Mitochondrial respiratory capacity and coupling control decline with age in human skeletal muscle

Abstract

Mitochondrial health is critical to physiological function, particularly in tissues with high ATP turnover, such as striated muscle. It has been postulated that derangements in skeletal muscle mitochondrial function contribute to impaired physical function in older adults. Here, we determined mitochondrial respiratory capacity and coupling control in skeletal muscle biopsies obtained from young and older adults. Twenty-four young (28 ± 7 yr) and thirty-one older (62 ± 8 yr) adults were studied. Mitochondrial respiration was determined in permeabilized myofibers from the vastus lateralis after the addition of substrates oligomycin and CCCP. Thereafter, mitochondrial coupling control was calculated. Maximal coupled respiration (respiration linked to ATP production) was lower in muscle from older vs. young subjects (P < 0.01), as was maximal uncoupled respiration (P = 0.06). Coupling control in response to the ATP synthase inhibitor oligomycin was lower in older adults (P < 0.05), as was the mitochondria flux control ratio, coupled respiration normalized to maximal uncoupled respiration (P < 0.05). Calculation of respiratory function revealed lower respiration linked to ATP production (P < 0.001) and greater reserve respiration (P < 0.01); i.e., respiratory capacity not used for phosphorylation in muscle from older adults. We conclude that skeletal muscle mitochondrial respiratory capacity and coupling control decline with age. Lower respiratory capacity and coupling efficiency result in a reduced capacity for ATP production in skeletal muscle of older adults.

advances in scientific understanding and clinical practice mean that we can now expect to live considerably longer than our ancestors. Since longevity per se has increased in recent decades, healthy aging has become a focus of gerontology researchers, with the aim of preserving quality of life and independence in elderly individuals. Skeletal muscle is particularly susceptible to advancing age. Reduced physical activity and sensitivity to anabolic stimuli are thought to result in sarcopenia, age-related muscle loss (6). Furthermore, numerous studies have shown that whole body maximal oxygen consumption (V̇o_{2 max}) declines with advancing age (3, 5, 20, 30, 33, 37). Moreover, reduced V̇o_{2 max} in older adults is often accompanied by a decline in skeletal muscle oxidative capacity (5, 20, 30, 33, 37). Thus, reduced muscle quality may be a component of sarcopenia that impacts functional capacity.

As the subcellular organelles responsible for the bulk of oxygen consumption and ATP production in vivo (29), the abundance and function of mitochondria within skeletal muscle are critical to contractile function. Previous research suggests that skeletal muscle mitochondrial function declines with advancing age (1, 3, 5, 17, 30, 31, 33, 35, 37, 38). Interestingly, though, others have shown that skeletal muscle mitochondrial respiration is similar in older men and women compared with cohorts of young men and women (10, 13, 16, 18, 28). Indeed, when matched for V̇o_{2 max} (18) or physical activity (16), skeletal muscle mitochondrial function is similar in young and older adults, suggesting that skeletal muscle mitochondrial dysfunction may not be an inherent component of senescence but rather a consequence of inactivity (27). Nevertheless, although detriments in skeletal muscle mitochondrial function may not be causal in the etiology of senescence, there is certainly strong evidence of an association (1, 3, 5, 7, 20, 30, 33, 37). Importantly, mitochondria are responsive to environmental stimuli (11, 12, 14). Moreover, greater ATP production rates of isolated skeletal muscle mitochondria from trained vs. sedentary older adults are associated with greater central and peripheral insulin sensitivity, and greater V̇o_{2 max} (17). This makes the mitochondrion an organelle of interest when considering strategies to augment metabolic function in humans, particularly in compromised populations such as the elderly.

In the present study, we set out to determine and compare skeletal muscle mitochondrial respiratory capacity and coupling control in a cohort of young adults and a cohort of older adults. Using high-resolution respirometry (HRR), we employed two respiratory protocols where specific substrate, inhibitor, and uncoupler titrations were utilized to investigate mitochondrial respiratory capacity and function within permeabilized myofiber bundles (32). Our purpose was to comprehensively compare mitochondrial respiratory capacity and coupling control in young and old skeletal muscle to ascertain whether one or both of these parameters decline with age. Our working hypothesis was that aging is associated with a decline in both skeletal muscle mitochondrial capacity and function.

MATERIALS AND METHODS

Human subjects.

Healthy adults (19–85 yr) were recruited for the present study by placing advertisements in local newspapers and flyers at local leisure centers and cafes. Study recruitment ran from January 2012 to March 2015. Subjects were in good health and free from any long-term ailments. All participants were recreationally active but not engaged in any form of structured exercise training. Prior to enrollment in the study, all subjects participated in a screening visit where blood was drawn for standard hepatic and renal function markers, after which, an oral glucose tolerance test was performed. Diabetic individuals were not included in the current study, nor were individuals who reported performing strenuous exercise training on three or more days per week. Young females recruited (<45 yr) were all premenopausal. Older females recruited (>55 yr) were all postmenopausal (>1 yr since the cessation of menses) and not taking any form of hormone replacement therapy. All participants were admitted to the Clinical Research Center at the University of Texas Medical Branch, Galveston, Texas, or the Clinical Research Center at the Reynolds Aging Center, University of Arkansas for Medical Science, Little Rock, Arkansas, for study visits. This study was approved by the Institutional Review Boards at the University of Texas Medical Branch and the University of Arkansas for Medical Science. All participants provided informed written consent prior to enrolment.

