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J Appl Physiol 105: 1889-1896, 2008. First published October 9, 2008; doi:10.1152/japplphysiol.90642.2008
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Effects of antioxidants on contracting spinotrapezius muscle microvascular oxygenation and blood flow in aged rats

Kyle F. Herspring,1 Leonardo F. Ferreira,2 Steven W. Copp,1 Brian S. Snyder,3 David C. Poole,1 and Timothy I. Musch1

1Departments of Kinesiology and Anatomy and Physiology, Kansas State University, Manhattan, Kansas; 2Department of Physiology, University of Kentucky, Lexington, Kentucky; and 3Department of Human Nutrition, Kansas State University, Manhattan, Kansas

Submitted 13 May 2008 ; accepted in final form 1 October 2008


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Aged rats exhibit a decreased muscle microvascular O2 partial pressure (PmvO2) at rest and during contractions compared with young rats. Age-related reductions in nitric oxide bioavailability due, in part, to elevated reactive O2 species, constrain muscle blood flow (Qm). Antioxidants may restore nitric oxide bioavailability, Qm, and ameliorate the reduced PmvO2. We tested the hypothesis that antioxidants would elevate Qm and, therefore, PmvO2 in aged rats. Spinotrapezius muscle PmvO2 and Qm were measured, and oxygen consumption (VmO2) was estimated in anesthetized male Fisher 344 x Brown Norway hybrid rats at rest and during 1-Hz contractions, before and after antioxidant intravenous infusion (76 mg/kg vitamin C and 52 mg/kg tempol). Before infusion, contractions evoked a biphasic PmvO2 that fell from 30.6 ± 0.9 Torr to a nadir of 16.8 ± 1.2 Torr with an "undershoot" of 2.8 ± 0.7 Torr below the subsequent steady-state (19.7 ± 1.2 Torr). The principal effect of antioxidants was to elevate baseline PmvO2 from 30.6 ± 0.9 to 35.7 ± 0.8 Torr (P < 0.05) and reduce or abolish the undershoot (P < 0.05). Antioxidants reduced Qm and VmO2 during contractions (P < 0.05), while decreasing force production 16.5% (P < 0.05) and elevating the force production-to-VmO2 ratio (0.92 ± 0.03 to 1.06 ± 0.6, P < 0.05). Thus antioxidants increased PmvO2 by altering the balance between muscle O2 delivery and VmO2 at rest and during contractions. It is likely that this effect arose from antioxidants reducing myocyte redox below the level optimal for contractile performance and directly (decreased tension) or indirectly (altered balance of vasoactive mediators) influencing O2 delivery and VmO2.

aging; antioxidant supplementation; muscle tension

MOST INDIVIDUALS EXPERIENCE significant age-related reductions in exercise tolerance after attaining adulthood that are associated with decrements in both maximal oxygen uptake and physical endurance capacity at submaximal work rates (2, 29, 33, 44). At rest, compared with their young counterparts, aged rats exhibit a 1) higher muscle capillary red blood cell velocity and flux (52); and 2) reduced partial pressure of oxygen in the resting muscle microvasculature (PmvO2) that is maintained throughout the rest-contractions transition (7). Thus a decreased PmvO2 driving pressure is present in aged rats compared with their younger counterparts, and one possible explanation for this phenomenon is age-related reductions in both tissue and/or vascular nitric oxide (NO) bioavailability (11, 20, 21, 55, 56, 58).

PmvO2 is dependent on the ratio of oxygen delivery to the muscle (QmO2) and muscle oxygen consumption (VmO2) (i.e., QmO2/VmO2). The maintenance of this ratio, and consequently PmvO2, during exercise is crucial to provide an adequate pressure head to drive O2 flux across the blood-myocyte interface and deliver O2 to the mitochondria during contractions (i.e., transition and steady state) (5, 6, 22, 45, 59). In young, healthy individuals, muscle blood flow (Qm) and QmO2 increase at the same rate or occasionally faster than VmO2, at least for the first 10–20 s, which prevents PmvO2 from falling (7–9). Beyond that first 10–20 s, the QmO2-to-VmO2 ratio does fall, and PmvO2 demonstrates an exponential decrease to the steady state, reflecting the increased fractional O2 extraction. The net effect of this behavior is that PmvO2 in young animals is maintained at higher levels across the rest-contraction transition, thereby improving mitochondrial O2 delivery, which permits more reliance on oxidative metabolism. However, aging is associated with an altered (i.e., lowered) PmvO2 profile (7) that potentially reflects peripheral vascular derangements. This reduction in O2 driving pressure is expected to slow VmO2 kinetics, increase the O2 deficit (19), and increase mitochondrial reactive oxygen species (ROS) production (13).

