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Why muscles matter

van der Zwaard, S.

2018

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citation for published version (APA)van der Zwaard, S. (2018). Why muscles matter: Optimizing sprint and endurance performance in athletes.

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5A

Maximal oxygen uptake is proportional to muscle fiber oxidative capacity

from chronic heart failure patients to professional cyclists

Van der Zwaard, S., de Ruiter C.J., Noordhof, D.A., Sterrenburg, R., Bloemers, F.W., de Koning, J.J., Jaspers R.T. & van der Laarse, W.J. Maximal oxygen uptake is proportional to muscle fiber oxidative capacity, from chronic heart failure patients to professional cyclists. J. Appl. Physiol. 121, 636–645 (2016).

Adapted from:

AbstractV̇O2max during whole-body exercise is presumably constrained by oxygen delivery to mitochondria rather than by mitochondria’s ability to consume oxygen. Humans and animals have been reported to exploit only 60-80% of their mitochondrial oxidative capacity at V̇O2max. However, ex vivo quantification of mitochondrial overcapacity is complicated by isolation or permeabilization procedures. An alternative method for estimating mitochondrial oxidative capacity is via enzyme histochemical quantification of succinate dehydrogenase (SDH) activity. We determined to what extent V̇O2max attained during cycling exercise differs from mitochondrial oxidative capacity predicted from SDH activity of m. vastus lateralis in chronic heart failure patients, healthy controls and cyclists. V̇O2max was assessed in 20 healthy subjects and 28 cyclists and SDH activity was determined from biopsy cryosections of m. vastus lateralis using quantitative histochemistry. Similar data from our laboratory of 14 chronic heart failure patients and 6 controls were included. Mitochondrial oxidative capacity was predicted from SDH activity using estimated skeletal muscle mass and the relationship between ex vivo fiber V̇O2max and SDH activity of isolated single muscle fibers and myocardial trabecula under hyperoxic conditions. Mitochondrial oxidative capacity predicted from SDH activity was related (r2=0.89, p<0.001) to V̇O2max measured during cycling in subjects with V̇O2max ranging from 9.8 to 79.0 mL∙kg-1∙min-1. V̇O2max measured during cycling was on average 90±14% of mitochondrial oxidative capacity. We conclude that human V̇O2max is related to mitochondrial oxidative capacity predicted from skeletal muscle SDH activity. Mitochondrial oxidative capacity is likely marginally limited by oxygen supply to mitochondria.

New and noteworthyV̇O2max during whole-body exercise is presumably constrained by oxygen delivery to mitochondria rather than by mitochondria's ability to consume oxygen. However, mitochondrial oxidative overcapacity remains unclear due to complicated isolation and permeabilization procedures. In the present study, human V̇O2max attained during cycling exercise is related and ~90% of mitochondrial oxidative capacity predicted from skeletal muscle succinate dehydrogenase activity. This mitochondrial oxidative overcapacity is substantially lower than previously reported from isolation and permeabilization procedures.

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IntroductionMaximal oxygen uptake (V̇O2max) is used to quantify cardiorespiratory fitness1. V̇O2max is critical for endurance performance1,2, but also predicts loss of independence in elderly3 and mortality in chronic patients and healthy subjects4. Moreover, V̇O2max has been widely used to assess effects of training interventions. Although V̇O2max is generally considered a strong predictor of physical performance, factors limiting V̇O2max are still subject to controversy.

Ever since A.V. Hill et al.5 have postulated the concept of V̇O2max in the 1920s, exercise physiologists have debated factors that limit V̇O2max

1,2,6–8. O2 flux may be limited by any factor related to the O2 pathway from atmosphere to the mitochondria. Hill et al.5 already speculated that V̇O2max is limited by the rate of O2 supply by the cardiorespiratory system. Currently, the consensus is that V̇O2max is constrained by oxygen delivery to the mitochondria and not by mitochondria’s ability to consume oxygen1,6–9. This presumption is supported by the observation that V̇O2max and performance increase when O2 supply is marginally enhanced by acute hyperoxia10,11 or blood reinfusion12. In addition, O2 supply limitations are less pronounced in exercises with smaller muscle groups13.

Even though it is presumed that mitochondria’s ability to consume oxygen is not limiting V̇O2max, mitochondrial volume density in locomotory muscles of animals is closely related to body-mass-specific V̇O2max (r2 = 0.97)14,15. Not only mitochondrial density, but also capillary density, heart’s pumping capacity and lung diffusion capacity are scaled in proportion to V̇O2max

14,15. These findings support the concept of “symmorphosis”, postulating that physiological determinants of the O2 cascade that contribute to V̇O2max are proportional16. Also in humans, V̇O2max is closely related to mitochondrial volume density (r = 0.82)17 and oxidative enzyme activity (r = 0.72-0.79)18,19 in locomotory muscles. Humans, however, have been suggested not to conform as closely to symmorphosis as other animal species because of their ‘excess’ mitochondrial oxidative capacity20,21. This ‘excess’ oxidative capacity is, however, less substantial considering that human bipedal locomotion involves less active muscle mass compared to animal quadrupedal locomotion21. Therefore, comparative data suggest that mitochondrial oxidative capacity scales with V̇O2max of an organism even though the extent of excess oxidative capacity remains unclear.

