Department of Integrative Biology & Physiology, Medical School, University of Minnesota, 6-125 Jackson Hall, 321 Church Street SE, Minneapolis, MN, 55455, USA.
Chemical Engineering & Materials Science, University of Minnesota, 151 Amundson Hall, 421 Washington Avenue SE, Minneapolis, MN, 55455, USA.
Skelet Muscle. 2023 May 19;13(1):9. doi: 10.1186/s13395-023-00318-y.
Duchenne muscular dystrophy (DMD) is caused by the lack of dystrophin, a cytoskeletal protein essential for the preservation of the structural integrity of the muscle cell membrane. DMD patients develop severe skeletal muscle weakness, degeneration, and early death. We tested here amphiphilic synthetic membrane stabilizers in mdx skeletal muscle fibers (flexor digitorum brevis; FDB) to determine their effectiveness in restoring contractile function in dystrophin-deficient live skeletal muscle fibers. After isolating FDB fibers via enzymatic digestion and trituration from thirty-three adult male mice (9 C57BL10, 24 mdx), these were plated on a laminin-coated coverslip and treated with poloxamer 188 (P188; PEO-PPO-PEO; 8400 g/mol), architecturally inverted triblock (PPO-PEO-PPO, 10,700 g/mol), and diblock (PEO-PPO-C, 4200 g/mol) copolymers. We assessed the twitch kinetics of sarcomere length (SL) and intracellular Ca transient by Fura-2AM by field stimulation (25 V, 0.2 Hz, 25 °C). Twitch contraction peak SL shortening of mdx FDB fibers was markedly depressed to 30% of the dystrophin-replete control FDB fibers from C57BL10 (P < 0.001). Compared to vehicle-treated mdx FDB fibers, copolymer treatment robustly and rapidly restored the twitch peak SL shortening (all P < 0.05) by P188 (15 μM = + 110%, 150 μM = + 220%), diblock (15 μM = + 50%, 150 μM = + 50%), and inverted triblock copolymer (15 μM = + 180%, 150 μM = + 90%). Twitch peak Ca transient from mdx FDB fibers was also depressed compared to C57BL10 FDB fibers (P < 0.001). P188 and inverted triblock copolymer treatment of mdx FDB fibers increased the twitch peak Ca transient (P < 0.001). This study shows synthetic block copolymers with varied architectures can rapidly and highly effectively enhance contractile function in live dystrophin-deficient skeletal muscle fibers.
杜氏肌营养不良症（DMD）是由于缺乏肌营养不良蛋白引起的，肌营养不良蛋白是维持肌肉细胞膜结构完整性所必需的细胞骨架蛋白。DMD 患者会出现严重的骨骼肌无力、退化和早逝。我们在这里测试了亲脂性合成膜稳定剂在 mdx 骨骼肌纤维（趾短屈肌；FDB）中的作用，以确定它们在恢复缺乏肌营养不良蛋白的活骨骼肌纤维的收缩功能方面的有效性。通过酶消化和研磨从 33 只成年雄性小鼠（9 只 C57BL10，24 只 mdx）中分离出 FDB 纤维后，将这些纤维铺在层粘连蛋白包被的盖玻片上，并分别用泊洛沙姆 188（P188；PEO-PPO-PEO；8400g/mol）、建筑倒置嵌段共聚物（PPO-PEO-PPO，10700g/mol）和二嵌段共聚物（PEO-PPO-C，4200g/mol）处理。通过场刺激（25V，0.2Hz，25°C）用 Fura-2AM 评估肌节长度（SL）和细胞内 Ca 瞬变的颤搐动力学。mdx FDB 纤维的颤搐收缩峰值 SL 缩短明显降低至 C57BL10 中肌营养不良蛋白充足的 FDB 纤维的 30%（P<0.001）。与 vehicle 处理的 mdx FDB 纤维相比，共聚物处理可快速而迅速地恢复颤搐峰值 SL 缩短（所有 P<0.05），泊洛沙姆 188（15μM=+110%，150μM=+220%），二嵌段（15μM=+50%，150μM=+50%）和倒置嵌段共聚物（15μM=+180%，150μM=+90%）。与 C57BL10 FDB 纤维相比，mdx FDB 纤维的颤搐峰值 Ca 瞬变也降低（P<0.001）。用泊洛沙姆 188 和倒置嵌段共聚物处理 mdx FDB 纤维可增加颤搐峰值 Ca 瞬变（P<0.001）。本研究表明，具有不同结构的合成嵌段共聚物可以快速而高度有效地增强缺乏肌营养不良蛋白的活骨骼肌纤维的收缩功能。