Skeletal muscle sample collection.

Participants reported to the Clinical Research Center the evening before each study. Following an overnight fast, a percutaneous biopsy of the vastus lateralis muscle was collected under local Lidocaine anesthesia using a suction-adapted 6-mm Bergström needle (2). To minimize subject-to-subject variation, the middle portion (the midpoint between the knee and the hip) of the vastus lateralis was sampled. One portion of muscle (∼25 mg) was immediately submerged in an ice-cold preservation buffer (BIOPS) containing (in mM) 10 CaK₂-EGTA, 7.23 K₂-EGTA, 20 imidazole, 20 taurine 50 K-MES, 0.5 dithiothreitol, 6.56 MgCl₂, 5.77 ATP, and 15 creatine phosphate (pH of 7.1). Samples were kept in ice-cold BIOPS buffer and immediately transferred to the laboratory.

Preparation of permeabilized myofibers.

Myofiber bundles were separated using sharp forceps in a six-well plate containing ice-cold BIOPS buffer. Myofiber bundles were dissected free of all visible blood and connective tissue. This procedure usually began within 1 h after muscle samples were colleced. Chemical permeabilization of the sacrolemmal membrane was achieved by incubating myofiber bundles in 2 ml of pH-adjusted (7.1) MIR05 buffer (in mM: 0.5 EGTA, 3 MgCl₂, 60 K-lactobionate, 20 taurine, 10 KH₂PO₄, 20 HEPES, and 110 sucrose, and 1 mg/ml essential fatty acid-free bovine serum albumin) containing 50 μg/ml saponin. Samples were agitated in MIR05 buffer containing saponin for 20–30 min at 4°C, after which, myofiber bundles were transferred to 2 ml of MIR05 buffer alone and agitated for 10–15 min to wash away any remaining saponin. Approximately 1–3 mg of permeabilized myofiber bundles were blotted on filter paper then weighed on a precision microbalance (Mettler-Toledo, Zaventem, Belgium) and immediately transferred to ice-cold BIOPS buffer for ∼5 min. Thereafter, myofiber bundles were placed in an Oxygraph-2k (O2K) respirometer chamber (Oroboros Instruments, Innsbruck, Austria) containing 2 ml of MIR05 buffer. All muscle samples were analyzed on the same day they were collected. The time from muscle biopsy collection to performing HRR analysis was typically 2–3 h. HRR analysis was performed in one of five O2K respirometers situated over two research sites.

HRR.

On the morning of each experiment, a background calibration was performed on each O2K polygraphic oxygen sensor (POS). This calibration was performed in MIR05 buffer at air saturation. Zero oxygen and instrumental background calibrations were performed at regular intervals throughout the data collection period (∼36 mo) using dithionite titrations. This ensured acceptable POS instrumental background and stability over time. Temperature was maintained at 37 ± 0.01°C by an electronic Peltier during all HRR experiments. O₂ concentration within the MIR05 buffer was recorded at 2- to 4-s intervals from which O₂ fluxes were calculated in the picomolar range (DatLab; Oroboros Instruments, Innsbruck, Austria). Once myofiber bundles had been placed in the O2K chamber, a gas phase was created. Around 5 ml of 99% O₂ was injected into each O2K chamber, and equilibration of that gas phase and MIR05 O₂ concentrations was monitored until an O₂ concentration of 400–450 μM was achieved in the MIR05 buffer; O₂ flux measurements were typically made when O₂ concentrations were in the range of 200–400 μM to minimize any O₂ dependency artifacts from the data.

Substrate uncoupler inhibitor titration (SUIT) protocols.

Mitochondrial respiratory capacity and function were interrogated by the sequential addition of substrates, inhibitors and uncouplers. Two specific SUIT protocols were employed here, Protocol A and Protocol B, which are schematically depicted in Fig. 1. These protocols were designed to study specific aspects of mitochondrial respiratory capacity and coupling control.

Fig. 1.

Schematic depiction of the substrate uncoupler inhibitor titration (SUIT) protocols employed in the current study. Substrates consisted of pyruvate (5 mM), octanoyl-l-carnitine (1.5 mM), malate (5 mM), and glutamate (10 mM) to maximally stimulate tricarboxylic cycle anaploresis (Protocols A and B). The mitochondrial coupling state following each titration is depicted above the Oxygraph trace; the corresponding respiratory state is depicted below the Oxygraph trace.

