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.

项目概要 2016年，心血管疾病占美国死亡人数的三分之一，并且是导致死亡的主要原因 全球（AHA，2019）。治疗仅限于通过小分子药物或药物进行对症干预 手术，但这些并不能解决心力衰竭的根本分子原因。其中一种机制是 肌球蛋白重链亚型表达失调。肌球蛋白重链 (MyHC) 是运动蛋白 将化学能转化为动能，产生肌肉收缩所需的力。在 在心脏中，表达三种 MyHC 同工型：a-MyHC、b-MyHC 和肌球蛋白重链 7b (MYH7b)。然而， 由于转录后外显子跳跃机制会产生 过早终止密码子。在蛋白质水平上，a-MyHC 和 b-MyHC 的比例严格控制在 10% a-MyHC 至 90% b-MyHC。在晚期心脏病中，a-MyHC 和 b-MyHC 的表达失调； a-MyHC 的比例降低至不可检测的水平，而 b-MyHC 表达增加至基本上 100%。这被认为是一种节省能量的补偿机制，因为 b-MyHC 的 ATP 速度较慢 周转率；然而，它会损害心脏的收缩功能，从而导致心源性死亡。 这种转录转变背后的机制尚不清楚。我们最近发现了这个表达 MYH7b RNA 和 b-MyHC（RNA 和蛋白质）的水平呈正相关，并且 MYH7b 中的变化 表达始终先于 b-MyHC 的表达。此外，通过反义基因敲低 MYH7b 寡核苷酸会导致 b-MyHC 表达减少。我们进行了RNA测序分析 从人源诱导多能干细胞分化而来的心肌细胞，MYH7b 水平降低 核糖核酸。这导致了 MYH7b 控制粘着斑表达的拟议途径的发现 激酶，导致 TEAD3 转录的相关变化，TEAD3 是一种转录增强因子， 增强 b-MyHC 的表达。我们将通过一系列救援实验来验证这条途径。 然后我们将使用基因操作来确定该途径的上位性，以确定每个基因的位置 驻留在路径中。最后，我们想要完全定义这条负责控制转录的途径 通过使用基于 RNA 的亲和纯化鉴定 MYH7b 的分子伴侣，对 b-MyHC 进行分析。我们将使用一个 识别蛋白质和核酸相互作用的综合方法，从而完全定义相互作用组 MYH7b。这项研究将对心脏生物学领域产生巨大影响，因为它将有助于解决古老的问题 心脏 MyHC 转录控制之谜。我假设 MYH7b 充当长非编码 心脏中的 RNA (lncMYH7b) 通过控制粘着斑水平来调节 b-MyHC 的转录 激酶和 TEAD3。在目标 1 中，我将验证并定义这条路径。在目标 2 中，我将确定分子伙伴 lncMYH7b。