Three-dimensionally engineered dystrophin-deficient mdx skeletal muscles and mdx mice were used to test the role of ROCK in DMD muscle function in vitro and in situ, respectively. The role of ARHGEF3, one of the RhoA guanine nucleotide exchange factors (GEFs), in RhoA/ROCK signalling and DMD pathology was examined by generating Arhgef3 knockout mdx mice. The role of RhoA/ROCK signalling in mediating the function of ARHGEF3 was determined by evaluating the effects of wild-type or GEF-inactive ARHGEF3 overexpression with ROCK inhibitor treatment. To gain more mechanistic insights, autophagy flux and the role of autophagy were assessed in various conditions with chloroquine.
Inhibition of ROCK with Y-27632 improved muscle force production in 3D-engineered mdx muscles (+25% from three independent experiments, P < 0.05) and in mice (+25%, P < 0.001). Unlike suggested by previous studies, this improvement was independent of muscle differentiation or quantity and instead related to increased muscle quality. We found that ARHGEF3 was elevated and responsible for RhoA/ROCK activation in mdx muscles, and that depleting ARHGEF3 in mdx mice restored muscle quality (up to +36%, P < 0.01) and morphology without affecting regeneration. Conversely, overexpressing ARHGEF3 further compromised mdx muscle quality (-13% vs. empty vector control, P < 0.01) in GEF activity- and ROCK-dependent manner. Notably, ARHGEF3/ROCK inhibition exerted the effects by rescuing autophagy which is commonly impaired in dystrophic muscles.
Our findings uncover a new pathological mechanism of muscle weakness in DMD involving the ARHGEF3-ROCK-autophagy pathway and the therapeutic potential of targeting ARHGEF3 in DMD.
方法:使用三维工程的肌营养不良蛋白缺乏的mdx骨骼肌和mdx小鼠,在体外和原位测试ROCK在DMD肌肉功能中的作用,分别。RhoA鸟嘌呤核苷酸交换因子(GEF)之一ARHGEF3的作用,在RhoA/ROCK中,通过产生Arhgef3敲除的mdx小鼠来检查信号传导和DMD病理学。RhoA/ROCK信号传导在介导ARHGEF3功能中的作用通过用ROCK抑制剂处理评估野生型或GEF无活性的ARHGEF3过表达的效果来确定。为了获得更多的机械见解,用氯喹在各种条件下评估自噬通量和自噬的作用。
结果:用Y-27632抑制ROCK改善了3D工程mdx肌肉的肌肉力量产生(三个独立实验的25%,P<0.05)和小鼠(+25%,P<0.001)。与以前的研究不同,这种改善与肌肉分化或数量无关,而与肌肉质量的提高有关.我们发现ARHGEF3在mdx肌肉中升高并负责RhoA/ROCK激活,并且在mdx小鼠中消耗ARHGEF3可以恢复肌肉质量(高达36%,P<0.01)和形态而不影响再生。相反,过表达ARHGEF3进一步损害mdx肌肉质量(-13%与空矢量控制,P<0.01)以GEF活动和ROCK依赖的方式。值得注意的是,ARHGEF3/ROCK抑制通过挽救在营养不良的肌肉中通常受损的自噬而发挥作用。
结论:我们的发现揭示了DMD中肌肉无力的新病理机制,涉及ARHGEF3-ROCK-自噬通路,以及在DMD中靶向ARHGEF3的治疗潜力。