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

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.

Keywords: ADP; bed rest; mitochondrial sensitivity; myosin heavy chains; skeletal muscle mitochondria.

FIGURE 1

ADP‐stimulated mitochondrial respiration. In the upper panels, respiration rates (JO₂, expressed as a percentage of maximal values) as a function of [ADP] in a typical subject PRE bed rest are shown. Data were fitted using three different mathematical models (see Section 2 and equations in the graphs). K_m indicates the [ADP] at 50% of JO_2max; K_m1 and K_m2 are the [ADP] values needed to stimulate the 50% of V_max1 and V_max2, respectively. In the lower panels, analysis of residuals showed an increased quality of the fitting for the superimposed and for the sequential two‐hyperbolic functions compared to the traditional MM kinetics equation. Goodness of fittings was evaluated by the r ² and the residual sum of squares (RSS) values. See text for further details.

FIGURE 2

Individual and mean (±SD) values of the sum of squared residuals (r ²) are shown in the graphs for the three different fitting approaches (Michaelis–Menten equation [MM], superimposed [SUP] and sequential [SEQ] two‐hyperbolic functions). See text for further details. NS, not significant; ****p < 0.0001.

FIGURE 3

Individual and mean (± SD) values of V_max1, V_max2, K_m1 and K_m2, obtained before (PRE) and after (POST) 10‐day horizontal bed rest by the sequential two‐hyperbolic function fitting approach are shown. Individual and mean (± SD) [ADP] values corresponding to the apparent K_m for a MM kinetics ([ADP] at 50% of JO_2max) are also shown. See text for further details. NS, not significant; ***p < 0.001; **p < 0.01.

FIGURE 4

An inverse linear relation between V_max1 and [ADP] values corresponding to 50% of maximal JO₂ was observed (left panel). A positive linear relation between V_max1 and the percentage of MyHC‐2A + 2X was also observed (right panel). Individual values obtained PRE and POST bed rest are shown. See text for further details.

FIGURE 5

A receiver operating characteristic curve (ROC) analysis was conducted in order to investigate the predictive capacity by MyHC 2A + 2X to discriminate when the value of [ADP] at 50% of JO_2max occurred in the domain of the first sequential hyperbolic function. See text for further details.

FIGURE 6

Data obtained in rat muscles. (a) Individual and mean (± SD) values of MyHC isoform percentages in soleus and tibialis anterior muscles. (b‐c) Individual and mean (± SD) values of maximal ADP‐stimulated mitochondrial respiration (JO_2max) and V_max1. (d) Individual and mean (± SD) values corresponding, for the sequential two‐hyperbolic equations, to the apparent K_m for a MM kinetics ([ADP] at 50% of JO_2max) in soleus and tibialis anterior. (e) A positive linear relation between V_max1 and the percentage of MyHC‐2A + 2X was also observed. Individual values are shown. See text for further details.