Zuccarelli Lucrezia, De Martino Maria, Filippi Antonio, Knapton Alice E, Thackray Benjamin D, Baldassarre Giovanni, Šimunič Boštjan, Pišot Rado, Sirago Giuseppe, Monti Elena, Narici Marco, Isola Miriam, Murray Andrew J, Lippe Giovanna, Grassi Bruno
Department of Medicine, University of Udine, Udine, Italy.
Department of Physiology, Neuroscience and Development, University of Cambridge, Cambridge, UK.
J Cachexia Sarcopenia Muscle. 2025 Jun;16(3):e13775. doi: 10.1002/jcsm.13775.
We 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 (JO) 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.
Isolated 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 JO versus [ADP] data a traditional Michaelis-Menten kinetics equation, with the calculation of the apparent K and maximal respiration (V), and two 'sequential' hyperbolic equations, yielding two K and V values. The two-hyperbolic equations were solved and the [ADP] value corresponding to 50% of JO 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.
The two hyperbolic equations provided an alternative fitting of data and identified two distinct phases of the JO versus [ADP] response: a first phase characterized by low V (V, 28 ± 10 pmol s mg) and apparent K (K, 62 ± 54 μM) and a second phase characterized by higher V (V, 61 ± 16 pmol s mg) and K (K, 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.
A novel mathematical approach (two sequential hyperbolic functions) for the fitting of JO 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 K and V values, was correlated with the percentage of less oxidative (Type 2A + 2X) MyHC isoforms. A second phase, characterized by high apparent K and V, was related to more oxidative (Type 1) MyHC isoforms.
我们最近证明，在10天不活动/模拟微重力暴露后，氧化代谢损伤位于线粒体功能的“上游”，这是通过体外测定的最大ADP刺激的线粒体呼吸（JO）来评估的。本研究的目的是通过一种旨在识别与纤维类型组成相关反应的替代方法，评估线粒体对亚最大[ADP]的敏感性。
在9名年轻男性进行10天水平卧床休息前后，通过高分辨率呼吸测定法分析分离的透化股外侧肌纤维。利用11次亚最大滴定的ADP（从12.5到10000μM）来评估复合体I+II相关的ADP敏感性。我们将传统的米氏动力学方程应用于JO与[ADP]的数据，计算表观K和最大呼吸（V），以及两个“连续”双曲线方程，得出两个K和V值。求解两个双曲线方程，并计算对应于JO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MyHC亚型百分比的大鼠骨骼肌样本进行了对照实验。
两个双曲线方程提供了数据的替代拟合，并确定了JO与[ADP]反应的两个不同阶段：第一阶段的特征是低V（V，28±10 pmol s mg）和表观K（K，62±54μM），第二阶段的特征是较高的V（V，61±16 pmol s mg）和K（K，1784±833μM）。在对具有不同MyHC亚型百分比的大鼠肌肉样本进行的对照实验中证实了这些数据。相关性和受试者工作特征分析表明，反应的两个阶段与MyHC亚型的百分比有关。
一种新的数学方法（两个连续双曲线函数）用于拟合通过高分辨率呼吸测定法在人类和大鼠的透化骨骼肌纤维上获得的JO与[ADP]数据，与传统的米氏动力学方程相比，提供了实验数据的替代拟合。这种替代模型允许识别反应中的两个不同阶段，这与纤维类型组成有关。第一阶段的特征是低表观K和V值，与氧化程度较低（2A+2X型）的MyHC亚型百分比相关。第二阶段的特征是高表观K和V，与氧化程度较高（1型）的MyHC亚型相关。
