Zhang Quan, Halle Jessica L, Counts Brittany R, Pi Min, Carson James A
Integrative Muscle Biology Laboratory, Division of Rehabilitation Sciences, College of Health ProfessionsUniversity of Tennessee Health Science Center, Memphis, Tennessee, United States.
Department of Medicine, University of Tennessee Health Science Center, Memphis, Tennessee, United States.
Am J Physiol Cell Physiol. 2024 Jul 1;327(1):C124-C139. doi: 10.1152/ajpcell.00237.2024. Epub 2024 May 20.
Protein synthesis regulation is critical for skeletal muscle hypertrophy, yet other established cellular processes are necessary for growth-related cellular remodeling. Autophagy has a well-acknowledged role in muscle quality control, but evidence for its role in myofiber hypertrophy remains equivocal. Both mammalian target of rapamycin complex I (mTORC1) and bone morphogenetic protein (BMP)-Smad1/5 (Sma and Mad proteins from and , respectively) signaling are reported regulators of myofiber hypertrophy; however, gaps remain in our understanding of how this regulation is integrated with growth processes and autophagy regulation. Therefore, we investigated the mTORC1 and Smad1/5 regulation of protein synthesis and autophagy flux during serum-stimulated myotube growth. Chronic serum stimulation experiments were performed on differentiated C2C12 myotubes incubated in differentiation medium [2% horse serum (HS)] or growth medium [5% fetal bovine serum (FBS)] for 48 h. Rapamycin or LDN193189 was dosed for 48 h to inhibit mTORC1 and BMP-Smad1/5 signaling, respectively. Acute serum stimulation was examined in differentiated myotubes. Protein synthesis was measured by puromycin incorporation. Bafilomycin A1 and immunoblotting for LC3B were used to assess autophagy flux. Chronic serum stimulation increased myotube diameter 22%, total protein 21%, total RNA 100%, and Smad1/5 phosphorylation 404% and suppressed autophagy flux. Rapamycin, but not LDN193189, blocked serum-induced myotube hypertrophy and the increase in total RNA. Acute serum stimulation increased protein synthesis 111%, Smad1/5 phosphorylation 559%, and rpS6 phosphorylation 117% and suppressed autophagy flux. Rapamycin increased autophagy flux during acute serum stimulation. These results provide evidence for mTORC1, but not BMP-Smad1/5, signaling being required for serum-induced myotube hypertrophy and autophagy flux by measuring LC3BII/I expression. Further investigation is warranted to examine the role of autophagy flux in myotube hypertrophy. The present study demonstrates that myotube hypertrophy caused by chronic serum stimulation requires mammalian target of rapamycin complex 1 (mTORC1) signaling but not bone morphogenetic protein (BMP)-Smad1/5 signaling. The suppression of autophagy flux was associated with serum-induced myotube hypertrophy and mTORC1 regulation of autophagy flux by measuring LC3BII/I expression. Rapamycin is widely investigated for beneficial effects in aging skeletal muscle and sarcopenia; our results provide evidence that rapamycin can regulate autophagy-related signaling during myotube growth, which could benefit skeletal muscle functional and metabolic health.
蛋白质合成调控对于骨骼肌肥大至关重要，然而其他已确立的细胞过程对于与生长相关的细胞重塑也是必需的。自噬在肌肉质量控制中具有公认的作用，但其在肌纤维肥大中的作用证据仍不明确。哺乳动物雷帕霉素靶蛋白复合物I（mTORC1）和骨形态发生蛋白（BMP）-Smad1/5（分别来自秀丽隐杆线虫和果蝇的Sma和Mad蛋白）信号传导均被报道为肌纤维肥大的调节因子；然而，我们对这种调节如何与生长过程和自噬调节整合的理解仍存在空白。因此，我们研究了血清刺激的肌管生长过程中mTORC1和Smad1/5对蛋白质合成和自噬通量的调节。对在分化培养基[2%马血清（HS）]或生长培养基[5%胎牛血清（FBS）]中孵育48小时的分化C2C12肌管进行慢性血清刺激实验。分别给予雷帕霉素或LDN193189 48小时以抑制mTORC1和BMP-Smad1/5信号传导。在分化的肌管中检测急性血清刺激。通过嘌呤霉素掺入法测量蛋白质合成。使用巴弗洛霉素A1和LC3B免疫印迹法评估自噬通量。慢性血清刺激使肌管直径增加22%，总蛋白增加21%，总RNA增加100%，Smad1/5磷酸化增加404%，并抑制自噬通量。雷帕霉素而非LDN193189阻断了血清诱导的肌管肥大和总RNA的增加。急性血清刺激使蛋白质合成增加111%，Smad1/5磷酸化增加559%，rpS6磷酸化增加117%，并抑制自噬通量。雷帕霉素在急性血清刺激期间增加了自噬通量。这些结果通过测量LC3BII/I表达为mTORC1而非BMP-Smad1/5信号传导是血清诱导的肌管肥大和自噬通量所必需提供了证据。有必要进一步研究以检查自噬通量在肌管肥大中的作用。本研究表明，慢性血清刺激引起的肌管肥大需要哺乳动物雷帕霉素靶蛋白复合物1（mTORC1）信号传导而非骨形态发生蛋白（BMP）-Smad1/5信号传导。通过测量LC3BII/I表达，自噬通量的抑制与血清诱导的肌管肥大和mTORC1对自噬通量的调节相关。雷帕霉素在衰老骨骼肌和肌肉减少症中的有益作用已得到广泛研究；我们的结果提供了证据表明雷帕霉素可以在肌管生长过程中调节自噬相关信号传导，这可能有益于骨骼肌功能和代谢健康。
