In vivo mitochondrial ATP production is improved in older adult skeletal muscle after a single dose of elamipretide in a randomized trial.

BACKGROUND: Loss of mitochondrial function contributes to fatigue, exercise intolerance and muscle weakness, and is a key factor in the disability that develops with age and a wide variety of chronic disorders. Here, we describe the impact of a first-in-class cardiolipin-binding compound that is targeted to mitochondria and improves oxidative phosphorylation capacity (Elamipretide, ELAM) in a randomized, double-blind, placebo-controlled clinical trial. METHODS: Non-invasive magnetic resonance and optical spectroscopy provided measures of mitochondrial capacity (ATPmax) with exercise and mitochondrial coupling (ATP supply per O2 uptake; P/O) at rest. The first dorsal interosseous (FDI) muscle was studied in 39 healthy older adult subjects (60 to 85 yrs of age; 46% female) who were enrolled based on the presence of poorly functioning mitochondria. We measured volitional fatigue resistance by force-time integral over repetitive muscle contractions. RESULTS: A single ELAM dose elevated mitochondrial energetic capacity in vivo relative to placebo (ΔATPmax; P = 0.055, %ΔATPmax; P = 0.045) immediately after a 2-hour infusion. No difference was found on day 7 after treatment, which is consistent with the half-life of ELAM in human blood. No significant changes were found in resting muscle mitochondrial coupling. Despite the increase in ATPmax there was no significant effect of treatment on fatigue resistance in the FDI. CONCLUSIONS: These results highlight that ELAM rapidly and reversibly elevates mitochondrial capacity after a single dose. This response represents the first demonstration of a pharmacological intervention that can reverse mitochondrial dysfunction in vivo immediately after treatment in aging human muscle.

Building strength, endurance, and mobility using an astaxanthin formulation with functional training in elderly.

BACKGROUND: Building both strength and endurance has been a challenge in exercise training in the elderly, but dietary supplements hold promise as agents for improving muscle adaptation. Here, we test a formulation of natural products (AX: astaxanthin, 12 mg and tocotrienol, 10 mg and zinc, 6 mg) with both anti-inflammatory and antioxidant properties in combination with exercise. We conducted a randomized, double-blind, placebo-controlled study of elderly subjects (65-82 years) on a daily oral dose with interval walking exercise on an incline treadmill. METHODS: Forty-two subjects were fed AX or placebo for 4 months and trained 3 months (3×/week for 40-60 min) with increasing intervals of incline walking. Strength was measured as maximal voluntary force (MVC) in ankle dorsiflexion exercise, and tibialis anterior muscle size (cross-sectional area, CSA) was determined from magnetic resonance imaging. RESULTS: Greater endurance (exercise time in incline walking, >50%) and distance in 6 min walk (>8%) accompanied training in both treatments. Increases in MVC by 14.4% (±6.2%, mean ± SEM, P < 0.02, paired t-test), CSA by 2.7% (±1.0%, P < 0.01), and specific force by 11.6% (MVC/CSA, ±6.0%, P = 0.05) were found with AX treatment, but no change was evident in these properties with placebo treatment (MVC, 2.9% ± 5.6%; CSA, 0.6% ± 1.2%; MVC/CSA, 2.4 ± 5.7%; P > 0.6 for all). CONCLUSIONS: The AX formulation improved muscle strength and CSA in healthy elderly in addition to the elevation in endurance and walking distance found with exercise training alone. Thus, the AX formulation in combination with a functional training programme uniquely improved muscle strength, endurance, and mobility in the elderly.

Impact of prolonged overfeeding on skeletal muscle mitochondria in healthy individuals.

