Transcription profiling of mouse denervated muscle from runx1f mutants vs. controls

In skeletal muscle, the pattern of electrical activity regulates the expression of proteins involved in synaptic transmission, contraction and metabolism. Disruptions in electrical activity, resulting from prolonged bed-rest, cast-immobilization or trauma, inevitably lead to muscle atrophy. The mechanisms that regulate muscle atrophy are poorly understood, but it seems likely that changes in gene expression play a key role in initiating and maintaining a muscle atrophy program. Previously, we found that Runx1, a transcription factor previously termed AML1, was substantially induced in muscle following denervation. More recently, we sought to determine whether this increase in Runx1 expression may be causally related to the morphological changes in skeletal muscle that accompany muscle disuse, notably muscle atrophy. We found that Runx1 is indeed required to sustain muscle and to minimize atrophy following denervation. Experiments described here are designed to identify the genes that are regulated by Runx1 in skeletal muscle with the particular goal of identifying genes that regulate muscle atrophy. We propose to use microarray analysis to identify genes, expressed in skeletal muscle, that are mis-regulated in mice lacking Runx1. We inactivated runx1 selectively in skeletal muscle and found that denervated myofibers in mutant mice atrophy far more (90% atrophy) than in wild-type mice (30% atrophy). We therefore reason that Runx1 activates and/or represses genes that are required to sustain muscle and to minimize atrophy. We generated MCK::cre; runx1f/- and runx1f/- control mice. In normal mice, an increase in runx1 expression is detected by two days after denervation and is maximal by five days after denervation. Muscle atrophy is first evident between one and two weeks after denervation. As we wish to avoid detecting global changes in gene expression that are associated with late stages of muscle atrophy, we plan to denervate muscle for three or five days and to compare gene expression in dissected innervated and denervated muscles from mutant and control mice. We will generate thirty samples for comparison-5 replicates per condition: Samples 1-3 from runx1f/- control mice. (1) innervated tibialis anterior muscles (TA); (2) 3-day-denervated TA; (3) 5-day-denervated TA. Samples 4-6 from MCK::cre; runx1f/- mice. (4) innervated TA; (5) 3-day-denervated TA; (6) 5-day-denervated TA. We obtain sufficient total RNA (10 micrograms) from each dissected muscle to avoid pooling samples. We will analyze adult mice of the same age (~six weeks after birth; most will be littermates) and sex-male. It is difficult to anticipate how many genes will be identified in this screen, as few target genes for Runx1 have been identified in any cell type and none in skeletal muscle. Moreover, although we would prefer to focus our attention on genes that are strongly dependent upon Runx1 expression (e.g. more than 5-fold difference in expression in wild-type and mutant mice), we do not know the extent to which target gene expression will depend upon Runx1. For these reasons, in these experiments, we will analyze expression from five "identical" samples, so that we can be confident that even small (e.g. three-fold) differences in expression can be reliably determined. Importantly, in order to confirm results obtained from the microarray data, we will use other assays (RNase protection) to measure RNA expression of candidate genes in innervated and denervated muscles of wild-type and mutant mice.在骨骼肌（skeletal muscle）中，电活动模式可调控参与突触传递、肌肉收缩及代谢相关蛋白的表达。长时间卧床、石膏固定或创伤所致的电活动紊乱，会不可避免地引发肌肉萎缩。目前人们对调控肌肉萎缩的分子机制尚不完全明确，但基因表达的改变似乎在启动和维持肌肉萎缩程序中发挥关键作用。此前我们发现，在去神经支配后的骨骼肌中，Runx1（一种曾被称为AML1的转录因子）的表达会显著升高。近期，我们旨在明确Runx1表达上调是否与肌肉失用（尤其是肌肉萎缩）伴随的骨骼肌形态学改变存在因果关联。我们发现，Runx1确实是维持骨骼肌功能、减轻去神经支配后肌肉萎缩所必需的。本研究描述的实验旨在鉴定骨骼肌中受Runx1调控的基因，核心目标为筛选出调控肌肉萎缩的相关基因。我们计划采用基因芯片分析（microarray analysis）来鉴定在Runx1敲除小鼠的骨骼肌中表达失调的基因。我们选择性地在骨骼肌中敲除了runx1，结果发现，突变小鼠的去神经支配肌纤维萎缩程度远高于野生型小鼠（突变组萎缩率达90%，野生型仅为30%）。据此我们推测，Runx1通过激活或抑制某些基因，这些基因对于维持骨骼肌功能并减轻肌肉萎缩是必需的。我们构建了MCK::cre; runx1f/-小鼠及runx1f/-对照小鼠。在正常小鼠中，去神经支配2天后即可检测到runx1表达上调，至5天时达到峰值。肌肉萎缩通常在去神经支配1至2周时首次显现。为避免检测到与肌肉萎缩晚期阶段相关的基因表达全局变化，我们计划将肌肉去神经支配时长设为3天或5天，并比较突变型与对照小鼠解剖分离的受神经支配和去神经支配肌的基因表达水平。我们将制备30份用于比较的样本，每个条件设置5个生物学重复：1-3号样本取自runx1f/-对照小鼠：(1) 受神经支配的胫前肌（tibialis anterior, TA）；(2) 去神经支配3天的TA；(3) 去神经支配5天的TA。4-6号样本取自MCK::cre; runx1f/-小鼠：(4) 受神经支配的TA；(5) 去神经支配3天的TA；(6) 去神经支配5天的TA。我们从每块解剖分离的肌肉中可获得足够的总RNA（10微克），因此无需合并样本。我们将选用相同周龄（出生后约6周，大部分为同窝仔鼠）的雄性成年小鼠进行实验。由于目前在任何细胞类型中鉴定出的Runx1靶基因寥寥无几，且骨骼肌中更是尚无报道，因此难以预估本次筛选可鉴定出多少个基因。此外，尽管我们希望优先关注受Runx1表达调控程度较强的基因（例如野生型与突变型小鼠间表达差异达5倍以上的基因），但目前我们尚不清楚靶基因的表达对Runx1的依赖程度。基于上述原因，在本次实验中，我们将对5份"重复"样本进行表达分析，以确保即便表达差异较小（例如3倍）也能得到可靠的检测结果。尤为重要的是，为验证基因芯片分析所得的结果，我们将采用其他检测方法（RNase保护实验（RNase protection assay））来检测野生型与突变型小鼠受神经支配肌与去神经支配肌中候选基因的RNA表达水平。