Leak respiration (state 2) supported primarily by electron flow through complex I of the respiratory chain (L_I), was achieved by the titration of 1.5 mM octanoyl-l-carnitine, 5 mM pyruvate, 2 mM malate, and 10 mM glutamate. Electron transfer was coupled to phosphorylation by the addition of 5 mM ADP, assessing phosphorylating state 3 respiration with electron transfer supported by Complex I (P_I). Succinate at 10mM was added to the O2K chamber to induce maximal phosphorylating (state 3) respiration with parallel electron input from Complex I and II (P_I+II). oligomycin at 5 μM was added (Protocol A only) to assess leak respiration when substrates and ADP were provided, but ATP synthase inhibited (L_I+II). cytochrome c (10 μM) was added to access the competence of the outer mitochondrial membrane (Protocol B only). The absence of a significant increase in respiratory flux following the addition of cytochrome c indicates that the outer mitochondrial membranes are largely intact. Any sample that showed a significant response to cytochrome c (an increase in respiration of more than 10% was not included in the final analysis) Finally, oxidative phosphorylation was uncoupled by the titration of carbonyl cyanide m-chlorophenyl hydrazone (CCCP) to a final concentration of 5 μM to access maximal electron transfer capacity (E) (Protocol B only). While we refer to respiration as being supported by complex I, complex II, or both, a fatty acid is also added with complex I substrates. This means that the electron transfer flavoprotein will also be providing reducing equivalents to the electron transfer chain at all respiratory states.

Protocols A and B were assayed concurrently in one O2K respirometer (i.e., Protocol A was run in one chamber and Protocol B in the other chamber). We chose this approach since both the inhibitor oligomycin (Protocol A) and the uncoupler CCCP (Protocol B) would be introduced into the O2K chambers. In our hands, we find respiration following the titration of CCCP to be lower in the presence of oligomycin, therefore no longer providing a measure of maximal respiratory capacity. Thus, we thought that it was imperative to assay state 4_O (L_I+II) respiration and maximal electron transfer capacity (E) in separate chambers. However, this means respiratory data were generated from a single sample and were not the average of technical replicates.

Respiratory flux data were selected from O2K traces and then exported to Microsoft Excel for further analysis. Respiration data were used for analysis only when a steady state was achieved. Following titration of substrates and ADP, 5–10 min typically elapsed before the next substrate/inhibitor/uncoupler was titrated to the O2K chamber. This allowed a minimum of 5 min of steady-state O₂ flux data to be averaged (∼150 data points when Datlab was recording at 2-s intervals) for each respiratory state. In the case of oligomycin, steady-state respiration is typically not achieved until 15–20 min after titration. Thus, ∼20 min was allowed to elapse following oligomycin titration, and only data from the last 5 min of this period was captured for final data analysis. For CCCP, We typically see a peak in respiration followed by a slow decline in respiration. This is likely due to O₂ dependency, at least in part, since this response is more pronounced in samples with high respiratory capacity >60 pmol·mg⁻¹·s⁻¹ when more than 3 mg of tissue is used. To minimize this, the respiration buffer was reoxygenated prior to CCCP titration. To further avoid any artifacts from O₂ dependency, respiratory flux data were captured immediately after a steady state was achieved following CCCP titration; 1–2 min of data was typical for maximal respiration after CCCP titration (30–60 data points when Datlab was recording at 2-s intervals).

Calculation of intrinsic mitochondrial function.

HRR permits the determination of numerous respiratory states within the same sample, from which respiratory control ratios/factors can be calculated (8, 9, 22). As these respirometry measurements are made in the same sample, and thus the same population of mitochondria, they present a robust means of calculating intrinsic mitochondrial function (mitochondrial quality), which is independent of mitochondria abundance.

The titration of saturating levels of ADP allowed the respiratory control ratio for ADP (RCR_ADP) to be calculated (Eq. 1). Similarly, the addition of saturating concentrations of succinate allowed the substrate control ratio for succinate (SCR_Succinate) to be calculated (Eq. 2):

$${\text{RCR}}_{\text{ADP}}={\mathrm{P}}_{\text{I}}/{\mathrm{L}}_{\text{I}}$$ (1)

$${\text{SCR}}_{\text{Succinate}}={\text{P}}_{\text{I}+\text{II}}/{\mathrm{P}}_{\text{I}}$$ (2)

Respiratory Protocol A specifically tested mitochondrial coupling control following oligomycin titration. The proportion of P_I+II respiration insensitive to oligomycin can be determined by calculating the flux control ratio for oligomycin (FCR_OM) (Eq. 3). Similarly, the proportion of P_I+II respiration sensitive to oligomycin can be determined by calculating the flux control factor for oligomycin (FCF_OM) (Eq. 4) (9):

$${\text{FCR}}_{\text{OM}}={\text{L}}_{\text{I}+\text{II}}/{\mathrm{L}}_{\text{I}}$$ (3)

$${\text{FCF}}_{\text{OM}}=1-{\text{FCR}}_{\text{OM}}$$ (4)

Respiratory Protocol B differed from Protocol A by determining outer membrane intactness (by cytochrome c titration) and maximal respiratory capacity (E) (by CCCP titration). As described by Gnaiger and colleagues (8, 9, 22), maximal respiration (E) in the uncoupled state (state 3_U) provides an internal gauge of the capacity of the electron transfer system and can therefore be used to normalize other respiratory states, which were measured within the capacity of the electron transfer system. These flux control ratios can be calculated for leak respiration (FCR_L) (Eq. 5), phosphorylating respiration with electron provision from complex I (FCR_PI) (Eq. 6), and complex I and II (FCR_PI+II) (Eq. 7):

$${\text{FCR}}_{\text{L}}=\text{L}/\text{E}$$ (5)

$${\text{FCR}}_{\text{PI}}={\mathrm{P}}_{\text{I}}/\text{E}$$ (6)

$${\text{FCR}}_{\text{PI}+\text{II}}={\text{P}}_{\text{I}+\text{II}}/\text{E}$$ (7)