AIMS/HYPOTHESES: Reduced mitochondrial capacity in skeletal muscle has been observed in obesity and type 2 diabetes. In humans, the aetiology of this abnormality is not well understood but the possibility that it is secondary to the stress of nutrient overload has been suggested. To test this hypothesis, we examined whether sustained overfeeding decreases skeletal muscle mitochondrial content or impairs function. METHODS: Twenty-six healthy volunteers (21 men, 5 women, age 25.3 ± 4.5 years, BMI 25.5 ± 2.4 kg/m2) underwent a supervised protocol consisting of 8 weeks of high-fat overfeeding (40% over baseline energy requirements). Before and after overfeeding, we measured systemic fuel oxidation by indirect calorimetry and performed skeletal muscle biopsies to measure mitochondrial gene expression, content and function in vitro. Mitochondrial function in vivo was measured by 31P NMR spectroscopy. RESULTS: With overfeeding, volunteers gained 7.7 ± 1.8 kg (% change 9.8 ± 2.3). Overfeeding increased fasting NEFA, LDL-cholesterol and insulin concentrations. Indirect calorimetry showed a shift towards greater reliance on lipid oxidation. In skeletal muscle tissue, overfeeding increased ceramide content, lipid droplet content and perilipin-2 mRNA expression. Phosphorylation of AMP-activated protein kinase was decreased. Overfeeding increased mRNA expression of certain genes coding for mitochondrial proteins (CS, OGDH, CPT1B, UCP3, ANT1). Despite the stress of nutrient overload, mitochondrial content and mitochondrial respiration in muscle did not change after overfeeding. Similarly, overfeeding had no effect on either the emission of reactive oxygen species or on mitochondrial function in vivo. CONCLUSIONS/INTERPRETATION: Skeletal muscle mitochondria are significantly resilient to nutrient overload. The lower skeletal muscle mitochondrial oxidative capacity in human obesity is likely to be caused by reasons other than nutrient overload per se. TRIAL REGISTRATION: ClinicalTrials.gov NCT01672632.

Pioglitazone-induced improvements in insulin sensitivity occur without concomitant changes in muscle mitochondrial function.

AIMS: Pioglitazone (Pio) is known to improve insulin sensitivity in skeletal muscle. However, the role of Pio in skeletal muscle lipid metabolism and skeletal muscle oxidative capacity is not clear. The aim of this study was to determine the effects of chronic Pio treatment on skeletal muscle mitochondrial activity in individuals with type 2 diabetes (T2D). MATERIALS AND METHODS: Twenty-four participants with T2D (13M/11F 53.38±2.1years; BMI 36.47±1.1kg/m2) were randomized to either a placebo (CON, n=8) or a pioglitazone (PIO, n=16) group. Following 12weeks of treatment, we measured insulin sensitivity by hyperinsulinemic-euglycemic clamp (clamp), metabolic flexibility by calculating the change in respiratory quotient (ΔRQ) during the steady state of the clamp, intra- and extra-myocellular lipid content (IMCL and EMCL, respectively) by 1H magnetic resonance spectroscopy (1H-MRS) and muscle maximal ATP synthetic capacity (ATPmax) by 31P-MRS. RESULTS: Following 12weeks of PIO treatment, insulin sensitivity (p<0.0005 vs. baseline) and metabolic flexibility (p<0.05 vs. CON) significantly increased. PIO treatment significantly decreased IMCL content and increased EMCL content in gastrocnemius, soleus and tibialis anterior muscles. ATPmax was unaffected by PIO treatment. CONCLUSIONS: These results suggest that 12weeks of pioglitazone treatment improves insulin sensitivity, metabolic flexibility and myocellular lipid distribution without any effect on maximal ATP synthetic capacity in skeletal muscle. Consequently, pioglitazone-induced enhancements in insulin responsiveness and fuel utilization are independent of mitochondrial function.

Effects of 12 Months of Caloric Restriction on Muscle Mitochondrial Function in Healthy Individuals.

