Investigating membrane repair capacity in facioscapulohumeral muscular dystrophy

摘要 面肩肱型肌营养不良症是一种常染色体显性遗传性肌肉疾病 由复杂的遗传和表观遗传机制引起。以前的研究表明，转录去- 由于D4 Z4区域的表观遗传变化导致双同源框蛋白4（DUX 4）的抑制， FSHD。表观遗传变化是由D4 Z4阵列从11-150个重复的收缩引起的， 未受影响个体中的1-10个重复单位到大约95%患者中的1-10个重复单位（FSHD 1），或 D4 Z4区域的表观遗传调节因子（FSHD 2）。DUX 4的表达导致下游的 分子和细胞的变化，这有助于疾病的进展。然而，细胞 引起FSHD的机制尚不清楚。在我们的初步研究中， FSHD成肌细胞与来自未受影响同胞的成肌细胞相比的缺陷。中有降 FSHD细胞中的DUX 4部分提高了修复能力。这种修复缺陷也被观察到， 来自FSHD样小鼠模型（FLExDUX 4）的骨骼肌。已知FSHD成肌细胞是 更容易受到氧化应激的影响。虽然活性氧（ROS）的调节起着重要的作用， 在肌膜修复中起重要作用，细胞中过度或延长的氧化应激导致 膜修复缺陷。我们的新发现提供了一个合理的联系， DUX 4（例如氧化应激）的错误调节和继发于减少的FSHD表型 膜修复能力。该研究的目的是进一步调查观察到的膜修复 缺陷和测试的假设，膜修复能力是由DUX 4水平调节， 肌纤维在目标1中，我们将确定DUX 4表达是否对肌膜细胞具有剂量依赖性作用。 使用骨骼肌特异性诱导小鼠模型和反义寡核苷酸修复缺陷 其可以调节FLExDXU 4小鼠中DUX 4的表达。修复缺陷的分子机制将 追究在目标2中，我们将评估DUX 4对FSHD患者肌膜修复的作用 使用FSHD的异种移植小鼠模型测量肌纤维。我们将生产人类肌纤维并测试它们的 肌膜修复能力，并确定AON介导的DUX 4敲低是否影响肌膜修复能力。 膜修复能力。这项研究将确定一种新的机制， 疾病表型的分子缺陷。此外，拟议的研究将确定是否 膜修复可以是一个适当的急性读数的治疗方法，旨在减少 DUX 4及其相关通路在FSHD骨骼肌中的作用。

ABSTRACT Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant muscle disorder caused by complex genetic and epigenetic mechanisms. Previous studies showed that transcription de- repression of double homeobox protein 4 (DUX4) due to epigenetic changes in the D4Z4 region causes FSHD. The epigenetic changes are caused by either contraction of the D4Z4 array from 11-150 repeat units in unaffected individuals to 1-10 repeat units in roughly 95% of patients (FSHD1), or mutations in epigenetic regulators of the D4Z4 region (FSHD2). The expression of DUX4 leads to downstream molecular and cellular changes, which contribute to disease progression. However the cellular mechanisms cause FSHD are not clear. In our preliminary studies we identified cell membrane repair deficit in FSHD myoblasts in comparison to myoblasts from unaffected siblings. Moderate reduction of DUX4 in the FSHD cells partially improved the repair capacity. This repair deficit was also observed in skeletal muscle from an FSHD-like mouse model (FLExDUX4). It is known that FSHD myoblasts are more susceptible to oxidative stress. While reactive oxygen species (ROS) regulation plays an important role in sarcolemmal membrane repair, excessive or prolonged oxidative stress in cells lead to membrane repair deficits. Our novel finding provides a plausible link between the molecular pathways mis-regulated by DUX4 (e.g. oxidative stress) and the FSHD phenotype secondary to reduced membrane repair capacity. The goal of the study is to further investigate the observed membrane repair deficit and test the hypothesis that the membrane repair capacity is modulated by DUX4 levels in myofibers. In aim 1, we will determine if DUX4 expression has a dose-dependent effect on sarcolemmal repair deficit using skeletal muscle-specific inducible mouse model and an antisense oligonucleotide that can modulate DUX4 expression in FLExDXU4 mice. Molecular mechanisms of repair deficits will be investigated. In aim 2, we will evaluate the effect of DUX4 on sarcolemmal repair in FSHD patient myofibers using a xenograft mouse model of FSHD. We will produce human myofibers and test their sarcolemmal repair ability and determine whether AON-mediated knockdown of DUX4 affects the membrane repair ability. The study will identify a novel mechanism, which links previously reported molecular deficits to the disease phenotypes. In addition, the proposed study will determine whether membrane repair can be an appropriate acute readout for therapeutic approaches that aim to reduce DUX4, and its related pathways, in FSHD skeletal muscle.