A novel aspect of the current study was that two separate respiratory protocols (A and B) were employed to investigate mitochondrial respiratory and coupling control in skeletal muscle of young and older adult humans. By combing the flux control ratio (Eq. 3) and factor (Eq. 4) for oligomycin (Protocol A) and the FCR (Eq. 7) (Protocol B), we calculated absolute L (leak) and P (phosphorylation) respiration. Collectively, the proportion of E that was L (Eq. 8), P (Eq. 9), or reserve (R) (Eq. 10) (excess respiratory capacity in the uncoupled state), could be calculated:

$${\text{L}}_{\left(\text{absolute}\right)}=\left({\text{P}}_{\text{I}+\text{II}}-\text{baseline}\hspace{0.17em}\hspace{0.17em}{\dot{\text{V}}\text{O}}_2\right)\times \left({\text{CCR}}_{\text{OM}}\right)\times \left({\text{P}}_{\text{I}+\text{II}}/\text{E}\right)$$ (8)

$${\text{P}}_{\left(\text{absolute}\right)}=\left({\text{P}}_{\text{I}+\text{II}}-\text{baseline}\hspace{0.17em}\hspace{0.17em}{\dot{\text{V}}\text{O}}_2\right)\times \left({\text{CCF}}_{\text{OM}}\right)\times \left({\text{P}}_{\text{I}+\text{II}}/\text{E}\right)$$ (9)

$$\text{R}=\text{E}-\left({\text{P}}_{\left(\text{absolute}\right)}+{\text{L}}_{\left(\text{absolute}\right)}\right)$$ (10)

Note: Baseline respiration is V̇o₂ prior to the addition of any substrate and, in the instance, is assumed to represent cytosolic nonmitochondrial respiration, given that in our hands baseline respiration in skeletal muscle is comparable to residual respiration following antimycin A titration.

Citrate synthase activity.

Five to ten milligrams of muscle tissue was homogenized in a glass tissue grinder in an ice-cold 175 mM KCl buffer containing 2 mM EDTA, 1% Triton, and a protease inhibitor cocktail (Sigma-Aldrich). Muscle homogenates were then centrifuged at 2,000 rpm at 4°C. Supernatants were stored at −80°C until analysis for citrate synthase (CS) activity and protein content.

Maximal CS activity was determined spectrophotometrically as per the method of Srere (34), with modifications. Briefly, muscle lysates were diluted 1:10 in a 110 mM Tris·HCl buffer (pH 8.1) containing 300 μM acetyl-CoA (lithium salt) and a 100 μM EtOH solution of (5,5′-dithiobis-2-nitrobenzoic acid) (DTNB). Thereafter, oxaloacetate was added to each well at a concentration of 500 μM. UV absorbance was recorded at 412 nM every 30 s for a total of 10 min at 37°C (BioTek Eon, Winooski, VT). The Δ/min in UV absorbance is proportional to the reaction of DTNB with free thiol groups (coenzyme A) following the condensation of oxaloacetate and acetyl-CoA. CS enzyme activity was calculated from the linear change in absorbance over time and expressed as micromoles per gram per minute.

Statistical analysis.

All data are presented as group means ± SE unless otherwise indicated. Data normality was tested using a Shapiro-Wilkes normality test. Differences between group means were detected using an unpaired Student's t-test. Relationships between respiratory capacity and function outcomes were determined by linear correlation. All statistical analysis was performed using GraphPad Prism v. 6 (GraphPad Software, La Jolla, CA). Statistical significance was accepted when P < 0.05.

RESULTS

Participants.

Fifty-five healthy adults (20 females and 35 males) ranging from 19 to 85 yr of age were studied over a 3-yr period. Participants' demographics are presented in Table 1. All participants in the young adult group were under the age of 45 yr (27 ± 5 yr, mean ± SD), and all participants in the older adult group were over the age of 50 yr (62 ± 9 yr, mean ± SD) (P < 0.001); 25% of participants in the young group were female, whereas 45% of paricipants in the older group were female. Older adults had significantly less lean body mass (P < 0.05) while having significantly more fat mass (P < 0.001) than their younger counterparts. In addition, BMI was significantly greater in the older adults than in the younger adults (P < 0.001).

Table 1.

Participants' demographics

	Young Adults (n = 24)	Older Adults (n = 31)	P
Age, yr	28 ± 7	62 ± 8	0.000
Male:Female	18:6	17:14
Height, cm	175 ± 10	170 ± 12	0.119
Weight, kg	79 ± 14	88 ± 18	0.035
Body mass index, kg/m2	26 ± 3	31 ± 6	0.000
Bone mineral content, kg	3.0 ± 0.5	2.8 ± 0.7	0.292
Fat-free mass, kg	53 ± 11	45 ± 15	0.029
Fat mass, kg	22 ± 7	39 ± 13	0.000
%Fat-free mass	70 ± 8	53 ± 14	0.000
%Fat mass	30 ± 8	47 ± 14	0.000

Values are group means ± SD.

Skeletal muscle respirometry.

