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

描述（由申请人提供）：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，这是一种常染色体显性遗传疾病，影响五百分之一的人，是青少年和年轻人心源性猝死的最常见原因。心脏 (c) MyBP-C 突变是 HCM 最常见的原因之一，迄今为止已描述了超过 149 种不同的突变。然而，尽管在确定 HCM 的遗传原因方面取得了进展，但任何单一 cMyBP-C 突变导致疾病的分子机制仍然未知。最近报道受影响的人类心肌中 cMyBP-C 的量减少，这表明受影响等位基因的功能性 cMyBP-C 丧失（即单倍剂量不足）是导致心功能障碍的常见因素。然而，影响蛋白质损害收缩功能或不正确折叠蛋白质的加工和降解导致异常细胞功能的其他可能性尚未消除。这些区别对于设计克服 HCM 的有效治疗策略至关重要，但尚未获得支持不同可能性的明确证据，部分原因是人类活检的可用性有限，部分原因是工程小鼠模型不能完全重现人类疾病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 HCM 大型动物模型来克服这些限制，该模型既与人类疾病表型非常相似，又具有已知的遗传原因。该突变是家养缅因猫 cMyBP-C 中的自发错义突变，导致密码子 31 (A31P) 处的丙氨酸被脯氨酸取代。该突变导致单个氨基酸取代，但导致 cMyBP-C 蛋白总量异常减少。由于人类心肌中错义 cMyBP-C 突变也有类似的 cMyBP-C 降低的报道，猫科动物 A31P 模型提供了一个独特的机会来区分被认为是人类疾病致病因素的三个主要因素，即单个氨基酸点突变的显性负面影响、基因剂量效应和细胞蛋白质折叠/运输缺陷。拟议的实验将检验以下假设：受影响的猫的肌节中 cMyBP-C 含量减少（即单倍剂量不足）会导致收缩缺陷，最终导致心脏功能障碍。该项目的具体目标是确定 1) 携带 A31P 突变的猫中总 cMyBP-C 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。总的来说，这些研究的结果将为 cMyBP-C 突变引起疾病的分子机制提供重要见解，并将通过开发独特的动物模型对 HCM 研究产生重大而持久的影响，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 相关的心肌病的治疗策略。 公共健康相关性：拟议的研究将调查心肌肌球蛋白结合蛋白-C (cMyBP-C) 中的 A31P 突变导致缅因猫（一种家猫品种）遗传性肥厚性心肌病 (HCM) 的基本致病机制。在人类中，估计每 500 人中就有 1 人患有 HCM，并且是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 HCM 的主要原因，因此预计在缅因猫中进行的研究将提供对人类疾病的见解，并最终有助于与 cMyBP-C 相关的 HCM 的疾病诊断、预后和治疗方面的进展。

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.