In aged individuals, Qm is sometimes found to be lower than that in young subjects during exercise (25, 38, 46, 47), and this reduction is thought to be related directly to compromised arteriolar vasomotor control (18, 41, 47). Production and release of NO from vascular endothelial cells is recognized as a contributor to the skeletal muscle hyperemia seen in healthy, young individuals during exercise, and the possibility exists that there may be an impaired NO-mediated vasodilation in aged individuals (31, 32, 53). In support of this notion, it has been shown that the relative contribution of NO to vascular control of skeletal muscle is reduced in aged individuals (11, 20, 21, 55, 56, 58), and thus decreased NO bioavailability may potentially explain the age-related reductions in the Qm response (18, 42, 47).

The mechanism(s) responsible for this decrease in NO bioavailability in aged individuals may result, in part, from an increased mitochondrial leakage of ROS (20, 24, 56). ROS can influence NO bioavailability: 1) ROS scavenge NO to produce peroxynitrite, which reduces NO bioavailability while simultaneously producing another ROS; and 2) ROS decrease the concentration of the NO synthase cofactor tetrahydrobiopterin, which is essential for the production of NO by NO synthase (26, 27). If either of these mechanisms contributes to the reduction in NO bioavailability in aged skeletal muscle, infusion of antioxidants may help restore NO bioavailability and endothelial (i.e., arteriolar) function (20, 55).

In the present investigation, we examined the role of antioxidant supplementation on QmO2 and PmvO2 across the rest-to-contractions transition in the spinotrapezius muscle of aged rats. Specifically, we tested the hypotheses that, after antioxidant supplementation: 1) the reduced PmvO2 normally found in the resting muscle of aged rats would be alleviated; 2) the elevated resting PmvO2 of aged rats found after antioxidant supplementation would be associated with an increase in resting Qm to the muscle; 3) there would be an increase in Qm to the contracting muscle, which would elevate the QmO2-to-VmO2 ratio and thus PmvO2 in the exercising steady state; and 4) the PmvO2 "undershoot" and prolonged PmvO2 kinetics that are found in the transition from rest to exercise in aged rats (7) would be reduced or eliminated.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Animals

Twenty old [26–30 mo, body mass 563 ± 85 (SD) g] male Fisher 344 x Brown Norway hybrid rats were used in this investigation. Rats were maintained on a 12:12-h light-dark cycle and received water and food ad libitum. Upon completion of the experiment, each rat was euthanized with pentobarbital sodium overdose. All protocols were approved by the Kansas State University Institutional Animal Care and Use Committee, conformed with guiding principles of the American Physiological Society, and were conducted according to the National Institutes of Health guidelines.

Surgical Preparation

All rats were anesthetized with pentobarbital sodium (50 mg kg ip, to effect) and placed on a heating pad to maintain a constant body temperature of 37–38°C. The carotid and tail (caudal) arteries were cannulated (polyethylene-50, Intra-Medic tubing; Clay Adams, Sparks, MD) for infusion of the phosphorescent probe palladium meso-tetra (4-carboxyphenyl) porphyrin dendrimer (R2; 15 mg/kg), monitoring of arterial blood pressure and heart rate (Digi-Med BPA model 200, Louisville, KY), blood withdrawal, and infusion of antioxidants. Catheter placement also permitted the measurement of resting and contracting Qm using the radiolabeled microsphere technique (37, 43). Skin and fascia around the middorsal region of the rat was reflected back to expose the right spinotrapezius muscle. Stainless steel electrodes were sutured to the rostral (cathode) and caudal (anode) region of the spinotrapezius using 6-0 sutures to ensure that electrode position remained unchanged throughout the experimental protocols.

Total Antioxidant Capacity

Blood was collected in EDTA tubes and centrifuged to obtain plasma samples, which were stored at –80°C until analyzed. Plasma was aliquoted in duplicate, and the total plasma antioxidant capacity was determined using a commercially available kit (no. K274-100 BioVision Total Antioxidant Capacity, Mountain View, CA). Briefly, Trolox was used to standardize all antioxidants, and antioxidant capacity was then measured in Trolox equivalents. Absorbance of samples and standards were analyzed at 570 nm (Bio-Tek, Winooski, VT).

