The Role of the Proteasome in Troponin related Cardiomyopathies

描述（由申请人提供）：尽管在心脏病的诊断和治疗方面取得了重大进展，心肌病仍然是发达国家最普遍的死亡原因。在所有家族性肥厚性心肌病（FHC）病例中，心肌肌钙蛋白T （cTnT）突变约占7%。携带这些与FHC相关的cTnT突变的患者显示出猝死的高发生率，而没有在其他蛋白质突变的FHC患者中看到的左心室心壁的典型增加。本研究的重点是蛋白酶体在肌钙蛋白相关心肌病中的作用。调控心脏蛋白酶体的分子机制及其在心肌病中的作用尚不清楚。表达与FHC相关的突变体（I79N） cTnT的转基因小鼠在蛋白酶体亚基的翻译后修饰和蛋白酶体的活性方面都发生了变化，但所研究的蛋白酶体亚基的表达没有发生显著变化。蛋白酶体磷酸化水平的变化伴随着所有三种20S和26S蛋白酶体活性的降低。了解蛋白酶体系统在心肌病中的重要性是至关重要的，以便能够正确地靶向这种关键的蛋白水解复合物，以实现未来心血管的益处。从I79N心脏分离的20S蛋白酶体中，与20S蛋白酶体相关的关键磷酸酶的数量也减少。这一点很重要，因为我们的研究结果还表明，心脏内与蛋白酶体复合物相关的激酶和磷酸酶是蛋白酶体活性的重要调节剂，心脏蛋白酶体与其他组织的蛋白酶体不同。根据我们的研究结果，我们假设：1)肌丝Ca2+敏感性的显著增加（>0.1pCa单位）有助于导致蛋白酶体功能障碍的细胞信号改变，从而导致泛素化蛋白增加和心功能障碍；2)肌钙蛋白中一些与FHC相关的突变直接影响这些蛋白被蛋白酶体降解的能力并改变蛋白酶体活性。3)肌钙蛋白相关性心肌病中，激酶和磷酸酶作为蛋白酶体复合物的关联蛋白；作为蛋白酶体亚蛋白质组的一部分，它们在调节蛋白酶体功能方面起着关键作用。为了研究这些假设，我们将研究三个具体目标：1)描述20S和26S蛋白酶体在肌钙蛋白相关心肌病中的作用，2)表征肌钙蛋白相关心肌病中蛋白酶体复合物的时间谱，以及3)表征心肌病诱导的26S蛋白酶体磷酸化变化。

DESCRIPTION (provided by applicant): Despite significant advances in diagnosis and treatment of heart diseases, cardiomyopathies remain the most prevalent cause of death in developed countries. Mutations in cardiac troponin T (cTnT) are responsible for ~7% of all familial hypertrophic cardiomyopathy (FHC) cases. Patients carrying these FHC related cTnT mutations show a high incidence of sudden death without the classical increase in the left ventricular heart wall seen in FHC patients with mutations in other proteins. This proposal focuses on the role of the proteasome in troponin related cardiomyopathies. The molecular mechanisms that regulate the cardiac proteasome and their role in cardiomyopathies are unknown. Transgenic mice expressing a mutant (I79N) cTnT that is associated with FHC exhibited changes in both post-translational modifications of proteasome subunits and the activity of the proteasome, but did not cause significant changes in the expression of the proteasome subunits investigated. Changes in phosphorylation levels of the proteasome were observed concomitant with decreases in all three 20S and 26S proteasome activities. It is critical to understand the importance of the proteasome system in cardiomyopathies to be able to properly target this key proteolytic complex for future cardiovascular benefit. The amount of a key phosphatase associated with the 20S proteasome is also decreased in 20S proteasomes isolated from I79N hearts. This is important since our results also suggest that the kinases and phosphatases associated with the proteasome complex inside the heart are important modulators of the proteasome activity, and that the cardiac proteasome is unlike proteasomes from other tissues. Based upon our results we hypothesize: 1) Significant increases in myofilament Ca2+-sensitivity (>0.1pCa units) contribute to cellular alterations in signaling that lead to proteasome dysfunction which results in increased ubiquitinated proteins and cardiac dysfunction, 2) Some FHC related mutations in troponin directly affect the ability of these proteins to be degraded by the proteasome and alter proteasome activity, and 3) Kinases and phosphatases function as associating proteins for proteasomal complexes in the Troponin-related cardiomyopathies; they play a critical role in modulating the proteasomal function as part of the proteasomal subproteome. To investigate these hypotheses we will investigate three specific aims: 1) To Delineate the Roles of the 20S and 26S Proteasomes in Troponin-related Cardiomyopathies, 2) To Characterize the Temporal Profile of the Proteasome complexes in Troponin-related Cardiomyopathies, and 3) To Characterize Cardiomyopathy Induced Phosphorylation Changes in 26S Proteasomes. PUBLIC HEALTH RELEVANCE: Familial hypertrophic cardiomyopathy (FHC) is the most common inherited heart disease with a prevalence of at least 0.2% and is the most common cause of sudden death in adolescents and young adults, especially in athletes. Dysfunction of the ubiquitin proteasome system (UPS) has been associated pressure overload induced hypertrophy, ischemia/reperfusion, and congestive heart failure. Investigating the role of the proteasome in FHC will give critical insights into proteasomal function in cardiomyopathies.