Guo Y, Petrof B J, Hussain S N
Critical Care Division, Royal Victoria Hospital, Room L3.05, 687 Pine Avenue West, Montréal, Québec H3A 1A1, Canada.
Muscle Nerve. 2001 Nov;24(11):1468-75. doi: 10.1002/mus.1170.
In skeletal muscle fibers, nitric oxide is synthesized by neuronal nitric oxide synthase (nNOS), which normally associates with the dystrophin complex in close proximity to the sarcolemma. Many reports have documented that very low levels of nNOS protein exist in muscle fibers of Duchenne muscular dystrophy (DMD) patients. In this study we investigated the functional significance of PIN (protein inhibitor of nNOS) in targeting of nNOS to the sarcolemma and the association between nNOS and the dystrophin complex in normal and dystrophic muscle fibers. Northern blotting for PIN mRNA in normal mouse muscles and muscles of mdx mice (an animal model of DMD) revealed a significant rise in PIN mRNA in dystrophic muscles compared with normal muscles. Immunohistochemical analysis showed that, in normal mouse muscle fibers, PIN expression was localized at the sarcolemma, peripheral nuclei, and the sarcoplasm. By comparison, PIN protein in muscles from mdx mice was more concentrated around the sarcolemma and central nuclei. The presence of PIN protein expression in muscles from mdx mice was evident despite the significant reduction in nNOS and dystrophin protein expressions in these fibers. In muscle sections of DMD patients, the absence of nNOS protein expression was accompanied by maintained PIN expression. Prominent PIN expression was also detectable in macrophages infiltrating dystrophic muscle fibers both in mdx mice and DMD patients. These results suggest that PIN expression in muscles from mdx mice and DMD patients is controlled by factors different from those involved in the regulation of nNOS and dystrophin. Moreover, our results indicate that PIN is not an integral component of the dystrophin complex inside skeletal muscle fibers.
在骨骼肌纤维中，一氧化氮由神经元型一氧化氮合酶（nNOS）合成，该酶通常与肌营养不良蛋白复合物在肌膜附近结合。许多报告记录了杜氏肌营养不良症（DMD）患者的肌肉纤维中存在极低水平的nNOS蛋白。在本研究中，我们调查了nNOS蛋白抑制剂（PIN）在将nNOS靶向至肌膜以及正常和营养不良性肌肉纤维中nNOS与肌营养不良蛋白复合物之间关联方面的功能意义。对正常小鼠肌肉和mdx小鼠（DMD动物模型）肌肉中的PIN mRNA进行Northern印迹分析显示，与正常肌肉相比，营养不良性肌肉中的PIN mRNA显著升高。免疫组织化学分析表明，在正常小鼠肌肉纤维中，PIN表达定位于肌膜、外周核和肌浆。相比之下，mdx小鼠肌肉中的PIN蛋白在肌膜和中央核周围更为集中。尽管这些纤维中的nNOS和肌营养不良蛋白表达显著降低，但mdx小鼠肌肉中仍明显存在PIN蛋白表达。在DMD患者的肌肉切片中，nNOS蛋白表达缺失，但PIN表达仍维持。在mdx小鼠和DMD患者的营养不良性肌肉纤维中浸润的巨噬细胞中也可检测到显著的PIN表达。这些结果表明，mdx小鼠和DMD患者肌肉中的PIN表达受不同于参与nNOS和肌营养不良蛋白调节的因素控制。此外，我们的结果表明，PIN不是骨骼肌纤维内肌营养不良蛋白复合物的组成部分。
