Inhibition of p53-MDM2 binding reduces senescent cell abundance and improves the adaptive responses of skeletal muscle from aged mice

Skeletal muscle adaptation to external stimuli, such as regeneration following injury and hypertrophy in response to resistance exercise, are blunted with advanced age. The accumulation of senescent cells, along with defects in myogenic progenitor cell (MPC) proliferation, have been strongly linked as contributing factors to age-associated impairment in muscle adaptation. p53 plays an integral role in all these processes, as upregulation of p53 causes apoptosis in senescent cells and prevents mitotic catastrophe in MPCs from old mice. The goal of this study was to determine if a novel pharmaceutical agent (BI01), which functions by upregulating p53 through inhibition of binding to MDM2, the primary p53 regulatory protein, improves muscle regeneration and hypertrophy in old mice. BI01 effectively reduced the number of senescent cells in vitro but had no effect on MPC survival or proliferation at a comparable dose. Following repeated oral gavage with 2 mg/kg of BI01 (OS) or vehicle (OV), old mice (24 months) underwent unilateral BaCl2 injury in the tibialis anterior (TA) muscle, with PBS injections serving as controls. After 7 days, satellite cell number was higher in the TA of OS compared to OV mice, as was the expression of genes involved in ATP production. By 35 days, old mice treated with BI01 displayed reduced senescent cell burden, enhanced regeneration (higher muscle mass and fiber cross-sectional area) and restoration of muscle function relative to OV mice. To examine the impact of 2 mg/kg BI01 on muscle hypertrophy, the plantaris muscle was subjected to 28 days of mechanical overload (MOV) in OS and OV mice. In response to MOV, OS mice had larger plantaris muscles and muscle fibers than OV mice, particularly type 2b + x fibers, associated with reduced senescent cells. Together our data show that BI01 is an effective senolytic agent that may also augment muscle metabolism to enhance muscle regeneration and hypertrophy in old mice. RNA-seq profiling from tibialis anterior skeletal muscle tissue of young and old mice with and without BaCl-induced muscle injury. A cohort of old mice were treated with a senolytic compound, BI01.

骨骼肌对外部刺激的适应性反应（如损伤后再生及抗阻运动诱导的肥大）会随衰老进程显著减弱。衰老细胞的积累，以及肌源性祖细胞（myogenic progenitor cell, MPC）增殖功能缺陷，被广泛认为是与年龄相关的骨骼肌适应性受损的关键诱因。p53在上述所有过程中均发挥核心作用：p53的上调可诱导衰老细胞凋亡，并阻止老年小鼠肌源性祖细胞发生有丝分裂灾难。
本研究旨在探究一种通过抑制p53与主要调控蛋白MDM2结合来实现p53上调的新型药剂BI01，能否改善老年小鼠的骨骼肌再生与肥大能力。
体外实验中，BI01可有效减少衰老细胞数量，但在同等剂量下对肌源性祖细胞的存活与增殖无显著影响。在对老年小鼠（24月龄）进行连续2 mg/kg BI01灌胃（OS组）或溶剂对照灌胃（OV组）处理后，对其单侧胫前肌（tibialis anterior, TA）实施氯化钡（BaCl2）损伤，以磷酸盐缓冲液（PBS）注射作为损伤对照。造模7天后，OS组小鼠胫前肌内的卫星细胞数量显著高于OV组，参与ATP生成的基因表达水平也同样升高。至造模35天时，与OV组相比，BI01处理组的老年小鼠衰老细胞负荷降低，骨骼肌再生能力增强（肌肉质量更高、肌纤维横截面积更大），且肌肉功能得到恢复。
为探究2 mg/kg BI01对骨骼肌肥大的影响，本研究对OS组与OV组小鼠的跖肌施加28天机械超负荷刺激（mechanical overload, MOV）。结果显示，在机械超负荷刺激下，OS组小鼠的跖肌及肌纤维体积显著大于OV组，尤其是2b+x型肌纤维，且该组小鼠体内衰老细胞数量更少。
综上，本研究数据表明BI01是一种有效的衰老细胞清除剂（senolytic agent），同时还可增强肌肉代谢，进而改善老年小鼠的骨骼肌再生与肥大能力。
本研究对年轻及老年小鼠（伴或不伴氯化钡诱导的骨骼肌损伤）的胫前肌组织进行了RNA测序分析，其中一组老年小鼠接受了衰老细胞清除化合物（senolytic compound）BI01处理。