Skeletal muscle mitochondrial respiration data from respiratory protocol A are presented in Fig. 2A. Respiratory fluxes in the leak (L_I) and phosphorylating (P_I) with electron provision through complex I were similar between young and older adults. Maximal phosphorylating respiration (P_I+II), when electrons were provided by both complexes I and II was significantly lower in older vs. young adults (46.6 ± 3.4 vs. 36.1 ± 2.2 pmol·s⁻¹·mg⁻¹, P < 0.05). Leak respiration with electron provision from both complexes I and II when ATP synthase was inhibited by oligomycin (L_I+II) was similar between groups Skeletal muscle CS activity, a proxy for mitochondrial protein abundance, is presented in Fig. 2B. While CS activity tended to be lower in older adults (24.3 ± 1.3 vs. 20.6 ± 1.5 μmol·mg⁻¹·min⁻¹), this was not a statistically significant finding (P = 0.1). Respirometry data from protocol A were normalized to muscle CS activity (Fig. 2C), a common approach to normalizing mitochondrial function data to mitochondrial density. Mitochondrial respiration, regardless of coupling state, was not different in young vs. older adults once normalized to CS activity, with the exception of L_I+II respiration. L_I+II respiration was greater in older adults than in young adults (0.71 ± 0.05 vs. 1.01 ± 0.14), which approached statistical significance (P = 0.06). Coupling/flux control ratios calculated from Protocol A are presented in Table 2. Both RCR_ADP and SCR_Succinate were similar in skeletal muscle mitochondria from young and older adults. The CCR_OM was significantly lower in older adults (P < 0.05).

Fig. 2.

A: skeletal muscle mitochondrial respiration determined in SUIT Protocol A. B: citrate synthase (CS) activity of muscle homogenates. C: mitochondrial respiration normalized to CS activity. *P < 0.05 vs. corresponding young adult group. ⁺Sufficient muscle tissue for CS activity measurements was available from n = 21 young adults and n = 21 older adults.

Table 2.

SUIT Protocol A respiratory control ratios

	Young Adults (n = 24)	Older Adults (n = 31)	P
RCRADP	2.43 ± 0.22	2.37 ± 0.18	0.810
SCRsuccinte	1.66 ± 0.07	1.56 ± 0.08	0.336
CCROM	0.36 ± 0.02	0.46 ± 0.03	0.021

Values are group means ± SE. RCR_ADP, respiratory control ratio for ADP; SCR_Succinate, substrate control ratio for succinate;CCR_OM, coupling control for oligomycin.

Mitochondrial RCR_ADP as a function of maximal coupled respiration (P_I+II) for all young (n = 24) and older (n = 31) adults is presented Fig. 3A. Mitochondrial RCR_ADP was significantly correlated with coupled respiration P_I+II. (R = 0.57, P < 0.001). Similarly, mitochondrial SCR_Succinate was significantly correlated with coupled respiration P_I+II. (R = 0.31, P < 0.05). Finally, CCR_OM was also significantly correlated with P_I+II (R = −0.38, P < 0.01). Collectively, these results suggest an association between mitochondrial respiratory capacity and coupling control.

Fig. 3.

Correlations between RCR_ADP and P_I+II, SCR_Succinate and P_I+II, and FCR_OM and P_I+II are presented in A, B and C, respectively. See Calculation of intrinsic mitochondrial function in materials and methods for definitions. Data from all participants (n = 49) were used for correlation analysis.

Mitochondrial respiratory data from protocol B are presented in Fig. 4A. Similar to protocol A, maximal coupled respiration P_I+II was significantly lower in older adults than in younger adults (55.3 ± 4.2 vs. 40.9 ± 3.1 pmol·s⁻¹·mg⁻¹, P < 0.01). Furthermore, maximal uncoupled respiration following CCCP titration (E) was also lower in older adults than in young adults (61.5 ± 4.6 vs. 50.7 ± 3.4 pmol·s⁻¹·mg⁻¹, P = 0.06). Respiratory flux in the uncoupled state (E) represents the respiratory capacity of the mitochondrial pool, since the inhibition of electron transfer and thus respiration by intramembrane proton accumulation is removed by the protonophore CCCP. In the current data set, E respiration strongly correlated with P_I+II in both younger (R = 0.95, P < 0.001) and older (R = 0.94, P < 0.001) adults (Fig. 4B), suggesting that the capacity for phosphorylation is related to the capacity for electron transfer (i.e., respiration). Thus, E respiratory flux presents an ideal internal normalization for leak and phosphorylating respiratory states (8, 9, 22). These flux control ratios (FCR) for protocol B are presented in Fig. 4C. FCR_PI+II, the flux control ratio for maximal coupled respiration (P_I+II/E) was significantly lower in older adults than in young adults (0.90 ± 0.02 vs. 0.80 ± 0.02, P < 0.01), indicating altered mitochondrial quality in skeletal muscle of older adults.

Fig. 4.

A: skeletal muscle mitochondrial respiration determined in SUIT Protocol B. B: correlation between P_I+II and E in both young (circles) and older (triangles) adults. C: respiratory flux control ratios calculated from SUIT Protocol B. **P < 0.01 vs. corresponding young adult group.