Context: The effects of caloric restriction (CR) on in vivo muscle mitochondrial function in humans are controversial. Objective: We evaluated muscle mitochondrial function and associated transcriptional profiles in nonobese humans after 12 months of CR. Design: Individuals from an ancillary study of the CALERIE 2 randomized controlled trial were assessed at baseline and 12 months after a 25% CR or ad libitum (control) diet. Setting: The study was performed at Pennington Biomedical Research Center in Baton Rouge, LA. Participants: Study participants included 51 (34 female subjects, 25 to 50 years of age) healthy nonobese individuals randomized to 1 of 2 groups (CR or control). Intervention: This study included 12 months of a 25% CR or ad libitum (control) diet. Main Outcomes: In vivo mitochondrial function [maximal ATP synthesis rate (ATPmax), ATPflux/O2 (P/O)] was determined by 31P-magnetic resonance spectroscopy and optical spectroscopy, and body composition was determined by dual-energy X-ray absorptiometry. In a subset of individuals, a muscle biopsy was performed for transcriptional profiling via quantitative reverse transcription polymerase chain reaction and microarrays. Results: Weight, body mass index (BMI), fat, and fat-free mass (P < 0.001 for all) significantly decreased at month 12 after CR vs control. In vivo ATPmax and P/O were unaffected by 12 months of CR. Targeted transcriptional profiling showed no effects on pathways involved in mitochondrial biogenesis, function, or oxidative stress. A subgroup analysis according to baseline P/O demonstrated that a higher (vs lower) P/O was associated with notable improvements in ATPmax and P/O after CR. Conclusions: In healthy nonobese humans, CR has no effect on muscle mitochondrial function; however, having a "more coupled" (versus "less coupled") phenotype enables CR-induced improvements in muscle mitochondrial function.

NAD+ repletion improves muscle function in muscular dystrophy and counters global PARylation.

Neuromuscular diseases are often caused by inherited mutations that lead to progressive skeletal muscle weakness and degeneration. In diverse populations of normal healthy mice, we observed correlations between the abundance of mRNA transcripts related to mitochondrial biogenesis, the dystrophin-sarcoglycan complex, and nicotinamide adenine dinucleotide (NAD+) synthesis, consistent with a potential role for the essential cofactor NAD+ in protecting muscle from metabolic and structural degeneration. Furthermore, the skeletal muscle transcriptomes of patients with Duchene's muscular dystrophy (DMD) and other muscle diseases were enriched for various poly[adenosine 5'-diphosphate (ADP)-ribose] polymerases (PARPs) and for nicotinamide N-methyltransferase (NNMT), enzymes that are major consumers of NAD+ and are involved in pleiotropic events, including inflammation. In the mdx mouse model of DMD, we observed significant reductions in muscle NAD+ levels, concurrent increases in PARP activity, and reduced expression of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme for NAD+ biosynthesis. Replenishing NAD+ stores with dietary nicotinamide riboside supplementation improved muscle function and heart pathology in mdx and mdx/Utr-/- mice and reversed pathology in Caenorhabditis elegans models of DMD. The effects of NAD+ repletion in mdx mice relied on the improvement in mitochondrial function and structural protein expression (α-dystrobrevin and δ-sarcoglycan) and on the reductions in general poly(ADP)-ribosylation, inflammation, and fibrosis. In combination, these studies suggest that the replenishment of NAD+ may benefit patients with muscular dystrophies or other neuromuscular degenerative conditions characterized by the PARP/NNMT gene expression signatures.

Differences in Mitochondrial Coupling Reveal a Novel Signature of Mitohormesis in Muscle of Healthy Individuals.

CONTEXT: Reduced mitochondrial coupling (ATP/O2 [P/O]) is associated with sedentariness and insulin resistance. Interpreting the physiological relevance of P/O measured in vitro is challenging. OBJECTIVE: To evaluate muscle mitochondrial function and associated transcriptional profiles in nonobese healthy individuals distinguished by their in vivo P/O. DESIGN: Individuals from an ancillary study of Comprehensive Assessment of Long-term Effects of Reducing Intake of Energy phase 2 were assessed at baseline. SETTING: The study was performed at Pennington Biomedical Research Center. PARTICIPANTS: Forty-seven (18 males, 26-50 y of age) sedentary, healthy nonobese individuals were divided into 2 groups based on their in vivo P/O. INTERVENTION: None. Main Outcome(s): Body composition by dual-energy x-ray absorptiometry, in vivo mitochondrial function (P/O and maximal ATP synthetic capacity) by 31P-magnetic resonance spectroscopy and optical spectroscopy were measured. A muscle biopsy was performed to measure fiber type, transcriptional profiling (microarray), and protein expressions. RESULTS: No differences in body composition, peak aerobic capacity, type I fiber content, or mitochondrial DNA copy number were observed between the 2 groups. Compared with the uncoupled group (lower P/O), the coupled group (higher P/O) had higher rates of maximal ATP synthetic capacity (maximal ATP synthetic capacity, P < .01). Transcriptomics analyses revealed higher expressions of genes involved in mitochondrial remodeling and the oxidative stress response in the coupled group. A trend for higher mitonuclear protein imbalance (P = .06) and an elevated mitochondrial unfolded protein response (heat shock protein 60 protein; P = .004) were also identified in the coupled group. CONCLUSIONS: Higher muscle mitochondrial coupling is accompanied by an overall elevation in mitochondrial function, a novel transcriptional signature of oxidative stress and mitochondrial remodeling and indications of an mitochondrial unfolded protein response.

