Function of Giant Sarcomere Matrix Proteins in Muscle

肌肉中巨肌节基质蛋白的功能

• 批准号: 9237049
• 负责人: Henk L. GRANZIER
• 金额: $ 38.38万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-01 至 2021-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9237049
• 关键词: Acute; Address; Alternative Splicing; Animal Model; Animals; Area; Attenuated; Calcium; Cardiac; Cardiac Myocytes; Cardiovascular system; Cavia; Cells; Characteristics; Chronic; Clinical; Complex; Cyclic GMP-Dependent Protein Kinases; DOCA; Data; Development; Dilated Cardiomyopathy; Disease; EFRAC; Elements; Extracellular Matrix; Failure; Follow-Up Studies; Functional disorder; Gene Targeting; Genetic; Genetic Models; Genetic Transcription; Heart; Heart Rate; Heart failure; Human; Immunoglobulin Domain; Knockout Mice; Left; Measures; Mechanics; Metformin; Microfilaments; Modeling; Molecular; Mus; Muscle; Mutation; Myocardial; Myosin Heavy Chains; Patients; Pharmaceutical Preparations; Pharmacology; Phenotype; Phosphorylation; Pilot Projects; Post-Translational Protein Processing; Protein Isoforms; Proteins; RNA Recognition Motif; RNA Splicing; Relaxation; Research; Role; Sarcomeres; Signal Pathway; Signal Transduction; Signaling Protein; Site; Stress; Therapeutic; Treatment Failure; Ventricular; Work; base; connectin; effective therapy; experimental study; heart cell; improved; in vivo; inhibitor/antagonist; insight; insulin sensitizing drugs; mouse model; novel; phosphoric diester hydrolase; pre-clinical; pressure; protein expression; transcriptome sequencing

Titin, the largest protein known, functions as a complex molecular spring that is a dominant contributor to passive myocardial stiffness. Importantly, titin’s stiffness can be tuned post-transcriptionally (by varying the expression ratio of the stiff N2B and more compliant N2BA titin isoforms) and post-translationally (e.g., via changes in protein kinase G (PKG) phosphorylation of titin). Titin’s stiffness is increased in heart failure with preserved ejection fraction (HFpEF), due to deranged titin phosphorylation, in particular hypo-phosphorylation of PKG sites. Currently no effective therapies for HFpEF exist. This application studies potential titin-based treatment options. For this work we have available several animal models of HFpEF: a genetic mouse model in which titin’s spring region is extended to a higher degree and passive stiffness is increased accordingly and pressure-overload (TAC/DOCA) induced mouse and guinea pig models that have diastolic dysfunction and deranged titin phosphorylation. The potential of existing drugs to ameliorate titin-based diastolic stiffening in HFpEF will be addressed in Aims 1 and 2. Metformin is an insulin sensitizing drug that has been shown to improve diastolic function in animal and human studies. Our pilot studies show that metformin rescues diastolic dysfunction and normalizes titin-based stiffness. The phosphodiesterase PDE9A was recently shown to reduce PKG activity and it is known that hypo-phosphorylation of titin’s PKG sites occurs in HFpEF. Hence we also study whether PDE9A inhibition (PDE9Ai) ameliorates diastolic dysfunction. Recent studies showed that titin mutations are causative for dilated cardiomyopathy (DCM), a prevalent form of HF characterized by progressive left ventricular (LV) dilation and systolic dysfunction. Aim 3 seeks to boost understanding of mechanisms by which titin can cause DCM. Through gene targeting we generated the first titin- based mouse model that under baseline conditions develops DCM, the N2BA-PEVK KO. In this model, PEVK sequences that are specific to the N2BA titin isoform were deleted and pilot studies show that this causes severe dilation and a reduction in ejection fraction. DCM-causing mechanisms will be studied at a preclinical stage (2 mo) and after the heart dilates (6 mo), using both an unbiased approach and a candidate approach that focusses on mechanisms that are unique to the N2BA isoform. A rescue experiment is included in which the stress on titin’s spring will be reduced by targeting the titin splicing factor RBM20. With its basic and clinically important research and its in-depth and integrative approach, this proposal seeks to continue our track record of cutting edge titin research. We anticipate that these studies will greatly improve understanding of the roles of titin in HFpEF and provide novel insights in therapeutic options. We also will study a novel mouse model with titin-based DCM and investigate mechanisms by which titin can cause DCM, an area that also needs urgent study. We have supportive pilot data and have an excellent research team in place.

肌动蛋白是已知的最大的蛋白质，其功能是一个复杂的分子弹簧，它是 被动心肌僵硬。重要的是，Titin的硬度可以在转录后进行调整(通过改变 僵硬的N2B和更顺从的N2BA的表达比率)和翻译后(例如，通过 蛋白激酶G(PKG)磷酸化的变化)。在心力衰竭患者中，Titin的僵硬程度随着 保留射血分数(HFpEF)，由于肌动蛋白磷酸化，特别是低磷酸化 PKG站点的数量。目前还没有有效的治疗HFpEF的方法。这个应用程序研究潜在的基于titin的 治疗方案。在这项工作中，我们提供了几种HFpEF的动物模型： 其中Titin的弹簧区域被延伸到更高的程度，并且被动刚度相应地增加 压力超负荷(TAC/DOCA)诱导的小鼠和豚鼠模型具有舒张期功能障碍和 错乱的肌动蛋白磷酸化。现有药物改善以肌钙蛋白为基础的舒张期硬化的潜力 HFpEF将在AIMS 1和2中解决。二甲双胍是一种胰岛素增敏药物，已被证明 改善动物和人体研究中的舒张期功能。我们的初步研究表明，二甲双胍挽救了舒张压 功能障碍，并使基于Titin的僵硬正常化。磷酸二酯酶PDE9A最近显示出减少 PKG活性，并且已知在HFpEF中发生了Titin的PKG位点的低磷酸化。因此我们也 研究PDE9A抑制剂(PDE9Ai)是否能改善舒张期功能障碍。 最近的研究表明，titin突变是扩张型心肌病(DCM)的病因，扩张型心肌病是一种流行的形式 以进行性左心室(LV)扩张和收缩功能障碍为特征的心衰。Aim 3寻求推动 了解肌动蛋白引起扩张性心肌病的机制。通过基因打靶，我们产生了第一个Titin- 在基线条件下建立DCM小鼠模型，建立N2BA-PEVK KO。在此模型中，PEVK 针对N2BA肌动蛋白亚型的序列被删除，初步研究表明，这会导致严重的 扩张和射血分数的减少。将在临床前阶段研究引起DCM的机制(2 Mo)和心脏扩张后(6个月)，使用无偏见的方法和聚焦的候选方法 关于N2BA亚型独有的机制。其中包括一项救援实验，在该实验中， 通过靶向Titin剪接因子RBM20，Titin的弹簧将会减少。 凭借其基础和临床重要研究以及深入和综合的方法，这项建议寻求 以延续我们在尖端技术研究方面的记录。我们预计这些研究将大大改善 了解Titin在HFpEF中的作用，并为治疗方案提供新的见解。我们也会研究 一种新的基于Titin的DCM小鼠模型的建立及其引起DCM的机制研究 这也需要迫切的研究。我们有支持性的试点数据，并有一支优秀的研究团队。