Murine 3D Engineered Tissue Model of Human MYBPC3 Mutations

描述（由申请人提供）：作为早期研究者R01提交的这项为期五年的提案，应用一种新型小鼠3D工程化心脏组织模型（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，HCM是一种人类患病率为1 / 500的疾病。疾病负担跨越新生儿到老年人，表现为HCM、心力衰竭和心脏骤停。在cMyBP-C中发现了140多个突变。这些突变如何影响心肌功能和HCM的发展在很大程度上仍然未知。我们已经开发了一种技术，从缺乏小鼠cMyBP-C的未成熟小鼠心肌细胞中产生工程化心肌，这些细胞尚未经历肥厚重塑。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，并确定施加应激源在HCM表型发展中的作用。在本提案中，我们将探讨hcm引起的人类cMyBP-C单氨基酸取代主要改变收缩功能的主要假设，即使在没有肥大的情况下，收缩功能严重改变的突变在响应环境应激时表现出更早和更明显的重塑。实验旨在实现三个主要目标：1 .确定在3D心脏组织模型（ECT）中生长的表达野生型人cMyBP-C的小鼠cMyBP-C缺陷心肌细胞的功能和分子表型；目的2：在ECT模型中表征人cMyBP-C错义突变急性表达对功能的影响；目标3：鉴定转基因表达ECT对机械负荷、电起搏或肾上腺素能刺激的适应性和非适应性功能和分子反应。我们在cMyBP-C中选择了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。本提案中所需的所有必要试剂和技术都在主要研究人员的实验室中得到了很好的建立。威斯康星大学麦迪逊分校广泛的资源和合作关系提供了一个优秀的环境，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.