Modulation of Ca Control of Cardiac Myofibrils

描述(申请人提供)：我们在这里提出的实验的长期目标是确定导致慢速骨骼肌钙蛋白I(SSTnI)亚型下调的机制(S)，这种机制发生在出生后的心脏成熟期间。初步数据支持我们的假设，即microRNA miR-208A参与沉默出生后心脏ssTnI的表达。我们的实验继续进行这一建议支持的研究，证明在转基因小鼠的成年心脏中表达ssTnI可以保护心肌免受常见应激的影响，包括家族性扩张型心肌病(DCM)。初步数据表明：1)肌肉特异性miR-208A、miR-1和miR-133a在发育中的心肌细胞中的表达增加，以及ii)208A-antagomiR下调心肌细胞中miR-208A的表达可诱导ssTnI的表达。我们的目标如下：目的1通过(A)研究miR-208a与甲状腺激素介导的ssTnI下调的相互作用，(B)分析miR-208a与其他共同调控的miRNAs在ssTnI基因调控中的作用，(C)研究ssTnI基因调控的转录机制，以及(D)评价ssTnI作为miRNA介导的直接调控靶点，以确定ssTnI和miR-208A介导的调控机制(S)。目的#2：确定miR-208A KO对细丝蛋白的修饰、对肌丝对钙的反应、对心肌细胞力学和钙的影响以及对在体心功能的相对影响。目的#3.确定miR-208A表达降低是否改善了与α-原肌球蛋白突变相关的扩张型心肌病(DCM)患者的心功能。我们实现目标1的方法包括：i)通过获得和丧失功能的方法分析ssTnI在新生儿心肌细胞中的表达，以剖析甲状腺相关信号在抑制ssTnI中的相对重要性；ii)通过与其他miR的共诱导，确定miR-208A作为ssTnI表达抑制因子的相对作用；iii)鉴定miR-208A靶向的转录因子，并确定它们的DNA结合和表达；以及iv)在ssTnI 3‘UTR中鉴定miR-208A的靶点。我们对AIMS#2的方法包括对miR208A-KO小鼠和对照组进行比较研究，以确定肌丝特性改变的功能影响，如亚型群体、翻译后修饰、PCA-力关系和跨桥动力学。通过研究心肌细胞水平上的钙瞬变和力学，以及原位跳动心脏对压力的反应动力学，包括那些由细丝特性变化主导的压力，对去皮心肌细胞的研究结果被整合到心肌功能中。在目标3中，我们的方法是确定与原肌球蛋白突变连锁的家族性DCM小鼠模型与miR208A-KO小鼠杂交是否能够挽救DCM表型。这里提出的实验填补了我们对心肌TnI亚型群体表达控制的一个重要空白，TnI是一种重要的细丝蛋白，并测试了一种治疗心脏疾病的重要方法。

DESCRIPTION (provided by applicant): Our long term goal in experiments proposed here is to identify the mechanism(s) responsible for the down-regulation of the slow skeletal troponin I (ssTnI) isoform, which occurs during post-natal maturation of the heart. Preliminary data underpin our hypothesis that the microRNA, miR-208a, is involved in silencing the cardiac expression of ssTnI after birth. Our experiments continue research supported by this proposal that demonstrated that expression of ssTnI in the adult heart of transgenic mice protects the myocardium against common stresses including familial dilated cardiomyopathy (DCM). Preliminary data demonstrate: i) increased expression of miR-208a with muscle specific miRs, miR-1 and miR-133a in developing cardiomyocytes, and ii) knockdown of miR-208a in cardiomyocytes by a 208a-antagomiR induces ssTnI expression. Our objectives are as follows: Aim #1 To determine mechanism(s) of ssTnI and miR-208a mediated regulation by (a) investigating interplay of miR-208a with thyroid hormone mediated down regulation of ssTnI, (b) analyzing the role of miR-208a in conjunction with other co-regulated miRNAs in ssTnI gene regulation, (c) investigating transcriptional mechanisms of ssTnI gene regulation, and (d) evaluating ssTnI as a direct target of miRNA- mediated regulation. Aim #2: To determine the relative impact of miR-208a KO on modifications of thin filament proteins, on myofilament response to Ca, on myocyte mechanics and Ca, and on in situ cardiac function. Aim #3. To determine whether reduced expression of miR-208a improves cardiac function in dilated cardiomyopathy (DCM) associated with an alpha-tropomyosin mutation. Our approach to Aim #1 includes i) analysis of expression of ssTnI in neonatal cardiac myocytes by gain and loss of function approaches to dissect the relative significance of thyroid related signaling in the repression of ssTnI; ii) determination the relative role of miR-208a as a repressor of ssTnI expression employing co-induction with other miRs; iii) identification of transcription factors targeted by miR-208a and determination of their DNA binding and expression; and iv) identification of targets of miR-208a in ssTnI 3'UTR. Our approaches to Aims #2 include studies comparing miR208a-KO mice to controls to determine the functional impact of altered myofilament properties as reflected in isoform population, post-translational modifications, pCa-force relations and cross- bridge kinetics. Findings from studies of skinned myocytes are integrated into myocardial function by studies of Ca2+ transients and mechanics at the level of cardiac myocytes and of dynamics of in situ beating hearts responding to stresses including those dominated by altered thin filament properties. In Aim #3 our approach is to determine whether cross-breeding a familial DCM mouse model linked to a tropomyosin mutation with the miR208a-KO mouse is able to rescue the DCM phenotype. Experiments proposed here fill a significant gap in our understanding of the control of expression of the isoform population of myocardial TnI, an essential thin filament protein, and test a significant therapeutic approach to cardiac disorders.