Molecular Mechanisms of Disease in a Novel Feline Model of Familial Hypertrophic

描述(申请人提供)：拟议研究的长期目标是了解肌球蛋白结合蛋白C(MyBP-C)突变导致肥厚性心肌病(HCM)的分子机制，HCM是一种常染色体显性遗传病，每500人中就有1人受到影响，是青少年和年轻人心源性猝死的最常见原因。心脏(C)MyBP-C的突变是肥厚性心肌病最常见的原因之一，迄今已描述了149个不同的突变。然而，尽管在确定肥厚型心肌炎的遗传原因方面取得了进展，但任何单个cMyBP-C突变导致疾病的分子机制(S)仍不清楚。最近有报道称，在受影响的人类心肌中cMyBP-C的数量减少，这表明受影响的等位基因(即单倍体功能不全)导致功能性cMyBP-C的丧失是导致心功能不全的常见因素。然而，受影响的蛋白质损害收缩功能或不正确折叠的蛋白质的加工和降解导致细胞功能异常的其他可能性尚未消除。这些区别对于设计有效的治疗策略来克服HCM至关重要，但尚未获得支持不同可能性的确凿证据，部分原因是人类活检的可获得性有限，部分原因是转基因小鼠模型不能完全概括人类疾病的表型或导致疾病的近端细胞过程。这里提出的实验将通过利用唯一自然发生的大型动物模型来克服这些限制，这种模型既与人类疾病表型非常相似，又具有已知的遗传原因。该突变是家养缅因州浣熊cMyBP-C的自发错义突变，导致密码子31(A31P)的丙氨酸替换。突变导致单一氨基酸替换，但导致cMyBP-C蛋白总量异常减少。由于人类心肌中cMyBP-C错义突变的cMyBP-C也有类似的下降，猫的A31P模型提供了一个独特的机会来区分人类疾病的三个主要因素，即单个氨基酸点突变的显性负效应、基因剂量效应和细胞蛋白折叠/运输缺陷。拟议的实验将检验一种假设，即受影响的猫的肌节中cMyBP-C的数量减少(即单倍体功能不全)会导致收缩缺陷，最终导致心脏功能障碍。该项目的具体目的是确定1)携带A31P突变的猫cMyBP-C总蛋白的表达水平和亚细胞定位，2)A31P突变对心肌细胞收缩特性的影响，以及3)A31P突变对心肌泛素-蛋白酶体(UPS)系统的影响。总而言之，这些研究的结果将为cMyBP-C突变致病的分子机制提供关键的见解，并将通过开发一种独特的动物模型来对HCM研究产生重大和持久的影响，该模型将成为基础研究的资源，并将有助于设计和测试与cMyBP-C相关的心肌病治疗策略。 公共卫生相关性：拟议的研究将探讨心肌肌球蛋白结合蛋白C(cMyBP-C)中的A31P突变导致缅因州肥厚性心肌病(HCM)的基本致病机制。在人类中，估计每500人中就有1人感染HCM，是导致青少年和年轻人猝死的主要原因。由于影响cMyBP-C的突变是人类肥厚型心肌炎的主要原因，预计拟议中的缅因州浣熊研究将提供对人类疾病的见解，并最终将有助于与cMyBP-C相关的HCM的疾病诊断、预后和治疗方面的进步。

DESCRIPTION (provided by applicant): The long-range goal of the proposed research is to understand the molecular mechanisms by which mutations in Myosin Binding Protein-C (MyBP-C) cause hypertrophic cardiomyopathy (HCM), an autosomal dominant disorder that affects 1 in 500 people and is the most common cause of sudden cardiac death in adolescents and young adults. Mutations in cardiac (c) MyBP-C are among the most frequent causes of HCM with >149 distinct mutations described so far. However, despite progress in identifying genetic causes of HCM, the molecular mechanism(s) by which any single cMyBP-C mutation causes disease are still unknown. Reduced amounts of cMyBP-C were recently reported in affected human myocardium, suggesting that loss of functional cMyBP-C from an affected allele (i.e., haploinsufficiency) is a common factor contributing to cardiac dysfunction. However, alternative possibilities that affected proteins impair contractile function or that processing and degradation of improperly folded proteins cause aberrant cell function have not been eliminated. These distinctions are critical for designing effective therapeutic strategies to overcome HCM, yet definitive evidence in support of the different possibilities has not been obtained in part because of the limited availability of human biopsies and in part because engineered mouse models do not fully recapitulate either the human disease phenotype or the proximal cell processes that lead to disease. Experiments proposed here will overcome these limitations by utilizing the only naturally occurring large animal model of HCM that both closely resembles the human disease phenotype and that has a known genetic cause. The mutation is a spontaneous missense mutation in cMyBP-C in domestic Maine Coon cats that results in a proline for alanine substitution at codon 31 (A31P). The mutation results in a single amino acid substitution, but causes an anomalous decrease in total amounts of cMyBP-C protein. Because similar decreases in cMyBP-C have been reported for missense cMyBP-C mutations in human myocardium, the feline A31P model offers a unique opportunity to distinguish between three primary factors proposed as causative in human disease i.e., dominant negative effects of a single amino acid point mutation, gene dosage effects, and cellular protein folding/trafficking defects. The proposed experiments will test the hypothesis that reduced amounts of cMyBP-C in sarcomeres (i.e., haploinsufficiency) of affected cats leads to contractile deficits that ultimately cause cardiac dysfunction. Specific aims of the project are to determine 1) the expression level and subcellular localization of total cMyBP-C protein in cats carrying the A31P mutation, 2) effects of the A31P mutation on myocyte contractile properties, and 3) effects of the A31P mutation on the myocardial ubiquitin-proteasome (UPS) system. Collectively, results from these studies will provide critical insights into molecular mechanisms by which mutations in cMyBP-C cause disease and will provide a significant and lasting impact on HCM research by developing a unique animal model that will be a resource for basic research and that will aid in the design and testing of therapeutic strategies for the treatment of cardiomyopathies linked to cMyBP-C. PUBLIC HEALTH RELEVANCE: The proposed studies will investigate basic pathogenic mechanisms by which the A31P mutation in the cardiac myosin binding protein-C (cMyBP-C) causes inherited hypertrophic cardiomyopathy (HCM) in Maine coon cats, a breed of domestic cat. In humans, HCM affects an estimated 1 in 500 people and is the leading cause of sudden death in adolescent and young adults. Because mutations affecting cMyBP-C are a leading cause of HCM in humans, it is anticipated that the proposed studies in Maine Coon cats will provide insights into human disease and will ultimately contribute to advances in disease diagnosis, prognosis, and treatment of HCM linked to cMyBP-C.