Muscle force, work and cost: a novel technique to revisit the Fenn effect.

Muscle produces force by forming cross-bridges, using energy released from ATP. While the magnitude and duration of force production primarily determine the energy requirement, nearly a century ago Fenn observed that muscle shortening or lengthening influenced energetic cost of contraction. When work is done by the muscle, the energy cost is increased and when work is done on the muscle the energy cost is reduced. However, the magnitude of the 'Fenn effect' and its mirror ('negative Fenn effect') have not been quantitatively resolved. We describe a new technique coupling magnetic resonance spectroscopy with an in vivo force clamp that can directly quantify the Fenn effect [E=I+W, energy liberated (E) equals the energy cost of isometric force production (I) plus the work done (W)] and the negative Fenn effect (E=I-W) for one muscle, the first dorsal interosseous (FDI). ATP cost was measured during a series of contractions, each of which occurred at a constant force and for a constant duration, thus constant force-time integral (FTI). In all subjects, as the FTI increased with load, there was a proportional linear increase in energy cost. In addition, the cost of producing force greatly increased when the muscle shortened, and was slightly reduced during lengthening contraction. These results, though limited to a single muscle, contraction velocity and muscle length change, do quantitatively support the Fenn effect. We speculate that they also suggest that an elastic element within the FDI muscle functions to preserve the force generated within the cross-bridges.

Skeletal Muscle Mitochondrial Function and Fatigability in Older Adults.

BACKGROUND: Fatigability increases while the capacity for mitochondrial energy production tends to decrease significantly with age. Thus, diminished mitochondrial function may contribute to higher levels of fatigability in older adults. METHODS: The relationship between fatigability and skeletal muscle mitochondrial function was examined in 30 participants aged 78.5 ± 5.0 years (47% female, 93% white), with a body mass index of 25.9 ± 2.7 kg/m(2) and usual gait-speed of 1.2 ± 0.2 m/s. Fatigability was defined using rating of perceived exertion (6-20 point Borg scale) after a 5-minute treadmill walk at 0.72 m/s. Phosphocreatine recovery in the quadriceps was measured using (31)P magnetic resonance spectroscopy and images of the quadriceps were captured to calculate quadriceps volume. ATPmax (mM ATP/s) and oxidative capacity of the quadriceps (ATPmax·Quadriceps volume) were calculated. Peak aerobic capacity (VO2peak) was measured using a modified Balke protocol. RESULTS: ATPmax·Quadriceps volume was associated with VO2peak and was 162.61mM ATP·mL/s lower (p = .03) in those with high (rating of perceived exertion ≥10) versus low (rating of perceived exertion ≤9) fatigability. Participants with high fatigability required a significantly higher proportion of VO2peak to walk at 0.72 m/s compared with those with low fatigability (58.7 ± 19.4% vs 44.9 ± 13.2%, p < .05). After adjustment for age and sex, higher ATPmax was associated with lower odds of having high fatigability (odds ratio: 0.34, 95% CI: 0.11-1.01, p = .05). CONCLUSIONS: Lower capacity for oxidative phosphorylation in the quadriceps, perhaps by contributing to lower VO2peak, is associated with higher fatigability in older adults.

