BackgroundWe recently demonstrated that following a 10‐day exposure to inactivity/simulated microgravity impairments of oxidative metabolism were located ‘upstream’ of mitochondrial function, as evaluated by maximal ADP‐stimulated mitochondrial respiration (JO2max) determined ex vivo. The aim of this study was to evaluate mitochondrial sensitivity to submaximal [ADP] by an alternative approach aimed at identifying responses associated with fibre type composition.MethodsIsolated permeabilized vastus lateralis fibres were analysed by high‐resolution respirometry in 9 young males before and after a 10‐day horizontal bed rest. Eleven submaximal titrations of ADP (from 12.5 to 10 000 μM) were utilized to assess complex I + II‐linked ADP sensitivity. We applied to JO2 versus [ADP] data a traditional Michaelis–Menten kinetics equation, with the calculation of the apparent Km and maximal respiration (Vmax), and two ‘sequential’ hyperbolic equations, yielding two Km and Vmax values. The two‐hyperbolic equations were solved and the [ADP] value corresponding to 50% of JO2max was calculated. Isoform expression of myosin heavy chains (MyHC) 1, 2A and 2X was also determined. Control experiments were also carried out on rat skeletal muscle samples with different percentages of MyHC isoforms.ResultsThe two hyperbolic equations provided an alternative fitting of data and identified two distinct phases of the JO2 versus [ADP] response: a first phase characterized by low Vmax (Vmax1, 28 ± 10 pmol s−1 mg−1) and apparent Km (Km1, 62 ± 54 μM) and a second phase characterized by higher Vmax (Vmax2, 61 ± 16 pmol s−1 mg−1) and Km (Km2, 1784 ± 833 μM). Data were confirmed in control experiments carried out in rat muscle samples with different percentages of MyHC isoforms. Correlation and receiver operating characteristics analyses suggest that the two phases of the response were related to the % of MyHC isoforms.ConclusionsA novel mathematical approach (two sequential hyperbolic functions) for the fitting of JO2 versus [ADP] data obtained by high‐resolution respirometry on permeabilized skeletal muscle fibres, obtained in humans and rats, provided an alternative fitting of the experimental data compared to the traditional Michaelis–Menten kinetics equation. This alternative model allowed the identification of two distinct phases in the responses, which were related to fibre type composition. A first phase, characterized by low apparent Km and Vmax values, was correlated with the percentage of less oxidative (Type 2A + 2X) MyHC isoforms. A second phase, characterized by high apparent Km and Vmax, was related to more oxidative (Type 1) MyHC isoforms.

中文翻译：

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卧床休息后线粒体对次极大 [ADP] 的敏感性：一种与纤维类型相关的新型两阶段方法


背景我们最近证明，在暴露于不活动/模拟微重力 10 天后，氧化代谢的微重力损伤位于线粒体功能的“上游”，通过体外测定的最大 ADP 刺激的线粒体呼吸 （JO2max） 进行评估。本研究的目的是通过一种旨在识别与纤维类型组成相关的反应的替代方法评估线粒体对次极大 [ADP] 的敏感性。方法通过高分辨率呼吸测定法分析 9 例年轻男性水平卧床休息 10 天前后分离的透化股外侧肌纤维。使用 ADP 的 11 次最大滴定 （从 12.5 到 10 000 μM） 来评估复合物 I + II 连接的 ADP 敏感性。我们将传统的 Michaelis-Menten 动力学方程应用于 JO2 与 [ADP] 数据，计算表观 Km 和最大呼吸 （Vmax），以及两个“连续”双曲方程，得出两个 Km 和 Vmax 值。求解双曲方程，并计算对应于 JO2max 的 50% 的 [ADP] 值。还测定了肌球蛋白重链 （MyHC） 1、 2A 和 2X 的亚型表达。还对具有不同百分比 MyHC 亚型的大鼠骨骼肌样品进行了对照实验。结果两个双曲线方程提供了数据的替代拟合，并确定了 JO2 与 [ADP] 响应的两个不同阶段：第一阶段以低 Vmax（Vmax1,28 ± 10 pmol s-1 mg-1）和表观 Km （Km1,62 ± 54 μM） 为特征，第二阶段以较高的 Vmax （Vmax2， 61 ± 16 pmol s-1 mg-1）和 Km （Km2， 1784 ± 833 μM）。在大鼠肌肉样品中用不同百分比的 MyHC 亚型进行的对照实验中证实了数据。 相关性和受试者作特征分析表明，反应的两个阶段与 MyHC 亚型的百分比相关。结论一种新颖的数学方法（两个连续双曲函数）用于拟合 JO2 与 [ADP] 数据，通过高分辨率呼吸测定法对人类和大鼠的透化骨骼肌纤维获得的数据，与传统的 Michaelis-Menten 动力学方程相比，提供了实验数据的替代拟合。这种替代模型允许在响应中识别出与纤维类型组成相关的两个不同阶段。第一阶段的特征是低表观 Km 和 Vmax 值，与低氧化性 （2A + 2X） MyHC 亚型的百分比相关。第二阶段以高表观 Km 和 Vmax 为特征，与更具氧化性 （1 型） MyHC 亚型有关。