ARTICLES

Role of skeletal muscles impairment and brain oxygenation in limiting oxidative metabolism during exercise after bed rest

Abstract

“Central” and “peripheral” limitations to oxidative metabolism during exercise were evaluated in 10 young males following a 35-day horizontal bed rest (BR). Incremental exercise (IE) and moderate- and heavy-intensity constant-load exercises (CLE) were carried out on a cycloergometer before and 1–2 days after BR. Pulmonary gas exchange, cardiac output (Q̇; by impedance cardiography), skeletal muscle (vastus lateralis), and brain (frontal cortex) oxygenation (by near-infrared spectroscopy) were determined. After BR, “peak” (values at exhaustion during IE) workload, peak O2 uptake (V̇o2peak), peak stroke volume, Q̇peak, and peak skeletal muscle O2 extraction were decreased (−18, −18, −22, −19, and −33%, respectively). The gas exchange threshold was ∼60% of V̇o2peak both before and after BR. At the highest workloads, brain oxygenation data suggest an increased O2 extraction, which was unaffected by BR. V̇o2 kinetics during CLE (same percentage of peak workload before and after BR) were slower (time constant of the “fundamental” component: 31.1 ± 2.0 s before vs. 40.0 ± 2.2 s after BR); the amplitude of the “slow component” was unaffected by BR, thus it would be greater, after BR, at the same absolute workload. A more pronounced “overshoot” of skeletal muscle O2 extraction during CLE was observed after BR, suggesting an impaired adjustment of skeletal muscle O2 delivery. The role of skeletal muscles in the impairment of oxidative metabolism during submaximal and maximal exercise after BR was identified. The reduced capacity of peak cardiovascular O2 delivery did not determine a “competition” for the available O2 between skeletal muscles and brain.

bed rest (BR) has been extensively utilized to simulate the effects of microgravity on body functions, including the adjustments to exercise (see e.g., the reviews Refs. 4, 9, 13, 19, 20, 36). In more general terms, BR studies allow us to evaluate the consequences on physiological functions deriving from extreme degrees of physical deconditioning. BR is known to lead, among other effects, to a reduction in maximal O2 uptake (V̇o2max) (9, 18, 20), which appears to be quantitatively related to the duration of the BR period (8). The relationship between the reduction in V̇o2max observed after BR and the changes observed during spaceflight, however, appears rather controversial. Whereas Levine et al. (33) observed no significant changes in V̇o2max, compared with preflight levels, during 9–14 days of spaceflight, according to Trappe et al. (43), cardiorespiratory responses during spaceflight are similar to those described after BR. In any case, the reduced V̇o2max after BR is associated with reduced maximal cardiac output (Q̇max) and O2 delivery (9, 18). According to Capelli et al. (8), after BR lasting for up to about 40 days, the percent decrease in Q̇max is higher than the percent decrease in V̇o2max. A substantially unchanged maximal capacity of O2 extraction, as evaluated by the calculated maximal systemic arterial-venous O2 concentration difference [C(a-v̄)o2max = V̇o2max/Q̇max] has indeed been described after BR (9, 18). This finding may appear surprising, considering that BR induces significant decreases in variables related to skeletal muscle, such as the volume density of mitochondria, oxidative enzyme activities, and capillary length (4, 18), which could affect C(a-v̄)o2 and V̇o2max. At least in part, the impairments related to skeletal muscle may be a consequence of an increased oxidative stress induced by BR (37). Possible explanations for this apparent discrepancy could lie, at least in part, in the problems associated with the determination of Q̇max by noninvasive methods in humans (44) and/or in the relatively minor contribution of “peripheral” (skeletal muscle) factors in limiting V̇o2max (12). Peripheral (skeletal muscle) limitations to oxidative metabolism after BR could impair variables of functional evaluation related to exercise tolerance, such as the efficiency of exercise, the “gas exchange threshold,” and the “fundamental” and the “slow” components of V̇o2 kinetics (28, 45), even without affecting systemic C(a-v̄)o2max. A systemic analysis of these variables after BR has not been conducted so far.

In recent years another potential “central” limitation to aerobic exercise performance has been identified, which could intervene in conditions characterized by a reduced maximal capacity of O2 delivery. This limitation is represented by an inadequate cerebral O2 delivery, which could induce cerebral hypoxia and, as a consequence, limit maximal exercise performance (35). Subudhi et al. (41), for example, observed during maximal exercise in hypobaric hypoxia a deoxygenation in cerebral tissue that apparently imposed a limit to maximal exercise, since its reversal was associated with an improved performance. Considering that Ferretti et al. (18) described an almost 40% decrease in the maximal capacity of O2 delivery after a 42-day BR, a condition of inadequate cerebral O2 delivery potentially limiting maximal aerobic performance could also be hypothesized in these experimental conditions.

The aims of the present study were to better define, after BR, the role of central (cardiovascular O2 delivery and brain oxygenation) and peripheral factors limiting maximal aerobic performance, as well as to perform a functional evaluation of oxidative metabolism more specifically aimed at submaximal workloads and at the skeletal muscle level. More specifically, we hypothesized, after a 35-day BR, an impaired cardiovascular performance, affecting cerebral oxygenation during maximal exercise; an impaired peak capacity of O2 extraction by anti-gravity skeletal muscles, which would contribute, together with the impaired peak capacity of O2 delivery, to the reduced V̇o2peak; a slower adjustment of pulmonary V̇o2 during exercise transitions, a more pronounced slow component of V̇o2 kinetics, and alterations in the matching between muscle O2 delivery and muscle V̇o2. Although we recognize the indirect nature of our measurements (see below), we also consider that their noninvasiveness allows them to be performed serially in the present, as well as in other, experimental models (including patients).

METHODS

Subjects.

