Correlation of Utrophin Levels with the Dystrophin Protein Complex and Muscle Fibre Regeneration in Duchenne and Becker Muscular Dystrophy Muscle Biopsies.
Correlation of Utrophin Levels with the Dystrophin Protein Complex and Muscle Fibre Regeneration in Duchenne and Becker Muscular Dystrophy Muscle Biopsies.
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DOI：
10.1371/journal.pone.0150818
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Tinsley J
中科院分区：
文献类型：
--
作者：
Janghra N;Morgan JE;Sewry CA;Wilson FX;Davies KE;Muntoni F;Tinsley J
杜氏肌营养不良症是一种严重的，目前无法治愈的进行性神经肌肉疾病，由DMD基因突变引起，导致无法产生肌营养不良蛋白。肌营养不良蛋白的缺乏导致肌肉纤维的损失和肌肉质量和功能的减少。肌营养不良蛋白缺陷小鼠模型的证据表明，通过遗传或药理学手段增加肌纤维肌膜中肌营养不良蛋白的水平显著降低了肌营养不良症的病理学。为了确定肌营养蛋白调节剂在临床试验中的功效，有必要在活检切片内逐个纤维地精确测量肌营养蛋白水平和其他生物标志物。我们的目的是开发强大的和可重复的染色和成像协议，以量化肌膜utrophin水平，肌膜肌营养不良蛋白复合物成员和活检切片内再生纤维的数量。我们定量肌膜肌营养不良蛋白在成熟和再生的纤维和再生的肌纤维的百分比，在肌肉活检杜氏，轻度贝克尔肌营养不良症和控制。进行荧光免疫染色，然后进行图像分析，以定量肌营养不良蛋白强度和肌膜处的β-肌营养不良蛋白聚糖和β-肌聚糖强度。胎儿和发育肌球蛋白的抗体用于识别再生肌纤维，从而准确计算活检中再生纤维的百分比。我们的研究结果表明，从贝克尔肌营养不良症患者的肌肉活检有较少数量的再生纤维和肌营养蛋白强度降低相比，从杜氏肌营养不良症患者的肌肉活检。特别令人感兴趣的是，我们首次表明，肌肉活检中再生肌纤维的百分比与Becker和Duchenne肌营养不良症患者的临床严重程度相关。正在进行的开发这些工具来量化肌膜肌营养蛋白和肌肉活检中的肌肉再生将是非常宝贵的，在未来的临床试验中评估肌营养蛋白调节剂的活性。
Duchenne muscular dystrophy is a severe and currently incurable progressive neuromuscular condition, caused by mutations in the DMD gene that result in the inability to produce dystrophin. Lack of dystrophin leads to loss of muscle fibres and a reduction in muscle mass and function. There is evidence from dystrophin-deficient mouse models that increasing levels of utrophin at the muscle fibre sarcolemma by genetic or pharmacological means significantly reduces the muscular dystrophy pathology. In order to determine the efficacy of utrophin modulators in clinical trials, it is necessary to accurately measure utrophin levels and other biomarkers on a fibre by fibre basis within a biopsy section. Our aim was to develop robust and reproducible staining and imaging protocols to quantify sarcolemma utrophin levels, sarcolemma dystrophin complex members and numbers of regenerating fibres within a biopsy section. We quantified sarcolemmal utrophin in mature and regenerating fibres and the percentage of regenerating muscle fibres, in muscle biopsies from Duchenne, the milder Becker muscular dystrophy and controls. Fluorescent immunostaining followed by image analysis was performed to quantify utrophin intensity and β-dystrogylcan and ɣ –sarcoglycan intensity at the sarcolemma. Antibodies to fetal and developmental myosins were used to identify regenerating muscle fibres allowing the accurate calculation of percentage regeneration fibres in the biopsy. Our results indicate that muscle biopsies from Becker muscular dystrophy patients have fewer numbers of regenerating fibres and reduced utrophin intensity compared to muscle biopsies from Duchenne muscular dystrophy patients. Of particular interest, we show for the first time that the percentage of regenerating muscle fibres within the muscle biopsy correlate with the clinical severity of Becker and Duchenne muscular dystrophy patients from whom the biopsy was taken. The ongoing development of these tools to quantify sarcolemmal utrophin and muscle regeneration in muscle biopsies will be invaluable for assessing utrophin modulator activity in future clinical trials.