A standard curve was prepared that spanned the range of measurements, and antioxidant capacity was calculated from the curve.

Measurement of PmvO2

PmvO2 measurements were made using a PMOD 1000 Frequency Domain Phosphorimeter (Oxygen Enterprises, Philadelphia, PA). After exposure of the right spinotrapezius, the common end of the bifurcated light guide was placed 2–3 mm above the medial region of the muscle, and excitation light focused on a ~2-mm-diameter circle. The phosphorescence quenching technique for measurement of PmvO2 is based upon the Stern-Volmer relationship (51), which describes the O2 dependence of phosphorescence decay in the presence of the phosphorescence probe. The phosphorescence signal (700 nm) was averaged over 10 x 200 ms intervals for all PmvO2 measurements, and these measurements were repeated every 2 s. The phosphorometer utilizes a sinusoidal modulation of the excitation light (524 nm) at frequencies between 100 Hz and 20 kHz, which allows phosphorescence lifetime measurements from 10 µs to ~2.5 ms. The PmvO2 measurement gives a volume-weighted value for PO2 that encompasses all vessels within the sampled volume. As most blood and plasma volume resides within the capillary and venular compartments, the PO2 measured is drawn toward, but is not synonymous with, venous PO2 (PvO2). Thus the exponential phosphorescence decay will necessarily represent the average decay profile for all vascular PO2 compartments within the muscle sampled.

Measurement of Spinotrapezius Qm and Vascular Conductance

Spinotrapezius Qm was determined in a subset of rats (n = 7) using the radionuclide-tagged microsphere technique, as described in detail by Musch and Terrell (43). The caudal artery catheter was attached to a Harvard pump (model 907, Cambridge, MA) for blood withdrawal at 0.25 ml/min. Two differentially labeled 15-µm microspheres (46Sc, 85Sr, New England Nuclear, Boston, MA) were injected through the carotid artery, and the catheter was flushed with saline. In random order and during steady-state contractions, one isotope was injected under control conditions, and the second isotope after antioxidant supplementation. In all animals, the right spinotrapezius was stimulated, and the left represented the resting condition. At the end of each experiment, the animal was euthanized, and both right and left spinotrapezius muscles, along with the right and left kidneys and selected hindlimb locomotory muscles, were removed. Tissue radioactivity was determined by a gamma scintillation counter (Packard Auto Gamma Spectrometer, Cobra model 5003), and Qm was determined by the reference sample method, as described by Ishise et al. (34). Vascular conductance was determined as Qm/mean arterial pressure (MAP) and presented as milliliters per minute per 100 grams. No animals demonstrated a >15% disagreement between right and left kidney Qm, which indicated acceptable microsphere mixing.

Measurement of PmvO2 Dynamics

The R2 probe was infused via the right carotid artery catheter ~15 min before the first contraction period. The spinotrapezius was kept moist continuously with Krebs-Henseleit solution. After PmvO2 was measured in the resting state, the spinotrapezius was stimulated at 1 Hz for 180 s (7–9 V, 2-ms pulse duration) using a Grass S88 stimulator (Quincy, MA). During contractions of the right spinotrapezius, the nonexposed contralateral (left) spinotrapezius was maintained in a resting (noncontracting) state. Continual measurements of PmvO2 were made in the right spinotrapezius and recorded every 2 s during the 180-s contraction period. Following the initial contraction period, each animal was given a minimum of 50 min for the right spinotrapezius muscle to recover. During this recovery period, animals were slowly (i.e., 1.5 ml/30 min) infused using a Harvard pump with either saline or a solution containing the antioxidants (ascorbic acid: 76 mg/kg, and tempol: 52 mg/kg; dissolved in 1.5-ml saline), and the original contraction protocol was repeated. The dosage of ascorbic acid utilized herein has demonstrated beneficial effects in diseased states (i.e., sepsis) by reducing the amount of circulating ROS (1, 57). The concentration of tempol used in the present investigation has exhibited beneficial cardiovascular effects (61).

Measurement of Spinotrapezius Force Production

In a separate group of aged rats (n = 4), the distal end of the right spinotrapezius was ligated and sutured to a stainless-steel wire horseshoe and attached to a nondistensible light weight (0.4 g) cable, linking the muscle to a force transducer. Baseline measurements were made with the spinotrapezius muscle stretched to optimal length (length at which maximum active tension is reached), which was obtained at ~4 g passive stretch. Muscle force production was measured and recorded continuously at rest and during contractions following the same procedure as those described above for Qm and PmvO2.