Ten young healthy untrained subjects [age: 23.9 ± 0.8 (mean ± SE) years; height: 179.5 ± 2.4 cm] volunteered for the study. They were evaluated before and after a 35-day horizontal BR without countermeasures, carried out within the 2007 ASI-OSMA Bed Rest Campaign (July–August 2007) at the Valdoltra Hospital (Ankaran, Slovenia). During BR, no deviations from the lying position were permitted, and subjects were continuously monitored by video cameras. Neither exercise nor muscle contraction tests were allowed. Measurements before BR were carried out during the last 2 days before the subjects were put to bed; measurements after BR were carried out during the first 2 days after the subjects arose from bed. To avoid weight gains due to the reduced activity level, the subjects followed a balanced diet during BR. Body mass (BM) and body mass index (BMI) of the subjects were, respectively, 77.2 ± 3.4 kg and 23.9 ± 0.9 kg/m2 before BR vs. 73.4 ± 3.2 kg and 22.8 ± 0.9 kg/m2 after BR; the ∼5% decreases in BM and BMI were statistically significant. Blood hemoglobin concentration was 14.5 ± 0.2 g/dl before BR and 14.6 ± 0.2 g/dl after BR (no significant difference).

The subjects were highly motivated to participate in the research, and their collaboration was excellent. All subjects were nonsmokers and underwent a complete physical examination and a cardiopulmonary stress test during the selection process. The subjects were informed about risks and discomfort associated with the experimental procedure before they gave their written consent to participate in the research program, which was approved by the ethical committees of the involved institutions. All tests were conducted under close medical supervision, and subjects were continuously monitored by 12-lead electrocardiography (ECG). After receiving a detailed explanation of all experimental procedures, subjects were allowed enough time to become familiar with the researchers and with the setup environment, as well as with the experimental protocol, by performing short preliminary practice runs.

Cycloergometric exercise protocol.

A mechanical flywheel cycle ergometer (Monark 818E; Stockholm, Sweden) was utilized. The following exercises were performed, both before and after BR. 1) An incremental exercise (IE) was performed to voluntary exhaustion. Before BR, 15–25 W/min increments were given after an initial 3 min of unloaded pedalling and 6 min at 60–90 W (the exact workloads were chosen on the basis of the estimated level of physical fitness of the subject). After BR, workload increments were 15 W/min, and the initial workload, following unloaded pedalling, was 45–75 W. Voluntary exhaustion was defined as the inability to maintain the pedalling frequency despite encouragement by the operators, as well as by maximal levels of self-perceived exertion using the validated Borg scale (5). Mean values of cardiovascular, ventilatory, gas exchange, and muscle and brain oxygenation variables (see Measurements) determined during the last 20 s of the exhausting load were considered “peak” values. 2) Three repetitions of 6-min constant-load exercise (CLE) of moderate intensity were performed at 60% of the “gas exchange threshold” (GET; see below) previously determined during IE; each repetition was separated by a 15- to 30-min recovery period. On-transitions were from unloaded pedalling to the imposed workload, which was attained in ∼3 s. 3) One repetition of a 6-min CLE of heavy intensity was performed at a workload corresponding to ∼50% of the difference between GET and V̇o2peak. Thus CLE before and after BR were matched in relative terms, that is, with respect to GET and V̇o2peak values determined in the two conditions. Both before and after BR, experiments were conducted on 2 consecutive days: IE during day 1 and CLE <GET and CLE >GET during day 2.

Measurements.

Pulmonary ventilation (V̇e; expressed in BTPS), O2 uptake (V̇o2), and CO2 output (V̇co2), both expressed in STPD, were determined breath by breath using a computerized metabolic cart (SensorMedics Vmax29c; Bilthoven, The Netherlands). Expiratory flow measurements were performed using a mass flow sensor (hot wire anemometer), calibrated before each experiment with a 3-liter syringe at three different flow rates. Tidal volume (Vt) and V̇e were calculated by integration of the flow tracings recorded at the mouth of the subject. V̇o2 and V̇co2 were determined by continuously monitoring Po2 and Pco2 at the mouth throughout the respiratory cycle and from established mass balance equations, after alignment of the expiratory volume and expiratory gases tracings and analog-to-digital conversion. Calibration of O2 and CO2 analyzers was performed before each experiment by using gas mixtures of known composition. Digital data were transmitted to a personal computer and stored on disk. Gas exchange ratio (R) was calculated as V̇co2/V̇o2. GET was determined using standard methods (2).

Arterial blood O2 saturation (SaO2) was continuously monitored by pulse oximetry (MasimoRAD-9; Masimo, Milan, Italy) at the ear lobe. Heart rate (HR) was determined by ECG. Stroke volume (SV) was estimated beat by beat using impedance cardiography (Physio Flow; Manatec, Paris, France). The accuracy of this device was previously evaluated during IE in healthy subjects against the direct Fick method (38); in that study the accuracy of the impedance cardiography method was found to be “acceptable” (38), although the confidence limits of the comparison between the two methods were rather wide. Cardiac output (Q̇) was calculated as HR × SV. Blood pressure (BP) was measured using a standard cuff sphygmomanometer. Severe hypertension (systolic BP value >250 Torr) or falling BP during exercise was considered a criterion for terminating the test. At rest, at the end of exercise, and at 1, 3, 5, and 7 min during the recovery period, blood lactate concentration ([La]b) was determined using an enzymatic method (Biosen 5030; EKF Diagnostic, Eppendorf Italia, Milano, Italy) on 20 μl of capillary blood obtained at the ear lobe.

