The contributions of excitation and contraction to muscle deterioration in a Drosophila model of CFL2 nemaline myopathy

兴奋和收缩对 CFL2 线状肌病果蝇模型肌肉退化的贡献

• 批准号: 10605858
• 负责人: Briana Christophers
• 金额: $ 5.11万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10605858
• 关键词: Actin-Binding Protein; Actins; Action Potentials; Address; Affect; Basic Science; Binding; Biochemical; Biology; Biopsy; Birth; Calcium; Calcium Signaling; Cell Maintenance; Cells; Characteristics; Childhood; Clinical; Clinical Treatment; Communication; Complement; Coupling; Cytoplasm; Data; Defect; Deterioration; Development; Diagnosis; Disease; Drosophila genus; Drosophila melanogaster; Electromyography; Electrons; Electrophysiology (science); Event; Exercise; Functional disorder; Genes; Growth; Histopathology; Human; Image Analysis; Imaging Techniques; Incidence; Ions; Knockout Mice; Larva; Life; Link; Literature; Live Birth; Mechanics; Membrane; Membrane Potentials; Microfilaments; Microscopy; Modeling; Molecular; Morphology; Motor Neurons; Mus; Muscle; Muscle Cells; Muscle Contraction; Muscle Weakness; Muscle function; Muscle hypotonia; Muscular Atrophy; Myopathy; Myosin ATPase; Nemaline Myopathies; Neuromuscular Junction; Neurons; Neurotransmitter Receptor; Organism; Output; Pathology; Patients; Pattern; Pediatrics; Phenotype; Physical shape; Physicians; Play; Preparation; Process; Protein Isoforms; Proteins; Regulation; Research; Research Personnel; Rod; Role; Sampling; Sarcomeres; Sarcoplasmic Reticulum; Scientist; Side; Signal Transduction; Skeletal Muscle; Structure; Synapses; System; Technical Expertise; Techniques; Testing; Training; Work; causal variant; cofilin; cofilin 2; confocal imaging; differential expression; early childhood; exercise intensity; exercise regimen; experimental study; fluorescence imaging; genetic manipulation; human disease; innovation; insight; interest; knock-down; neuronal patterning; neurotransmitter release; novel therapeutics; perinatal period; postnatal; postsynaptic; receptor; response; skeletal muscle weakness; transcriptome sequencing; ultra high resolution

PROJECT SUMMARY Nemaline myopathy (NM) is a skeletal muscle disease hallmarked by muscle weakness with an incidence of 1 in 50,000 live births. On histopathology, there is an obvious presence of actin accumulations in disrupted muscle. The causative mutations identified thus far are in genes critical for actin filament structure within the muscle, yet the molecular mechanisms for how alteration of these proteins leads to NM pathology is not well understood. Cofilin-2, which is important for actin filament severing, is one such affected actin-binding protein. This cofilin isoform is the predominant form in postnatal and mature skeletal muscle; its function has mainly been studied with respect to actin at the sarcomere, the muscle’s main contractile unit. Cofilin-2 is known to biochemically bind more readily to cytoplasmic non-sarcomeric actin than sarcomeric actin, but the impact of this characteristic on NM progression has not been studied. A Drosophila model of muscle-specific cofilin (DmCFL) knockdown was shown by our lab to have progressive muscular defects linked to sarcomere addition during growth. I analyzed RNA sequencing data produced from muscle-enriched preparation of the DmCFL knockdown model and found that genes associated with excitation-contraction coupling (ECC) are differentially expressed. ECC is the process by which signals from the motor neuron are communicated to the muscle ultimately leading to contraction. My preliminary data show disordered actin organization at the muscle side of the neuromuscular junction (NMJ), which is where the muscle receives signals from the motor neuron. Based on the literature and these preliminary findings, I hypothesize that cofilin regulation of non-sarcomeric actin is critical for the proper NMJ and contraction machinery structure needed for ECC prior to muscle deterioration. To address this hypothesis, I will use the DmCFL knockdown model to analyze the impact of decreased cofilin on the NMJ signal transduction (Aim 1) and muscle contraction (Aim 2). The former will be accomplished by using molecular, microscopy, and electrophysiological techniques to analyze changes in NMJ protein localization, morphology, and function at the muscle (Aim 1A) and motor neuron (Aim 1B). I will compare the morphological findings from Drosophila larval muscle to those from cofilin-2 knockout mouse muscle samples. Contraction will be assessed using fluorescent imaging techniques targeted to the calcium signaling machinery (Aim 2A). Using a modified exercise approach (Aim 2B), I will discover how exercise intensity influences contraction and phenotype progression in DmCFL knockdown larva. These experiments will collectively provide insight into the status of the NMJ and contractile activity in cofilin NM while leveraging the simplicity yet high level of evolutionary conservation of Drosophila. In answering this question relevant to a clinical disease that typically manifests early in life, I will further develop the technical skills and scientific reasoning needed as a cell and developmental biologist. These studies will complement my clinical activities as I train to become a well-rounded physician-scientist and independent investigator interested in pediatrics.

