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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.

项目总结