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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要线状肌病 (NM) 是一种以肌肉无力为特征的骨骼肌疾病，发病率为 1 在 50,000 名活产儿中，在组织病理学上，受损的肌肉中明显存在肌动蛋白积聚。迄今为止发现的致病突变位于对肌肉内肌动蛋白丝结构至关重要的基因中，但这些蛋白质的改变如何导致 NM 病理的分子机制尚不清楚。 Cofilin-2 对肌动蛋白丝的切断很重要，它就是一种受影响的肌动蛋白结合蛋白。同工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。就生化而言，肌节的肌动蛋白是肌肉的主要收缩单位 Cofilin-2。与肌节肌动蛋白相比，更容易与细胞质非肌节肌动蛋白结合，但这一特性的影响尚未研究肌肉特异性丝切蛋白 (DmCFL) 敲低的果蝇模型。我们的实验室表明，在生长过程中存在与肌节添加相关的进行性肌肉缺陷 I。分析了 DmCFL 敲低模型的肌肉富集制剂产生的 RNA 测序数据并发现与兴奋-收缩耦合（ECC）相关的基因存在差异表达。来自运动神经的信号传递到肌肉的过程，最终导致我的初步数据显示神经肌肉肌肉侧的肌动蛋白组织紊乱。根据文献和研究，连接点（NMJ）是肌肉接收运动神经信号的地方。这些初步发现表明，丝切蛋白对非肌节肌动蛋白的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。为了解决这个假设，我将使用 DmCFL 敲低模型来分析 cofilin 减少对 NMJ 信号转导（目标 1）和肌肉收缩（目标 2）将通过前者来完成。使用分子、显微镜和电生理技术分析 NMJ 蛋白的变化我将比较肌肉（目标 1A）和运动神经（目标 1B）的定位、形态和功能。果蝇幼虫肌肉与 cofilin-2 敲除小鼠肌肉样本的形态学发现。将使用针对钙信号机制的荧光成像技术来评估合同（目标 2A）。使用修改后的锻炼方法（目标 2B），我将发现锻炼强度如何影响。这些实验将共同提供 DmCFL 敲低幼虫的收缩和表型进展。洞察 NMJ 的状态和 cofilin NM 的收缩活动，同时利用简单而高效的方法在回答与临床疾病相关的问题时，果蝇的进化保守水平。这通常在生命早期表现出来，我将进一步发展技术技能和科学推理作为一名细胞和发育生物学家，这些研究将补充我训练时的临床活动。成为一名对儿科感兴趣的全面的医师科学家和独立研究者。