At V̇O2max, animals have been suggested to exploit only 60-80% of their mitochondrial oxidative capacity22. This estimation results from comparisons of in situ measured V̇O2max with electrically stimulated muscle and ex vivo mitochondrial oxidative capacity that is quantified from isolated mitochondria by polarographic measures of muscle mitochondrial respiration22. In humans, mitochondrial oxidative capacity has been quantified from isolated mitochondria or permeabilized muscle fibers obtained from biopsy samples20,23. Such experiments show that at V̇O2max during cycling exercise humans use only 64-73% of the mitochondrial oxidative capacity in active lower limb muscles20,23. The excess mitochondrial oxidative capacity is higher than what is expected based on the mitochondrial oxygen tension during maximal exercise (3.1 Torr based on myoglobin saturation)24 and the Michaelis constant for oxygen of the mitochondria (0.5 Torr)25. This discrepancy may be explained by mitochondrial inhibition, for instance by nitric oxide26, or by methodological issues related to isolation or permeabilization procedures27. Isolation of mitochondria alters mitochondrial morphology and function, which is presumably less of a problem in permeabilized fibers, but the incubation buffers differ from in vivo conditions27. An alternative method to determine mitochondrial oxidative capacity is quantitative histochemistry

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Maximal oxygen uptake is proportional to muscle fiber oxidative capacity

of succinate dehydrogenase (SDH) activity, a citric acid cycle enzyme and complex II of the electron transport chain. SDH activity obtained from homogenized muscle tissue strongly relates to mitochondrial content (similar to other mitochondrial biomarkers e.g. citrate synthase, complex I and IV activity)28 as well as mitochondrial oxidative capacity in permeabilized fibers (similar to complex IV activity)28. Here we determined SDH activity by quantitative enzyme histochemistry as this method allows for muscle fiber specific comparison with other histochemical assays (e.g. myosin heavy chain typing, capillary density and muscle fiber size). This method has previously been calibrated showing that SDH activity is proportionally related to ex vivo V̇O2max in intact single muscle fibers and myocardial trabeculae in hyperoxia29,30. The predictive relationship has only been investigated for SDH activity, but illustrates that SDH activity provides a quantitative measure of mitochondrial oxidative capacity even though it may not be rate limiting for the maximal flux through the Krebs cycle18. Therefore, within a muscle biopsy, mitochondrial oxidative capacity of individual muscle fibers can be estimated from SDH activity by quantitative enzyme histochemistry, avoiding isolation or permeabilization procedures.

The aim of this study was to quantify to what extent mitochondrial oxidative capacity predicted from SDH activity in biopsies of m. vastus lateralis differs from V̇O2max attained during cycling exercise in chronic heart failure patients, healthy subjects and elite cyclists. We hypothesized that in humans SDH activity of m. vastus lateralis is related to cycling V̇O2maxand that humans exploit 60-80% of their mitochondrial oxidative capacity.

Methods

Subjects48 subjects (20 healthy untrained subjects and 28 cyclists ranging from recreationally trained to professional) volunteered to participate in this study (Table 5.1). Cyclists competed at (inter)national level, except for 4 amateur cyclists. Previously published data from our laboratory of 14 chronic heart failure (CHF) patients and 6 healthy subjects31 were re-examined (see details below). Therefore, 68 subjects were included in our analysis. Prior to participation, experimental procedures and risks of the study were explained and all subjects provided written informed consent. The study was conducted according to the principles of the Declaration of Helsinki and was approved by the medical ethics committee of the VU medical center, Amsterdam, the Netherlands (NL43423.029.13 and NL49060.029.14).

Whole-body V̇O2max during cyclingSubjects performed a maximal incremental exercise test on a cycle ergometer to voluntary exhaustion, despite verbal encouragement (respiratory exchange ratio of 1.20 ± 0.07). V̇O2peak was calculated as the highest 30-s value achieved during the maximum-effort incremental test and is considered a valid index of V̇O2max in subjects exercising to their limit of exercise tolerance32. Respiratory data were analyzed breath-by-breath using open circuit spirometry and expressed at STPD (Cosmed Quark CPET, Cosmed S. R. L., Rome, Italy). Prior to every test, the gas analyzer and volume transducer were calibrated according to manufacturer’s instructions. Subjects were instructed to avoid strenuous exercise and alcohol consumption within 24 hours before the test and caffeinated beverages and meals within 3 hours before the test.

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Notes:

Table 5.1. Participant characteristics

Values are means±SD; n, no. of subjects. CHF, chronic heart failure; BMI, body mass index; V̇O2max, maximal oxygen uptake per kg body mass; SHD, succinate dehydrogenase; A660, change of absorbance at 660 nm. Tquadriceps, temperature quadriceps at maximal exercise. p <0.01, significantly different from CHF patients (*) and control subjects (#). †Data for 6 controls and 14 CHF patients were obtained from Bekedam et al.31.

Skeletal muscle biopsyBiopsy samples were obtained from the m. vastus lateralis using a modified Bergström needle technique. The vastus lateralis muscle (part of the m. quadriceps femoris) acts as knee extensor and for cycling exercise is predominantly involved in the pushing phase and power-producing phase of the pedal cycle33. Biopsy sites were locally anesthetized with a 2% lidocaine solution and an incision of < 1 cm was made through the skin and fascia latae. The biopsy needle was inserted ~15 cm above the patella to a depth of approximately 4 cm. Biopsy samples were carefully removed and aligned according to their muscle fiber arrangement using a magnifying glass. Subsequently, samples were frozen in liquid nitrogen. After freezing, biopsy samples were placed in a cryostat and cut in 10 μm thick sections at -20°C. Sections were collected on polylysine-coated slides.

