Changes in Protein Fluxes in Skeletal Muscle During Sequential Stages of Muscle Regeneration After Acute Injury in Male Mice

Background - Changes in protein turnover play an important role in dynamic physiological processes, including skeletal muscle regeneration, which occurs as an essential part of tissue repair after injury. The inability of muscle tissue to recapitulate this regenerative process can lead to pathology and clinical symptoms in various musculoskeletal diseases, including muscular dystrophies and pathological atrophy.

Methods - Here, we employed a workflow that couples deuterated water (2H2O) administration with tandem mass spectrometry (MS) to systematically measure in-vivo protein turnover rates across the muscle proteome in 8-week-old male C57BL6/J mice. We compared the turnover kinetics of over 100 proteins in response to cardiotoxin (CTX) induced muscle damage and regeneration at unique sequential stages along the regeneration timeline. This analysis is compared to gene expression data from mRNA-sequencing (mRNA-seq) from the same tissue.

Results - The data reveals quantitative protein flux signatures in response to necrotic damage, in addition to sequential differences in cell proliferation, energy metabolism, and contractile gene expression. Interestingly, the mRNA changes correlated poorly with changes in protein synthesis rates, consistent with post-transcriptional control mechanisms.

Conclusions - In summary, the experiments described here reveal the signatures and timing of protein flux changes during skeletal muscle regeneration, as well as the inability of mRNA expression measurements to reveal changes in directly measured protein turnover rates. The results of this work described here provide a better understanding of the muscle regeneration process and could help to identify potential biomarkers or therapeutic targets.

背景——蛋白质周转（protein turnover）的动态变化在多种生理过程中发挥关键作用，其中包括骨骼肌再生（skeletal muscle regeneration）：后者是机体损伤后组织修复的核心环节。若肌肉组织无法完成该再生程序，则会引发多种肌肉骨骼疾病的病理改变与临床症状，此类疾病涵盖肌营养不良症（muscular dystrophies）与病理性萎缩（pathological atrophy）。
方法——本研究采用了将氘代水（2H2O）给药与串联质谱（tandem mass spectrometry, MS）联用的实验流程，以系统性测定8周龄雄性C57BL6/J小鼠肌肉蛋白质组的体内蛋白质周转速率。我们对比了超过100种蛋白质的周转动力学特征，以观察其在肌肉再生时间轴上不同独特时序阶段中，响应心脏毒素（cardiotoxin, CTX）诱导的肌肉损伤与再生过程的变化，并将该分析结果与同一组织的mRNA测序（mRNA-sequencing, mRNA-seq）基因表达数据进行比对。
结果——本研究数据揭示了响应坏死性损伤的蛋白质通量（protein flux）定量特征，同时展现了细胞增殖、能量代谢与收缩基因表达在时序上的差异。值得注意的是，mRNA水平的变化与蛋白质合成速率的变化相关性极低，这与转录后调控机制（post-transcriptional control mechanisms）的作用相符。
结论——综上，本研究所描述的实验揭示了骨骼肌再生过程中蛋白质通量变化的特征与时序规律，同时证实了mRNA表达检测无法反映直接测定的蛋白质周转速率的变化。本研究结果有助于加深对肌肉再生过程的理解，并可为潜在生物标志物或治疗靶点的筛选提供参考依据。