Nuclear receptor/microRNA circuitry links muscle fiber type to energy metabolism.

The mechanisms involved in the coordinate regulation of the metabolic and structural programs controlling muscle fitness and endurance are unknown. Recently, the nuclear receptor PPARß/δ was shown to activate muscle endurance programs in transgenic mice. In contrast, muscle-specific transgenic overexpression of the related nuclear receptor, PPARα, results in reduced capacity for endurance exercise. We took advantage of the divergent actions of PPARß/δ and PPARα to explore the downstream regulatory circuitry that orchestrates the programs linking muscle fiber type with energy metabolism. Our results indicate that, in addition to the well-established role in transcriptional control of muscle metabolic genes, PPARß/δ and PPARα participate in programs that exert opposing actions upon the type I fiber program through a distinct muscle microRNA (miRNA) network, dependent on the actions of another nuclear receptor, estrogen-related receptor γ (ERRγ). Gain-of-function and loss-of-function strategies in mice, together with assessment of muscle biopsies from humans, demonstrated that type I muscle fiber proportion is increased via the stimulatory actions of ERRγ on the expression of miR-499 and miR-208b. This nuclear receptor/miRNA regulatory circuit shows promise for the identification of therapeutic targets aimed at maintaining muscle fitness in a variety of chronic disease states, such as obesity, skeletal myopathies, and heart failure.

Skeletal muscle mitochondrial energetics are associated with maximal aerobic capacity and walking speed in older adults.

BACKGROUND: Lower ambulatory performance with aging may be related to a reduced oxidative capacity within skeletal muscle. This study examined the associations between skeletal muscle mitochondrial capacity and efficiency with walking performance in a group of older adults. METHODS: Thirty-seven older adults (mean age 78 years; 21 men and 16 women) completed an aerobic capacity (VO2 peak) test and measurement of preferred walking speed over 400 m. Maximal coupled (State 3; St3) mitochondrial respiration was determined by high-resolution respirometry in saponin-permeabilized myofibers obtained from percutanous biopsies of vastus lateralis (n = 22). Maximal phosphorylation capacity (ATPmax) of vastus lateralis was determined in vivo by (31)P magnetic resonance spectroscopy (n = 30). Quadriceps contractile volume was determined by magnetic resonance imaging. Mitochondrial efficiency (max ATP production/max O2 consumption) was characterized using ATPmax per St3 respiration (ATPmax/St3). RESULTS: In vitro St3 respiration was significantly correlated with in vivo ATPmax (r (2) = .47, p = .004). Total oxidative capacity of the quadriceps (St3*quadriceps contractile volume) was a determinant of VO2 peak (r (2) = .33, p = .006). ATPmax (r (2) = .158, p = .03) and VO2 peak (r (2) = .475, p < .0001) were correlated with preferred walking speed. Inclusion of both ATPmax/St3 and VO2 peak in a multiple linear regression model improved the prediction of preferred walking speed (r (2) = .647, p < .0001), suggesting that mitochondrial efficiency is an important determinant for preferred walking speed. CONCLUSIONS: Lower mitochondrial capacity and efficiency were both associated with slower walking speed within a group of older participants with a wide range of function. In addition to aerobic capacity, lower mitochondrial capacity and efficiency likely play roles in slowing gait speed with age.

Exercise efficiency is reduced by mitochondrial uncoupling in the elderly.