SDH histochemistrySDH activity was determined using quantitative histochemistry, which has been described in detail elsewhere31,34. In short, biopsy sections were incubated at 37°C in a medium consisting of 0.4 mM tetranitroblue tetrazolium (Sigma, St. Louis, MO), 75 mM sodium succinate, 5 mM sodium azide, and 37.5 mM sodium phosphate buffer, pH 7.634. Biopsy sections were incubated for 20 minutes. Images were made with ×10 or ×20 objectives and absorbance was measured by microdensitometry35 with an interference filter at 660 nm34 using NIH ImageJ (https://imagej.nih.gov/ij/). Weighed average SDH activity was determined from spatially averaged SDH activity of over 40 randomly selected individual cells, including the subsarcolemma mitochondria. The randomly selected cells mirrored the distribution of high oxidative (i.e. type I) and low oxidative (i.e. type II) fibers within the biopsy section (i.e. SDH activity of individual fibers can be used to discriminate between fiber types similar to myofibrillar ATPase activity)36. Reproducibility of quantitative SDH histochemistry was assessed by comparison of absorbance values of two subsequent measurements of the same muscle tissue.

Temperature affects SDH activity in rats, mice and fish34,37, but the effect of temperature on SDH activity has not been assessed in humans. In the present study, we assessed the relationship

CHF patients Controls Cyclistsn 14 26 † 28Age (year) 61 ± 10 38 ± 11 * 25 ± 7 *#

Body mass (kg) 78.0 ± 10.2 90.5 ± 11.6 * 77.4 ± 8.1 #

BMI 25.6 ± 2.7 26.9 ± 2.2 22.4 ± 1.9 *#

Muscle mass (kg) 27.2 ± 4.7 33.9 ± 4.2 * 32.6 ± 2.3 *Muscle mass as percentage body mass (%) 34.7 ± 2.9 37.5 ± 2.8 * 42.3 ± 2.1 *#

V̇O2max (mL∙kg-1∙min-1) 19.2 ± 4.5 40.1 ± 7.2 * 62.7 ± 7.9 *#

SDH activity at 37°C (ΔA660∙μm-1∙s-1) 8.6 ± 1.8 ×10-6 13.7 ± 2.0 ×10-6 * 17.9 ± 2.1 ×10-6 *#

SDH activity at Tquadriceps (ΔA660∙μm-1∙s-1) 8.4 ± 1.7 ×10-6 16.8 ± 2.4 ×10-6 * 21.9 ± 2.6 ×10-6 *#

Fiber V̇O2max (nmol∙mm-3∙s-1) 0.051 ± 0.01 0.101 ± 0.015 * 0.132 ± 0.015 *#

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Maximal oxygen uptake is proportional to muscle fiber oxidative capacity

between incubation temperature and SDH activity in human muscle tissue sections that were incubated at 32°C, 37°C and 42°C (Figure 5.3). Subsequently, the temperature quotient Q10 - the increase in SDH activity with a 10°C increase in temperature - was determined.

Temperature-adjusted SDH activityFor comparison of SDH activity to whole-body V̇O2max, SDH incubation temperature should equal quadriceps muscle temperature at maximal exercise. Previously, similar data on SDH activity and V̇O2max during cycling have been obtained in 14 chronic heart failure (CHF) patients and 6 healthy controls, but SDH activity was not adjusted to muscle temperature at maximal exercise. In the present study, SDH activity was adjusted to temperature using the Q10 for human tissue and estimated quadriceps muscle temperature at maximal effort, being 36.5°C in CHF patients38 and ~41°C in healthy subjects and cyclists39.

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Figure 5.1. Succinate dehydrogenase (SDH) staining using quantitative histochemistry. A) healthy control subject. B) cyclist. Cross sections of the human vastus lateralis muscle incubated for SDH activity at 37°C. Section thickness 10 μm, incubation time 20 min. Scale bar 100 μm.

Figure 5.2. Reproducibility of SDH activity measure- ments. SDH activity was measured on two separate occasions without incubation () and after 20 min incubation in high oxidative fibers () and low oxida- tive fibers () of the same muscle tissue. The solid line represents the line of identity. SDH activity of 120 muscle fibers in total was determined from absorbance measurements at 660 nm (A660). Values are mean ± SD.

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Figure 5.3. Relationship between SDH activity and incubation temperature. Average SDH activity of high and low oxidative fibers was measured after 20 min incubation () at 32, 37 and 42°C. The SDH activity was significantly different between 32, 37 and 42 °C (*p < 0.001). SDH activity of 240 muscle fibers in total was determined from absorbance measurements at 660 nm. Values are mean ± SD.

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Skeletal muscle fiber V̇O2max

Previously, it has been shown that paired determination of SDH activity by quantitative histochemistry and ex vivo maximum rate of oxygen consumption of intact single fibers under hyperoxic conditions are proportionally related in Xenopus (both determined at 20°C)30 and rat myocardial trabeculae (both determined at 38°C)29. Therefore, skeletal muscle fiber V̇O2max (in nmol∙mm-3∙s-1) was calculated as 6,000 × the temperature-adjusted SDH staining rate (in change of absorbance at 660 nm per μm section thickness per s incubation time) as described previously29,30.