Oxygenation changes in vastus lateralis muscle and brain (frontal cortex) were evaluated using near-infrared spectroscopy (NIRS) (6, 15). For skeletal muscle, a portable NIR single-distance continuous-wave photometer (HEO-100; Omron, Kyoto, Japan) was used. The instrument provides separate measurements of concentration changes of deoxygenated hemoglobin (Hb) and myoglobin (Mb), expressed in arbitrary units. Concentration changes of oxygenated Hb + Mb {Δ[oxy(Hb+Mb)]} and deoxygenated Hb+Mb {Δ[deoxy(Hb+Mb)]}, with respect to an initial value arbitrarily set equal to zero, were also calculated and expressed in arbitrary units. The sum of the two variables {Δ[oxy(Hb+Mb) + deoxy(Hb+Mb)]} is related to changes in the total Hb volume in the muscle region of interest. As in previous studies (see e.g., Refs. 11, 16, 22, 23, 30, 32) Δ[deoxy(Hb+Mb)] was taken as an estimate of skeletal muscle fractional O2 extraction, because this variable, unlike Δ[oxy(Hb+Mb)], is relatively insensitive to changes in blood volume (14, 23). Because these data are expressed in arbitrary units, a “physiological calibration” of Δ[deoxy(Hb+Mb)] values was performed after exercise was terminated: data obtained during the exercise protocol were expressed as a percentage of the values determined by obtaining a maximal deoxygenation of muscle, by pressure cuff inflation at 300–350 Torr at the root of the thigh (subjects in the sitting position on the cycloergometer) for a few minutes until Δ[deoxy(Hb+Mb)] increase reached a plateau. For more technical details of the measurement, see Grassi et al. (22, 23).

For brain oxygenation, a different single-distance continuous-wave NIRS instrument (Oxymon, Artinis, The Netherlands) was used. Headsets held an NIR emitter (laser light at 780 and 850 nm) and detector pair over the right frontal cortex region of the forehead; optodes were held in place by a plastic spacer with fixed optode distance. Spacing between optodes was 4.5 cm, corresponding to a penetration depth of ∼2.5 cm. The Beer-Lambert law was used to calculate micromolar (μM) changes in tissue oxygenation {changes in concentration of oxyhemoglobin (Δ[O2Hb]) and deoxyhemoglobin (Δ[HHb])} by using received optical densities and a differential path length factor of 5.93 [for more technical details on the measurement, see Subudhi et al. (40)]. Total Hb (Δ[O2Hb+HHb]) was taken as an index of changes in regional blood volume. Data were recorded at 10 Hz. Measurements obtained during exercise were normalized as changes from an initial value arbitrarily defined as 0 μM.

Reliability of tissue oxygenation indices obtained by NIRS, evaluated using the intraclass correlation coefficient for repeated measurements on the same subject during different days, was recently found to be very high for both brain and skeletal muscle (40); for brain measurements, those authors used the same instrument used in the present study. NIRS measurements in cerebral (25) and muscle tissue (46) have been shown to be well correlated with local venous O2 saturation. Subudhi et al. (42) recently demonstrated that brain deoxygenation during high-intensity exercise occurs across the cortex and thus also directly affects the motor areas that regulate central motor drive.

Kinetics analysis.

o2 kinetics were evaluated during the transitions from unloaded pedalling to CLE. Breath-by-breath V̇o2 values obtained in the various repetitions of CLE at the same workload were time-aligned and then superimposed for each subject. Average V̇o2 values every 10 s were calculated and used for kinetics analysis. V̇o2 data obtained during the first 20 s of the transition [corresponding to the “cardiodynamic” phase (45)] were excluded from the analysis. Thus V̇o2 kinetics analysis dealt with the “phase 2” (or fundamental component) of the response, which should more closely reflect gas exchange kinetics occurring at the skeletal muscle level (24, 39).

To mathematically evaluate the V̇o2 kinetics, data were fitted using a function of the type

y(t)=yBAS+Af[1e(xTDf)/τf](1)
Parameter values (TDf, τf) were determined that yielded the lowest sum of squared residuals. In Eq. 1, yBAS indicates the baseline value; Af, the amplitude between yBAS and the steady-state value during the fundamental component of the kinetics; TDf, the time delay; and τf, the time constant of the function for the fundamental component.

To check the presence of a slow component of the V̇o2 kinetics (28, 45), data were also fitted using a function of the type

y(t)=yBAS+Af[1e(xTDf)/τf]+As[1e(xTDs)/τs](2)
In Eq. 2, As, TDs, and τs indicate, respectively, the amplitude, time delay, and time constant of the slow component of the kinetics. The equation that best fitted the experimental data was determined by F-test (see Statistical analysis). That is to say, when Eq. 2 provided a better fit of the data, a slow component of V̇o2 kinetics was present, superimposed on the fundamental component. The slow component, however, did not always follow an exponential function, being sometimes linearly related to the time of exercise; moreover, its τs values appear devoid of physiological significance. Thus the actual amplitude of the slow component (A′s) was estimated as the difference between an average V̇o2 value calculated during the last 20 s of the exercise period and the asymptotic value of the fundamental component. The contribution of As to the total amplitude (Atot) of the response (A′s/Atot) was then calculated (23, 39).

Δ[Deoxy(Hb+Mb)] kinetics were evaluated during IE and CLE. For IE, average data obtained during the last 10 s of each workload were calculated and retained for analysis. As described by Ferreira et al. (16), the Δ[deoxy(Hb+Mb)] vs. workload relationship was fitted by a sigmoidal function of the type

y(x)=yBAS+A/[1+e(c+dx)](3)
In Eq. 3, yBAS indicates the baseline; A, the amplitude of the response; and c, a constant dependent on d, the slope of the sigmoid, where c/d gives the x value corresponding to (yBAS + A)/2.

For the transitions from unloaded pedalling to CLE, Δ[deoxy(Hb+Mb)] data obtained in the various repetitions of CLE at the same workload were time-aligned and then superimposed for each subject. Average values every second were calculated and retained for analysis. To evaluate the Δ[deoxy(Hb+Mb)] kinetics and estimate the amplitude of its “overshoot” (17), data were fitted by a double exponential function of the type

y(t)=yBAS+Au[1e(xTDu)/τu]Ad[1e(xTDd)/τd](4)
In Eq. 4, yBAS indicates the baseline; Au, the amplitude of the upward component between yBAS and the transient steady-state value reached in the first seconds of the kinetics; TDu, the time delay; and τu, the time constant of the function for the upward component. Ad, TDd, and τd indicate, respectively, the amplitude, time delay, and time constant of the downward component.