A reduction in exercise efficiency accompanies ageing in humans. Here we evaluated the impact of changes in the contractile-coupling and mitochondrial-coupling efficiencies on the reduction in exercise efficiency in the elderly. Nine adult (mean, 38.8 years old) and 40 elderly subjects (mean, 68.8 years old) performed a cycle ergometer test to measure O2 uptake and leg power output up to the aerobic limit ( ). Reduced leg power output per unit O2 uptake was reflected in a drop in delta efficiency (εD) from 0.27 ± 0.01 (mean ± SEM) in adults to 0.22 ± 0.01 in the elderly group. Similar declines with age were apparent for both the leg power output at and the ATP generation capacity (ATPmax) determined in vivo using (31)P magnetic resonance spectroscopy. These similar declines resulted in unchanged contractile-coupling efficiency values (εC) in the adult (0.50 ± 0.05) versus the elderly group (0.58 ± 0.04) and agreed with independent measures of muscle contractile-coupling efficiency in human quadriceps (0.5). The mitochondrial-coupling efficiency calculated from the ratio of delta to contractile-coupling efficiencies in the adults (εD/εC = 0.58 ± 0.08) corresponded to values for well-coupled mitochondria (0.6); however, εD/εC was significantly lower in the elderly subjects (0.44 ± 0.03). Conversion of ATPmax per mitochondrial volume (ATPmax/Vv[mt,f]) reported in these groups into thermodynamic units confirmed this drop in mitochondrial-coupling efficiency from 0.57 ± 0.08 in adults to 0.41 ± 0.03 in elderly subjects. Thus, two independent methods revealed that reduced mitochondrial-coupling efficiency was a key part of the drop in exercise efficiency in these elderly subjects and may be an important part of the loss of exercise performance with age.

Ectopic lipid accumulation and reduced glucose tolerance in elderly adults are accompanied by altered skeletal muscle mitochondrial activity.

CONTEXT: Aging is associated with insulin resistance and unfavorable changes in body composition including increased fat accumulation, particularly in visceral and ectopic depots. Recent studies suggest that skeletal muscle mitochondrial activity may underlie some age-associated metabolic abnormalities. OBJECTIVE: Our objective was to measure mitochondrial capacity and coupling of the vastus lateralis muscle in elderly and young adults using novel in vivo approaches and relate mitochondrial activity to metabolic characteristics. DESIGN: This was a cross-sectional study. PARTICIPANTS AND INTERVENTION: Fourteen sedentary young (seven males and seven females, 20-34 yr of age) and 15 sedentary elderly (seven males and eight females, 70-84 yr of age) nonobese subjects selected for similar body weight underwent measures of body composition by magnetic resonance imaging and dual-energy x-ray absorptiometry, oral glucose tolerance, and in vivo mitochondrial activity by (31)P magnetic resonance and optical spectroscopy. Muscle biopsy was carried out in the same muscle to measure mitochondrial content, antioxidant activity, fiber type, and markers of mitochondrial biogenesis. RESULTS: Elderly volunteers had reduced mitochondrial capacity (P = 0.05) and a trend for decreased coupling efficiency (P = 0.08) despite similar mitochondrial content and fiber type distribution. This was accompanied by greater whole-body oxidative stress (P = 0.007), less skeletal muscle mass (P < 0.001), more adipose tissue in all depots (P ≤ 0.002) except intramyocellular (P = 0.72), and lower glucose tolerance (P = 0.07). CONCLUSIONS: Elderly adults show evidence of altered mitochondrial activity along with increased adiposity, oxidative stress, and reduced glucose tolerance, independent of obesity. We propose that mild uncoupling may be induced secondary to age-associated oxidative stress as a mechanism to dissipate the proton-motive force and protect against further reactive oxygen species production and damage.

Skeletal muscle NAMPT is induced by exercise in humans.

In mammals, nicotinamide phosphoribosyltransferase (NAMPT) is responsible for the first and rate-limiting step in the conversion of nicotinamide to nicotinamide adenine dinucleotide (NAD+). NAD+ is an obligate cosubstrate for mammalian sirtuin-1 (SIRT1), a deacetylase that activates peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha), which in turn can activate mitochondrial biogenesis. Given that mitochondrial biogenesis is activated by exercise, we hypothesized that exercise would increase NAMPT expression, as a potential mechanism leading to increased mitochondrial content in muscle. A cross-sectional analysis of human subjects showed that athletes had about a twofold higher skeletal muscle NAMPT protein expression compared with sedentary obese, nonobese, and type 2 diabetic subjects (P < 0.05). NAMPT protein correlated with mitochondrial content as estimated by complex III protein content (R(2) = 0.28, P < 0.01), MRS-measured maximal ATP synthesis (R(2) = 0.37, P = 0.002), and Vo(2max) (R(2) = 0.63, P < 0.0001). In an exercise intervention study, NAMPT protein increased by 127% in sedentary nonobese subjects after 3 wk of exercise training (P < 0.01). Treatment of primary human myotubes with forskolin, a cAMP signaling pathway activator, resulted in an approximately 2.5-fold increase in NAMPT protein expression, whereas treatment with ionomycin had no effect. Activation of AMPK via AICAR resulted in an approximately 3.4-fold increase in NAMPT mRNA (P < 0.05) as well as modest increases in NAMPT protein (P < 0.05) and mitochondrial content (P < 0.05). These results demonstrate that exercise increases skeletal muscle NAMPT expression and that NAMPT correlates with mitochondrial content. Further studies are necessary to elucidate the pathways regulating NAMPT as well as its downstream effects.