Mitochondrial oxidative capacity predicted from SDH activityOxidative capacity of mitochondria was predicted from temperature-adjusted SDH activity, total skeletal muscle mass (see details below) and body mass (Equation 5.1). This prediction involves the following assumptions: 1) that all oxygen at V̇O2max is consumed by skeletal muscle mitochondria, 2) that all mitochondria are active at their V̇O2max during maximal cycling exercise and 3) that SDH activity of the vastus lateralis locomotor muscle represents SDH activity of whole body musculature. As these assumptions can be debated (see details below), another prediction of mitochondrial oxidative capacity is made that also accounts for 1) differences in SDH activity between arm and leg muscles, 2) active skeletal muscle mass during maximal cycling exercise and 3) oxygen consumption of other organs (see details below).

(5.1)

where mitochondrial oxidative capacity is calculated at STPD (in mL∙kg-1∙min-1), SDH is weighed succinate dehydrogenase activity of m. vastus lateralis adjusted for temperature (in ΔA660∙μm-1∙s-1), Vm is molar volume of oxygen at STPD (22.4 L∙mol-1), δ is muscle density (1.04 kg∙L-1), Mm is skeletal muscle mass (kg) and Mb is body mass (kg).

Skeletal muscle massTotal skeletal muscle mass (including respiratory muscles) was estimated from an anthropometric regression model that has been shown to predict total skeletal mass obtained from whole-body magnetic resonance imaging in 244 nonobese subjects40:

(5.2)

where Mb is body mass (kg), Ht is height (m), sex = 1 for males or 0 for female, ethnicity = -1.2 for Asian (one CHF patient) and 0 for Caucasian.

SDH activity of skeletal muscle In animals, mean mitochondrial volume density of whole-body skeletal musculature is not significantly different from mitochondrial volume density of the hind limb musculature21. In

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Maximal oxygen uptake is proportional to muscle fiber oxidative capacity

humans, however, mitochondrial volume density of arm and leg musculature may differ due to bipedal locomotion. In untrained subjects SDH activity of the deltoid muscle was 80% of the SDH activity in m. vastus lateralis41, whereas in leg-trained subjects this was 65%41. Therefore we assume that SDH activity of arm musculature is 80% of m. vastus lateralis SDH activity in healthy controls and CHF patients and 65% in cyclists. Here, arm muscle mass accounts for 20% of total skeletal muscle mass (i.e. ~6kg)42.

Active skeletal muscle during cycling exercise During maximal leg cycling exercise, mitochondria in the arm musculature are likely not active at their V̇O2max. In healthy subjects, oxygen uptake of the arms during leg pedaling at maximal effort (26.1 mL∙kgmuscle

-1∙min-1) yielded approximately 35% of V̇O2max of the arms attained during arm cranking (77.8 mL∙kgmuscle

-1∙min-1)43. Therefore, we assume that during maximal leg cycling exercise mitochondria in the arm musculature are active at 35% of their V̇O2max.

Oxygen consumption of other organsAt maximal effort, oxygen consumption by the brain was assumed to be similar to resting conditions, approximating 20% of measured oxygen consumption in rest (~1 mL∙kg-1∙min-1; cf. Ref44). Maximal myocardial oxygen consumption has been estimated to be 0.45 mM∙s-1 45,46, that is 670 mL∙kgheart mass

-1∙min-1 at core temperature during maximal exercise and ~3 mL∙kgbody

mass-1∙min-1 using an average heart mass of 380 g in males and 330 g in females47. Note that oxygen

consumption of other organs such as the liver, kidneys, and digestive system is neglected but is assumed to be less than in resting conditions (< 2 mL∙kg-1∙min-1; cf. Ref 44).

(5.3)

where V̇O2,rest is oxygen consumption at rest, V̇O2max,heart is 670 mL∙kgheart mass-1∙min-1, Mh is heart

mass (330 g in females, 380 g in males). SDH = 1/5 × 0.35 × (SDHarm/SDHleg) × SDHVL

+ 4/5 × SDHVL, that is SDH activity of arm musculature is not fully active during maximal cycling exercise (35%) and is lower than leg SDH activity (65% in cyclists, 80% in controls and CHF patients) and accounts for ~20% of whole-body skeletal muscle mass, with SDHVL is SDH activity determined from biopsy samples of the m. vastus lateralis.

StatisticsAll data are presented as individual values or as mean±SD, unless otherwise indicated. Differences between groups were assessed by one-way ANOVAs (between factor group: CHF patients, controls, cyclists). Differences in SDH activity with incubation temperature were assessed by repeated measures ANOVA (within factor temperature: 32, 37 and 42°C). Post hoc comparisons with Bonferroni correction were performed to detect differences between groups and temperatures. The relationships between SDH-activity and whole-body V̇O2max, SDH-activity and V̇O2max normalized for skeletal muscle mass and between mitochondrial oxidative capacity and whole-body V̇O2max were assessed by linear regressions. To check whether regression lines differed from the line of identity or regression line from literature, differences in

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slope and intercept were tested by confidence intervals of the regression coefficients. Differences were considered to be significant if p < 0.05.

ResultsTable 5.1 summarizes average age, body mass, body mass index (BMI), calculated skeletal muscle mass, V̇O2max, SDH activity at 37°C, SDH activity adjusted for temperature, and calculated muscle fiber V̇O2max of CHF patients, healthy controls and cyclists. Even though skeletal muscle mass as percentage body mass was lower in controls compared to cyclists, the absolute skeletal muscle mass was similar in controls and cyclists, which was likely due to a higher body mass and BMI in the control group.