Statistical analysis.

Results are means ± SE. The statistical significance of differences between two means was checked by Student's t-test (two tailed, paired analysis). The statistical significance of differences between more than two means was checked using one-way repeated-measurements analysis of variance (ANOVA); a Tukey's post hoc test was used when significant differences emerged. Data fitting by exponential functions was performed using the least-squares residuals method. Comparisons between fittings with different exponential models were carried out using the F-test. The level of significance was set at P < 0.05. Statistical analyses were carried out using a commercially available software package (Prism 4.0; GraphPad, San Diego, CA).

RESULTS

Mean steady-state values of the main variables obtained during CLE <GET and CLE >GET and at exhaustion (peak values) are given in Table 1. As discussed above, submaximal workloads were matched before and after BR in relative terms, that is, with respect to peak workload determined in the two conditions. Expressed as a percentage of peak workload, the chosen workloads corresponded, for CLE <GET, to 33.0 ± 2.2% before BR vs. 30.4 ± 1.2% after BR, and for CLE >GET, to 81.5 ± 1.3% before BR vs. 79.7 ± 0.6% after BR (no significant differences). As a consequence of the different workloads, V̇o2 and V̇co2 values were higher before BR, for both CLE <GET and CLE >GET. R values were higher after BR. This may suggest a higher reliance on carbohydrates as fuel, a shift of muscle phenotype toward less oxidative fibers (see also the higher [La]b), and/or a relative hyperventilation [see also higher end-tidal Po2 (PetO2) and lower end-tidal Pco2 (PetCO2)]. For both workloads, no differences were observed, before and after BR, in V̇e, Vt, respiratory frequency (fR), SaO2, and rate of perceived exertion.

Table 1. Steady-state main variables before and after BR during CLE <GET and CLE >GET and at exhaustion (peak values)

Ẇ, Wo2, l/mino2, ml·kg−1·min−1co2, l/minRe, l/minVt, LitersfRSaO2, %PetO2, TorrPetCO2, Torr[La]b, mMΔ[Deoxy (Hb+Mb)], % of IschemiaRPE
CLE <GET
    Before BR77.5 ± 3.81.31 ± 0.0716.6 ± 0.91.14 ± 0.070.87 ± 0.0231.8 ± 2.01.53 ± 0.1021.5 ± 1.097.4 ± 0.3101.4 ± 1.443.1 ± 0.71.7 ± 0.220.4 ± 3.48.7 ± 0.6
    After BR59.3* ± 2.41.09* ± 0.0514.9* ± 0.60.98* ± 0.050.90 ± 0.0128.7* ± 1.21.35* ± 0.0922.4 ± 1.197.4 ± 0.3105.8* ± 0.941.1* ± 0.62.1 ± 0.312.4 ± 2.09.6 ± 0.6
CLE >GET
    Before BR195.7 ± 11.52.76 ± 0.1836.1 ± 2.52.82 ± 0.191.02 ± 0.0170.1 ± 3.22.48 ± 0.1729.1 ± 1.396.1 ± 0.9105.3 ± 1.244.3 ± 1.16.0 ± 0.465.4 ± 3.413.0 ± 1.0
    After BR157.0* ± 7.52.42* ± 0.0733.3 ± 1.32.62* ± 0.071.09* ± 0.0176.6 ± 2.82.37 ± 0.1132.9* ± 1.696.7 ± 0.4114.5* ± 1.938.8* ± 1.88.0* ± 0.444.1* ± 3.415.0 ± 0.6
Peak
    Before BR240.0 ± 13.83.20 ± 0.1841.3 ± 2.63.65 ± 0.231.14 ± 0.0297.4 ± 6.52.63 ± 0.1537.5 ± 2.393.3* ± 0.8111.7 ± 2.241.9 ± 1.99.3 ± 0.761.6 ± 4.216.7 ± 0.6
    After BR197.5* ± 10.32.63* ± 0.1136.2* ± 1.63.17* ± 0.171.20* ± 0.0296.0 ± 4.42.60 ± 0.1537.5 ± 1.395.6* ± 0.6119.8* ± 1.737.1* ± 1.812.1* ± 0.741.3* ± 2.817.3 ± 0.3

Mean (±SE) values of workload (Ẇ), O2 uptake (V̇o2), CO2 output (V̇co2), pulmonary gas exchange ratio (R), pulmonary ventilation (V̇e, tidal volume (Vt), respiratory frequency (fR), arterial blood O2 saturation (Sao2), O2 end-tidal partial pressure (PetO2), CO2 end-tidal partial pressure (PetCO2), blood lactate concentration ([La]b), concentration change of deoxygenated hemoglobin and myoglobin {Δ[deoxy(Hb+Mb)]}, and rate of perceived exertion (RPE; Borg's scale) obtained before and after bed rest (BR) for constant-load exercise (CLE) below (<GET) and above gas exchange threshold (>GET) and at exhaustion (peak values).

*P < 0.05, significantly different from the value obtained before BR.

Peak workload and V̇o2peak were significantly lower after BR. When V̇o2peak was expressed per unit of body mass, the percentage decrease, after vs. before BR, was slightly less (−12 ± 3%) compared with that found when the variable was expressed in liters per minute (−18 ± 2%). V̇e peak, fR peak, and Vt peak were not affected by BR. Both before and after BR, SaO2 peak values were lower than those found at submaximal workload; SaO2 peak decrease was more pronounced before BR. R peak and [La]b peak were slightly higher after BR.

Mean GET values, expressed as absolute V̇o2 values and as a percentage of V̇o2peak, are shown in Fig. 1, together with V̇o2peak. GET (l/min of V̇o2) was ∼20% lower after BR; percentagewise, the decrease was very similar to that described for V̇o2peak. Expressed as a percentage of V̇o2peak, GET was not significantly different in the two conditions, being in both cases ∼60%.

Fig. 1.

