Multi-Modal Metabolic Spectroscopy: Transformational innovations in in vivo diagnostic tools, developed in our lab, fill the gap in experimental tools to permit quantitative measurement of the key mass and energy fluxes (ATP and O2) governed by the mitochondria, as illustrated in Figure 1. We have shown how to use these fluxes to characterize key mitochondrial functions: the capacities for phosphorylation (ATPmax) and oxidation (O2max) as well as the coupling efficiency (ATPase/O2 uptake in Figure 1). These innovations provide a new window on the cell that shows – for the first time – substantial variation in mitochondria in response to disease and age well before onset of symptoms or cell energy failure such as ATP loss. These advances now make these measurements possible and highlight human muscles as an ideal system to characterize mitochondrial dysfunction and determine the links to disease and degenerative processes such as aging.
Mitochondrial Quality and Quantity
The importance of these in vivo diagnostic tools is that they are the first non-invasive tools to measure not only the mitochondrial functional properties but also important energetic fluxes such as glycolysis and cell energetic markers such as ATP (1, 2). This ability to reveal important aspects of mitochondrial dysfunction with disease and age is shown in Figure 2 by the variation in coupling (quality: P/O) and capacity (quantity: O2max) that is revealed by these new tools. The top panel shows that P/O – the efficiency of oxidative phosphorylation – declines between adult and elderly groups (1). Earlier in vivo studies have provided evidence (but not direct quantitative measurement) of changes in coupling with thyroid function and activation of uncoupling protein. In vitro studies indicate that uncoupling is an early stage in the apoptotic process leading to the cell loss that underlies sarcopenia (6), (12). Thus a measurement of mitochondrial coupling provides insight into a central mitochondrial property in cell pathology leading to cell death.
The second new development – in vivo measurement of O2max – is shown in the lower panel of Fig. 2. This measure of mitochondrial oxidative capacity directly parallels mitochondrial content in a range of human muscles to reveal a constant O2max per mitochondrial volume. The dashed line in this figure is the relationship found in a survey of mammalian muscles (8) indicating a constant O2max per mitochondrial content that agrees with our measurement in human muscle. Also clear in Fig. 2 is that these new methods are sensitive enough to identify the nature of mitochondrial defects and their impact on cell function in an individual muscle. We are now taking advantage of this sensitivity to pair in vivo diagnostic measures with analysis of muscle biopsy tissue to begin a research program that will analyze the biochemical basis of the mitochondrial functional declines in vivo with age. Another current research goal is to then use these tools to test how mitochondrial functional and adaptive changes are linked to the ATP depletion that leads to the cell loss in aging with insight into degenerative disorders in general.
The breadth of impact is quite wide since mitochondrial dysfunction leading to cell loss is ubiquitous (muscle wasting, neurodegeneration, and heart failure) and is a leading cause not only of disability (exercise intolerance, loss of cognition and motor control) but also mortality. Thus both scientific and medical communities will be affected by the findings of this work. New translational connections between the basic biology of the cell and disease symptoms will likely be revealed by these innovative in vivo measurements. This approach could also prompt a fundamental shift in focus for medical diagnosis (e.g., radiologists). Current diagnostic practices detect anatomic changes of well-developed disease. Shifting diagnosis to early functional signs prior to symptom onset would drastically alter diagnostic practice to focus on halting early disease progression rather than treatment to ameliorate symptoms. Rehabilitation specialists would also benefit by providing insights into interventions for halting early functional changes and reversing later stages showing energetic failure (ATP loss). The end result is an approach with impacts that will transform not only our understanding of the functional variation of mitochondria but also provide new tools for disease diagnosis, new insights into disease progression, and new interventions for halting or reversing disease symptoms. To achieve these new insights on a wide scale, we propose to disseminate our approach to sites involved in human aging research.
The Approach: Mitochondrial Function Protocol
Our key innovation in this proposal is a function test that measures O2 and ATP fluxes and yields mitochondrial phosphorylation (ATPmax) and oxidative capacities (O2max), as well as energy coupling (P/O) in muscles. Our protocol combines optical (OS) and MR spectroscopy (MRS) to measure the contents and dynamics of key energy compounds as shown in Figure 3. This protocol is used with blood flow occlusion to measure P/O (Ischemia Protocol) and exercise to determine ATPmax and O2max (Exercise Protocol). Three phases are used to make these determinations:
An Aerobic rest period is used for quantitation of the concentration of the key energetic compounds (e.g. PCr, shown below the x-axis).
A Dynamics phase is used to perturb [PCr], [Mb-O2], and [Hb-O2]. Ischemia is used to generate the dynamics in these compounds to yield ATP flux and O2 uptake for the P/O determination (see below for full description). An exercise bout is used to drop PCr for the ATPmax determination.
