Modulation of Ca Control of Cardiac Myofibrils

心肌原纤维 Ca2+ 控制的调节

• 批准号: 8321473
• 负责人: MADHU GUPTA
• 金额: $ 39.85万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-18 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8321473
• 关键词: 3' Untranslated Regions; Acidosis; Address; Adrenergic Agents; Adult; Affect; Animal Model; Birth; Breeding; Calcium; Cardiac; Cardiac Myocytes; Cardiomyopathies; Connexin 43; DNA Binding; Data; Depressed mood; Dilated Cardiomyopathy; Down-Regulation; Embryo; Embryonic Heart; Experimental Models; Fetal Heart; Functional RNA; Functional disorder; Gene Expression; Gene Expression Regulation; Genetic; Goals; Growth; Heart; Heart Diseases; Heart failure; Hypothyroidism; In Situ; Indium; Kinetics; Knock-out; Lead; Life; Link; Mechanics; Mediating; MicroRNAs; Microfilaments; Modification; Mus; Muscle; Muscle Cells; Muscle Contraction; Mutation; Myocardial; Myocardium; Myofibrils; Myosin ATPase; Neonatal; Patients; Phenotype; Phosphorylation; Phosphotransferases; Population; Post-Translational Protein Processing; Property; Protein Isoforms; Proteins; Proteolysis; Rattus; Regenerative Medicine; Regulation; Relative (related person); Repression; Research Proposals; Research Support; Resistance; Role; Signal Transduction; Skeletal Muscle; Skin; Stress; Testing; Therapeutic; Thin Filament; Thyroid Gland; Thyroid Hormones; Transgenic Mice; Transgenic Organisms; Tropomyosin; Troponin; Troponin I; adrenergic; alpha Tropomyosin; angiogenesis; cardiogenesis; design; fetal; improved; in vivo; loss of function; mouse model; postnatal; prevent; research study; response; skeletal; transcription factor; translational medicine

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.

描述（由申请人提供）：我们在这里提出的实验的长期目标是确定在出生后心脏成熟过程中发生的慢速骨骼肌钙蛋白I （ssTnI）亚型下调的机制。初步数据支持了我们的假设，即microRNA miR-208a参与了出生后ssTnI在心脏表达的沉默。我们的实验继续支持该提案的研究，证明转基因小鼠成年心脏中ssTnI的表达可以保护心肌免受包括家族性扩张性心肌病（DCM）在内的常见应激。初步数据表明：i)在发育中的心肌细胞中，miR-208a与肌肉特异性miRs、miR-1和miR-133a的表达增加，ii) 208a-antagomiR在心肌细胞中敲低miR-208a可诱导ssTnI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与其他共调控的miRNA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)评估ssTnI作为miRNA介导的调控的直接靶点。目的2：确定miR-208a KO对细丝蛋白修饰的相对影响，对肌丝对Ca的反应，对肌细胞力学和Ca以及对原位心功能的影响。目标# 3。确定miR-208a表达降低是否能改善与α -原肌球蛋白突变相关的扩张型心肌病（DCM）的心功能。我们针对Aim #1的方法包括i)通过功能获得和功能丧失方法分析新生儿心肌细胞中ssTnI的表达，以剖析甲状腺相关信号在ssTnI抑制中的相对重要性；ii)通过与其他mir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靶向转录因子并测定其DNA结合和表达；iv)鉴定miR-208a在ssTnI 3'UTR中的靶点。我们对目标#2的研究方法包括将miR208a-KO小鼠与对照组进行比较，以确定肌丝特性改变对功能的影响，这反映在异构体群体、翻译后修饰、pca -力关系和过桥动力学中。皮肤肌细胞的研究结果通过心肌细胞水平的Ca2+瞬态和力学研究，以及原位跳动心脏对应力的响应动力学，包括那些由细丝特性改变主导的应力，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相关的家族性DCM小鼠模型与miR208a-KO小鼠杂交是否能够挽救DCM表型。本文提出的实验填补了我们对心肌TnI（一种必需的细丝蛋白）异构体群体表达控制的理解的重大空白，并测试了一种重要的心脏疾病治疗方法。