Fig. 1.Mean (±SE) values of peak O2 uptake (V̇o2peak), gas exchange threshold (GET), and GET/V̇o2peak obtained before and after bed rest (BR). See text for further details. *P < 0.05.


Mean values of HR, SV, and Q̇ obtained at rest, during unloaded pedalling, during CLE <GET and CLE >GET, and at peak workload are shown in Fig. 2. At rest and at all submaximal workloads, HR was significantly higher and SV was significantly lower after BR, whereas Q̇ was unaffected. At peak workload, SV peak and Q̇ peak values were significantly (by ∼20%) lower after BR, whereas HR peak was not significantly different in the two conditions.

Fig. 2.

Fig. 2.Mean (±SE) values of heart rate (HR), stroke volume (SV), and cardiac output (Q̇) obtained at rest, during unloaded pedalling, during constant-load exercise (CLE) below (<GET) and above GET (>GET), and at peak workload. *P < 0.05, before vs. after BR at the same relative workload. See text for further details.


Mean values of the NIRS-obtained muscle oxygenation variable {Δ[deoxy(Hb+Mb)]}, which was used to evaluate fractional O2 extraction, are shown as a function of workload during IE in Fig. 3. Δ[Deoxy(Hb+Mb)] data are expressed as a percentage of those obtained during a transient limb ischemia induced at the end of the test (see methods). To obtain this figure, we grouped individual Δ[deoxy(Hb+Mb)] data for discrete workload intervals. The sigmoidal functions (see Eq. 3) that were used to fit the data are also shown in Fig. 3. In both conditions, Δ[deoxy(Hb+Mb)] increased as a function of workload, showing a plateau at ∼70% of peak workload, that is, above ∼200 W before BR and above ∼150 W after BR. At workloads lower than ∼125 W, Δ[deoxy(Hb+Mb)] values were not different in the two conditions. Δ[Deoxy(Hb+Mb)] peak values were significantly lower after (41 ± 3%) vs. before BR (63 ± 3%).

Fig. 3.

Fig. 3.Mean (±SE) values of the near-infrared spectroscopy (NIRS)-obtained muscle oxygenation variable {Δ[deoxy(Hb+Mb)]}, which was used to evaluate fractional O2 extraction, are shown as a function of workload during the incremental exercise (IE). Δ[Deoxy(Hb+Mb)] data are expressed as a percentage of those obtained during a transient limb ischemia induced at the end of the test. Individual Δ[deoxy(Hb+Mb)] data were grouped for discrete workload intervals. The sigmoidal functions (Eq. 3) used to fit the data are also shown. See text for further details.


NIRS-obtained brain oxygenation data during IE are shown in Figs. 4 and 5. In Fig. 4, mean values of individual Δ[O2Hb], Δ[HHb], and Δ[O2Hb+HHb] data, grouped for discrete workload intervals, are reported as a fraction of the peak workload obtained before BR (workload/peak workload before BR). Mean peak values are shown in Fig. 5. Before BR, Δ[O2Hb] increased at submaximal workloads, whereas it did not significantly change from the baseline after BR; in both conditions, Δ[O2Hb] peak values were not significantly different from the baseline. In both conditions, Δ[HHb] did not increase from the baseline until ∼75% of peak workload, after which the variable showed a significant increase; Δ[HHb] peak values were not significantly different in the two conditions. Similar to Δ[O2Hb], Δ[O2Hb+HHb] at submaximal workload was higher before BR; in both conditions, Δ[O2Hb+HHb] increased substantially after ∼75% of peak workload; peak values were not significantly different in the two conditions.

Fig. 4.

Fig. 4.NIRS-obtained brain oxygenation data obtained during the IE. Mean (±SE) values of individual changes in the concentration of oxyhemoglobin (Δ[O2Hb]), deoxyhemoglobin (Δ[HHb]), and total hemoglobin (Δ[O2Hb+HHb]), grouped for discrete workload intervals, are reported as a fraction of peak workload obtained before BR. *P < 0.05, before vs. after BR at the same relative workload. See text for further details.


Fig. 5.

Fig. 5.Mean (±SE) peak values of Δ[O2Hb], Δ[HHb], and Δ[O2Hb+HHb] before and after BR. See text for further details.


Typical examples of pulmonary V̇o2 kinetics in a representative subject are shown in Fig. 6. Mean values of kinetics parameters are presented in Table 2. Gain of the fundamental component (Gain f) was calculated as Af/Δworkload; Δworkload was calculated as the imposed workload minus 15 W, considered to correspond to unloaded pedalling (the baseline of the investigated transition). Gain of the slow component (Gain s) was calculated as Atot/Δworkload. τf values during CLE <GET were significantly higher after BR. During CLE >GET, the difference between τf before and after BR did not reach statistical significance. In both conditions, τf values during CLE <GET were not significantly different from those obtained during CLE >GET. If we consider data obtained during CLE <GET and CLE >GET together, the difference between τf before (31.1 ± 2.0 s) and after BR (40.0 ± 2.2 s) was highly significant (P < 0.01). The data demonstrate a slower fundamental component of V̇o2 kinetics following BR. Both before and after BR, no subject showed a slow component of V̇o2 kinetics during CLE <GET, whereas all subjects showed a slow component during CLE >GET. As for A′s/Atot, no significant differences were observed before and after BR. Thus, at the same relative workload, BR did not affect the amplitude of the slow component of V̇o2 kinetics. This means that for the same absolute workload, the amplitude of the slow component would be greater after BR. Af and A′s were significantly lower after BR as a consequence of the lower workload. Gain f and Gain s were slightly higher after vs. before BR, although the difference did not reach statistical significance. Thus cycling efficiency at submaximal workloads was not significantly affected by BR. τf and A′s/Atot values are also shown in Fig. 7.

Fig. 6.

