The Role of the Proteasome in Troponin related Cardiomyopathies

描述(申请人提供)：尽管心脏病的诊断和治疗取得了重大进展，但在发达国家，心肌病仍然是最常见的死亡原因。心肌肌钙蛋白T(CTnT)基因突变占家族性肥厚型心肌病(FHC)患者总数的7%。携带这些FHC相关cTnT突变的患者显示出猝死的高发生率，而不像其他蛋白质突变的FHC患者那样出现典型的左室心壁增厚。这项建议侧重于蛋白酶体在肌钙蛋白相关的心肌病中的作用。调控心肌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(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，但没有引起所研究的蛋白酶体亚基的表达显著变化。观察到蛋白酶体的磷酸化水平的变化伴随着三个20S和26S蛋白酶体活性的下降。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一关键的蛋白分解复合体，从而使未来的心血管受益。在从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了。这一点很重要，因为我们的结果还表明，与心脏内蛋白酶体复合体相关的激酶和磷酸酶是蛋白酶体活性的重要调节器，心脏蛋白酶体不同于其他组织的蛋白酶体。根据我们的结果，我们假设：1)肌丝钙敏感性(&gt；0.1pCa单位)的显著增加有助于细胞内导致蛋白酶体功能障碍的信号变化，从而导致泛素化蛋白增加和心功能障碍；2)肌钙蛋白中的一些FHC相关突变直接影响这些蛋白被蛋白酶体降解并改变蛋白酶体活性的能力；3)在肌钙蛋白相关的心肌病中，激酶和磷酸酶作为蛋白酶体复合体的结合蛋白发挥作用；它们作为蛋白酶体亚蛋白质组的一部分，在调节蛋白酶体的功能中发挥关键作用。为了研究这些假说，我们将研究三个特定的目标：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)表征蛋白酶体复合体在肌钙蛋白相关心肌病中的时间分布，以及3)表征心肌病引起的26S蛋白酶体的磷酸化变化。 公共卫生相关性：家族性肥厚性心肌病(FHC)是最常见的遗传性心脏病，患病率至少为0.2%，是青少年和年轻人，尤其是运动员猝死的最常见原因。泛素蛋白酶体系统(UPS)功能障碍与压力超负荷所致的肥厚、缺血/再灌注和充血性心力衰竭相关。研究蛋白酶体在FHC中的作用将使我们对蛋白酶体在心肌疾病中的功能有重要的了解。

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.