Histochemical method for SDH activityFigure 5.1 shows muscle fiber cross-sections of m. vastus lateralis that were incubated for SDH using quantitative enzyme histochemistry. SDH activity was significantly different between CHF patients, controls and cyclists.

Reproducibility was determined from two measurements of mean SDH absorbance without incubation and in high oxidative (i.e. type I) muscle fibers and low oxidative (i.e. type II) muscle fibers36 after 20 minutes of incubation (Figure 5.2). It is concluded that average SDH absorbance values were similar for both measurements.

The relationship between SDH absorbance and incubation temperature in human muscle tissue is shown in Figure 5.3. Mean absorbance values of high and low oxidative fibers were significantly different between 32°C, 37°C and 42°C (p<0.001). At 42°C, SDH absorbance is 1.76 times higher compared to that at 32°C. Hence, SDH enzyme activity of all subjects was adjusted for muscle temperature using a Q10 of 1.76.

Relationship between SDH activity and whole-body V̇O2max

Figure 5.4 shows that SDH activity adjusted for muscle temperature is closely related to whole-body V̇O2max normalized to body mass (r2 = 0.81, p<0.001) and whole-body V̇O2maxnormalized to skeletal muscle mass (r2 = 0.83, p<0.001). Without adjustment for muscle temperature, correlation coefficients are slightly lower for V̇O2max normalized to body mass (r2 = 0.79, p<0.001) and V̇O2max normalized to skeletal muscle mass (r2 = 0.80, p<0.001). These results indicate that SDH activity is proportional to the V̇O2max in humans, irrespective of training status (V̇O2max ranging from 9.8 to 79.0 mL∙kg-1∙min-1). Normalization of V̇O2max to skeletal muscle mass allows direct comparison with ex vivo measurements from intact animal myocytes in hyperoxic Tyrode solution (represented by the solid line; see methods). In humans, the relationship between temperature-adjusted SDH activity and V̇O2max differed significantly from the predicted relationship from ex vivo measurements of animal fibers in hyperoxic conditions (slope < 7.6 × 106, CI = [5.3 × 106 - 6.7 × 106], p<0.05; intercept = 0, p>0.05)29,30. Therefore, at V̇O2max, the oxidative enzyme activity is not fully exploited. Note that V̇O2max normalized to skeletal muscle mass may even provide an overestimation of the true value, because it also includes oxygen uptake by tissues other than skeletal muscles (Figure 5.4B).

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Maximal oxygen uptake is proportional to muscle fiber oxidative capacity

Figure 5.4. Relationship between SDH activity and whole-body maximal oxygen uptake (V̇O2max) obtained during incremental cycling exercise. A) V̇O2max normalized to body mass; B) V̇O2max normalized to skeletal muscle mass. Data is displayed for CHF patients (), controls () and cyclists () and the group averages (large open symbols). In B, the solid line represents the relationship between V̇O2max and SDH activity from ex vivo measurements of intact animal fibers in hyperoxia (see text). The glycogen-depleted biopsy reported by Bekedam et al.31 was excluded.

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Relationship between mitochondrial oxidative capacity and whole-body V̇O2max The relationship between body-mass specific V̇O2max during leg cycling and mitochondrial oxidative capacity from temperature-adjusted SDH activity and total skeletal muscle mass is shown in Figure 5.5A. In CHF patients, controls, and cyclists, mitochondrial oxidative capacity is proportionally related to V̇O2max during cycling (r2 = 0.88-0.89, p<0.001). The relationship between V̇O2max and mitochondrial capacity based on Equation 5.1 significantly differed from the line of identity (slope<1, CI = [0.72 - 0.86], p<0.05; intercept = 0, p>0.05) and indicates that subjects do not fully use (~85%) the oxidative enzyme capacity at V̇O2max during cycling exercise. Note that in Figure 5.5A it is assumed that all oxygen at V̇O2max is consumed by skeletal muscle mitochondria, that all mitochondria are active at their V̇O2max during maximal cycling exercise and that SDH activity of m. vastus lateralis represents SDH activity of whole body musculature. Figure 5.5B displays the relationship between V̇O2max and mitochondrial oxidative capacity based on Equation 5.3. This prediction suggests that V̇O2max during cycling is 90±14% of the mitochondrial oxidative capacity. Even though the relationship in Figure 5.5B tended to differ from the line of identity, the slope was not significantly different from 1 (slope = 1, CI = [0.84 - 1.00], p>0.05; intercept = 0, p>0.05). Note that there is still considerable individual variation between mitochondrial oxidative capacity (Equation 5.3) and measured V̇O2max during cycling (CV = 15.2%), which is also illustrated by lower correlation coefficients in separate subgroups (CHF patients r=0.47, p=0.107; healthy controls r=0.61, p<0.001, cyclists r=0.67, p<0.001). Mitochondrial oxidative overcapacity did not differ between CHF patients, controls, and cyclists (p=0.108).

DiscussionThe present study showed that SDH activity and mitochondrial oxidative capacity predicted from SDH activity are related to V̇O2max measured during cycling (r2 = 0.81, p<0.001 and r2 = 0.89, p<0.001, respectively) across chronic heart failure patients, healthy untrained controls and cyclists (V̇O2max ranging from 9.8 to 79.0 mL∙kg-1∙min-1). During cycling exercise, V̇O2max was on average 90±14% of the mitochondrial oxidative capacity, indicating that humans do not fully exploit their oxidative enzyme capacity.

