Modulators of Cardiomyopathic Diseases

 描述(由申请人提供)：这项工作的长期目标是确定心肌细胞内与心肌疾病发展有关的成分。这项建议侧重于心肌肌钙蛋白C(CTNC)，它是收缩装置的开关，也是心肌细胞内主要的细胞内钙缓冲。为了了解肌钙蛋白复合体在体内的调节特性及其与控制心脏病的异常细胞内钙离子处理的联系，这一提议具有相当大的健康相关性。与肥厚型(HCM)和扩张型(DCM)心肌病表型结局相关的细丝调节复合体(原肌球蛋白、肌钙蛋白T和肌钙蛋白I)的突变被认为是通过改变cTNC的钙结合特性来间接扰乱心肌收缩。然而，cTNC突变体影响钙敏感收缩反应的效果尚未在活体中测试其致病能力。指导这一提议的中心假设是，cTNC N末端钙结合亲和力的变化单独可以在体内引起心脏重构。我们进一步假设，消融专用的高保真激酶有可能逆转由钙敏化的HCM连锁cTNC突变体造成的过度收缩状态。目的1将评估细丝中cTNC钙结合亲和力的直接变化作为心肌病发生的关键决定因素。这一目的验证了这样的假设，即在N-结构域(调节)增加cTNC与钙结合亲和力的cTNC突变体可以引发舒张性功能障碍，导致肥厚性心肌病；而设计的降低cTNC钙结合亲和力的突变体将概括出一种DCM的表型。心脏病理生理学、生物物理学和生化方法将被用来剖析cTNC突变在我们新培育的敲入(KI)小鼠中的作用。这些cTNC突变体对钙结合动力学的相反影响将在发生独特的心脏重塑之前进行早期研究。此外，本项目还将进一步将TNNC1(cTNC编码基因)定义为心肌病易感基因。目标2将建立有关肌丝钙敏感性正常化的机制和潜在的治疗联系。本研究的目的是探讨有条件地去除一种特殊的肌松解酶是否能纠正肌丝钙离子反应，减少过度收缩表型，改善心脏松弛，从而逆转症状后的肥厚性心肌病。在Ki cTNC-HCM心脏中有条件地移除该激酶的后果将作为时间的函数进行监测。最后一个目标可以作为开发靶向治疗的概念验证，目的是调节专门的肌瘤激酶的活性。这里产生的新概念将定义cTNC在启动和调节一类心肌病中的作用，从而为开发新的量身定制的治疗方法开辟道路。

 DESCRIPTION (provided by applicant): The long-term goal of this work is to identify components inside the cardiac cell that are involved with the development of cardiomyopathic diseases. This proposal focuses on cardiac troponin C (cTnC), the on-off switch of the contractile apparatus and a major cardiomyocyte intracellular Ca2+ buffer. Aimed at understanding the regulatory properties of the troponin complex in vivo and its link to abnormal intracellular Ca2+ handling governing heart disease, this proposal is of considerable health relevance. Mutations in the regulatory complex of the thin filament (tropomyosin, troponin T and troponin I) associated with phenotypic outcomes of hypertrophic (HCM) and dilated (DCM) cardiomyopathies are suggested to indirectly disrupt cardiac muscle contraction by altering the Ca2+-binding properties of cTnC. However, effects of cTnC mutants that influence Ca2+-sensitive contractile responses have yet to be tested for their pathogenic capacity in living organisms. The central hypothesis guiding this proposal is that changes in cTnC N-terminus Ca2+-binding affinity, alone, can evoke cardiac remodeling in vivo. We further posit that ablation of a dedicated high-fidelity kinase has the potential to reverse the hypercontractile state imposed by Ca2+-sensitizing HCM-linked cTnC mutants. Aim 1 will evaluate direct changes in cTnC Ca2+-binding affinity in the thin filament as a critical determinant underlying cardiomyopathic development. This Aim tests the hypothesis that cTnC mutants increasing Ca2+-binding affinity in the N-domain (regulatory) can instigate diastolic dysfunction, leading to HCM; while a designed mutant decreasing cTnC Ca2+- binding affinity will recapitulate a DCM-reminiscent phenotype. Cardiac patho-physiological, biophysical and biochemical approaches will be used to dissect the role of cTnC mutations in our newly developed knock-in (KI) mice. The antithetical effects that these cTnC mutants exert on Ca2+-binding dynamics will be investigated early, prior to development of distinctive cardiac remodeling. In addition, this projet will further define TNNC1 (cTnC- encoding gene) as a cardiomyopathy-susceptibility gene. Aim 2 will establish mechanistic and potential therapeutic links regarding normalization of myofilament Ca2+-sensitivity. This Aim examines whether conditional removal of a dedicated sarcomeric kinase will correct the myofilament Ca2+ response, diminish the hypercontractile phenotype and improve cardiac relaxation, thus reversing post-symptomatic HCM disease. The consequences of conditional removal of this kinase in KI cTnC-HCM hearts will be monitored as a function of time. The last aim can serve as a proof-of-concept for the development of targeted therapies aimed at modulating the activity of dedicated sarcomeric kinases. The novel concepts generated here will define the role of cTnC in initiating and modulating one class of cardiomyopathies, thus opening avenues for development of new tailored therapeutic approaches.