Mitochondrial function in vivo: spectroscopy provides window on cellular energetics

Abstract

Mitochondria integrate the key metabolic fluxes in the cell. This role places this organelle at the center of cellular energetics and, hence, mitochondrial dysfunction underlies a growing number of human disorders and age-related degenerative diseases. Here we present novel analytical and technical methods for evaluating mitochondrial metabolism and (dys)function in human muscle in vivo. Three innovations involving advances in optical spectroscopy (OS) and magnetic resonance spectroscopy (MRS) permit quantifying key compounds in energy metabolism to yield mitochondrial oxidation and phosphorylation fluxes. The first of these uses analytical methods applied to optical spectra to measure hemoglobin (Hb) and myoglobin (Mb) oxygenation states and relative contents ([Hb]/[Mb]) to determine mitochondrial respiration (O2 uptake) in vivo. The second uses MRS methods to quantify key high-energy compounds (creatine phosphate, PCr, and adenosine triphosphate, ATP) to determine mitochondrial phosphorylation (ATP flux) in vivo. The third involves a functional test that combines these spectroscopic approaches to determine mitochondrial energy coupling (ATP/O2), phosphorylation capacity (ATP(max)) and oxidative capacity (O2max) of muscle. These new developments in optical and MR tools allow us to determine the function and capacity of mitochondria noninvasively in order to identify specific defects in vivo that are associated with disease in human and animal muscle. The clinical implication of this unique diagnostic probe is the insight into the nature and extent of dysfunction in metabolic and degenerative disorders, as well as the ability to follow the impact of interventions designed to reverse these disorders.

FIGURE 1:

Diagram of the key energy fluxes in the cell and the magnetic resonance spectroscopic (MRS) and optical spectroscopic (OS) methods used to determine fluxes. ATP flux from mitochondria to the cell ATPases is measured by MRS. The figure in the lower left shows the changes in PCr that are the basis for determining the net ATP demand of the cell that must be met by mitochondrial ATP synthesis. Oxygen flux from capillary to mitochondrial oxidation is determineded from OS measurements of oxy- and deoxy- contents of myoglobin (Mb) and hemoglobin (Hb). The figure in the lower right illustrates the changes in Mb- and Hb-O₂ saturation that underlie the O₂ uptake determination.

FIGURE 2:

, A) Diagram of the 3-phase protocol for measuring mitochondrial function in muscle in vivo. B) Flow diagram of the measurements that define mitochondrial function (ATP/O₂) and capacities (ATP_max and O₂max). Variables in brackets ([]) are chemical concentrations and delta (Δ) denotes the change in these concentrations during the Dynamics phase; τ_PCr is the time constant of PCr recovery.

FIGURE 3:

Optical measurements showing for Hb-O₂ saturation (upper panel) and Mb-O₂ saturation (lower panel) during air breathing (0–3 min), breathing of 100% O₂ (4–8 min), ischemia (9–23 min), and the recovery after tourniquet release (>24 min). The horizontal dashed lines show the calibration end-points: 100% O₂ saturation at 8 min for the Hb-O₂ peak and 27 min for the Mb-O₂ peak, and 0% O₂ saturation at the end of ischemia at 23 min for both peaks.

FIGURE 4:

Comparison of ATP flux, O₂ uptake rates, and P/O in the FDI of adult and elderly subjects from ref [14]. Horizontal solid bars represent the group means and * denotes difference between mean values of the muscles at P<0.05. The horizontal dashed line in the P/O panel defines the theoretical value for well-coupled mitochondria [52, 53, 63]

FIGURE 5:

ATP concentration ([ATP]) as a function of mitochondrial coupling (P/O) in the FDI of adult and elderly subjects from ref [14].

FIGURE 6:

Maximum oxygen uptake of muscle measured in vivo vs. mitochondrial content reported for these human muscles. The dashed line represents a constant maximum O₂ uptake per mitochondrial volume (4 ml O₂ (ml mito⁻¹ min⁻¹)), as reported for animals exercising at the aerobic capacity [61]. Maximum oxygen uptake of muscle is determined using Eq. 4 using data from [14, 21, 64]. Muscle mitochondrial content from [21, 65, 66]. Muscle abbreviations: human first dorsal interosseus (FDI), vastus lateralis (VL) and tibialis anterior (TA).