Absolute rates of leak (L_absolute), coupled (P_absolute), and reserve (R_absolute) respiration were calculated according to equations 8, 9 and 10, respectively, and are presented in Fig. 5A. L_(absolute) was similar between young and older adults (15.9 ± 1.2 vs. 16.1 ± 1.7 pmol·s⁻¹·mg⁻¹). In contrast, P_(absolute) was significantly lower in older adults (34.1 ± 4.3 vs. 17.7 ± 2.0 pmol·s⁻¹·mg⁻¹, P < 0.001). Furthermore, R_absolute was significantly greater in older adults than in young adults (10.1 ± 1.1 vs. 6.2 ± 1.5 pmol·s⁻¹·mg⁻¹, P < 0.05). Absolute respiratory rates expressed as a percentage of total mitochondrial respiration (E-baseline respiration) are presented in Fig. 5B as an index of mitochondrial quality. Leak respiration accounted for a similar proportion of total mitochondrial respiration in skeletal muscle young and older adults (29 ± 2 vs. 36 ± 3%). Coupled respiration represented a significantly greater proportion of total mitochondrial respiration in younger vs. older adults (59 ± 3 vs. 39 ± 3%, P < 0.001). Reserve respiratory capacity accounted for a significantly smaller proportion of total mitochondrial respiration in younger vs. older adults (12 ± 3 vs. 24 ± 3%, P < 0.01). Collectively, these data further demonstrate that the quality of mitochondria is diminished with advancing age.

Fig. 5.

A: absolute skeletal muscle mitochondrial respiration in the leak and coupled state, as well as reserve respiratory capacity. B: absolute respiration rates expressed as %maximum mitochondrial respiration. *P < 0.05 vs. corresponding young adult group; **P < 0.01 vs. corresponding young adult group; ***P < 0.001 vs. corresponding young adult group.

Although we did not set out to study whether there were sex difference in skeletal mitochondrial function, in a subanalysis of men and women, P_absolute was similar between young men (35.2 ± 4.4 pmol·s⁻¹·mg⁻¹, n = 13) and young women (28.7 ± 2.2 pmol·s⁻¹·mg⁻¹, n = 3) and between older men (17.6 ± 2.7 pmol·s⁻¹·mg⁻¹, n = 12) and older women (18.0 ± 2.7 pmol·s⁻¹·mg⁻¹, n = 5). Moreover, P_absolute was 50% lower in older men vs. young men, and 63% lower in older women vs. young women, suggesting that mitochondrial respiratory capacity declines in skeletal muscle with age in both men and women.

DISCUSSION

Altered skeletal muscle mitochondrial function is thought to be a component of senescence, i.e., cellular aging (39). However, whether aging results in a reduction in mitochondrial density, a decline in mitochondrial quality, or both, in human skeletal muscle remains unclear. The purpose of the current study was to comprehensively compare mitochondrial respiratory capacity and coupling control in large cohorts of healthy younger and healthy older adults. Our findings suggest that both the respiratory capacity and coupling control of skeletal muscle mitochondria decline with advancing age. Specifically, we show that respiratory capacity and the proportion of respiration coupled to phosphorylation are lower in older adults, suggesting that the capacity of skeletal muscle mitochondria to produce electrochemical potential and to couple this to ADP phosphorylation is diminished with advancing age Furthermore, correlation analysis revealed a relationship between mitochondrial respiratory capacity and coupling control, supporting the notion that as respiratory capacity declines coupling control is also lost. To the best of our knowledge, this is the first study to document concurrent reductions in skeletal muscle mitochondrial respiratory capacity and coupling control with advancing age in a large cohort of healthy humans.

Although significant effort has been placed on determining the impact of aging on skeletal muscle bioenergetics, the numerous analytical techniques employed to assay mitochondrial function have somewhat addled the literature. Several studies have shown reduced respiration or ATP production with advancing age in mitochondria isolated from skeletal muscle biopsies by homogenization and differential centrifugation (3, 33, 35, 38). More recent data suggest that isolation of mitochondria from skeletal muscle has deleterious effects on mitochondrial morphology and function (25). In the context of aging, this analytical artifact may be an issue, since mitochondria isolated from the skeletal muscle of older rodents appear more susceptible to the damage caused by extraction than those of younger rodents (23). Moreover, Rasmussen et al. (26) reported that the yield of mitochondria extracted from a similar amount of skeletal muscle was lower in older (37 ± 7%) than in young adults (47 ± 5%), suggesting that altered muscle composition with advancing age may have an impact upon the yield of mitochondria extracted. However, in the current study we show in permeabilized muscle fibers, where the entire mitochondrial pool is studied in situ, that aging is associated with a decline in mitochondrial respiratory capacity, in line with respiration (3, 38) and ATP production rate measurements made in isolated mitochondria (17, 33, 35).

