Genomic Profiling and Physiological Approaches to Understand Aquaporins and their Role in ROS Signalling within Skeletal Muscle
Genomic Profiling and Physiological Approaches to Understand Aquaporins and their Role in ROS Signalling within Skeletal Muscle
复制标题
了解水通道蛋白及其在骨骼肌内 ROS 信号传导中的作用的基因组分析和生理学方法
DOI：
10.1096/fasebj.2020.34.s1.02026
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Staunton C
中科院分区：
文献类型：
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作者：
Staunton C
水通道蛋白（AQP）通道是一个完整的膜蛋白家族，显示有助于过氧化氢（H2O2）跨膜移动[1]。它们在骨骼肌中的作用是非常重要的，因为H2O2和其他活性氧（ROS）的细胞内产生是响应于肌肉收缩而发生的。这些ROS启动信号传导过程并激活许多转录因子[2，3]。我们的研究[4]以及其他人的研究已经确定了H2O2在肌肉中的来源、汇和功能，但H2O2如何从肌肉中的起源部位被确切地转运还从未被研究过。我们假设水通道蛋白是调节H2O2通过骨骼肌膜运动的主要途径。通过RNAseq（150bp-读取深度> 280 M簇/泳道，n = 5）检查来自对照成年（6 - 8个月）和老年（26个月）小鼠以及来自腓神经挤压后的小鼠的胫骨前肌（TA）肌肉的RNA转录物。还用水通道蛋白抑制剂处理单个肌肉肌管，并用AAV6-HyPer2转染以确定不同AQP同种型对细胞内H2O2含量的作用，并使用溶胀测定法确定H2O渗透性[5，6] ARNASeq数据鉴定了TA肌肉中存在的8种AQP mRNA转录物。神经夹伤后AQP4和AQP1含量降低，AQP3含量升高（n = 5，P <0.01），免疫学结果证实了这一点。目前正在研究这些水通道蛋白的定位和作用，以确定它们是否具有保护功能或有助于与年龄相关的骨骼肌衰退。当AAV6-Hyper2转染的肌纤维暴露于10 μ M H2O2时，记录到细胞内H2O2水平呈指数增加。用AQP阻断剂HgCl2（30 μ M）、TEA（3mM）或布美他尼（5 μ M）预处理肌细胞，可显著降低细胞内H2O2含量的增加速率。这些数据表明水通道蛋白调节骨骼肌H2O2渗透性的能力。用80mOsm溶液与药理学AQP1抑制剂联合培养细胞也显示出细胞体积增加速率的显著降低。总之，这些数据表明AQPs在肌肉中具有潜在的重要作用。使用AAV-shRNA敲低特定AQP的进一步工作正在用于鉴定在骨骼肌中起作用的主要AQP。支持或资助信息本研究得到MRC和NIA（AG051442）的支持。1Bienert，G.P.， J.K. Schjoerring，以及 T.P. Jahn，《生物化学和生物物理学报》，2006年。 1758（8）：p. 994 - 1003.2Jackson，M.J. Mol Aspects Med，2016. 50：p. 33 - 40.3杰克逊，M.J.和 a.麦卡德尔 J Physiol，2011. 589（Pt 9）：第2139 - 45.4页，Staunton，C.A.，等。科学报告，2019年。 9（1）：p. 14461.5 Kumagai，K.，et al. Osteoarthritis and Carcinoma，2016. 24（10）：p. 1786 - 1794.6 Wibberley，A.，等PLoS One，2015. 10（6）：第e0127889页。
Aquaporin (AQP) channels are a family of integral membrane proteins shown to aid the movement of hydrogen peroxide (H202) across membranes [1]. Their role in skeletal muscle is of key importance since intracellular generation of H202and other reactive oxygen species (ROS) occurs in response to muscle contractions. These ROS initiate signalling processes and activate of a number of transcription factors [2, 3]. During ageing, excessive levels of ROS have pathological effects, inducing muscle degradation, loss of neuromuscular junction integrity and loss of neuromuscular transmission.Our studies [4], as well as those of others, has identified the sources, sinks and functions of H202in muscle but exactly how H202is transported from its site of origin within muscle has never been examined. We hypothesize that (AQPs) are the primary means of regulating H202movement through skeletal muscle membranes. This study has therefore examined expression of AQP isoforms in muscle and how they may change with age.RNA transcripts from Tibialis Anterior (TA) muscle from control adult (6–8 months), and old (26month) mice, and from mice following peroneal nerve crush, were examined by RNAseq (150bp‐read depth >280 M clusters per lane, n=5). Individual muscle myotubes were also treated pharmacologically with aquaporin inhibitors and transfected with AAV6‐HyPer2 to determine the role of different AQP isoforms on intracellular H202content and H20 permeability was determined using the swell assay [5, 6].RNAseq data identified 8 AQP mRNA transcripts present in TA muscle. A decrease in AQP4 and AQP1 and an increase in AQP3 contents were found following nerve crush (n=5, P<0.01) and this was confirmed by immunological techniques. The localisation and roles of these aquaporins is currently being investigated to determine whether they have protective functions or contribute to the age‐related decline skeletal muscle.An exponential increase in intracellular H2O2levels was recorded when AAV6‐Hyper2 transfected muscle fibres were exposed to 10μM H2O2. Pre‐treatment of muscle cells with AQP blockers HgCl2(30μM), TEA (3mM) or Bumetanide (5μM) substantially decreased the rate of increase in intracellular H202content. These data demonstrate the ability of aquaporins to regulate skeletal muscle H2O2permeability. Challenging cells with an 80mOsm solution in conjunction with pharmacological AQP1 inhibitors also demonstrated a significant reduction in the rate of increase in cell volume.In summary, these data demonstrate a potential important role for AQPs in muscle. Further work using AAV‐shRNA to knockdown specific AQPs is being utilised to identify the major AQPs that function in skeletal muscle.Support or Funding InformationThis study was supported by the MRC and NIA (AG051442).1Bienert, G.P., J.K. Schjoerring, and T.P. Jahn, Biochim Biophys Acta, 2006. 1758 ( 8): p. 994– 1003.2Jackson, M.J. Mol Aspects Med, 2016. 50: p. 33– 40.3Jackson, M.J. and A. McArdle. J Physiol, 2011. 589 ( Pt 9): p. 2139– 45.4Staunton, C.A., et al. Scientific Reports, 2019. 9 ( 1): p. 14461.5Kumagai, K., et al. Osteoarthritis and Cartilage, 2016. 24 ( 10): p. 1786– 1794.6Wibberley, A., et al. PLoS One, 2015. 10 ( 6): p. e0127889.
体积敏感Cl－电流在兔膝前交叉韧带横断中的早期激活
DOI：
--
发表时间：
2018
期刊：
影响因子：
--
作者：
K. Kumagai;F. Toyoda;T. Maeda;H. Tanigawa;N. Okumura;H. Matsuura;S. Imai
通讯作者：
S. Imai
HyPer2 成像揭示体内去神经和老化骨骼肌纤维中过氧化氢的时间和异质变化
影响因子：
4.6
作者：
C. Staunton;E. Owen;N. Pollock;A. Vasilaki;R. Barrett;A. Mcardle;M. Jackson
通讯作者：
M. Jackson