Department of Musculoskeletal Biology, Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, United Kingdom.
Antioxid Redox Signal. 2013 Feb 20;18(6):603-21. doi: 10.1089/ars.2012.4623. Epub 2012 Dec 6.
The sources of cytosolic superoxide in skeletal muscle have not been defined. This study examined the subcellular sites that contribute to cytosolic superoxide in mature single muscle fibers at rest and during contractile activity.
Isolated fibers from mouse flexor digitorum brevis loaded with superoxide and nitric-oxide-sensitive fluorescent probes, specific pathway inhibitors and immunolocalization techniques were used to identify subcellular sites contributing to cytosolic superoxide. Treatment with the electron transport chain complex III inhibitor, antimycin A, but not the complex I inhibitor, rotenone, caused increased cytosolic superoxide through release from the mitochondrial intermembrane space via voltage-dependent anion or Bax channels, but inhibition of these channels did not affect contraction-induced increases in cytosolic superoxide. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitors decreased cytosolic superoxide at rest and following contractions. Protein and mRNA expression of NADPH oxidase subunits was demonstrated in single fibers. NOX2, NOX4, and p22(phox) subunits localized to the sarcolemma and transverse tubules; NOX4 was additionally expressed in mitochondria. Regulatory p40(phox) and p67(phox) proteins were found in the cytoplasm of resting fibers, but following contractions, p40(phox) appeared to translocate to the sarcolemma.
Superoxide and other reactive oxygen species generated by skeletal muscle are important regulators of muscle force production and adaptations to contractions. This study has defined the relative contribution of mitochondrial and cytosolic sources of superoxide within the cytosol of single muscle fibers at rest and during contractions.
Muscle mitochondria do not modulate cytosolic superoxide in skeletal muscle but NADPH oxidase is a major contributor both at rest and during contractions.
骨骼肌细胞浆中超氧化物的来源尚未确定。本研究检测了在休息和收缩活动期间成熟的单个肌纤维细胞浆中超氧化物的亚细胞来源。
使用从小鼠屈趾短肌分离的纤维，加载超氧化物和一氧化氮敏感的荧光探针、特定途径抑制剂和免疫定位技术，以确定对细胞浆中超氧化物有贡献的亚细胞部位。用电子传递链复合物 III 抑制剂抗霉素 A 处理，但不是复合物 I 抑制剂鱼藤酮处理，会通过电压依赖性阴离子或 Bax 通道从线粒体膜间隙中释放，从而导致细胞浆中超氧化物增加，但抑制这些通道不会影响收缩引起的细胞浆中超氧化物增加。烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶抑制剂减少了休息时和收缩后的细胞浆中超氧化物。单个纤维中证明了 NADPH 氧化酶亚基的蛋白和 mRNA 表达。NOX2、NOX4 和 p22(phox)亚基定位于肌膜和横管；NOX4 还在线粒体中表达。调节 p40(phox)和 p67(phox)蛋白存在于休息纤维的细胞质中，但在收缩后，p40(phox)似乎向肌膜转移。
骨骼肌产生的超氧化物和其他活性氧是肌肉力量产生和收缩适应的重要调节因子。本研究定义了在休息和收缩期间单个肌纤维细胞浆中，线粒体和细胞浆中超氧化物的相对来源。
肌肉线粒体不会调节骨骼肌中的细胞浆中超氧化物，但 NADPH 氧化酶是休息和收缩期间的主要来源。
