Catherine E. Amara

Mild mitochondrial uncoupling impacts cellular aging in human muscles in vivo

Faster aging is predicted in more active tissues and animals because of greater reactive oxygen species generation. Yet age-related cell loss is greater in less active cell types, such as type II muscle fibers. Mitochondrial uncoupling has been proposed as a mechanism that reduces reactive oxygen species production and could account for this paradox between longevity and activity. We distinguished these hypotheses by using innovative optical and magnetic resonance spectroscopic methods applied to noninvasively measured ATP synthesis and O2 uptake in vivo in human muscle. Here we show that mitochondrial function is unchanged with age in mildly uncoupled tibialis anterior muscle (75% type I) despite a high respiratory rate in adults. In contrast, substantial uncoupling and loss of cellular [ATP] indicative of mitochondrial dysfunction with age was found in the lower respiring and well coupled first dorsal interosseus (43–50% type II) of the same subjects. These results reject respiration rate as the sole factor impacting the tempo of cellular aging. Instead, they support mild uncoupling as a mechanism protecting mitochondrial function and contributing to the paradoxical longevity of the most active muscle fibers.

Measuring central-chemoreflex sensitivity in man: rebreathing and steady-state methods compared.

We compared the central-chemoreflex sensitivities estimated from steady-state tests with those estimated from rebreathing tests in five subjects. In one laboratory, each subject underwent nine dynamic end-tidal forcing experiments. Three repetitions of 3, 6 and 9 mmHg step changes in the end-tidal partial pressure of carbon dioxide, from a pre-step partial pressure 1.5 mmHg above resting, were used to establish four points of the steady-state ventilatory response to carbon dioxide. In another laboratory, each subject underwent two rebreathing experiments, one using Reads rebreathing technique and the other a modified rebreathing method which included a prior hyperventilation. The central-chemoreflex sensitivities, estimated from the slopes of the ventilatory responses to carbon dioxide using different combinations of the four steady-state points. were compared to those estimated from the slopes of the rebreathing responses. The steady-state sensitivities were significantly lower than the Read rebreathing sensitivities. The ratio of modified rebreathing sensitivities to steady-state sensitivities was closest to one when steady-state sensitivities were estimated from the two middle points of the ventilatory responses. The mean (SE) ratio of the sensitivities was 1.22 (0.21) in this case. We identify a number of factors that may affect the estimation of central-chemoreflex sensitivity using each technique. These include a maximum limit of the ventilation response at high partial pressures of carbon dioxide, an inability to sustain high ventilation for the duration of the steady-state tests and the inclusion of parts of the ventilatory response whose carbon dioxide partial pressures lie below the central-chemoreflex threshold. We conclude that the modified rebreathing method provides the best estimate of central-chemoreflex sensitivity of the three methods.

Mitochondrial function, fibre types and ageing: new insights from human muscle in vivo

Mitochondrial changes are at the centre of a wide range of maladies, including diabetes, neurodegeneration and ageing‐related dysfunctions. Here we describe innovative optical and magnetic resonance spectroscopic methods that non‐invasively measure key mitochondrial fluxes, ATP synthesis and O2 uptake, to permit the determination of mitochondrial coupling efficiency in vivo (P/O: half the ratio of ATP flux to O2 uptake). Three new insights result. First, mitochondrial coupling can be measured in vivo with the rigor of a biochemical determination and provides a gold standard to define well‐coupled mitochondria (P/O ≈ 2.5). Second, mitochondrial coupling differs substantially among muscles in healthy adults, from values reflective of well‐coupled oxidative phosphorylation in a hand muscle (P/O = 2.7) to mild uncoupling in a leg muscle (P/O = 2.0). Third, these coupling differences have an important impact on cell ageing. We found substantial uncoupling and loss of cellular [ATP] in a hand muscle indicative of mitochondrial dysfunction with age. In contrast, stable mitochondrial function was found in a leg muscle, which supports the notion that mild uncoupling is protective against mitochondrial damage with age. Thus, greater mitochondrial dysfunction is evident in muscles with higher type II muscle fibre content, which may be at the root of the preferential loss of type II fibres found in the elderly. Our results demonstrate that mitochondrial function and the tempo of ageing varies among human muscles in the same individual. These technical advances, in combination with the range of mitochondrial properties available in human muscles, provide an ideal system for studying mitochondrial function in normal tissue and the link between mitochondrial defects and cell pathology in disease.

