Creating Transformational Tools For Human and Animal In Vivo Studies of Disease
University of Washington
UW - South Lake Union
In Vivo Diagnostic Tools
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Here we present transformational innovations in in vivo diagnostic tools that 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 show 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) (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. We describe advances that 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. What is needed now is to relate these in vivo measurements to classic in vitro measurements that have defined mitochondrial function in the past (Aim #1).
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 The Approach Our approach is to use muscle as a model system that provides a unique window into the cell energy network while revealing the underlying mechanisms common to all tissues. We will make paired in vivo and in vitro measurements in the human vastus lateralis muscle for goal #1 and in vivo measurements across a range of muscle with differing tempos of aging to achieve goal #2. With these new tools evaluated against classic in vitro measures, we will emulate our success in disseminating our approach at the Pennington Biomedical Research Center in Baton Rouge (see attached letter of support) by adding 3 new research sites to achieve goal #3: University of Florida, University of Pittsburgh, and the National Institute of Aging’s Baltimore Longitudinal Study (see attached letters of support).
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 by 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.
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 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).
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.
1. Amara CE, Marcinek DJ, Shankland EG, Schenkman KA, Arakaki LS, and Conley KE. Mitochondrial function in vivo: spectroscopy provides window on cellular energetics. Methods 46: 312-318, 2008. |
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 (Aim #3). 
Phosphorylation capacity: ATPmax is determined from the PCr recovery time constant (tau) after depletion by exercise as shown in Figure 5: 
Oxidative capacity: The O2max or maximum O2 uptake is determined from the phosphorylation capacity (ATPmax) and the coupling of mitochondria (ATP/O2):