Ion Current-Based Proteomic Profiling for Understanding the Inhibitory Effect of Tumor Necrosis Factor Alpha on Myogenic Differentiation

Despite a demonstrated role for TNF-α
in promoting muscle
wasting and cachexia, the associated molecular mechanisms and signaling
pathways of myoblast differentiation dysregulated by TNF-α remain
poorly understood. This study presents well-controlled proteomic profiling
as a means to investigate the mechanisms of TNF-α-regulated
myogenic differentiation. Primary human muscle precursor cells (MPCs)
cultured in growth medium (GM), differentiation medium (DM) to induce
myogenic differentiation, and DM with 20 ng/mL of TNF-α (n = 5/group) were comparatively analyzed by an ion current-based
quantitative platform consisting of reproducible sample preparation/on-pellet
digestion, a long-column nano-LC separation, and ion current-based
differential analysis. The inhibition of myogenic differentiation
by TNF-α was confirmed by reduced formation of multinucleated
myotubes and the recovered expression of altered myogenic proteins
such as MYOD and myogenin during myogenic differentiation. Functional
analysis and validation by immunoassay analysis suggested that the
cooperation of NF-κB and STAT proteins is responsible for dysregulated
differentiation in MPCs by TNF-α treatment. Increased MHC class
I components such as HLA-A, HLA-B, HLA-C, and beta-2-microglobulin
were also observed in cultures in DM treated with TNF-α. Interestingly,
inhibition of the cholesterol biosynthesis pathway during myogenic
differentiation induced by serum starvation was not recovered by TNF-α
treatment, which combined with previous reports, implies that this
process may be an early event of myogenesis. This finding could lay
the foundation for the potential use of statins in modulating myogenesis
through cholesterol, for example, in stem cell-based myocardial infarction
treatment, where differentiation of myoblasts and stem cells into
force-generating mature muscle cells is a key step to the therapeutic
capacity. In conclusion, the landscapes of altered transcription regulators,
metabolic processes, and signaling pathways in MPCs are revealed in
the regulation of myogenic differentiation by TNF-α, which is
valuable for myogenic cellular therapeutics.

尽管已有研究证实肿瘤坏死因子α（TNF-α）可促进肌肉萎缩与恶病质，但TNF-α介导的成肌细胞分化失调相关的分子机制与信号通路仍未被充分阐明。本研究采用严格可控的蛋白质组学分析技术，探究TNF-α调控肌源性分化的具体机制。本研究对三组人原代肌肉前体细胞（MPCs）进行比较分析：分别培养于生长培养基（GM）、诱导肌源性分化的分化培养基（DM），以及添加20 ng/mL TNF-α的分化培养基（每组n=5）。分析采用基于离子电流的定量平台，该平台包含可重复的样品制备/颗粒内酶解步骤、长柱纳米液相色谱（nano-LC）分离及基于离子电流的差异分析流程。TNF-α对肌源性分化的抑制作用已得到验证：肌源性分化过程中，多核肌管的形成量减少，且MYOD、肌细胞生成素（myogenin）等异常表达的肌源性蛋白的水平得以恢复。功能分析与免疫测定验证结果显示，核因子κB（NF-κB）与信号转导与转录激活因子（STAT）的协同作用，是TNF-α处理后MPCs分化失调的原因。在添加TNF-α的DM培养体系中，还观察到主要组织相容性复合体I类（MHC I）分子的表达上调，包括HLA-A、HLA-B、HLA-C及β2-微球蛋白。有趣的是，血清饥饿诱导肌源性分化过程中，胆固醇生物合成通路的抑制并未因TNF-α处理而得到逆转；结合既往研究，这一结果提示该过程可能是肌发生的早期事件。该发现为他汀类药物通过胆固醇途径调控肌发生的潜在应用奠定了基础，例如在基于干细胞的心肌梗死治疗中：成肌细胞与干细胞分化为可产生收缩力的成熟肌细胞，是实现治疗效果的关键步骤。综上，本研究揭示了TNF-α调控MPCs肌源性分化过程中，转录调控因子、代谢过程及信号通路的整体变化特征，这一结果对肌源性细胞治疗具有重要参考价值。