Mitochondrial Sensitivity to Submaximal [ADP] Following Bed Rest: A Novel Two-Phase Approach Associated With Fibre Types

IF 9.4 1区医学 Q1 GERIATRICS & GERONTOLOGY

Journal of Cachexia Sarcopenia and Muscle Pub Date : 2025-04-25 DOI:10.1002/jcsm.13775

Lucrezia Zuccarelli, Maria De Martino, Antonio Filippi, Alice E. Knapton, Benjamin D. Thackray, Giovanni Baldassarre, Boštjan Šimunič, Rado Pišot, Giuseppe Sirago, Elena Monti, Marco Narici, Miriam Isola, Andrew J. Murray, Giovanna Lippe, Bruno Grassi

{"title":"Mitochondrial Sensitivity to Submaximal [ADP] Following Bed Rest: A Novel Two-Phase Approach Associated With Fibre Types","authors":"Lucrezia Zuccarelli, Maria De Martino, Antonio Filippi, Alice E. Knapton, Benjamin D. Thackray, Giovanni Baldassarre, Boštjan Šimunič, Rado Pišot, Giuseppe Sirago, Elena Monti, Marco Narici, Miriam Isola, Andrew J. Murray, Giovanna Lippe, Bruno Grassi","doi":"10.1002/jcsm.13775","DOIUrl":null,"url":null,"abstract":"<div>\n \n \n <section>\n \n <h3> Background</h3>\n \n <p>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<sub>2max</sub>) 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.</p>\n </section>\n \n <section>\n \n <h3> Methods</h3>\n \n <p>Isolated permeabilized <i>vastus lateralis</i> 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<sub>2</sub> versus [ADP] data a traditional Michaelis–Menten kinetics equation, with the calculation of the apparent K<sub>m</sub> and maximal respiration (V<sub>max</sub>), and two ‘sequential’ hyperbolic equations, yielding two K<sub>m</sub> and V<sub>max</sub> values. The two-hyperbolic equations were solved and the [ADP] value corresponding to 50% of JO<sub>2max</sub> 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.</p>\n </section>\n \n <section>\n \n <h3> Results</h3>\n \n <p>The two hyperbolic equations provided an alternative fitting of data and identified two distinct phases of the JO<sub>2</sub> versus [ADP] response: a first phase characterized by low V<sub>max</sub> (V<sub>max1</sub>, 28 ± 10 pmol s<sup>−1</sup> mg<sup>−1</sup>) and apparent K<sub>m</sub> (K<sub>m1</sub>, 62 ± 54 μM) and a second phase characterized by higher V<sub>max</sub> (V<sub>max2</sub>, 61 ± 16 pmol s<sup>−1</sup> mg<sup>−1</sup>) and K<sub>m</sub> (K<sub>m2</sub>, 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.</p>\n </section>\n \n <section>\n \n <h3> Conclusions</h3>\n \n <p>A novel mathematical approach (two sequential hyperbolic functions) for the fitting of JO<sub>2</sub> 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<sub>m</sub> and V<sub>max</sub> values, was correlated with the percentage of less oxidative (Type 2A + 2X) MyHC isoforms. A second phase, characterized by high apparent K<sub>m</sub> and V<sub>max</sub>, was related to more oxidative (Type 1) MyHC isoforms.</p>\n </section>\n </div>","PeriodicalId":48911,"journal":{"name":"Journal of Cachexia Sarcopenia and Muscle","volume":"16 3","pages":""},"PeriodicalIF":9.4000,"publicationDate":"2025-04-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jcsm.13775","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of Cachexia Sarcopenia and Muscle","FirstCategoryId":"3","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/jcsm.13775","RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"GERIATRICS & GERONTOLOGY","Score":null,"Total":0}

引用次数: 0

Abstract

Background

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_2max) 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.

Methods

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 JO₂ versus [ADP] data a traditional Michaelis–Menten kinetics equation, with the calculation of the apparent K_m and maximal respiration (V_max), and two ‘sequential’ hyperbolic equations, yielding two K_m and V_max values. The two-hyperbolic equations were solved and the [ADP] value corresponding to 50% of JO_2max 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.

Results

The 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 V_max (V_max1, 28 ± 10 pmol s⁻¹ mg⁻¹) and apparent K_m (K_m1, 62 ± 54 μM) and a second phase characterized by higher V_max (V_max2, 61 ± 16 pmol s⁻¹ mg⁻¹) and K_m (K_m2, 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.

Conclusions

A 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 K_m and V_max values, was correlated with the percentage of less oxidative (Type 2A + 2X) MyHC isoforms. A second phase, characterized by high apparent K_m and V_max, was related to more oxidative (Type 1) MyHC isoforms.

Abstract Image

查看原文本刊更多论文

卧床休息后对亚最大值[ADP]的线粒体敏感性：一种与纤维类型相关的新型两阶段方法

我们最近证明，在10天的不活动/模拟微重力暴露后，氧化代谢损伤位于线粒体功能的“上游”，通过体外测定最大adp刺激的线粒体呼吸（JO2max）来评估。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来评估线粒体对亚最大值[ADP]的敏感性。方法采用高分辨率呼吸仪对9例年轻男性在水平卧床休息前后分离的渗透股外侧肌纤维进行分析。采用11次ADP亚最大值滴定（12.5 ~ 10000 μM）来评估配合物I + ii连接ADP的敏感性。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“顺序”双曲方程，得到两个Km和Vmax值。求解双双曲方程，计算出50% JO2max对应的[ADP]值。肌球蛋白重链（MyHC） 1、2A和2X的异构体表达也被检测。同时对不同百分比MyHC亚型的大鼠骨骼肌样本进行对照实验。结果两个双曲方程提供了另一种拟合数据，并确定了JO2与[ADP]反应的两个不同阶段：第一阶段具有低Vmax （Vmax1, 28±10 pmol s−1 mg−1）和表观Km (Km1, 62±54 μM)，第二阶段具有较高的Vmax （Vmax2, 61±16 pmol s−1 mg−1）和Km （Km2, 1784±833 μM）。数据在具有不同百分比MyHC亚型的大鼠肌肉样本中进行的对照实验中得到证实。相关性和受者操作特征分析表明，这两个阶段的反应与MyHC亚型的百分比有关。与传统的Michaelis-Menten动力学方程相比，一种新的数学方法（两个顺序双曲函数）可用于拟合人类和大鼠渗透性骨骼肌纤维高分辨率呼吸测量获得的JO2与[ADP]数据，为实验数据提供了另一种拟合方法。这种替代模型允许在响应中识别两个不同的阶段，这与纤维类型组成有关。第一阶段以低表观Km和Vmax值为特征，与较少氧化（2A + 2X型）MyHC亚型的百分比相关。第二阶段，以高表观Km和Vmax为特征，与更多的氧化（1型）MyHC亚型有关。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

求助全文

约1分钟内获得全文求助全文

来源期刊

Journal of Cachexia Sarcopenia and Muscle MEDICINE, GENERAL & INTERNAL-

CiteScore

13.30

自引率

12.40%

发文量

234

审稿时长

16 weeks

期刊介绍： The Journal of Cachexia, Sarcopenia and Muscle is a peer-reviewed international journal dedicated to publishing materials related to cachexia and sarcopenia, as well as body composition and its physiological and pathophysiological changes across the lifespan and in response to various illnesses from all fields of life sciences. The journal aims to provide a reliable resource for professionals interested in related research or involved in the clinical care of affected patients, such as those suffering from AIDS, cancer, chronic heart failure, chronic lung disease, liver cirrhosis, chronic kidney failure, rheumatoid arthritis, or sepsis.