Calculations

Curve fitting.   The dynamics of PmvO2 were described by means of a nonlinear regression analysis using a commercial software package (SIGMAPLOT 9.0; Systat Software, Point Richmond, CA). Curve fitting of the PmvO2 results, i.e., selection of one vs. two component models, was determined according to three criteria: 1) the coefficient of determination (r2); 2) the sum of the squared residuals; and 3) visual inspection. The equations used to fit the PmvO2 kinetics are shown below.

Formula

Formula
where t is time after the initiation of contractions. Baseline (BL) corresponds to precontraction PmvO2 and {Delta}1PmvO2 and {Delta}2PmvO2 are the amplitudes of the primary and secondary component PmvO2 responses, respectively. Time constants for the primary and secondary components were designated as {tau}1 and {tau}2, and TD1 and TD2 and were the independent time delays of the respective responses. When the response profile closely approximated a monoexponential the primary component was used to fit the data. The two-component model was used whenever an "undershoot" was evident, and the criteria listed above mitigated the more complex model.

VmO2.   VmO2 was estimated as previously described (6, 40). Arterial O2 content (CaO2) was measured directly (carotid arterial blood), and effluent venous O2 content (CvO2) was approximated from the PmvO2 using the rat dissociation curve [n = 2.6 (Hill coefficient)], the measured hemoglobin (Hb) concentration, P50 (PO2 at which Hb is 50% saturated with O2) of 38 Torr, and an O2 carrying capacity of 1.34 ml O2/g Hb (40). Steady-state measurements of spinotrapezius Qm made at rest and during muscle contractions were then used to estimate VmO2 using the Fick equation [i.e., VmO2 = Qm x (CaO2 – CvO2)]. In individual animals (n = 6/13), where Qm measurements were not made, the mean Qm value was used to estimate VmO2.

Stability and Reproducibility of the Spinotrapezius Preparation

Our laboratory has shown previously that the isolated spinotrapezius muscle preparation is physiologically stable and viable following the completion of this surgery (3). In addition, we have observed previously with the spinotrapezius preparation that we can retain reproducible PmvO2 results during the transition from rest to muscle contractions when a minimum of 20 min of recovery is allowed between exercise bouts (B. J. Behnke, P. McDonough, T. I. Musch, D. C. Poole, unpublished observations). We reaffirmed the stability and reproducibility of the spinotrapezius preparation in the present investigation by examining the PmvO2 kinetics in 16 animals using the initial and subsequent contraction paradigm. Results demonstrated that reproducibility from the initial to the second contraction bout was excellent. Specifically, there was no change qualitatively in the PmvO2 profile nor quantitatively (P > 0.05) in any of the variables or parameters measured.

Statistical Analysis

The effects of the antioxidant supplementation on PmvO2, Qm, and force production were analyzed by means of paired t-tests. Statistical significance was accepted at P < 0.05. Data are reported as means ± SD for body mass and mean ± SE where statistical inferences are derived (i.e., all other data).


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Antioxidant Capacity

The antioxidant infusion increased total antioxidant capacity ~2.5-fold (P < 0.001).

PmvO2

Similar to previous findings in our laboratory (7), the aged rats demonstrated a biphasic PmvO2 profile during the transition from rest to steady-state contractions that consisted of a significant undershoot followed by a rise in the PmvO2 to the steady-state level (Fig. 1). Following the infusion of antioxidants, PmvO2 at rest increased significantly from 30.6 ± 0.9 to 35.7 ± 0.8 Torr (P < 0.05, Fig. 1, Table 1). Moreover, the PmvO2 profile found following the onset of contractions was changed significantly, as the undershoot found in the control condition was either greatly reduced or eliminated (Figs. 1 and 2, Table 1). Consequently, after antioxidant supplementation, the PmvO2 rest-to-contractions profile resembled closely that found previously in young animals (Fig. 1, Table 1; Ref. 7). The infusion of antioxidants also changed the magnitude of the PmvO2 response as the reduction in PmvO2 from rest to steady-state contractions increased from 10.2 ± 1.5 to 17.8 ± 1.2 Torr (P < 0.01, Table 1). This increase in the magnitude of the PmvO2 response was primarily due to the increase in PmvO2 found at rest following the infusion of antioxidants (Fig. 1, Table 1), as the PmvO2 measured during steady-state contractions was not different following supplementation (19.7 ± 1.2 vs. 17.7 ± 1.0 Torr, P > 0.05, Table 1).


