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Abstract：
Myasthenia gravis (MG) is a rare, treatable, antibody-mediated disease characterized by fatigable muscle weakness of extraocular muscles (EOMs) and non-ocular skeletal muscles. The antibodies are directed against muscle-endplate proteins, most frequently the acetylcholine receptor (AChR) alpha-subunit. Although most MG patients respond to immunosuppressive treatment, some individuals, frequently with African-genetic ancestry, develop treatment-resistant ophthalmoplegia (OP-MG). Although the underlying pathogenetic mechanisms of OP-MG remain unknown, experimental rodent models of MG showed upregulation of genes involved in oxidative metabolism in muscles. EOMs are highly dependent on oxidative metabolism. We opportunistically sampled EOM-tendons of two rare OP-MG patients (and non-MG controls) undergoing re-alignment surgery, and established ocular fibroblast cultures. Metabolic assays were performed on these live cells to assess real-time differences in energy metabolism. To study the cellular bioenergetic profiles in the context of MG, we exposed the cultures to homologous 5% MG sera for 24 h, vs. growth media, from two independent MG patients (with circulating AChR-antibodies) and five controls without MG, and estimated the fold change in oxygen consumption rates in response to three compounds which inhibit different mitochondrial chain complexes. Quantitative PCR (qPCR) was performed in cells before and after MG sera exposure, to assess transcript levels of mitochondrial genes, PDK4, ANGPTL4 and UCP3, which were altered in experimental MG. In response to the mitochondrial stressors, basal oxidative metabolism parameters were similar between OP-MG and control fibroblasts (p = 0.81). However, after exposure to MG sera, bioenergetic parameters (oxygen consumption rate as an indicator of oxidative phosphorylation; extracellular acidification rate as an indicator of glycolysis), were induced to higher levels in OP-MG fibroblasts compared to controls (2.6-fold vs 1.5-fold; p = 0.031) without evidence of mitochondrial insufficiency in the OP-MG ocular fibroblasts. In support of the bioenergetic responses to the same MG sera, gene transcripts of PDK4 and ANGPLT4 in ocular fibroblasts also showed significant upregulation (p ≤ 0.041), but similarly in OP-MG and control cases. Taken together we showed similar basal and metabolic adaptive responses after exposure to mitochondrial inhibitors in ocular fibroblasts derived from OP-MG cases and controls, although the OP-MG cells showed greater activation in response to MG conditions. These pilot results in orbital-derived tissues provide support for myasthenic-induced changes in cellular metabolism and evidence that orbital fibroblasts may be useful for dynamic bioenergetic assessments.
摘要：
重症肌无力（MG）是一种罕见的，可治疗,抗体介导的疾病，其特征是眼外肌（EOM）和非眼部骨骼肌的易疲劳肌无力。这些抗体针对肌肉终板蛋白，最常见的是乙酰胆碱受体（AChR）α亚基。尽管大多数MG患者对免疫抑制治疗有反应，一些人,经常有非洲遗传血统，开发治疗抗性眼肌麻痹（OP-MG）。尽管OP-MG的潜在致病机制尚不清楚，MG的实验性啮齿动物模型显示参与肌肉氧化代谢的基因上调。EOM高度依赖于氧化代谢。我们对两名罕见的OP-MG患者（和非MG对照）进行重新对准手术的EOM肌腱进行了机会采样，并建立了眼成纤维细胞培养物。对这些活细胞进行代谢测定以评估能量代谢的实时差异。为了研究MG背景下的细胞生物能量谱，我们将培养物暴露于同源5%MG血清24小时，vs.生长培养基,来自两名独立的MG患者（具有循环AChR抗体）和五名无MG的对照，并估计了响应于抑制不同线粒体链复合物的三种化合物的耗氧率的倍数变化。在MG血清暴露前后的细胞中进行定量PCR（qPCR），评估线粒体基因的转录水平，PDK4，ANGPTL4和UCP3在实验性MG中发生了变化。为了应对线粒体应激源，OP-MG和对照成纤维细胞的基础氧化代谢参数相似(p=0.81).然而,暴露于MG血清后，生物能量参数（耗氧率作为氧化磷酸化的指标；细胞外酸化率作为糖酵解的指标），与对照组相比，OP-MG成纤维细胞被诱导至更高的水平（2.6倍vs1.5倍；p=0.031），而OP-MG眼成纤维细胞中没有线粒体功能不全的证据。为了支持对相同MG血清的生物能量反应，眼成纤维细胞中PDK4和ANGPLT4的基因转录物也显示出显着的上调（p≤0.041），但在OP-MG和对照病例中相似。总之，我们在暴露于来自OP-MG病例和对照的眼成纤维细胞中的线粒体抑制剂后显示出相似的基础和代谢适应性反应。尽管OP-MG细胞对MG条件的反应显示出更大的活化。这些轨道衍生组织的先导结果为肌无力诱导的细胞代谢变化提供了支持，并证明了轨道成纤维细胞可能用于动态生物能量评估。