Symmorphosis in humans: mitochondrial oxidative capacity is proportionally related to V̇O2max Our results confirmed that V̇O2max scales proportionally with SDH activity (r2=0.81) and mitochondrial oxidative capacity (r2=0.89) across heart failure patients, healthy controls, and cyclists. These results are in line with previous cross-sectional studies that have shown that in heterogeneous healthy populations the body-mass-specific V̇O2max is related to SDH activity (r=0.79)19 and mitochondrial volume density (r=0.82)17. Note that in more homogeneous populations, correlations between SDH activity and V̇O2max were found to be substantially lower (r=0.23)48. Our subgroups also showed lower correlations, which in combination with our coefficient of variation indicated considerable individual differences in mitochondrial oxidative overcapacity (see Unexplained variance of mitochondrial oxidative capacity and V̇O2max). However, mitochondrial oxidative overcapacity did not differ between CHF patients, controls, and cyclists, which supports existence of symmorphosis in humans. CHF impairs V̇O2max and

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Figure 5.5. Relationship between mitochondrial oxidative capacity predicted from SDH activity and skeletal muscle mass and the measured V̇O2max during incremental cycling exercise. A) mitochondrial oxidative capacity based on Equation 5.1; B) mitochondrial oxidative capacity based on Equation 5.3. Data is displayed for CHF patients (), controls () and cyclists () and the group averages are shown (large open symbols). In A, mitochon- drial oxidative capacity is predicted based on the assumption that all oxygen is consumed by skeletal muscle mitochondria and that SDH activity of the vastus lateralis locomotory muscle represents SDH activity of whole body musculature. In B, mitochondrial oxidative capacity takes into account 1) the differences in SDH activity between arm and leg muscles, 2) active skeletal muscle mass at maximal cycling exercise and 3) oxygen consumption of other organs (see text). The glycogen-depleted biopsy reported by Bekedam et al.31 was excluded.

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mitochondrial oxidative capacity due to the limited cardiac output49, as a result of reduced O2 flux to the mitochondria from impaired red blood cell (RBC) flux and velocity, reduced percentage of flowing capillaries supporting RBC flux during rest and exercise, impaired Q̇O2/V̇O2max matching, low microvascular PO2 and high fractional oxygen extraction49–52. Our results indicate that these impairments in both V̇O2max and mitochondrial oxidative capacity are proportional in CHF patients and therefore support existence of symmorphosis in humans. Also trained cyclists did conform to the proportional relationship between V̇O2max and mitochondrial oxidative capacity. In humans, however, individual increases in V̇O2max induced by endurance training are generally not proportional to changes in oxidative enzyme activity of the skeletal muscle involved in endurance training (cf. Ref 53,54). This discrepancy may be explained by the time course of (acute) training adaptations. For instance, V̇O2max tends to increase by 15-30% during the first 2-3 months of endurance training, while 40-50% increases in V̇O2max may occur over 9-24 months of training54. However, concomitant changes in the citric acid cycle and respiratory chain enzymes are much faster, displaying half-lives in the order of 1-3 weeks53,54. Therefore, increases in V̇O2max and mitochondrial capacity are likely disproportional during acute training adaptations (<9 months), but may become proportional with chronic training adaptations (>9 months), such as in our trained cyclists with an extensive training history (120 months on average). Also note that trainability of V̇O2max may markedly vary between subjects and is strongly related to heritability55. Thus, mitochondrial oxidative capacity scales with V̇O2max and largely accounts for differences in body-mass-specific V̇O2max in this heterogeneous population.

Animals conform to the concept of symmorphosis assuming that all parts of the O2 pathway that contribute to V̇O2max are designed proportionally16. Across sedentary and athletic animal species, it was shown that mitochondrial volume density is proportional to body-mass-specific V̇O2max explaining 97% of its variance14. Athletic animals presented a 2.5-fold greater body-mass-specific V̇O2max, which was matched by a 2.5-fold larger mitochondrial volume density in their muscles14. Therefore, the maximal rate of O2 consumption by skeletal muscle has been considered to be invariant across animals (4-5 mL O2∙mL-1 mitochondria∙min-1)14. Our cross-sectional observation suggests that also in a heterogeneous population of human subjects, V̇O2max was matched by mitochondrial oxidative capacity: both V̇O2max and SDH or predicted oxidative capacity were ~1.5-fold greater in cyclists compared to controls, ~3-fold greater in cyclists compared to CHF patients and ~2-fold greater in controls compared to CHF patients. Our results contradict previous findings56, which challenged the existence of symmorphosis in humans and suggested that the relationship between V̇O2max during cycling and mitochondrial oxidative capacity (from permeabilized fibers) varied with exercise training status. In this previous study56, measurements in untrained subjects did conform to the concept of symmorphosis, while trained subjects did not. However, this conclusion is based on data of only 4 trained subjects and on a smaller observed range of V̇O2max (31-66 mL∙kg-1∙min-1). In the present study, mitochondrial oxidative capacity is proportional to V̇O2max explaining 81-89% of its variance when observing a larger range of V̇O2max (9.8-79.0 mL∙kg-1∙min-1). Hence, similar to animals, a higher oxygen uptake demand is met by increasing mitochondrial enzyme activity, while oxidative capacity per mitochondria remains invariant across human individuals as well. Note that the observed marginal mitochondrial oxidative overcapacity does not challenge the concept of symmorphosis, as small excess in mitochondrial capacity at maximal exercise may serve as a safety factor16. Thus, our data support the concept of symmorphosis in a heterogeneous population of human subjects

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indicating that mitochondrial oxidative capacity is proportional to V̇O2max.

