Murine 3D Engineered Tissue Model of Human MYBPC3 Mutations

描述（由申请人提供）：作为早期研究者R 01提交的这份为期五年的提案应用了一种新型小鼠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，HCM是一种人类患病率为1/500的疾病。疾病的负担从新生儿到老年人，表现为HCM、心力衰竭和心脏骤停。在cMyBP-C中已经鉴定出超过140种突变。这些突变如何影响心肌功能和HCM的发展仍然在很大程度上未知。我们已经开发了一种技术，从缺乏鼠cMyBP-C的未成熟小鼠心肌细胞中产生工程化心肌，这些心肌细胞尚未经历肥厚性重塑。在这个系统中，我们可以表达特定的人类突变cMyBP-C，测试收缩功能，并确定应用的应激源在HCM表型发展中的作用。在这项提案中，我们将探讨的主要假设，HCM引起的单一氨基酸取代在人类cMyBP-C主要改变收缩功能，即使在没有肥大，和突变与严重改变收缩功能表现出更早和更明显的重塑，以应对环境压力。提出实验以追求三个主要目的：1：定义在3D心脏组织模型（ECT）中生长的表达野生型人cMyBP-C的鼠cMyBP-C缺陷型心肌细胞的功能和分子表型;目的2：表征ECT模型中人cMyBP-C错义突变的急性表达的功能影响;以及目的3：转基因表达ECT对机械负荷、电起搏或肾上腺素能刺激的适应性和适应不良功能和分子反应的鉴定。我们选择了cMyBP-C中的12个突变，这些突变跨越了从新生儿到成人的发病年龄和疾病严重程度。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。本提案中要求的所有必要试剂和技术均已在主要研究者实验室内得到充分确立。威斯康星大学麦迪逊分校的广泛资源和合作关系提供了一个出色的环境，并提高了成功实现这些目标的可能性。

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.