Murine 3D Engineered Tissue Model of Human MYBPC3 Mutations

人类 MYBPC3 突变的小鼠 3D 工程组织模型

• 批准号: 8669119
• 负责人: John Carter Ralphe
• 金额: $ 36.87万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-13 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8669119
• 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

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提交的五年提案应用了一种新型的小鼠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突变是肥厚性心肌病（HCM）的主要遗传原因之一，这种疾病是人类患病率为500的疾病。疾病的负担跨越了新生婴儿对老年人的负担，并且表现为HCM，心脏失败，心脏失败，突然心脏抑制。在CMYBP-C中已经确定了140多个突变。这些突变如何影响心肌功能，而HCM的发展仍然很大程度上是未知的。我们已经开发了一种技术，可以从尚未经历肥厚性重塑的鼠CMYBP-C中产生工程性的心肌。在该系统中，我们可以表达特定的人类突变体CMYBP-C，测试收缩功能，并定义应用应激源在HCM表型发展中的作用。在该提案中，我们将探讨人类CMYBP-C中引起HCM的单个氨基酸取代的主要假设，即使在没有肥大的情况下，也主要改变了收缩功能，并且具有严重变化的收缩功能的突变表现出了早期，并且在响应对环境压力的响应后更明显地重塑。提出了实验来追求三个原则：1：定义鼠CMYBP-C缺乏表达野生型人类CMYBP-C的功能和分子表型在3D心脏组织模型（ECT）中生长；目标2：表征ECT模型中人CMYBP-C错义突变的急性表达的功能影响；和目标3：鉴定转基因ECT对机械负荷，电气起搏或肾上腺素能刺激的适应性和不良适应性功能和分子反应。我们已经选择了CMYBP-C中的十二个突变，这些突变跨越了从新生儿到成年人的发病年龄，以及疾病的严重程度。从这些实验中得出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。本提案中所需的所有必要试剂和技术都是在原则研究人员实验室中确定的。威斯康星大学麦迪逊分校的广泛资源和协作关系为成功实现这些目标的可能性提供了杰出的环境。