Reactive oxygen species (ROS) are a key output of the skeletal muscle mitochondrial information processing system both at rest and during exercise. In skeletal muscle, mitochondrial ROS release depends on multiple factors; however, fiber-type specific differences remain ambiguous in part owing to the use of mitochondria from mammalian muscle that consist of mixed fibers. To elucidate fiber-type specific differences, we used mitochondria isolated from rainbow trout (Oncorhynchus mykiss) red and white skeletal muscles that consist of spatially distinct essentially pure red and white fibers. We first characterized the assay conditions for measuring ROS production (as H₂O₂) in isolated fish red and white skeletal muscle mitochondria (RMM and WMM) and thereafter compared the rates of emission during oxidation of different substrates and the responses to mitochondrial electron transport system (ETS) pharmacological modulators. Our results showed that H₂O₂ emission rates by RMM and WMM can be quantified using the same protein concentration and composition of the Amplex UltraRed-horseradish peroxidase (AUR-HRP) detection system. For both RMM and WMM, protein normalized H₂O₂ emission rates were highest at the lowest protein concentration tested and decreased exponentially thereafter. However, the absolute values of H₂O₂ emission rates depended on the calibration curves used to convert fluorescent signals to H₂O₂ while the trends depended on the normalization strategy. We found substantial qualitative and quantitative differences between RMM and WMM in the H₂O₂ emission rates depending on the substrates being oxidized and their concentrations. Similarly, pharmacological modulators of the ETS altered the magnitudes and trends of the H₂O₂ emission differently in RMM and WMM. While comparable concentrations of substrates elicited maximal albeit quantitively different emission rates in RMM and WMM, different concentrations of pharmacological ETS modulators may be required for maximal H₂O₂ emission rates depending on muscle fiber-type. Taken together, our study suggests that biochemical differences exist in RMM compared with WMM that alter substrate oxidation and responses to ETS modulators resulting in fiber-type specific mitochondrial H₂O₂ emission rates.

活性氧（ROS）是骨骼肌线粒体信息处理系统在休息和运动时的关键输出。在骨骼肌中，线粒体 ROS 释放取决于多种因素；然而，纤维类型的具体差异仍然不明确，部分原因是使用了由混合纤维组成的哺乳动物肌肉的线粒体。为了阐明纤维类型的具体差异，我们使用从虹鳟鱼（Oncorhynchus mykiss）红色和白色骨骼肌中分离出的线粒体，这些骨骼肌由空间上不同的基本上纯的红色和白色纤维组成。我们首先描述了测量离体鱼红色和白色骨骼肌线粒体（RMM 和 WMM）中 ROS 产生（如 H ₂ O ₂ ）的测定条件，然后比较了不同底物氧化过程中的发射速率以及对线粒体电子传递的响应系统（ETS）药理调节剂。我们的结果表明，可以使用 Amplex UltraRed-辣根过氧化物酶 (AUR-HRP) 检测系统的相同蛋白质浓度和组成来量化 RMM 和 WMM 的H ₂ O _{2排放率。}对于RMM 和WMM 而言，蛋白质归一化H ₂ O ₂排放率在测试的最低蛋白质浓度下最高，此后呈指数下降。_{然而，H 2} O ₂发射率的绝对值取决于用于将荧光信号转换为H ₂ O ₂的校准曲线，而趋势取决于归一化策略。我们发现 RMM 和 WMM 之间的 H ₂ O ₂排放速率存在显着的定性和定量差异，具体取决于被氧化的底物及其浓度。类似地，ETS 的药理调节剂在 RMM 和 WMM 中不同地改变了 H ₂ O _{2排放的幅度和趋势。}虽然相当浓度的底物在RMM和WMM中引起最大尽管数量上不同的排放速率，但根据肌纤维类型，最大H ₂ O ₂排放速率可能需要不同浓度的药理学ETS调节剂。总而言之，我们的研究表明，与 WMM 相比，RMM 中存在生化差异，这些差异改变了底物氧化和对 ETS 调节剂的反应，从而导致纤维类型特定的线粒体 H ₂ O ₂排放率。