Fig. 6.Typical examples of pulmonary V̇o2 kinetics in a representative subject are shown for CLE <GET and CLE >GET before and after BR. The exponential (Eq. 1) or double-exponential functions (Eq. 2) fitting the data are also shown. Vertical hatched line indicates exercise onset; horizontal hatched lines indicate the amplitudes of the total response (Atot) and the actual amplitude of the slow component (A′s). τf is the time constant of the fundamental component. See text for further details.


Table 2.o2 kinetics parameters for CLE <GET and CLE >GET before and after BR

yBAS, l/minTDfTf, sAf, sGain f, l/minA′s, ml·min−1·W−1Gain s, l/minA′s/Atot, ml·min−1·W−1
CLE <GET
    Before BR0.609 ± 0.03917.8 ± 2.430.3 ± 2.20.684 ± 0.06110.8 ± 0.6
    After BR0.543 ± 0.02615.3 ± 2.440.7* ± 3.50.546* ± 0.03112.4 ± 0.4
CLE >GET
    Before BR0.559 ± 0.03319.4 ± 2.932.0 ± 3.51.800 ± 0.1599.9 ± 0.50.374 ± 0.06311.9 ± 0.50.17 ± 0.02
    After BR0.602 ± 0.02916.1 ± 2.539.4 ± 2.81.481* ± 0.18410.4 ± 0.30.352 ± 0.03313.0 ± 0.30.20 ± 0.02

Mean (±SE) values of baseline (yBAS); time delay (TDf), time constant (τf), amplitude (Af), and gain (Gain f) of the fundamental component; actual amplitude (A′s) and gain (Gain s) of the slow component; and total amplitude of the response (Atot).

*P < 0.05, significantly different from the corresponding value obtained before BR. See text for further details.

Fig. 7.

Fig. 7.Mean (±SE) values of τf for CLE <GET, CLE >GET, and A′s/Atot before and after BR. See text for further details. *P < 0.05.


Typical examples of Δ[deoxy(Hb+Mb)] kinetics in a representative subject are shown in Fig. 8. TD and τ of the initial exponential increase (TDu and τu, respectively) were not affected by BR. For CLE <GET, TDu and τu were, respectively, 12.9 ± 1.0 and 8.5 ± 1.5 s before BR and 13.9 ± 1.0 and 10.7 ± 1.4 s after BR. For CLE >GET, TDu and τu were, respectively, 9.3 ± 0.8 and 5.8 ± 0.5 s before BR and 8.6 ± 0.6 and 8.8 ± 0.4 s after BR. Expressed as a percentage of Δ[deoxy(Hb+Mb)] changes during ischemia, the overshoot of the Δ[deoxy(Hb+Mb)] kinetics (17) (Ad in Eq. 4) was significantly more pronounced after BR, for both CLE <GET (5 ± 1% before BR vs. 12 ± 1% after BR) and CLE >GET (3 ± 1% before BR vs. 13 ± 2% after BR).

Fig. 8.

Fig. 8.Typical examples of Δ[deoxy(Hb+Mb)] kinetics in a representative subject are shown for CLE <GET and CLE >GET before and after BR. The double-exponential functions (Eq. 4) fitting the data are also shown. Δ[Deoxy(Hb+Mb)] data are expressed as a percentage of Δ[deoxy(Hb+Mb)] changes during ischemia. Vertical hatched line indicates exercise onset; horizontal hatched lines indicate the overshoot of the Δ[deoxy(Hb+Mb)] kinetics (Ad in Eq. 4), which was significantly more pronounced after BR, for both CLE <GET and CLE >GET. See text for further details.


DISCUSSION

In the present study a battery of noninvasive tests was used to perform a functional evaluation of oxidative metabolism before and after a 35-day horizontal BR. Some of these tests were mainly related to central limiting factors, whereas others were mainly aimed at the skeletal muscle level. More specifically, we intended to evaluate the role of skeletal muscle impairment and brain oxygenation in limiting oxidative metabolism during submaximal and maximal exercise after BR. The variables evaluated are discussed below.

o2peak and the GET.

Both V̇o2peak and GET were lower after BR. These variables are important determinants of exercise tolerance, and a quantitative evaluation of their impairment after BR has an obvious functional relevance. Considering the duration of the present BR (35 days), the observed decrease in V̇o2peak is in line with literature data (8). To the best of our knowledge, GET had not been specifically analyzed in previous BR studies. In the present study, the GET decrease after BR was very similar, percentagewise, to that described for V̇o2peak such that GET/V̇o2peak (∼60%) was unchanged. Thus, even if the capacity to sustain exhausting aerobic work is impaired after BR, the subjects should be able to sustain for relatively prolonged periods of time the same fraction of their V̇o2peak. Although the factors limiting V̇o2max are the result of an integration between central and peripheral mechanisms and variables, in normal subjects, in normoxia, and during whole body exercise, V̇o2peak appears to be quantitatively mainly limited by the maximal capacity of O2 delivery to the exercising muscles by the cardiovascular system (see e.g., Ref. 12). Thus determination of this variable should mainly evaluate the central (cardiovascular) limitations to oxidative metabolism. They were significantly affected by BR, as also confirmed by the substantially lower Q̇ peak values (see below).

Cardiovascular adjustments to exercise and systemic O2 delivery.

Blood hemoglobin concentration was unaffected by BR. The subjects showed some degree of arterial desaturation at peak workload; the desaturation was less pronounced after BR. For cardiovascular variables, the results of the present study substantially confirm literature data (8, 9, 18, 20): higher HR and lower SV, at the same submaximal workload, after BR; Q̇ at submaximal workload unaffected by BR; and SV peak and Q̇ peak ∼20% lower after BR, suggesting a significantly impaired maximal capacity of systemic O2 delivery.

Cerebral oxygenation.