Figure 1
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  		Fig. 1. Time course of change in spinotrapezius O2 partial pressure [microvascular O2 partial pressure (PmvO2)] following the onset of contractions (vertical line at 0 s) measured before (dashed line) and after antioxidant supplementation (solid line) for a representative animal. Note pronounced "undershoot" of PmvO2 that drives PmvO2 below subsequent end-contraction values.

 

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  		Table 1. Microvascular PO2 values and model (PmvO2) parameters measured at rest and during contractions in the right spinotrapezius muscle

 

Figure 2
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  		Fig. 2. Effect of antioxidant supplementation on the undershoot (see Fig. 1 for visual identification of undershoots) of PmvO2 in spinotrapezius muscle (n = 16). Note in nearly all cases the undershoot either decreased toward or became zero. Animals that illustrated the same response before and after antioxidant supplementation are illustrated by one line. *Before vs. after supplementation (P < 0.05).

 
Qm and Conductance

Since PmvO2 is dependent on the relationship of QmO2 to VmO2, changes in Qm exert a commanding influence on PmvO2 during the transition from rest to muscle contractions. Qm measured in the resting left spinotrapezius muscle was not significantly altered following antioxidant supplementation (see Table 4). In contrast, Qm in the contracting right spinotrapezius muscle decreased significantly from 157 ± 28 to 91 ± 15 ml·min–1·100 g–1 following the infusion of antioxidants. Antioxidant supplementation affected the resting hindlimb locomotor Qm somewhat differently however. Specifically, at rest, Qm was either increased (i.e., soleus) or tended to increase following antioxidant supplementation (Table 2).


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  		Table 2. Blood flows measured at rest in various hindlimb locomotor muscles

 
Antioxidant supplementation lowered MAP significantly from 131 ± 5 to 107 ± 4 mmHg (P < 0.05). When spinotrapezius Qm was normalized to MAP and expressed as conductance, antioxidant supplementation had no significant effect at rest, but conductance was decreased during contractions (see Table 4). In contrast, for the majority of hindlimb locomotor muscles, vascular conductance was substantially elevated following the infusion of antioxidants (Table 3).


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  		Table 3. Resting conductance of selected hindlimb locomotor muscles

 
VmO2

Since PmvO2 is dependent on the QmO2-to-VmO2 relationship, alterations in VmO2 could also have significant effects on PmvO2 during the rest-to-contractions transition. Although antioxidant supplementation had no significant effect on the CaO2, we found that the infusion of antioxidants produced a significant increase in the CvO2 estimated from PmvO2. This resulted in a significant decrease in the arteriovenous O2 difference and hence VmO2 following antioxidant supplementation (Table 4).


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  		Table 4. Oxygen transport variables calculated for the right contracting spinotrapezius and left resting spinotrapezius muscle

 
Following antioxidant supplementation, VmO2 was reduced during steady-state contractions (Table 4). This antioxidant-induced decrease of VmO2 was associated with a reduction in Qm in the absence of significant alterations in CaO2, and estimated CvO2, and arteriovenous O2 difference (Table 4).

Muscle Force Production

Because antioxidant supplementation reduced Qm and VmO2 significantly, it was important to determine the effects of antioxidant supplementation on muscle force production. Antioxidant supplementation produced a significant decrease in the amount of force that was generated in the contracting right spinotrapezius muscle during a constant voltage stimulus (Fig. 3). The ratio of the right contracting spinotrapezius muscle force production to VmO2 demonstrated a significant increase (0.92 ± 0.03 and 1.06 ± 0.6, respectively, P < 0.05) in the face of reduced VmO2 after antioxidant supplementation.


Figure 3
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  		Fig. 3. Muscle force production produced by the contracting right spinotrapezius muscle before (bullet) and after antioxidant supplementation ({circ}) (n = 4). Values are means ± SE. *Before vs. after supplementation (P < 0.05).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
To our knowledge, the present investigation was the first to examine the effects of antioxidants (tempol and ascorbic acid) on the PmvO2 kinetics in contracting muscle of aged rats. The principal novel findings of our investigation were that, following antioxidant supplementation: 1) the baseline PmvO2 in the resting spinotrapezius muscle was significantly increased, but Qm remained unchanged; 2) the PmvO2 undershoot found following the onset of contractions during the transition from rest to contractions was significantly decreased, but Qm during steady-state contractions was significantly reduced; and 3) muscle force production during contractions was significantly impaired.

