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

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.