Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo, Oslo, Norway.
PLoS One. 2012;7(3):e33203. doi: 10.1371/journal.pone.0033203. Epub 2012 Mar 22.
Physical exercise leads to substantial adaptive responses in skeletal muscles and plays a central role in a healthy life style. Since exercise induces major systemic responses, underlying cellular mechanisms are difficult to study in vivo. It was therefore desirable to develop an in vitro model that would resemble training in cultured human myotubes.
Electrical pulse stimulation (EPS) was applied to adherent human myotubes. Cellular contents of ATP, phosphocreatine (PCr) and lactate were determined. Glucose and oleic acid metabolism were studied using radio-labeled substrates, and gene expression was analyzed using real-time RT-PCR. Mitochondrial content and function were measured by live imaging and determination of citrate synthase activity, respectively. Protein expression was assessed by electrophoresis and immunoblotting.
High-frequency, acute EPS increased deoxyglucose uptake and lactate production, while cell contents of both ATP and PCr decreased. Chronic, low-frequency EPS increased oxidative capacity of cultured myotubes by increasing glucose metabolism (uptake and oxidation) and complete fatty acid oxidation. mRNA expression level of pyruvate dehydrogenase complex 4 (PDK4) was significantly increased in EPS-treated cells, while mRNA expressions of interleukin 6 (IL-6), cytochrome C and carnitin palmitoyl transferase b (CPT1b) also tended to increase. Intensity of MitoTracker®Red FM was doubled after 48 h of chronic, low-frequency EPS. Protein expression of a slow fiber type marker (MHCI) was increased in EPS-treated cells.
Our results imply that in vitro EPS (acute, high-frequent as well as chronic, low-frequent) of human myotubes may be used to study effects of exercise.
体育锻炼会引起骨骼肌的显著适应性反应，在健康的生活方式中起着核心作用。由于运动引起了主要的全身反应，因此很难在体内研究潜在的细胞机制。因此，开发一种类似于在培养的人类肌管中进行训练的体外模型是很有必要的。
对贴壁的人类肌管进行电脉冲刺激（EPS）。测定细胞内的 ATP、磷酸肌酸（PCr）和乳酸含量。使用放射性标记的底物研究葡萄糖和油酸的代谢，并用实时 RT-PCR 分析基因表达。通过活细胞成像和测定柠檬酸合酶活性分别测量线粒体含量和功能。通过电泳和免疫印迹评估蛋白质表达。
高频、急性 EPS 增加了脱氧葡萄糖的摄取和乳酸的产生，而 ATP 和 PCr 的细胞含量都降低了。慢性、低频 EPS 通过增加葡萄糖代谢（摄取和氧化）和完全脂肪酸氧化来增加培养的肌管的氧化能力。EPS 处理的细胞中丙酮酸脱氢酶复合物 4（PDK4）的 mRNA 表达水平显著增加，而白细胞介素 6（IL-6）、细胞色素 C 和肉毒碱棕榈酰转移酶 b（CPT1b）的 mRNA 表达也有增加的趋势。慢性、低频 EPS 48 小时后，MitoTracker®Red FM 的强度增加了一倍。EPS 处理的细胞中慢肌纤维型标志物（MHCI）的蛋白表达增加。
我们的结果表明，体外 EPS（急性、高频和慢性、低频）可用于研究运动的影响。