Mitochondrial Dysfunction: Impact on Exercise Performance and Cellular Aging

Innovative noninvasive methods open a new window on the cell in vivo. This window reveals that the tempo of mitochondrial dysfunction with age varies among muscles and in proportion to Type II muscle fiber content. Exercise training can reverse age-related dysfunction, thereby providing an intervention to slow the pace of aging and disability in the elderly.

Lung function in older humans: the contribution of body composition, physical activity and smoking

An allometric model was used to determine the important factors related to the decline in forced expiratory volume (FEV1.0) across ages 55-86 years in independently living men and women. Measurements were available from a randomized sample of 181 men and 203 women residing in London, Ontario, Canada. The effects of height, age, sex, adiposity, fat free mass (FFM), grip strength and physical activity (PA) on FEV1.0 were assessed using an allometric model to test the hypothesis that sex differences in lung function would be due in part to sex-related differences in the aforementioned variables and would therefore be eliminated by our analysis. The following model was linearized and parameters were identified using standard multiple regression: FEV1.0 = heightβ1·FFMβ2 ·grip strengthβ3 ·PAβ4 ·exp (β0 + β5age + β6sex + β7smoking + β8%body fat)·∈. Results indicate that the amount of FFM and heavy intensity physical activity participated in by the elderly may be more important in influencing forced expiratory function than previously recognized. In addition, results from this study have confirmed the importance of age and height in the prediction of FEV1.0 and demonstrated a negative effect of smoking on lung function. Individuals with a greater FFM and physical activity level tended to be associated with an above average lung function performance. The cross-sectional rate of decline in FEV1.0 determined from our model was ≈12% per decade.

Wavelength Shift Analysis: A Simple Method to Determine the Contribution of Hemoglobin and Myoglobin to In Vivo Optical Spectra

The ability to quantify the contributions of hemoglobin (Hb) and myoglobin (Mb) to in vivo optical spectra has many applications for clinical and research use such as noninvasive measurement of local tissue O2 uptake rates and regional blood content. Recent work has demonstrated an approach to independently measure oxygen saturations of Hb and Mb in optical spectra collected in vivo. However, the utility of this approach is limited without information on tissue concentrations of these species. Here we describe a strategy to quantify the contributions of Hb and Mb to in vivo optical spectra. We have found that the peak position of the deoxy-heme peak around 760 nm in the optical spectra of the deoxygenated tissue is a linear function of the relative contributions of Hb and Mb to the optical spectra. Therefore, analysis of this peak position, hereafter referred to as wavelength shift analysis, reveals the relative concentration of Hb to Mb in solutions and intact tissue. Biochemical analysis of muscle homogenates confirmed that the wavelength shift of the combined Hb/Mb peak in in vivo spectra reflects the ratio of concentrations (Hb/Mb) in muscle. The importance of quantifying the Hb contribution is illustrated by our data demonstrating that Hb accounts for approximately 80% of the optical signal in mouse skeletal muscle but only approximately 20% in human skeletal muscle. This advance will facilitate comparison of the metabolic properties between individual muscles and provides a fully noninvasive approach to measuring local respiration that can be adapted for clinical use.

Mitochondrial function in vivo: Spectroscopy provides window on cellular energetics

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.

