Unloading during skeletal muscle regeneration retards iNOS-expressing macrophage recruitment and perturbs satellite...

After skeletal muscle injury, unloading disturbs the regenerative process of injured myofibers, in a manner highly attributed to impairment of macrophage functions. However, the effect of unloading on the spatiotemporal context of proinflammatory macrophage recruitment and satellite cell accumulation within the damaged area remains unclear. This study focused on macrophages expressing inducible nitric oxide synthase (iNOS) that synthesize nitric oxide, a key regulator of muscle regeneration, and compared the continuous hindlimb unloading (HU) by tail suspension versus weight-bearing (WB) after skeletal muscle crush injury in rats. We found that in the WB group, the recruitment of iNOS⁺ proinflammatory macrophages into the injured site gradually increased until their peak number at 48 h post-injury. In the HU group, the accumulation of iNOS⁺ macrophages until 48 h after injury was significantly less than that in the WB group and continued to increase at 72 h. In accordance with attenuated and/or delayed iNOS⁺ macrophage recruitment, whole iNOS expression at 24 and 48 h after injury was weakened by unloading. Additionally, in the HU group, satellite cell content of dystrophin-positive non-injured areas diminished at 48 h after injury, and the numbers of activated satellite cells within the regenerating area at 72 and 96 h post-injury were significantly smaller than those in the WB group. These findings suggest that muscle regeneration under unloading conditions results in attenuated and/or delayed recruitment of iNOS⁺ macrophages and lower iNOS expression in the early phase after muscle injury, leading to perturbed satellite cell accumulation and muscle regeneration.