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.

Mitochondrial dysfunction and age.

PURPOSE OF REVIEW: Mitochondrial dysfunction is commonly thought to result from oxidative damage that leads to defects in the electron transport chain (ETC). In this review, we highlight new research indicating that there are early changes in mitochondrial function that precede ETC defects and are reversible thereby providing the possibility of slowing the tempo of mitochondrial aging and cell death. RECENT FINDINGS: Increased mitochondrial uncoupling - reduced adenosine triphosphate (ATP) produced per O2 uptake - and cell ATP depletion are evident in human muscle nearly a decade before accumulation of irreversible DNA damage that causes ETC defects. New evidence points to reduction in activators of biogenesis (e.g. PGC-1alpha) and to degradation of mitochondria allowing accumulation of molecular and membrane damage in aged mitochondria. The early dysfunction appears to be reversible based on improved mitochondrial function in vivo and elevated gene expression levels after exercise training. SUMMARY: New molecular and in vivo findings regarding the onset and reversibility of mitochondrial dysfunction with age indicate the potential: 1) for diagnostic tools to identify patients at risk for severe irreversible defects later in life; and 2) of an intervention to delay the tempo of aging and improve the quality of life of the elderly.

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 O(2) 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.

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 O(2) uptake, to permit the determination of mitochondrial coupling efficiency in vivo (P/O: half the ratio of ATP flux to O(2) 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 approximately 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.

Reduced mitochondrial coupling in vivo alters cellular energetics in aged mouse skeletal muscle.

The mitochondrial theory of ageing proposes that the accumulation of oxidative damage to mitochondria leads to mitochondrial dysfunction and tissue degeneration with age. However, no consensus has emerged regarding the effects of ageing on mitochondrial function, particularly for mitochondrial coupling (P/O). One of the main barriers to a better understanding of the effects of ageing on coupling has been the lack of in vivo approaches to measure P/O. We use optical and magnetic resonance spectroscopy to independently quantify mitochondrial ATP synthesis and O2 uptake to determine in vivo P/O. Resting ATP demand (equal to ATP synthesis) was lower in the skeletal muscle of 30-month-old C57Bl/6 mice compared to 7-month-old controls (21.9 +/- 1.5 versus 13.6 +/- 1.7 nmol ATP (g tissue)(-1) s(-1), P = 0.01). In contrast, there was no difference in the resting rates of O2 uptake between the groups (5.4 +/- 0.6 versus 8.4 +/- 1.6 nmol O2 (g tissue)(-1) s(-1)). These results indicate a nearly 50% reduction in the mitochondrial P/O in the aged animals (2.05 +/- 0.07 versus 1.05 +/- 0.36, P = 0.02). The higher resting ADP (30.8 +/- 6.8 versus 58.0 +/- 9.5 micromol g(-1), P = 0.05) and decreased energy charge (ATP/ADP) (274 +/- 70 versus 84 +/- 16, P = 0.03) in the aged mice is consistent with an impairment of oxidative ATP synthesis. Despite the reduced P/O, uncoupling protein 3 protein levels were not different in the muscles of the two groups. These results demonstrate reduced mitochondrial coupling in aged skeletal muscle that alters cellular metabolism and energetics.