While numerous studies have reported a decline in skeletal muscle mitochondrial function with advancing age (1, 3, 5, 15, 17, 20, 30, 31, 33, 35, 37), there is disagreement in the literature (10, 13, 16, 18, 28). These discrepancies are likely attributable, in part, to heterogeneity in the physical activity levels and fitness of study cohorts. Indeed, when matched for physical activity levels (16) or V̇o_{2 max} (18), skeletal muscle mitochondrial function is not different in young and older adults. Furthermore, low-functioning but not high-functioning elders have significantly lower skeletal muscle mitochondrial respiratory capacity than younger adults (15), further demonstrating the association between physical activity and skeletal muscle mitochondrial capacity. By way of example, Hutter et al. (13) recently showed that 2 wk of immobilization decreased muscle CS activity and mitochondrial respiration in both young and older adults, something that was reversed following 6 wk of aerobic cycle training, underscoring the important role of contraction in mediating skeletal muscle oxidative capacity. A limitation of the current study is that we did not directly measure the physical fitness of participants. However, we did not recruit people who were engaged in vigorous exercise training; thus, considered these participants to healthy and physically active, but not trained.

In the current study, both men and women were recruited. Previous studies had shown no sex differences in quadriceps muscle oxidative capacity in vivo in young and older adults (16), no sex-related difference in mitochondria isolated from quadriceps muscle of young and older individuals (3), no difference in respiratory capacity in gastrocnemius muscle fibers or CS activity in gastrocnemius muscle homogenates in older men and women (36), and no sex differences in quadriceps muscle fiber respiration (per mg tissue or when normalized to CS activity) in cohorts of both young and older adults (13). However, it should be noted that others have shown a sex effect of skeletal muscle oxidative capacity, where cytochrome c oxidase (but not CS) enzyme activity was lower in quadriceps muscle of women but not men in cohorts of young, middle-aged, and older adults (30). However, these researchers (30) found a similar decline in quadriceps muscle cytochrome c oxidase and CS enzyme activity with age in men and women. In a subanalysis of our current data, mitochondrial respiratory capacity was similar between men and women in both the young and older groups and declined with age in both sexes.

Of the studies that suggest an association between aging and reduced skeletal muscle mitochondrial function (1, 3, 5, 15, 17, 20, 30, 31, 33, 35, 37), skeletal muscle mitochondrial function was determined by assaying the activity of mitochondrial enzymes (20, 30, 31, 37), ATP flux or phosphocreatine recovery in vivo (1, 5, 20), respiration in isolated mitochondria (3, 38), or ATP production rates in isolated mitochondria (17, 33, 35). However, these measurements will likely be influenced significantly by mitochondrial volume density. In the current study, when normalized to CS activity, a robust proxy of skeletal muscle mitochondrial volume density (19), P_I+II was not different between young and older adults. Thus, our current data support the notion that reduced skeletal muscle mitochondrial respiratory capacity associated with aging is attributable, at least in part, to a reduction in mitochondrial abundance.

Multiple biochemical assays, stereological approaches, and gene/protein abundance measurements have been used as surrogates of mitochondrial abundance, some of which may be more meritorious than others (19). Moreover, normalizing ATP production or V̇o₂ measurements to surrogates of mitochondrial density to determine mitochondrial quality relies on the assumption that the muscle biopsy used was completely homogenous. High-resolution respirometry permits the sequential titrations of substrates, inhibitors, and/or uncouplers; coupling/flux control ratios to be calculated thereafter. Since these ratios reflect the response of the entire mitochondrial pool within the sample to saturating levels of a substrate, inhibitor, or uncoupler, they are independent of mitochondrial density and therefore provide an index of mitochondrial quality (8, 9, 22).

To address whether aging was associated with altered mitochondrial quality, we determined mitochondrial flux control ratios. We found that the mitochondrial flux control ratio was significantly lower in skeletal muscle from older adults. These data suggest that, per mitochondrion, maximal coupled respiration represents a lower proportion of mitochondrial respiratory capacity in older adults. Thus, in addition to reduced respiratory capacity, mitochondrial efficiency appears diminished with advancing age.

Using permeabilized myofiber preparations, we provide evidence supporting the notion that skeletal muscle mitochondrial respiratory capacity and coupling control decline with advancing age. In addition, correlation analysis revealed associations between maximal coupled respiration (P_I+II) and mitochondrial coupling control. Specifically, the RCR_ADP and SCR_succinate both correlated with P_I+II (R = 0.38, P = 0.009 and R = 0.34, P = 0.02, respectively), as did the FCR_OM (R = −0.36, P = 0.01), further demonstrating a link between mitochondrial respiratory capacity and coupling control. Collectively, these data support the supposition that, in the context of aging, skeletal muscle mitochondrial respiratory capacity and coupling control are related, where a decline in mitochondrial respiratory capacity may be accompanied by a reduced coupling control.

To further investigate the deficits in mitochondrial quality seen in skeletal muscle of older adults, we developed formulae encompassing the FCR_OM and FCF_OM determined in Protocol A, along with P_I+II and the FCR_PI+II determined in Protocol B. These formulae were used to calculate the absolute rates of phosphorylating and leak respiration. The rationale behind calculating these absolute respiratory rates comes from the observation that electron transfer is a fundamental property of the electron transfer system. Theoretically, this would lead to accumulation of protons within the mitochondrial membranes that would eventually arrest electron transfer and respiration. However, the inner mitochondrial membrane is inherently leaky to protons, permitting a basal or leak respiratory rate (i.e., state 2/state 4_O) where ATP is not produced. Although we refer to P_I+II as maximal coupled respiration, this may not be completely accurate. Indeed, in the current study, 36–46% of P_I+II in skeletal muscle was insensitive to oligomycin (Table 2), which is in line with previously published data (10). This suggests that leak respiration may represent a proportion of P_I+II, at least in these experimental settings where supraphysiological concentrations of substrates are provided.

