Murine 3D Engineered Tissue Model of Human MYBPC3 Mutations

人类 MYBPC3 突变的小鼠 3D 工程组织模型

基本信息

批准号：
8484866
负责人：
John Carter Ralphe
金额：
$ 35.82万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-06-13 至 2015-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8484866
关键词：
3-Dimensional Ablation Achievement Actins Acute Adenoviruses Adrenergic Agents Adult Affect Age Age of Onset Amino Acid Substitution Angiotensin Receptor Arrhythmia Calcium Cardiac Cardiac Myocytes Cardiac Myosins Cell Survival Child Childhood Code Data Development Disease Elderly Engineering Environment Environmental Risk Factor Familial Hypertrophic Cardiomyopathy Genes Genetic Heart Arrest Heart failure Human Hypertrophic Cardiomyopathy Hypertrophy Immunoblotting Immunohistochemistry Individual Infant Kinetics Laboratories Lead Length Link Mechanics Missense Mutation Modeling Molecular Mus Muscle Muscle Cells Muscle function Mutation Myocardium Myosin ATPase Neonatal Newborn Infant Phenotype Phosphorylation Site Physiological Point Mutation Prevalence Primary Myocardial Diseases Progressive Disease Property Protein Binding Proteins Reagent Research Personnel Resources Role Sarcomeres Severities Severity of illness Signal Pathway Staging Stress System Techniques Testing Thick Filament Time Tissue Engineering Tissue Model Tissues Transgenes Universities Wild Type Mouse Wisconsin adrenergic burden of illness connectin genetic regulatory protein human disease innovation molecular phenotype mutant myosin-binding protein C novel research study response screening stressor tissue culture tool vector control

项目摘要

DESCRIPTION (provided by applicant): This five year proposal submitted as an Early Stage Investigator R01 applies a novel mouse 3D engineered cardiac tissue model (ECT) to study the effects of mutations in the human myosin binding protein C (cMyBP-C). cMyBP-C mutations are one of the leading genetic causes of hypertrophic cardiomyopathy (HCM), a disease with a human prevalence of 1 in 500. The burden of disease spans newborn infants to older adults, and manifests with HCM, heart failure, and sudden cardiac arrest. Over 140 mutations have been identified in cMyBP-C. How these mutations affect cardiac muscle function and the development of HCM remains largely unknown. We have developed a technique to produce engineered myocardium from immature mouse cardiomyocytes deficient in murine cMyBP-C that have yet to undergo hypertrophic remodeling. Within this system we can express specific human mutant cMyBP-C, test contractile function, and define the role of applied stressors in the development of the HCM phenotype. In this proposal we will explore the main hypotheses that HCM-causing single amino acid substitutions in human cMyBP-C primarily alter contractile function, even in the absence of hypertrophy, and that mutations with severely altered contractile function demonstrate earlier and more pronounced remodeling in response to environmental stress. Experiments are proposed to pursue three principle aims: 1: Defining the functional and molecular phenotype of murine cMyBP- C deficient cardiomyocytes expressing wild-type human cMyBP-C grown in the 3D cardiac tissue model (ECT); Aim 2: Characterization of the functional impact of acute expression of human cMyBP-C missense mutations in the ECT model; and Aim 3: Identification of adaptive and maladaptive functional and molecular responses of the transgene-expressing ECT to mechanical load, electrical pacing, or adrenergic stimulation. We have selected twelve mutations in cMyBP-C that span both the age of onset from newborns to adults, and disease severity. Data derived from these experiments will extend our understanding of human cMyBP-C HCM and identify potential environmental factors that influence development of the HCM phenotype. All of the necessary reagents and techniques required in this proposal are well established within the Principle Investigators laboratory. The extensive resources and collaborative relationships at the University of Wisconsin-Madison offer an outstanding environment and enhance the likelihood of successful achievement of these aims.

描述（由申请人提供）：作为早期研究者 R01 提交的这份为期五年的提案应用了新型小鼠 3D 工程心脏组织模型 (ECT) 来研究人肌球蛋白结合蛋白 C (cMyBP-C) 突变的影响。 cMyBP-C 突变是肥厚型心肌病 (HCM) 的主要遗传原因之一，这种疾病的人类患病率为五百分之一。疾病负担涵盖新生儿到老年人，表现为 HCM、心力衰竭和突发性心脏病。心脏停搏。 cMyBP-C 中已鉴定出 140 多个突变。这些突变如何影响心肌功能和 HCM 的发展仍然很大程度上未知。我们开发了一种技术，可以利用缺乏小鼠 cMyBP-C 且尚未进行肥厚重塑的未成熟小鼠心肌细胞生产工程化心肌。在这个系统中，我们可以表达特定的人类突变体 cMyBP-C，测试收缩功能，并定义施加的应激源在 HCM 表型发展中的作用。在本提案中，我们将探讨主要假设，即即使在没有肥大的情况下，人类 cMyBP-C 中引起 HCM 的单氨基酸取代也主要改变收缩功能，并且收缩功能严重改变的突变表现出更早、更明显的重塑反应环境压力。实验旨在实现三个主要目标： 1：定义在 3D 心脏组织模型 (ECT) 中生长的表达野生型人类 cMyBP-C 的小鼠 cMyBP-C 缺陷心肌细胞的功能和分子表型；目标 2：表征 ECT 模型中人类 cMyBP-C 错义突变急性表达的功能影响；目标 3：鉴定表达转基因的 ECT 对机械负荷、电起搏或肾上腺素刺激的适应性和适应不良功能和分子反应。我们选择了 cMyBP-C 中的 12 种突变，涵盖从新生儿到成人的发病年龄以及疾病严重程度。从这些实验中获得的数据将扩展我们对人类 cMyBP-C HCM 的理解，并确定影响 HCM 表型发展的潜在环境因素。本提案所需的所有必要试剂和技术均已在主要研究人员实验室内得到完善。威斯康星大学麦迪逊分校广泛的资源和合作关系提供了出色的环境，并提高了成功实现这些目标的可能性。