A Recovery phase yields the phosphorylation capacity (ATPmax) from [PCr] recovery. These measurements are then used to derive the oxidative capacity (O2max) of mitochondria in vivo.
Ischemia Protocol: Mitochondrial coupling (P/O)
Our in vivo determination of P/O involves application of a tourniquet to occlude blood flow. We have used this protocol for over 15 yrs with no adverse effects in hundreds of subjects and it is commonly and safely used in surgery for avoiding venous pooling prior to ischemia. We monitor ATP flux (PCr breakdown) by 31P MR spectroscopy and O2 (Mb and Hb desaturation) flux by optical spectroscopy, as shown in the lower panels of Fig. 1. These fluxes are shown in Figure 4 in the context of mitochondrial oxidative phosphorylation with electron transport chain (ETC) flux setting the oxidation rate (O2 uptake) and proton gradient (H+) resulting in the membrane potential (∆y) that drives phosphorylation (ATP supply). ∆y also drives ATP transport into the cytoplasm (by the adenine nucleotide transporter, ANT) and is important in setting the ATP level in the cell.
O2 flux: The protocol starts by occlusion of blood flow by a tourniquet that blocks O2 delivery and results in a drop in oxy-myoglobin (Mb-O2) and oxy-hemoglobin (Hb-O2) saturations as shown in the lower right panel of Figure 1. These saturation changes combined with the respective Mb and Hb concentrations yield the O2 uptake in muscle (see recent papers (10, 11) for details of methods).
ATP flux: Our P/O determination is made once the muscle is anoxic. The left lower panel of Fig. 1 shows our MRS method for measuring this ATP flux (ATPase) based on the changes in PCr that accompany anoxia in the muscle via the creatine kinase reaction:
PCr + ADP + H+ <---> ATP + Cr
where ADP, H+ and Cr are the adenosine diphosphate, proton and free creatine levels in muscle. Glycolytic ATP supply is eliminated from our measurement but determined from the spectral information as a control (see (2) for details). The first in vivo measurement of mitochondrial coupling in a single human muscle is shown in the upper plot in Fig. 2 for the FDI (hand muscle) of adult (19-50 yrs old) and elderly (65-80 yrs old) subjects (2). The P/O values in the FDI muscle of adults (2.7+0.1, n=10) overlap the value for well-coupled mitochondria in vitro (P/O=2.5 (4)) with a small variance among subjects (coefficient of variation (CV) =7%) (2). In contrast, every elderly subject showed some reduction in P/O and thereby uncoupling. At the Pennington Biomedical Research Center our approach is highly reproducible for the P/O measurement of the vastus lateralis with a CV of 8% (n=10, unpublished data). Thus we have a quantitative and very sensitive measure of mitochondrial coupling in vivo that rivals in vitro determinations on isolated tissues and reveals significant change in mitochondrial function with age in vivo.
Exercise Protocol: Mitochondrial Capacities - ATPmax and O2max
Phosphorylation capacity: ATPmax is determined from the PCr recovery time constant (tau) after depletion by exercise as shown in Figure 5:
ATPmax = [PCr]rest/tau.
where [PCr]rest is the PCr level in muscle at rest. This measure of ATPmax agrees well with independent measurements of mitochondrial oxidative phosphorylation capacity (5). We have found excellent reliability of our approach in a collaborating site, the Pennington Biomedical Research Center. Two trials of our ATPmax test had a CV of +6% (n=15) and a correlation of r=0.86 with a slope that parallels the identity line. At a different field strength (2T) and magnet vendor (GE) we have found similarly good reproducibility in our ATPmax determinations with repeated measures on the same subject agreeing to within +11% (3). Thus, with careful standardization of the protocol, training of the local staff, and quality control checks, the ATPmax determination has good reliability at different field strengths (1.5, 2 and 3T), with different magnet vendors (GE, Bruker, Philips) and at different study sites (UW, PBRC).
Oxidative capacity: The O2max or maximum O2 uptake is determined from the phosphorylation capacity (ATPmax) and the coupling of mitochondria (ATP/O2):
O2max = (ATPmax) / (ATP/O2).
Figure 7 shows the relationship between the functional (O2max) and structural measures (% mitochondrial content) in 5 human muscles reported in the literature. This plot demonstrates that in vivo measurement of mitochondrial oxidative capacity (O2max) is directly proportional to the reported mitochondrial content in a range of human muscles. Remarkably, these data fall along a dashed line that defines the maximum oxygen consumption per mitochondrial volume determined in exercising animals (>10 species) (8). These results show that human muscles have the same maximum oxygen consumption per mitochondrial volume as found in a range of mammalian species. Thus we have a functional measure of mitochondrial oxidative capacity that can be determined in vivo and non-invasively in adult and elderly muscle.
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