Brain (frontal cortex) oxygenation measurements obtained by NIRS in the present study suggest an increased O2 extraction at the tissue level at the highest workloads. These effects were not different before vs. after BR. As pointed out in the review by Nybo and Rasmussen (35), in normal conditions this increased O2 extraction could be attributed, in the presence of increased neuronal activity and metabolic needs by the tissue, to a decreased cerebral blood flow, a consequence of the hyperventilation-induced hypocapnia. According to Nybo and Rasmussen (35), it is unlikely that this increased O2 extraction could determine, in normal conditions, “critical” Po2 levels in some areas of the brain, potentially representing a central limitation to maximal exercise tolerance. This would happen, on the other hand, in hypoxic conditions, in which the enhanced hyperventilation would further decrease arterial Pco2 (PaCO2) and cerebral blood flow (1, 27, 40, 41). A similar phenomenon would also occur in subjects with severe exercise-induced arterial hypoxemia (34). In our study, peak systemic convective O2 delivery was substantially reduced after BR, mainly as a consequence of the lower Q̇ peak. Our subjects showed some degree of exercise-induced arterial desaturation, both before and after BR; the magnitude of this phenomenon, however, should not have substantially affected arterial Po2 (PaO2) and O2 delivery. Moreover, the arterial desaturation was slightly less pronounced after BR. Although during exercise PetCO2 is a rather poor surrogate of PaCO2, exercise hyperventilation, as estimated from the measured PetCO2 (see Table 1), was slightly more pronounced after BR, but the magnitude of this effect (PetCO2 of ∼37 Torr at peak workload after BR) should not significantly affect cerebral blood flow (which was not determined in the present study). In strict terms, an increased O2 extraction is suggested by NIRS by the observation of increased deoxygenation, decreased oxygenation, and unchanged oxy- plus deoxygenation signals (23, 40). In the present study we observed, at peak exercise, increased deoxygenation, unchanged oxygenation, and increased oxy- plus deoxygenation signals. This suggests either increased O2 extraction or vasodilation at the tissue level (or a combination of the two). The important point is that these effects were not affected by BR (see Figs. 4 and 5). It appears therefore unlikely that cerebral deoxygenation could represent a limiting factor to exercise performance after BR. Thus one of the hypotheses of the present study was not confirmed: after BR, a significant “competition” between skeletal muscles and brain for the available O2 seems unlikely. In more general terms, it can be concluded that in the absence of a significantly reduced cerebral blood flow, deriving from the hypoxic-induced hyperventilation and hypocapnia, a relatively lower peak systemic convective O2 delivery, as observed after BR, does not significantly impair cerebral oxygenation and does not limit exercise tolerance. If also confirmed in pathological conditions characterized by a lowered peak systemic O2 delivery, this concept could have a significant functional relevance.

Peak capacity of O2 extraction by skeletal muscle.

The peak capacity of fractional O2 extraction by an anti-gravity skeletal muscle (vastus lateralis) was estimated using NIRS. This method yields tissue oxygenation variables that are the result of the balance between V̇o2 and O2 delivery in the tissue under consideration, being therefore conceptually similar to fractional O2 extraction. In the present study, as well as in others (11, 16, 22, 23, 30, 32), fractional O2 extraction was estimated by Δ[deoxy(Hb+Mb)]. Δ[Deoxy(Hb+Mb)] peak values were lower after BR, suggesting a significantly impaired peak capacity of O2 extraction by the investigated muscle. This observation appears compatible with data showing BR-induced alterations in skeletal muscle, such as a decreased volume density of mitochondria, oxidative enzyme activities, and capillary length (18). Our data appear to be a “mirror image” of those by Kalliokowski et al. (29), reporting an increased O2 extraction by skeletal muscle following endurance training. Thus endurance training would increase, whereas profound deconditioning would decrease, the peak capacity of O2 extraction by skeletal muscles.

Kinetics of skeletal muscle O2 extraction during IE and CLE.

As discussed above, Δ[deoxy(Hb+Mb)] is the result of the balance between muscle V̇o2 and muscle O2 delivery. For the same workload, and for the same V̇o2O2 (the V̇o2 vs. workload relationship was substantially unaffected by BR, as suggested by the unchanged efficiency of cycling), a higher Δ[deoxy(Hb+Mb)] would indicate a reduced reliance on blood flow and an enhanced reliance on O2 extraction to sustain the needed V̇o2 (see also Ref. 32). The opposite would be true for lower Δ[deoxy(Hb+Mb)] values for the same workload. In the present study, Δ[deoxy(Hb+Mb)] at submaximal workloads (lower than ∼70–80% of peak) were the same before and after BR (see Fig. 3). Thus the latter did not affect, in the investigated muscle, the matching of O2 delivery and V̇o2 at submaximal workloads. Δ[Deoxy(Hb+Mb)], however, showed a plateau at a lower absolute workload after BR, suggesting a reduced peak capacity of O2 extraction (see above).

For Δ[deoxy(Hb+Mb)] kinetics during CLE, two points can be made. 1) TDu, that is, the time at the onset of exercise during which Δ[deoxy(Hb+Mb)] does not change compared with the baseline level, was not affected by BR. The unchanged Δ[deoxy(Hb+Mb)] suggests a tight balance between muscle O2 delivery and muscle V̇o2 during this phase of the transition (23). 2) Ad, the parameter evaluating the overshoot of muscle O2 extraction during the metabolic transition (17), was significantly greater after BR, for both CLE <GET and CLE >GET. According to Ferreira et al. (17), this overshoot in muscle O2 extraction occurs when muscle O2 delivery shows an initial plateau (after the initial fast response, mainly induced by the muscle pump effect), whereas muscle V̇o2 continues its monoexponential increase. As such, the overshoot is a sign of a relatively inadequate muscle O2 delivery vs. muscle V̇o2, which may be associated with reduced microvascular O2 pressures, and thus with a lower blood-to-myocyte “driving force” for peripheral O2 diffusion. This phenomenon can affect oxidative metabolism and may be responsible, at least in part, for the slower V̇o2 kinetics observed after BR.

Kinetics of adjustment of skeletal muscle oxidative metabolism during exercise transitions (V̇o2 kinetics).