Mitochondrial oxidative overcapacity estimated from SDH activityOur subjects used on average 90% of their mitochondrial capacity during maximal cycling exercise, suggesting that mitochondrial oxidative capacity exceeded O2 use by ~11%. Based on the relationship between mitochondrial respiration and cellular PO2, one could calculate at what percentage of their maximal capacity the mitochondria operate during dynamic exercise. Assuming first order Michaelis Menten kinetics for mitochondrial oxygen consumption25, the maximum obtainable V̇O2 relative to the theoretical V̇O2max (under hyperoxic conditions) is:

(5.4)

where Km is the Michaelis constant for oxygen of the mitochondria (0.5 Torr)25 and PO2 is the mitochondrial oxygen tension (3.1 Torr during maximal cycling exercise estimated from myoglobin saturation and the P50 of myoglobin for oxygen)24. Following Equation 5.4, V̇O2 / V̇O2max = 0.86. Therefore, during maximal cycling exercise, the mitochondria should theoretically operate at ~86% of their maximal oxidative capacity, which agrees well with reported values reported in the present study. Further mitochondrial inhibition (e.g. by NO) is therefore not necessary to explain the reported oxidative overcapacity. It should be noted that Equation 5.4 assumes homogeneous oxygen tension inside the muscle cells, which is physiologically impossible (for further details see Refs57,58). Note that the use of mitochondrial oxidative capacity (90 ± 14%) is similar to the exploited mitochondrial oxidative capacity (90 ± 15%) when based on the assumption that skeletal muscle mass accounts for ~90% of the total oxygen consumption at maximal exercise14 (i.e. instead of estimating oxygen consumption by the heart and brain). Moreover, considering that V̇O2max is 5-10% higher during running versus leg cycling59,60 and with arm musculature being fully active, the exploited mitochondrial oxidative capacity during running is estimated to be comparable to leg cycling (86 ± 13% and 90 ± 14% respectively). Therefore, mitochondrial oxidative overcapacity of the present study appears to be robust and agrees well with mitochondrial overcapacity calculated from cellular oxygen tension.

Mitochondrial oxidative overcapacity with enhanced oxygen supply Our results indicate that circumventing limitations to V̇O2max can increase V̇O2max on average by ~11% at most. What happens to maximal oxygen uptake when limitations of O2 supply are reduced? As the O2 pathway from atmosphere to mitochondria is an in-series system, it is evident that every step significantly impacts V̇O2max

8. Acute interventions that enhance O2

supply have shown to increase V̇O2max. Hyperoxia, for instance, by increasing inspired O2 fraction (FiO2) increased whole body V̇O2max with 2-5%11 and leg V̇O2max with 8%10 by enhancing oxygen diffusion at the lungs and from microvessels to mitochondria10,11. Blood reinfusion of 900-1350 mL elevated oxygen carrying capacity of the blood and thereby increased V̇O2max by 4-9%12. Moreover, V̇O2max of one leg derived from blood flow and arterio-venous O2 differences measurements was 5-14% higher in one-legged cycling compared to two-legged cycling exercise, because of the higher leg blood flow in one-legged exercise61. The lower leg blood flow

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during maximal exercise with two legs compared to one leg is likely due to vasoconstriction of active muscles that is required to avoid hypotension and increase transit time at the muscle capillaries13,61. The theoretical model of Wagner, linking lungs, circulation and muscles to V̇O2max, predicts that an isolated 20% increase in ventilation, FiO2, lung diffusion capacity, muscle O2 diffusion conductance, hemoglobin concentration or blood flow will increase V̇O2max by only 1.3-5%6,62. Enhancement of oxygen supply can also be studied together with noninvasive assessment of post-exercise PCr recovery kinetics with 31P-MRS63–65, as PCr recovery kinetics have been used to estimate mitochondrial oxidative capacity66. Previous studies have shown that enhanced oxygen supply with hyperoxia63 and reactive hyperemia induced by cuff occlusion65 improved both PCr recovery kinetics and estimated mitochondrial respiration rate, however, the quantitative interpretation of muscle mitochondrial capacity remains difficult (for review see67). Therefore, evidence indicates that increases in whole-body V̇O2max due to enhanced O2 supply are generally within a 10% range and agree well with our findings of excess mitochondrial capacity.