Higher Mitochondrial Respiration and Uncoupling with Reduced Electron Transport Chain Content in Vivo in Muscle of Sedentary Versus Active Subjects

OBJECTIVE This study investigated the disparity between muscle metabolic rate and mitochondrial metabolism in human muscle of sedentary vs. active individuals. RESEARCH DESIGN AND METHODS Chronic activity level was characterized by a physical activity questionnaire and a triaxial accelerometer as well as a maximal oxygen uptake test. The ATP and O(2) fluxes and mitochondrial coupling (ATP/O(2) or P/O) in resting muscle as well as mitochondrial capacity (ATP(max)) were determined in vivo in human vastus lateralis muscle using magnetic resonance and optical spectroscopy on 24 sedentary and seven active subjects. Muscle biopsies were analyzed for electron transport chain content (using complex III as a representative marker) and mitochondrial proteins associated with antioxidant protection. RESULTS Sedentary muscle had lower electron transport chain complex content (65% of the active group) in proportion to the reduction in ATP(max) (0.69 ± 0.07 vs. 1.07 ± 0.06 mM sec(-1)) as compared with active subjects. This lower ATP(max) paired with an unchanged O(2) flux in resting muscle between groups resulted in a doubling of O(2) flux per ATP(max) (3.3 ± 0.3 vs. 1.7 ± 0.2 μM O(2) per mM ATP) that reflected mitochondrial uncoupling (P/O = 1.41 ± 0.1 vs. 2.1 ± 0.3) and greater UCP3/complex III (6.0 ± 0.7 vs. 3.8 ± 0.3) in sedentary vs. active subjects. CONCLUSION A smaller mitochondrial pool serving the same O(2) flux resulted in elevated mitochondrial respiration in sedentary muscle. In addition, uncoupling contributed to this higher mitochondrial respiration. This finding resolves the paradox of stable muscle metabolism but greater mitochondrial respiration in muscle of inactive vs. active subjects.

Synthetic signal injection using inductive coupling.

Conversion of MR signals into units of metabolite concentration requires a very high level of diligence to account for the numerous parameters and transformations that affect the proportionality between the quantity of excited nuclei in the acquisition volume and the integrated area of the corresponding peak in the spectrum. We describe a method that eases this burden with respect to the transformations that occur during and following data acquisition. The conceptual approach is similar to the ERETIC method, which uses a pre-calibrated, artificial reference signal as a calibration factor to accomplish the conversion. The distinguishing feature of our method is that the artificial signal is introduced strictly via induction, rather than radiation. We tested a prototype probe that includes a second RF coil rigidly positioned close to the receive coil so that there was constant mutual inductance between them. The artificial signal was transmitted through the second RF coil and acquired by the receive coil in parallel with the real signal. Our results demonstrate that the calibration factor is immune to changes in sample resistance. This is a key advantage because it removes the cumbersome requirement that coil loading conditions be the same for the calibration sample as for experimental samples. The method should be adaptable to human studies and could allow more practical and accurate quantification of metabolite content.

Modelling the Influence of Fat‐Free Mass and Physical Activity on the Decline in Maximal Oxygen Uptake with Age in Older Humans

The purpose of this study was to use an allometric model (maximal oxygen uptake (V̇O2,max) = FFMb1 × PAb2 × exp(b0 + b3 age + b4 sex) ×ε) to determine the influence of fat‐free mass (FFM), physical activity (PA), sex and age on V̇O2,max in older men (n = 152) and women (n = 146) aged 55‐86 years. V̇O2,max was measured during a fatigue‐limited treadmill test, FFM was determined from skinfold thickness and physical activity by the Minnesota Leisure Time Physical Activity questionnaire. The model was linearised by taking the natural logarithm of V̇O2,max, FFM and physical activity. Variables were selected using multiple linear regression (P < 0.05). The sex variable was not significant (P = 0.062). The model explained 72.1% of the variance in V̇O2,max. Significant individual coefficients were incorporated into the model yielding the following expression: V̇O2,max= FFM0.971 × PA0.026 × exp(‐2.48 – 0.015age). Therefore, FFM and physical activity were significant factors contributing to the changes in V̇O2,max with age. In addition, controlling for FFM and physical activity abolished sex differences in V̇O2,max. The rate of decline in V̇O2,max (after accounting for FFM and physical activity) with age, was approximately 15% per decade.