BackgroundDelocalization of neuronal nitric oxide synthase (nNOS) from the sarcolemma leads to functional muscle ischemia. This contributes to the pathogenesis in cachexia, aging and muscular dystrophy. Mutations in the gene encoding dystrophin result in Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). In many BMD patients and DMD patients that have been converted to BMD by gene therapy, sarcolemmal nNOS is missing due to the lack of dystrophin nNOS-binding domain.MethodsDystrophin spectrin-like repeats 16 and 17 (R16/17) is the sarcolemmal nNOS localization domain. Here we explored whether R16/17 protein therapy can restore nNOS to the sarcolemma and prevent functional ischemia in transgenic mice which expressed an R16/17-deleted human micro-dystrophin gene in the dystrophic muscle. The palmitoylated R16/17.GFP fusion protein was conjugated to various cell-penetrating peptides and produced in the baculovirus-insect cell system. The best fusion protein was delivered to the transgenic mice and functional muscle ischemia was quantified.ResultsAmong five candidate cell-penetrating peptides, the mutant HIV trans-acting activator of transcription (TAT) protein transduction domain (mTAT) was the best in transferring the R16/17.GFP protein to the muscle. Systemic delivery of the mTAT.R16/17.GFP protein to micro-dystrophin transgenic mice successfully restored sarcolemmal nNOS without inducing T cell infiltration. More importantly, R16/17 protein therapy effectively prevented treadmill challenge-induced force loss and improved muscle perfusion during contraction.ConclusionsOur results suggest that R16/17 protein delivery is a highly promising therapy for muscle diseases involving sarcolemmal nNOS delocalizaton.

中文翻译：

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肌营养不良蛋白 R16/17 蛋白疗法可恢复反式肌膜 nNOS 并改善肌肉灌注和功能

背景来自肌膜的神经元一氧化氮合酶 (nNOS) 的离域导致功能性肌肉缺血。这有助于恶病质、衰老和肌营养不良的发病机制。编码肌营养不良蛋白的基因突变导致杜氏肌营养不良症 (DMD) 和贝克尔肌营养不良症 (BMD)。在许多 BMD 患者和通过基因治疗转化为 BMD 的 DMD 患者中，由于缺乏抗肌萎缩蛋白 nNOS 结合域，肌膜 nNOS 缺失。领域。在这里，我们探讨了 R16/17 蛋白疗法是否可以恢复肌膜的 nNOS 并预防在营养不良肌肉中表达 R16/17 缺失的人类微肌养蛋白基因的转基因小鼠的功能性缺血。棕榈酰化的 R16/17。GFP 融合蛋白与各种细胞穿透肽结合并在杆状病毒-昆虫细胞系统中产生。将最佳融合蛋白递送至转基因小鼠并量化功能性肌肉缺血。 结果在五种候选细胞穿透肽中，突变型HIV反式转录激活因子（TAT）蛋白转导结构域（mTAT）在转移R16时效果最好。 /17.GFP 蛋白质到肌肉。mTAT.R16/17.GFP 蛋白向微肌养蛋白转基因小鼠的全身递送成功地恢复了肌膜 nNOS，而不会诱导 T 细胞浸润。更重要的是，R16/17 蛋白质疗法有效地防止了跑步机挑战引起的力量损失并改善了收缩期间的肌肉灌注。