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 线状肌病（NM）是一种以肌无力为特征的骨骼肌疾病，发病率为1 在50，000个活产婴儿中。在组织病理学上，有一个明显的存在，肌动蛋白的积累，在破碎的肌肉。 到目前为止，已确定的致病突变发生在对肌肉内肌动蛋白丝结构至关重要的基因中， 这些蛋白质的改变如何导致NM病理学的分子机制还没有被很好地理解。 Cofilin-2是一种受影响的肌动蛋白结合蛋白，它对肌动蛋白丝切断很重要。这个cofilin 同种型是出生后和成熟骨骼肌中的主要形式，其功能主要被研究 相对于肌节处的肌动蛋白，肌肉的主要收缩单位。Cofilin-2在生物化学上 比肌节肌动蛋白更容易与细胞质的非肌节肌动蛋白结合，但这种特性的影响 对NM进展的影响尚未研究。肌肉特异性cofilin（DmCFL）基因敲低的果蝇模型 我们的实验室显示，在生长过程中，与肌节增加有关的进行性肌肉缺陷。我 分析从DmCFL敲低模型的肌肉富集制备物产生的RNA测序数据 并发现与兴奋-收缩偶联（ECC）相关的基因差异表达。ECC是 来自运动神经元的信号传递到肌肉的过程，最终导致 收缩。我的初步数据显示，在神经肌肉的肌肉侧， 连接（NMJ），这是肌肉从运动神经元接收信号的地方。基于文献和 根据这些初步的发现，我推测非肌节肌动蛋白的cofilin调节对于 在肌肉退化之前，ECC需要适当的NMJ和收缩机械结构。到 为了解决这一假设，我将使用DmCFL敲低模型来分析减少的cofilin对 NMJ信号转导（Aim 1）和肌肉收缩（Aim 2）。前者将通过以下方式实现： 使用分子、显微镜和电生理技术分析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 果蝇幼虫肌肉的形态学发现与cofilin-2敲除小鼠肌肉样品的形态学发现。 收缩将使用针对钙信号机制的荧光成像技术进行评估 (Aim 2A）。使用一种改进的锻炼方法（目标2B），我将发现锻炼强度如何影响 DmCFL敲除幼虫的收缩和表型进展。这些实验将共同提供 深入了解NMJ的状态和cofilin NM中的收缩活性，同时利用简单而高的 果蝇的进化保守水平。在回答这个与临床疾病相关的问题时， 这通常体现在生命的早期，我将进一步发展的技术技能和科学推理 作为一个细胞和发育生物学家。这些研究将补充我的临床活动，因为我训练 成为一个全面的医生，科学家和独立的研究人员感兴趣的儿科。