Understanding the Role of the Non-coding Variant of MYH7b in the Regulation of Beta Myosin Heavy Chain

了解 MYH7b 非编码变体在β肌球蛋白重链调节中的作用

• 批准号: 10383133
• 负责人: Lindsey Broadwell
• 金额: $ 2.39万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2021-12-06
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10383133
• 关键词: ATP Hydrolysis; Address; Affect; Affinity Chromatography; Alternative Splicing; Antisense Oligonucleotides; Binding; Binding Proteins; Biological; Biology; Brain; Cardiac; Cardiac Death; Cardiac Myocytes; Cardiac Myosins; Cardiovascular Diseases; Cause of Death; Cessation of life; Chemicals; Contracts; Diagnosis; Down-Regulation; Enhancers; Equilibrium; Exons; Family; Focal Adhesion Kinase 1; Foundations; Gene Expression; Genes; Genetic Epistasis; Genetic Transcription; Head; Heart; Heart Diseases; Heart failure; Human; Human Genome Project; Immunoprecipitation; Intervention; Introns; Kinetics; Lead; Mammals; MicroRNAs; Microfilaments; Molecular; Molecular Motors; Motor; Muscle Contraction; Muscle Proteins; Muscle Spindles; Myosin ATPase; Myosin Heavy Chains; Myosin Phosphatase Pathway; Nonmuscle Myosin Type IIA; Nonmuscle Myosin Type IIB; Nonsense Codon; Nucleic Acids; Operative Surgical Procedures; Organ; Pathway interactions; Patients; Pattern; Personal Communication; Pharmaceutical Preparations; Phosphotransferases; Protein Isoforms; Proteins; Protocols documentation; RNA; Regulation; Research; Research Project Grants; Rod; Role; Series; Skeletal Muscle; Striated Muscles; Therapeutic; Thick Filament; Tissues; Transcript; Transcriptional Regulation; United States; Untranslated RNA; Variant; base; beta-Myosin; enhancing factor; exon skipping; experimental study; genetic manipulation; heart function; human old age (65+); induced pluripotent stem cell; knock-down; non-muscle myosin heavy chain-B; novel; orbit muscle; overexpression; preservation; small molecule; transcription factor; transcriptome sequencing

PROJECT SUMMARY Cardiovascular disease accounted for 1 in 3 deaths in the USA in 2016 and was the leading cause of death worldwide (AHA, 2019). Treatment is limited to symptomatic interventions through small molecule drugs or surgery, but these do not address the underlying molecular causes of heart failure. One such mechanism is the dysregulation of myosin heavy chain isoform expression. Myosin heavy chains (MyHC) are the motor proteins that convert chemical energy into kinetic energy to produce the force necessary for muscle contraction. In the heart, three MyHC isoforms are expressed: a-MyHC, b-MyHC, and Myosin Heavy Chain 7b (MYH7b). However, MYH7b is not translated to protein due to a post-transcriptional exon-skipping mechanism that produces a premature stop codon. At the protein level, a-MyHC and b-MyHC exist in a carefully controlled ratio of 10% a-MyHC to 90% b-MyHC. In late-stage heart diseases, the expression of a-MyHC and b-MyHC is dysregulated; the proportion of a-MyHC is reduced to undetectable levels, while b-MyHC expression increases to essentially 100%. This is thought to be a compensatory mechanism to conserve energy, as b-MyHC has a slower ATP- turnover rate; however, it compromises the contractile function of the heart and can thus lead to cardiac death. The mechanism behind this transcriptional shift is not understood. We have recently discovered that expression levels of MYH7b RNA and b-MyHC (both RNA and protein) positively correlate, and that changes in MYH7b expression always precede those of b-MyHC. Furthermore, knockdown of MYH7b by anti-sense oligonucleotides causes a decrease in b-MyHC expression. We have performed RNA-sequencing analysis in cardiomyocytes differentiated from human-derived induced pluripotent stem cells with reduced levels of MYH7b RNA. This lead to a discovery of a proposed pathway where MYH7b controls the expression of focal adhesion kinase, leading to a correlating change in the transcription of TEAD3, a transcription enhancer factor that enhances the expression of b-MyHC. We will validate this pathway through a series of rescue experiments. Then we will determine the epistasis of the pathway using genetic manipulations to determine where each gene resides in the pathway. Finally, we want to fully define this pathway responsible for controlling the transcription of b-MyHC by identifying the molecular partners of MYH7b, using RNA-based affinity purification. We will use a comprehensive approach to identify both protein and nucleic-acid interactions, thus fully defining the interactome of MYH7b. This research will have a large impact on the field of cardiac biology, as it will help solve the age-old puzzle of MyHC transcriptional control in the heart. I hypothesize that MYH7b is acting as a long non-coding RNA (lncMYH7b) in the heart to regulate the transcription of b-MyHC through controlling levels of focal adhesion kinase and TEAD3. In Aim 1 I will validate and define this pathway. In Aim 2, I will identify the molecular partners of lncMYH7b.

项目总结 2016年，心血管疾病占美国死亡人数的三分之一，是主要的死亡原因 全球(AHA，2019年)。治疗仅限于通过小分子药物或 手术，但这些并不能解决心力衰竭的潜在分子原因。一种这样的机制是 肌球蛋白重链亚型表达失调。肌球蛋白重链(MyHC)是马达蛋白 将化学能转化为动能，以产生肌肉收缩所需的力量。在 心脏，表达三种MyHC亚型：a-MyHC、b-MyHC和肌球蛋白重链7b(MYH7b)。然而， MYH7b没有翻译成蛋白质，这是因为转录后外显子跳过机制产生了一种 过早终止密码子。在蛋白质水平上，a-MyHC和b-MyHC以10%的精心控制的比例存在 A-MyHC降至90%b-MyHC。在晚期心脏病中，a-MyHC和b-MyHC的表达失衡； A-MyHC的比例降低到检测不到的水平，而b-MyHC的表达增加到基本上 100%这被认为是一种保存能量的补偿机制，因为b-MyHC具有较慢的ATP- 周转率；然而，它损害心脏的收缩功能，从而可能导致心源性死亡。 这种转录变化背后的机制尚不清楚。我们最近发现，这句话 MYH7b RNA和b-MyHC(RNA和蛋白质)的水平呈正相关，MYH7b的变化 B-MyHC的表达总是先于b-MyHC。此外，MYH7b的反义敲除 寡核苷酸导致b-MyHC表达下降。我们已经进行了RNA测序分析 MYH7b表达水平降低的人源性诱导多能干细胞分化为心肌细胞 核糖核酸。这导致了一个被提出的途径的发现，其中MYH7b控制着焦点黏附的表达 激酶，导致转录增强因子TEAD3转录的相关变化 增强b-MyHC的表达。我们将通过一系列救援实验来验证这一途径。 然后，我们将使用基因操作来确定每个基因的位置，从而确定该路径的上位性 驻留在路径中。最后，我们想要完全定义负责控制转录的这条途径 通过基于RNA的亲和纯化，鉴定MYH7b的分子伴侣，从而获得b-MyHC。我们将使用 确定蛋白质和核酸相互作用的综合方法，从而充分定义相互作用组 MYH7b。这项研究将对心脏生物学领域产生很大的影响，因为它将有助于解决 心脏中MyHC转录调控之谜。我假设MYH7b是作为一个长的非编码 心脏中的RNA(LncMYH7b)通过控制局部黏附水平来调节b-MyHC的转录 蛋白激酶和TEAD3。在目标1中，我将验证和定义这条途径。在目标2中，我将确定分子伙伴 IncMYH7b。