Transcriptional analysis of genes regulating the mitochondrial dNTP pool in muscle cells

Mutations in genes involved in dNTP metabolism can lead to tissue-specific mitochondrial depletion syndromes (MDS), likely because the expression of key enzymes is reduced to critical levels in post mitotic cells. Our goal was to establish an in vitro skeletal muscle cell model to study the muscle specificity of MDS associated with mitochondrial dNTP pool imbalance. We performed a comprehensive analysis at the mRNA level of enzymes and transporters responsible for dNTP pool imbalance in muscle cells in vitro and in vivo. Agilent Mouse Oligo Arrays 4x44K were utilized to examine expression levels in proliferating and differentiated C2C12 cells as well as in the mouse EDL (fast glycolytic) and soleus (slow oxidative) muscles. The comparison of mRNA expression profiles supports the reliability of our in vitro cell system. Proliferating mouse C2C12 myoblasts were collected at about 50 percent confluence. Myoblasts were then induced to differentiate in vitro into myotubes that were harvested after 96 hours and further purified to reduce the contribution of mononucleated cells present in the culture. Gene expression in C2C12 myoblasts and myotubes was compared with fully differentiated muscle fibers in vivo. To this aim, the extensor digitorum longus (EDL) and soleus hind limb muscles were isolated from adult CD1 mice. These muscles were selected because they have different metabolic (glycolytic vs. oxidative) and twitching (fast vs. slow) properties. Triplicate total RNA samples were submitted to gene expression profiling.参与dNTP代谢的基因发生突变，可引发组织特异性线粒体耗竭综合征（mitochondrial depletion syndromes, MDS），其潜在机制可能是有丝分裂后细胞内关键酶的表达水平被下调至临界阈值。本研究旨在构建体外骨骼肌细胞模型，以探究与线粒体dNTP库失衡相关的MDS的肌肉组织特异性。我们对体外及体内肌肉细胞中参与dNTP库失衡的酶与转运蛋白开展了mRNA水平的综合分析。本研究采用安捷伦小鼠寡核苷酸芯片4x44K（Agilent Mouse Oligo Arrays 4x44K），检测增殖及分化后的C2C12细胞，以及小鼠趾长伸肌（extensor digitorum longus, EDL，快速糖酵解型）和比目鱼肌（soleus，慢速氧化型）中的基因表达水平。对mRNA表达谱的比较验证了本体外细胞模型的可靠性。我们在细胞融合度约50%时收集增殖中的小鼠C2C12成肌细胞。随后诱导成肌细胞体外分化为肌管，于分化96小时后收集肌管，并进一步纯化以降低培养体系中单核细胞的干扰。我们将C2C12成肌细胞及肌管的基因表达，与体内完全分化的肌纤维进行比较。为此，我们从成年CD1小鼠体内分离后肢趾长伸肌（extensor digitorum longus, EDL）及比目鱼肌（soleus）。选择这两种肌肉是因为二者具有不同的代谢（糖酵解 vs. 氧化）及收缩（快速 vs. 慢速）特性。我们将三份生物学重复的总RNA样本用于基因表达谱分析。