cTnT在老年II型肌纤维慢性去神经萎缩中的作用及机制研究

cTnT was found to be expressed in some skeletal muscle myopathy, which indicates that abnormally elevated cTnT is potentially a pathological phenomena. cTnT is likely to be associated with sarcopenia which is caused mainly by muscle chronic denervation. Yet the research on cTnT and skeletal muscle chronic denervation in aging is rare. Our early stage work found that expression and enrichment of cTnT at NMJ is only found in elderly mice’s type II muscle fiber. Also we found PKA-RIα decreased, while PKA-RIIα increased. The former plays a key role in NMJ stability while the role of the latter is still unknown at NMJ. Given that cTnT is a known dual-AKAP, we propose the hypothesis: cTnT destroyed the dynamic balance of PKA-RI/PKA-RII which is closely related with NMJ stability, thus causing NMJ impairment and promoting muscle atrophy with aging. We plan to use qRT-PCR, Western blot, AChR pulldown, Immunofluorescence staining, confocal microscopy, and co-immunoprecipitation, to compare the level of cTnT and PKA in NMJ of type II muscle fiber in young, old, and very old mice. And by regulating cTnT expression with adeno-associated virus infection and shRNA, and by regulating PKA activity and expression, we expect to elucidate the role of cTnT in the chronic denervation of type II muscle fiber and the underlying molecular mechanism, which will provide new ideas and potential targets of intervention for sarcopenia.

已有研究发现cTnT在病变骨骼肌组织中表达明显升高，提示异常升高的cTnT是一种病理现象，很可能与慢性去神经导致的老年肌少症密切相关(II型肌纤维去神经发生更早更严重)，但是目前未见cTnT和骨骼肌慢性去神经萎缩的研究。前期工作发现：老年小鼠II型肌纤维NMJ部位cTnT信号较年轻小鼠显著增强，而维持NMJ稳定的PKA-RIα减弱、PKA-RIIα增强。cTnT是一种双向激酶锚定蛋白，我们提出假说：cTnT破坏了和NMJ稳定密切相关的PKA-RI/PKA-RII平衡，导致NMJ结构功能损伤，促进肌肉萎缩。我们采用AChR Pulldown、免疫荧光染色、激光共聚焦、免疫共沉淀、腺相关病毒感染、shRNA等方法，在不同年龄小鼠NMJ部位比较cTnT及PKA的差异，通过调控cTnT和PKA，明确cTnT在老年II型肌纤维慢性去神经时的作用及分子机制，为老年肌少症防治提供新的思路和潜在干预靶点。

老年肌少症是一种与增龄相关的进行性、广泛性肌量减少和（或）肌强度下降及肌肉生理功能减退，从而导致老年人功能状态下降的一种疾病。它明显增加老年人跌倒和骨折的风险，严重降低生活质量、影响预期寿命。但迄今为止有效治疗方法仍然是抗阻力锻炼和营养支持，因此加强肌少症发病机制研究，寻找致病关键因子，将会给肌少症的药物研发提供可靠的理论依据。本研究：1) 观察cTnT在老年II型肌纤维及NMJ中的表达水平，通过调控cTnT表达，观察肌纤维功能变化和NMJ结构、功能改变，分析cTnT在老年II型肌纤维慢性去神经萎缩中的作用；2) 进一步研究cTnT和II型肌纤维NMJ部位PKA-RI/PKA-RII信号分子的结合情况，观察cTnT水平变化时、信号分子PKA-RI/PKA-RII的平衡改变，以及调控PKA-RI和PKA-RII表达水平时NMJ结构及功能变化，从而明确cTnT促进老年II型肌纤维萎缩的分子机制。结果发现：cTnT在老年II型肌纤维NMJ部位表达明显升高，而在I型肌纤维基本没有显示；老年II型肌纤维NMJ部位，PKA-RIIa信号增强，而PKA-RIa几乎没有信号，PKA-RIa/PKA-RIIa平衡失调与老年II型肌纤维萎缩有关；cTnT和PKA RIa、PKA RIIa的结合是竞争性的，cTnT通过与PKA RIIa结合，竞争性抑制了其与PKA RIa的结合；抑制cTnT表达后，老年II型肌纤维的损伤受到抑制、肌肉功能得到了部分恢复；cTnT通过PKA信号途径调控老年II型肌纤维NMJ功能，抑制老年肌肉萎缩现象。实验结果说明：cTnT-PKA信号途径有可能是一个新的治疗靶点，干预这个信号通路可以改善老年II型肌纤维NMJ功能，从而改善老年肌肉运动功能，延缓老年肌少症进展。实验结果为老年肌少症发病机制研究提供补充，为临床研制老年肌少症治疗药物提供理论依据。

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