In the current study, absolute respiration in the coupled state (phosphorylation) was twofold greater in young adults compared with older adults (P < 0.001). This indicates that the ATP producing capacity per milligram of muscle is significantly diminished in older adults, which is in agreement with bioluminescence measurements made in isolated mitochondria (17, 33) and ³¹P-MNR measurements in vivo (1). Absolute rates of leak respiration were similar between young and older adults. This is particularly interesting, since when considering the CCR_OM one may conclude that leak respiration is greater per milligram of muscle in older adults; however, this does not hold true when absolute rates of leak respiration are calculated. Thus, it would appear that diminished capacity for phosphorylation as opposed to increased leak respiration is the dominant phenotype of mitochondria from aged skeletal muscle. Furthermore, we found that the reserve respiratory capacity was significantly greater in skeletal muscle mitochondria from older adults (P < 0.01), suggesting that mitochondria from older adults are unable to use as much of their respiratory capacity to producing ATP.

The difference in absolute phosphorylating and reserve respiration per milligram of tissue between young and older adults seen here are likely influenced mitochondrial abundance. Therefore, we normalized these respiratory fluxes to the capacity of the electron transfer system. While the proportion of maximal respiration linked to leak respiration was similar in young and older adults (29 ± 3 vs. 36 ± 3%), respiration linked to phosphorylation was markedly diminished in the muscle of older adults (59 ± 3 vs. 39 ± 3%, P < 0.001). Furthermore, reserve respiratory capacity accounted for a greater percentage of maximal mitochondrial respiration in skeletal muscle of older adults (12 ± 3 vs. 24 ± 3%, P < 0.01), further demonstrating a decline in mitochondrial quality in skeletal muscle of older adults. These data highlight the inherent differences in mitochondrial coupling control in skeletal muscle from older individuals.

While the notion of developing an index of bioenergetic function to diagnose various pathologies has been proposed previously, this method (4) relies on the analysis of cells, which may not be the most relevant sample type, particularly with regard to muscle (24). In our view, the instrumental stability offered by O₂ electrode respirometers, coupled with the ability to determine O₂ flux in permeabilized muscle biopsy samples in a stirred buffer (21) make the approach described here more appropriate for prospective human biopsy studies. Since we monitored instrumental performance over the entire study period, we believe our data to be quantitative. Therefore, we suggest that the approach described here is a robust means of diagnosing deficits in skeletal muscle mitochondrial capacity and coupling control in a prospective human study.

In summary, we have presented data that demonstrate that reduced skeletal muscle respiratory capacity and coupling control are associated with aging. Furthermore, we have shown that skeletal muscle respiratory capacity and coupling control are related, where reduced respiratory capacity was accompanied by reduced coupling control. Finally, by combining two respiratory function protocols, absolute rates of leak and coupled and reserve mitochondrial respiration were calculated, revealing profound reductions in the ATP-producing capacity of muscle from older adults. We propose that this novel approach for calculating mitochondrial respiratory capacity yields quantitative data that may be of diagnostic value.

GRANTS

This work was supported by the following National Institutes of Health (NIH) and Shriners of North America (Shrine) grants: RO1 AG-033761 to E. Børsheim, RO1 AR-049877 to B. B. Rasmussen, 5RO1 1CA127971 to M. Sheffield-Moore, P30 AG-024832 to E. Volpi, and 84090 (Shriners of North America) to L. S. Sidossis. The majority of participants were screened and studied at the Clinical Research Center at UTMB, which is supported by the Institute for Translational Sciences [supported in part by a Clinical and Translational Science Award (UL1 TR-000071) from the National Center for Advancing Translational Sciences, NIH]. Studies at Reynold's Center on Aging at UAMS were supported by NIH P30 AG-028718. C. Porter was supported in part by an Interdisciplinary Rehabilitation Research Postdoctoral Training Grant (H133P110012) from the National Institute of Disability and Rehabilitation Research and the Department of Education. N. M. Hurren, M. V. Cotter, and E. Børsheim are partly supported by funding from the Arkansas Biosciences Institute, the major research component of the Arkansas Tobacco Settlement Proceeds Act of 2000.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: C.P., E.L.D., W.J.D., M.S.-M., E.V., L.S.S., B.B.R., and E.B. conception and design of research; C.P., N.M.H., M.V.C., N.B., P.T.R., E.L.D., W.J.D., D.T., E.V., and E.B. performed experiments; C.P., M.V.C., N.B., and D.T. analyzed data; C.P., M.V.C., and D.T. interpreted results of experiments; C.P. prepared figures; C.P. drafted manuscript; C.P., N.M.H., M.V.C., N.B., P.T.R., E.L.D., W.J.D., M.S.-M., E.V., L.S.S., B.B.R., and E.B. edited and revised manuscript; C.P., N.M.H., M.V.C., N.B., P.T.R., E.L.D., W.J.D., D.T., M.S.-M., E.V., L.S.S., B.B.R., and E.B. approved final version of manuscript.