项目摘要 Nemaline肌病（NM）是一种骨骼肌肉疾病，其标志是肌肉无力，有1例 在50,000个活产。在组织病理学上，肌动蛋白在肌肉中存在明显存在。 迄今为止鉴定出的结构突变是对肌肉内肌动蛋白丝结构至关重要的基因，但是 这些蛋白质改变如何导致NM病理的分子机制尚不清楚。 Cofilin-2对于肌动蛋白丝的切断很重要，它是这种影响的肌动蛋白结合蛋白。这个cofilin 同工型是产后和成熟骨骼肌的主要形式。它的功能主要是研究 关于肌肉的肌动蛋白，肌肉的主要收缩单元。 Cofilin-2在生化上是已知的 比肉瘤肌动蛋白更容易结合细胞质非骨肌动蛋白，但是这种特征的影响 关于NM的进展尚未研究。肌肉特异性Cofilin（DMCFL）敲低的果蝇模型 我们的实验室表明，在生长过程中具有与肌节添加有关的渐进性肌肉缺陷。我 分析了由DMCFL敲低模型的肌肉增强制备产生的RNA测序数据 并发现与兴奋 - 收缩偶联（ECC）相关的基因被不同表达。 ECC是 电动机神经元信号传达给肌肉的过程最终导致 收缩。我的初步数据显示神经肌肉的肌肉方面无序的肌动蛋白组织 交界处（NMJ），这是肌肉从运动神经元接收信号的地方。基于文学和 这些初步发现，我假设Cofilin的非骨肌动蛋白调节对 在确定肌肉之前，ECC需要适当的NMJ和收缩机械结构。到 解决这一假设，我将使用DMCFL敲低模型来分析Cofilin对降低的影响 NMJ信号转移（AIM 1）和肌肉合同（AIM 2）。前者将通过 使用分子，显微镜和电生理技术来分析NMJ蛋白的变化 肌肉（AIM 1A）和运动神经元（AIM 1B）的定位，形态和功能。我将比较 从果蝇幼虫肌肉到Cofilin-2基因敲除小鼠肌肉样本的形态学发现。 将使用针对钙信号机械的荧光成像技术评估收缩 （AIM 2A）。使用改良的运动方法（AIM 2B），我将发现运动强度如何影响 DMCFL敲低幼虫的收缩和表型进展。这些实验将集体提供 洞悉Cofilin NM中NMJ和收缩活动的状态，同时利用简单而高 果蝇的进化保守水平。在回答与临床疾病有关的问题时 这通常是生命早期的表现，我将进一步发展技术技能和科学推理 需要作为细胞和发育生物学家。当我训练时，这些研究将补充我的临床活动 成为一名全面的身体科学家和独立研究者，对儿科感兴趣。