表明，一个优化的生物工程平台可生成合同的千分尺 在弹性体底物上缩放二维心肌组织（称为M2D）。 M2D组织展示 协调的，单轴收缩，强大的肌原纤维对齐以及对收缩的预期响应 激动剂/对手。此外，我的初步数据表明，M2D组织可以修改 RNA转染，使收缩蛋白的突变替代> 90％。我假设M2D 技术将实现机械确定失调的收缩速度和工作量关系 与对照组相比，在心肌病患者IPSCM中，这些分析将启用 由于厚与细丝突变引起的收缩缺陷的亚分类，该突变将预测对 收缩功能的药理调制。第一个目标测试了M2D系统的能力 在患者IPSCM肌肉组织（MYH7，MYBPC3）与薄的患者IPSCM肌肉组织中区分收缩失调 （TNNT2）与对照组相比，总共10例患者IPSC系中的细丝肌膜基因变异。修改的 RNA转染将用作其他模型，因为我们能够实现非常高的转染 M2D系统的效率。肌原纤维对齐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者中进行评估 患者并控制肌肉组织。第二个目标将测试厚丝与细丝变体IPSCM是否具有 与肌球蛋白抑制剂MYK-461的收缩失调的差异逆转。实施 M2D技术在存在肌节基因变体的情况下询问收缩功能 通过启用收缩的分析，可转化用于患者特异性心肌细胞的精确分析 具有可调节工作量的生理微环境中的表型。另外，实施 新技术将是从我的K08到未来R01提案的主要策略。