The fundamental component of pulmonary V̇o2 kinetics reflects rather closely V̇o2 kinetics occurring at the skeletal muscle level (24, 39). Differently from V̇o2peak, V̇o2 kinetics during transitions to moderate-intensity exercise appear mainly limited by the capacity by skeletal muscles to utilize the O2 they receive from the cardiovascular system (21). Thus determination of V̇o2 kinetics is considered a functional evaluation tool mainly aimed at the skeletal muscle level (21). V̇o2 kinetics are correlated with the size of the “O2 deficit”: a slower V̇o2 kinetics, as observed in the present study after BR, causes a greater O2 deficit and would then be negatively associated with exercise tolerance (21). A slower V̇o2 kinetics also would be associated with a lower level of “metabolic stability,” that is, with more pronounced decreases in phosphocreatine concentration ([PCr]) and in the cytosolic phosphorylation potential, as well as with more pronounced increases in [Pi], [ADP], [AMP], and [IMP] for a given increase in V̇o2 at steady state (48). A lower level of metabolic stability is considered negatively associated with exercise tolerance (48).

The finding of a slower fundamental component of pulmonary V̇o2 kinetics in the present study is compatible with the larger O2 deficit described by Convertino et al. (10) after a much shorter (7 days) BR. These authors measured V̇o2 using the Douglas bags method and did not perform a formal analysis of the fundamental and the slow component (see below) of pulmonary V̇o2 kinetics. Moreover, Convertino et al. (10) tested subjects at the same absolute workload, before vs. after BR, so that the relative workload was likely different in the two conditions. V̇o2 kinetics are known to be markedly affected by relative workload (28, 45). In the present study, V̇o2 kinetics were evaluated at the same relative workload, for both moderate- (<GET) and heavy-intensity exercise (>GET).

During CLE >GET, the slow component of V̇o2 kinetics causes the attainment of V̇o2 values that are higher than those derived from the extrapolation of the V̇o2 vs. workload relationship for CLE <GET (28, 45). This suggests a reduced efficiency of skeletal muscle oxidative metabolism or of muscle contractions in general. Although the causes of the slow component are still debated (28), the phenomenon is traditionally considered to derive from a progressive recruitment of aerobically less efficient type 2 fibers (31), which would explain the “excess V̇o2” with respect to the constant external workload. On the other hand, the slow component of V̇o2 kinetics could be related to metabolic factors that induce muscle fatigue (28, 45, 47). Within this scenario, muscles would become less efficient because they are approaching the metabolic characteristics of fatigue. The slow component of V̇o2 kinetics could then be associated with (or be a consequence of) a lower level of “metabolic stability” (see above).

Although BR does not seem to induce, in humans, significant changes of fiber type distribution (13), immobilization usually determines a transition in the expression of myosin heavy chain isoforms toward the less oxidative type IIx (26). Prolonged BR also could impair muscle fiber recruitment patterns by the central nervous system (13), and, as shown in the present study, it could impair skeletal muscle oxidative metabolism. On the basis of such premises, we hypothesized an increased amplitude of the slow component after BR. At first sight this hypothesis was not confirmed by the obtained results, which demonstrated an unchanged amplitude. It must be considered, however, that in the present study, V̇o2 kinetics were evaluated, before and after BR, at the same relative workload (with respect to V̇o2peak and GET). Thus, after BR, it is reasonable to hypothesize, for the same absolute workload above GET, a greater amplitude of the slow component, which would contribute to the impaired exercise tolerance. This hypothesis is in agreement with previous studies (3, 7), which described, following endurance training, a lower amplitude of the slow component when the subjects, after training, were tested at the same absolute workload, but no changes when the subjects were tested at the same relative workload.

Conclusions.

In young subjects exposed to a 35-day horizontal BR, we observed significant decreases in V̇o2peak, associated with a significant decrease in the peak capacity of cardiovascular O2 delivery, as well as with an impaired peak capacity of fractional O2 extraction by anti-gravity muscles. The reduced peak capacity of cardiovascular O2 delivery, however, did not seem to determine a competition between the central nervous system and skeletal muscles for the available O2. GET, expressed as a percentage of V̇o2peak, was unaffected by BR. Cycling efficiency (oxidative metabolism) at submaximal workloads was also unaffected by BR. The findings during moderate and heavy constant-load exercises (slower fundamental component of V̇o2 kinetics, higher amplitude of the slow component for the same absolute workload, and enhanced overshoot in the kinetics of skeletal muscle O2 extraction) suggest an impairment of both microvascular O2 delivery and muscular O2 utilization after BR, which should affect exercise tolerance at submaximal workloads.

Skeletal muscles contribute to the impairment of oxidative metabolism following BR. Thus skeletal muscle oxidative metabolism can be added to the list of functions/variables [muscle mass, fiber size, muscle force, “explosive” power, motor coordination, and others (9, 13, 19, 20, 36)] that are affected by prolonged BR. These finding may be relevant in terms of exercise tolerance at both maximal and submaximal workloads, for the definition of countermeasures aimed at reversing skeletal muscle deterioration in microgravity, and, in more general terms, for a better understanding of the effects of profound deconditioning.

GRANTS

Financial support by the Agenzia Spaziale Italiana (ASI-OSMA Contract I/007/06/0-Workpackage 1B-32-1) is acknowledged.

DISCLOSURES

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

ACKNOWLEDGMENTS

We are grateful to Dr. Bostijan Simunic (University of Primorska, Koper, Slovenia), Prof. Gianni Biolo, and Dr. Francesco Agostini (University of Trieste, Italy) for the excellent organization and coordination of the Valdoltra 2007 bed rest campaign, as well as to the subjects who enthusiastically agreed to participate in our experiments. We also thank Dr. Federico Casamassima and Angelo Colombini for technical assistance.

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AUTHOR NOTES

  • Address for reprint requests and other correspondence: B. Grassi, Dipartimento di Scienze e Tecnologie Biomediche, Piazzale M. Kolbe 4, I-33100 Udine, Italy (e-mail: ).