Effects of Antioxidants on PmvO2, Qm, and Conductance at Rest

The resting baseline PmvO2 found in the spinotrapezius was significantly increased following antioxidant supplementation. This elevation in PmvO2 was hypothesized to result from elevation of Qm and vascular conductance, consequent to a decrease in ROS concentration and an increased NO bioavailability. However, Qm and spinotrapezius conductance remained either unchanged or even decreased slightly in contrast to other locomotor muscles (Tables 2 and 3). Therefore, with respect to the primary muscle of interest, the spinotrapezius, the present results do not support the hypothesis.

Skeletal muscles vary in their fiber-type composition and function. In this regard, the spinotrapezius muscle contains all of the basic fiber types (41% type I, 7% type IIa, 35% type IIb, and 17% IId/x) found in rats (15), while other muscles may only contain two or three of these predominant fiber types. For example, in the rat, the soleus contains primarily type I fibers, ~84% (15), while other locomotor muscles from the hindlimb contain a variety of fiber types ranging widely in their distribution. We found increases in resting Qm and conductance in several different hindlimb locomotor muscles following antioxidant supplementation (Tables 2 and 3) in a manner that did not appear to be fiber-type dependent. In marked contrast, resting Qm and conductance remained unchanged in the spinotrapezius muscle following the infusion of antioxidants. The mechanisms responsible for these changes in resting hindlimb Qm and conductance found after antioxidant supplementation are unclear at this time. However, sympathetic activation and/or withdrawal have a commanding effect on Qm and conductance (29), and the possibility exists that muscle-specific changes in sympathetic vascular tone might have resulted from infusion of antioxidants (61) and thus have contributed to the present results.

The present investigation demonstrated clearly that antioxidant supplementation produced an increase in the resting PmvO2 of the spinotrapezius muscle without any significant changes in resting Qm or conductance to the muscle. Thus the resting metabolic rate (VmO2) was estimated to decrease ~25%.

Effects of Antioxidants on the Transition of PmvO2 from Rest to Exercise

There are three distinct phases during the transition from rest to exercise in the PmvO2 profile that have been described previously for the spinotrapezius muscle in young rats (9). There is an initial TD at the onset of contractions, where the PmvO2 profile either remains constant (lasting ~10–20 s) or increases slightly. This has been interpreted as QmO2 increasing sufficiently to meet the rising VmO2 demands (9).

In the present investigation, the TD before antioxidant supplementation was consistent with that demonstrated previously in aged rats (7). We expected initially that the antioxidant supplementation would produce an increase in the TD due to an increased bioavailability of NO, thereby allowing an increased NO-mediated vasodilation. However, we were surprised to find that the TD was unaffected by antioxidant supplementation (Fig. 1, Table 1), and we attributed this finding to a preservation of the QmO2-to-VmO2 ratio, albeit at different absolute levels of QmO2 and VmO2.

In young, healthy animals after the onset of contractions subsequent to the TD, the PmvO2 profile is described by an exponential decrease much akin to the solid line in Fig. 1 (see also Ref. 8). This dynamic phase represents an interval when VmO2 is still increasing and doing so out of proportion to QmO2 until the steady state is reached (7, 9). Our laboratory has demonstrated that disease states, such as chronic heart failure (17) and diabetes (8), as well as aged individuals (7), express a biphasic post-TD response that is characterized by an undershoot, or a temporal mismatch between QmO2 and VmO2 that is subsequently rectified, presumably as QmO2 increases later in the contraction bout (i.e., the QmO2 response is sluggish). The occurrence of this undershoot in aged individuals in the present investigation is consistent with those previous findings (Figs. 1 and 2). Before the infusion of antioxidants, all of the aged rats (i.e., 16 of 16) demonstrated an undershoot in the present investigation, which was qualitatively and quantitatively similar to those found previously (7, 8, 17). This "inadequate" QmO2 response may occur consequent to an impaired NO-mediated vasodilation.