Unexplained variance of mitochondrial oxidative capacity and V̇O2max

Even though V̇O2max and mitochondrial oxidative capacity are related, there are still considerable individual differences in mitochondrial oxidative overcapacity. These individual differences may arise from methodological errors in estimating mitochondrial oxidative capacity. For instance, the location of biopsy sampling affects fiber type distribution by 2-3% and since SDH activity is closely related to fiber type36 this could also affect weighed SDH activity. Nonetheless, we showed that SDH activity from quantitative histochemistry is highly reproducible (Figure 5.2). We could not account for individual variation in muscle temperature, which in healthy controls is likely to be 1-1.5%39,68 and ~3.5% in CHF patients38. Even though skeletal muscle mass was estimated from an anthropometric model40, the estimated skeletal mass as percentage of body mass (34.7% in CHF patients, 37.5% in controls and 42.3% in cyclists) was in agreement with skeletal muscle mass derived from MRI measurements in subgroups of 468 subjects with similar age (33.8%, 39.1% and 42.3% respectively)69. Errors in oxygen consumption by the brain and heart (e.g. due to differences in heart mass and mitochondrial volume density between CHF patients, controls and cyclists) may have attributed to the unexplained variance between measured V̇O2max and mitochondrial oxidative capacity. However, note that maximal oxygen consumption of the heart (~3 mL∙kg-1∙min-1) and oxygen consumption of the brain (~1 mL∙kg-1∙min-1) only marginally contributed to the mitochondrial oxidative capacity in control subjects and cyclists. Moreover, we could not account for individual variation in SDH activity differences between arm and leg musculature and submaximally active mitochondria in arm musculature during leg cycling exercise. V̇O2max has shown to be obtained reproducibly within our laboratory (ICC=0.98, p<0.001; unpublished results) and all subjects terminated exercise due to voluntary exhaustion. Although V̇O2peak attained during the maximum-effort incremental test to voluntary exhaustion is considered a valid index of V̇O2max

32 that is not different from V̇O2peak attained during supra-maximal exercise70, small differences in maximal effort may have contributed to individual differences in mitochondrial oxidative overcapacity, as underestimates of V̇O2max may have occurred in some subjects. Taken together, unexplained variance between mitochondrial oxidative capacity and V̇O2max is only 11% (r2 = 0.89) in the present study, and therefore it is likely that possible effects of methodological errors are (partially) cancelled out in our group of human subjects.

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A portion of unexplained variance between mitochondrial capacity and V̇O2max may also be attributed to physiological differences between individuals. These differences may arise from limitations to the convective O2 supply, for instance arterial and venous saturation, systemic hematocrit, hemoglobin content, cardiac output and end-diastolic heart volume1,2,8. Also, limitations to diffusive O2 supply may explain individual differences in mitochondrial oxidative overcapacity at maximal exercise. Oxygen diffusion at the muscle level is determined by oxygen transport from sarcolemma to mitochondria by myoglobin and by blood-myocyte O2 flux set by the oxygen tension gradient between the capillaries and sarcolemma, which depends on (changes in) red blood cell (RBC) velocity and capillary hematocrit (i.e. RBC flux), on capillary density and the proportion of flowing capillaries1,8,49,71. Moreover, differences in mitochondrial oxidative overcapacity may be investigated in light of individual fiber type distribution as the vasomotor control may be modulated as a function of fiber type and/or oxidative capacity of the muscle fibers51,72, displaying better Q̇O2/V̇O2 matching and thus higher microvascular PO2 in high versus low oxidative muscle fibers or muscle parts51. Furthermore, individual differences may arise from the effectiveness in energy distribution within the muscle cells, which depends on metabolite facilitated diffusion (i.e. oxygen transport by the oxy-deoxy myoglobin shuttle and ATP diffusion by the creatine kinase shuttle) and on membrane potential conduction via the mitochondrial reticulum73. Since SDH activity using quantitative enzyme histochemistry is determined in individual muscle fibers, it can be related to other fiber specific variables (e.g. myosin heavy chain type and muscle fiber size, capillary density, diffusion distance or myoglobin concentration). Future studies should investigate whether differences in determinants of convective and diffusive O2 supply or energy distribution may also account for part of the unexplained variance in the relationship between mitochondrial oxidative capacity and V̇O2max, i.e. explain differences between individual subjects with similar V̇O2max but different SDH activity.

Use of mitochondrial oxidative overcapacityEven though oxygen use during maximal cycling exercise is on average ~90% of the oxidative capacity, a greater proportion of the mitochondrial oxidative capacity may be used when oxygen supply is not limiting. For instance, during exercise with smaller muscle groups (without cardiac output limitation), blood flow heterogeneity may facilitate matching of oxygen supply to oxygen demand in the active muscle groups, while this does not come at the expense of Q̇O2/V̇O2 matching in another region51,72. Also at submaximal exercise intensities, a higher mitochondrial oxidative capacity may enable mitochondria to operate at a lower percentage of their maximal oxidative rate for a given oxygen consumption. Consequently, following Michealis Menten kinetics of mitochondria, V̇O2 kinetics will be faster and result in glycogen sparing and faster adaptations of steady state during submaximal exercise. In addition, lower substrate concentrations are required and mitochondria are less likely to become hypoxic, thereby reducing damage from oxidative stress induced by hypoxia. Moreover, hypoxia in the muscle fibers will likely occur at higher exercise intensity and therefore could increase intensity at the lactate or ventilatory threshold (i.e. intensity at which aerobic ATP resynthesis can no longer match ATP use in the working muscles), which is an important determinant of endurance performance in subjects with similar V̇O2max

1. Therefore, with a higher mitochondrial oxidative capacity, a higher rate of ATP production may be sustained during endurance performance74. As mitochondria conform to Michaelis Menten kinetics, it is highly unlikely that mitochondria operate at 100% of their maximal oxidative

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capacity, as this requires very high intracellular PO2 (that would likely require an increase in the P50 of the blood and enhanced diffusion capacity in the lungs) and extremely high substrate concentrations (that potentially could have an osmotic effect similar to lactate accumulation and thereby increase blood viscosity).

ConclusionsHuman maximal oxygen uptake attained during cycling exercise is related to mitochondrial oxidative capacity predicted from skeletal muscle succinate dehydrogenase activity. Measured whole-body V̇O2max is ~90% of the mitochondrial oxidative capacity, which can be explained by limited oxygen supply to muscle mitochondria.

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