Functional genomics in zebrafish to disset the pathogenesis of myofibrillar myopathies

为了在该联盟内开发针对人类肌原纤维肌病（MFM）的新型靶向治疗策略，我们在第一个资助期内的研究旨在阐明在斑马鱼模型中使用正向和反向遗传方法将已知MFM突变转化为肌病表型的遗传基础和机制。对已知MFM疾病基因的功能丧失研究表明，MFM基因缺陷的斑马鱼在没有蛋白质聚集迹象的情况下发生心脏和骨骼肌肌病，这表明聚集不是斑马鱼肌病的主要触发因素。我们发现，有针对性地耗尽推定的MFM疾病基因内确定的财团（如Strumpelin，KIAA 1033）导致严重的肌病表型影响，心脏和骨骼肌，在斑马鱼胚胎。此外，我们产生了过表达选定的人类MFM突变瞬时或诱导和稳定（转基因）的方式斑马鱼。转基因株系现在将使我们能够评估MFM的病理生理学为选定的人类突变。在第二个资助期内，我们将专注于详细描述新的MFM疾病基因，Nexilin，确定了这个财团。该项目的具体目标1是从功能、结构、分子和生物力学方面表征我们在第一个资助期产生的Nexilin敲除小鼠，以及稳定过表达与肌病相关的人Nexilin突变的斑马鱼。具体目标2是在选定的功能丧失和转基因斑马鱼MFM疾病模型中，破译MFM疾病基因缺陷对肌纤维功能和结构的直接影响。最后，具体目标3是评估最近在该联盟内通过各种方法确定的潜在新型MFM疾病基因对斑马鱼心脏和骨骼肌功能的影响/相关性。

In an attempt to develop within this consortium novel targeted treatment strategies for human myofibrillar myopathies (MFMs) our research within the first funding period aimed to elucidate the genetic basis and the mechanisms that translate known MFM mutations into the myopathic phenotype using forward and reverse genetic approaches in the zebrafish model. Loss-of-function studies of known MFM disease genes reveal that MFM genedeficient zebrafish develop heart and skeletal muscle myopathies without signs of protein aggregations, suggesting that aggregates are not the main trigger of myopathy in the zebrafish. We find that targeted depletion of putative MFM disease genes identified within this consortium (e.g. Strumpellin, KIAA1033) lead to severe myopathic phenotypes affecting both, heart and skeletal muscle, in zebrafish embryos. Additionally, we generated zebrafish that overexpress selected human MFM mutations transiently or in an inducible and stable (transgenic) manner. The transgenic lines will now allow us to evaluate MFM pathophysiology for selected human mutations.Within the second funding period we will focus on the detailed characterization of the novel MFM disease gene, Nexilin, identified by this consortium. Specific aim 1 of this project is to functionally, structurally, molecularly as well as biomechanically characterize Nexilinknockout mice which we generated during the first funding period, as well as zebrafish stably overexpressing human Nexilin mutations associated with myopathies. Specific aim 2 is to decipher the direct effects of MFM disease gene deficiency on myofiber function and structure in selected loss-of-function and transgenic zebrafish MFM disease models. Finally, specific aim 3 is to evaluate the impact/relevance of potential novel MFM disease genes recently identified within this consortium by various approaches on heart and skeletal muscle function in zebrafish.