Thus, following the TD and preceding attainment of the steady state, the overall PmvO2 was reduced significantly in aged rats compared with their younger counterparts (7). These reductions in PmvO2 represent a decreased O2 driving pressure from the muscle microvascular space to the mitochondria during the transition from rest to contractions. Previous studies support the notion that there is less NO bioavailability during muscle contractions (i.e., exercise) when aged individuals are compared with their younger counterparts, and it is believed that this decreased NO may be mechanistically related to the diminished Qm response found in these individuals (11, 20, 21, 55, 56, 58). It should be noted that previous studies have not examined the effects of antioxidants on the PmvO2 profile. We originally anticipated that the infusion of ascorbic acid would increase the bioavailability of NO, thereby amplifying the vasodilatory response found in the contracting muscle (1, 57). In addition, we anticipated that the infusion of tempol would decrease sympathetic vasoconstrictor output, thereby enhancing the Qm response to the exercising muscle (61). Contrary to our expectations, we found that contracting spinotrapezius Qm was actually reduced, which was not consistent with our hypothesis.

The PmvO2 profile reaches a steady state > 60 s after the onset of contractions, and this condition is believed to constitute attainment of steady-state levels of both QmO2 and VmO2. Our findings indicate that antioxidant supplementation had no significant effect on the contracting steady-state PmvO2. However, due to the increased resting PmvO2 found after antioxidant supplementation and the reduced contracting Qm, there was a significant increase in the amplitude of the change in PmvO2 response (Fig. 1, Table 1).

Other Potential Mediators of the PmvO2 Profile

Previous studies support the notion that endothelium-mediated vasodilation is reduced in aged skeletal muscle, and that this reduction can be ascribed, in part, to an increase in ROS found in this population (20, 24, 56). The primary sources of increased ROS during exercise are thought to be skeletal muscle mitochondria and NAD(P)H oxidase (12, 35, 36). In the present investigation, we anticipated that the infusion of antioxidants and the associated reduction of ROS would produce an increase in NO bioavailability (20, 55). Accordingly, we expected the PmvO2 profile to be significantly modified, and we anticipated that this modification would be mediated primarily by changes in QmO2 (i.e., increased). Contrary to our expectations following antioxidant supplementation, we found that Qm (and, therefore, QmO2) was significantly decreased during steady-state contractions. Moreover, we found that the calculated VmO2 was also reduced, contrary to the anticipated beneficial effects on the electron transport chain. Given these findings, the likelihood that increased NO bioavailability may have competitively inhibited the binding of O2 in the electron transport chain must be considered (14). Although the infusion of antioxidants may have reduced ROS in the contracting muscle and thereby increased the bioavailability of NO, in turn this increased NO concentration may have compromised mitochondrial function and hence VmO2. Since the Qm response found in skeletal muscle is tightly coupled to oxidative metabolism (i.e., VmO2) under exercising (contracting) steady-state conditions (16), the probability exists that any potential increase in vasodilatory function was effectively offset by reductions in oxidative metabolism (Table 4), with the net effect that both Qm (QmO2) and VmO2 were reduced.

There also may have been direct effects of NO on skeletal muscle contractile function that, in addition to decreased VmO2, may have impaired muscle force production. Accordingly, previous studies have shown that high concentrations of NO can significantly inhibit muscle force production (48, 49). The mechanistic factors associated with this observation are not fully understood at this time, but Reid and Durham (49) have suggested that muscle-derived ROS and NO can modulate force production via the redox status of the muscle. This relationship has been schematized as a bell-shaped process (Fig. 4). According to Reid and Durham, in contrast to the young, healthy individual whose redox state places them just to the left of the optimal muscle force production on the bell curve, their aged counterparts might be placed to the right of optimal muscle force production due to elevated concentrations of ROS, which has increased their oxidized state (49). It was our belief that antioxidant supplementation would shift the redox state in aged individuals such that their muscle biochemical environment would be closer to that which would normally be found in younger individuals (i.e., closer to optimal/maximal muscle force production). Contrary to our original belief, antioxidant supplementation significantly reduced muscle force production. Due to the effect of antioxidants on ROS, it is likely our protocol actually produced a larger-than-anticipated swing toward the reduced state, one consequence of which was a decreased muscle force production (4). This effect is likely to be coupled to the lowered VmO2 after the infusion of antioxidants. Therefore, the contracting spinotrapezius muscle produced less force at a lower energy (VmO2) cost. In support of this hypothesis, we found that the ratio between force production and VmO2 actually increased ~15%, which means that the contracting spinotrapezius muscle was able to increase its force production per unit of VmO2 (i.e., contracting more efficiently) after antioxidant supplementation. This process may have facilitated the improved temporal QmO2-to-VmO2 ratio during the transition from rest-to-exercise and is consistent with the elevated transient PmvO2 profile found in the present investigation. The reduced vascular conductance in the antioxidant condition likely resulted, in part, from the decreased force production, which is supported by the fall in estimated VmO2. There may also have been an altered balance in vasoactive mediators (e.g., decreased hydrogen peroxide, increased NO) that reduced the overall vasodilatory response to contractions.


Figure 4
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  		Fig. 4. Biphasic response of muscle force production coupled with redox state. Open circle is the muscle force production by aged rats before antioxidant supplementation. Solid circle is the muscle force production by aged rats after antioxidant supplementation. Young healthy rats are indicated by the cross. [Drawn from the concept described by Reid and Durham (49)].

 
Methodological Considerations

First, electrical stimulation produces a fiber recruitment pattern that is dissimilar to that found in vivo and may result in a very different response from that which occurs during voluntary exercise. Notwithstanding this departure from voluntary contractions, the PmvO2 profile so obtained agrees closely with the profile of muscle effluent PvO2 seen in studies of humans performing exercise (4, 23).

Second, with respect to phosphorescence quenching measurement of PmvO2, blood pH and temperature are the principal physicochemical changes that can distort the measurement itself (39), and neither was changed by the experimental protocol. However, if some sequela of the antioxidant treatment, for example, increased NO bioavailability, altered HbO2 affinity estimation of blood O2 content and thus VmO2 could be affected. At very high NO concentrations, Stepuro and Zinchuk's (54) data demonstrate a small, although significant, increase in HbO2 affinity (decreased P50). Whereas it is doubtful that this was a consideration in the present investigation, any such effect would act to accentuate the fall in estimated VmO2 above that reported herein.

Third, as qualified in METHODS, PmvO2, which was used to approximate PvO2 and estimate VmO2, is not synonymous with PvO2. It is appropriate, therefore, to consider what error might arise from this procedure. During the steady-state of contractions, mean PmvO2 was ~20 Torr, corresponding to a fractional arterial-venous O2 extraction of 0.83. This value is close to that measured systemically in the mixed venous blood of maximally exercising rats (28) and approximates that measured from effluent muscle venous blood from humans performing maximal knee-extensor exercise (50). Based upon these comparisons and the very low PmvO2 (and the high fractional extraction this represents), it is physiologically and mathematically not possible for PmvO2 to exceed PvO2 substantially.

Fourth, the effects of repeated muscle contractions and the possibility of increasing muscle fatigue must be considered. Repeated contractions in aged rats may exacerbate the depletion of intramuscular phosphocreatine and glycogen stores. Depletion of these finite energy sources will ultimately lead to muscle fatigue (60). In the present investigation, however, throughout each trial, the contracting spinotrapezius muscle force production did not fall significantly from minute 0 to minute 4, and independent trials demonstrated that this contraction protocol could be continued >30 min without discernable fatigue (K. H. Herspring, S. W. Copp, D. C. Poole, and T. I. Musch, unpublished observations). Thus there are no indications that fatigue secondary to the control bout of contractions could account for the decreased force production seen in the antioxidant trials. Other methodological considerations regarding the spinotrapezius muscle preparation have been discussed in detail previously (3, 9, 10).

Conclusion

Antioxidant supplementation alters the balance between QmO2 and VmO2 at rest and following the onset of muscle contractions, which modifies the microvascular PmvO2 profile. Specifically, antioxidants substantially elevate PmvO2 at rest and for the first 60–100 s of contractions, which improves the potential for diffusive blood-myocyte flux. This effect arises, in part, from the unanticipated fall in muscle force production consequent to antioxidant supplementation, rather than an elevated QmO2.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
ACKNOWLEDGMENTS
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This work was supported by American Heart Association Heartland Affiliate Grant 070090Z.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
REFERENCES
 
We thank Drs. Craig A. Harms and Mark D. Haub for significant contributions to this investigation, and also Sue Hageman and Scott Hahn for technical assistance during this study, without which this investigation would not have been possible.


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. I. Musch, Dept. of Anatomy and Physiology, College of Veterinary Medicine, Kansas State Univ., Manhattan, KS 66506-5802 (e-mail: musch{at}vet.ksu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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