The effects of antioxidants on microvascular oxygenation and blood flow in skeletal muscle of young rats

Abstract

Alterations of skeletal muscle redox state via antioxidant supplementation have the potential to impact contractile function and vascular smooth muscle tone. The effects of antioxidants on the regulation of muscle O₂ delivery–O₂ utilization () matching (which sets the microvascular partial pressure of O₂; ) in young healthy muscle are not known. Therefore, the purpose of this study was to test the effects of acute antioxidant supplementation on rat spinotrapezius muscle force production, blood flow, and (phosphorescence quenching). Anaesthetized male Fischer 344 × Brown Norway rats (6–8 months old) had their right spinotrapezius muscles either exposed for measurement of blood flow and (n= 13) or exteriorized for measurement of muscle force production (n= 6). Electrically stimulated 1 Hz twitch contractions (∼7–9 V) were elicited for 180 s, and measurements were made before and after acute intra-arterial antioxidant supplementation (76 mg kg⁻¹ ascorbic acid, 52 mg kg⁻¹ tempol) dissolved in saline and infused over 30 min. The principal effects of antioxidants were a ∼25% decrease (P < 0.05) in contracting spinotrapezius muscle force production concurrent with reductions in muscle blood flow and at rest and during contractions (P < 0.05 for both). Antioxidant supplementation reduced the resting baseline (before, 29.9 ± 1.2 mmHg; after, 25.6 ± 1.3 mmHg; P < 0.05), and this magnitude of depression was sustained throughout the rest-to-exercise transition (steady-state value before, 16.4 ± 0.7 mmHg; after, 13.6 ± 0.9 mmHg; P < 0.05). In addition, the time constant of the decrease was reduced after antioxidant supplementation (before, 23.4 ± 4.3 s; after, 15.6 ± 2.7 s; P < 0.05). These results demonstrate that antioxidant supplementation significantly impacts the control of in young rats at rest and during contractions.

Muscle contractions increase mitochondrial oxygen (O₂) delivery and O₂ utilization (). A consequence of this elevated muscle is increased generation of reactive O₂ species (ROS). Low-level increases of ROS during contractions in healthy muscle are imperative for optimal physiological function of the skeletal muscle contractile apparatus (Reid et al. 1993) and the regulation of vascular smooth muscle tone (Rubanyi & Vanhoutte, 1986). However, when ROS accumulation, either acutely or chronically, exceeds the buffering capacity of the endogenous antioxidant system, physiological fatigue and/or pathological dysfunction may result (Hamilton et al. 2001; Chen et al. 2005). Consequently, therapeutic antioxidant supplementation has been considered as a potential treatment to curtail or prevent the adverse effects of ROS accumulation on skeletal muscle contractile function and muscle O₂ delivery (; Eskurza et al. 2004; Tyml et al. 2005; Herspring et al. 2008; Kelly et al. 2009).

Reid and colleagues (1993; Reid, 2001) have developed a conceptual model relating muscle contractile performance (isometric force) to myocyte reduction–oxidation (redox) state. For unfatigued young healthy muscle, low-level increases in ROS during contractile activity shift the redox balance towards a more oxidized state and increase isometric force production potential. In contrast, myocyte exposure to a reducing agent (i.e. exogenous antioxidants) decreases isometric force potential (Fig. 2 of Reid, 2001). These effects may be related via redox regulation of the sarcoplasmic reticulum (SR) ryanodine calcium-release channels (Anzai et al. 2000) and/or SR calcium-dependent (SERCA) ATPase pump activity (Daiho & Kanazawa, 1994). In addition, the myofibrils themselves are directly impacted by redox modification (Haycock et al. 1996; Andrade et al. 1998). A further consideration is the potential for alterations in the redox state to significantly impact the control of the skeletal muscle blood flow, either directly through alterations in the control of the vascular smooth muscle (Rubanyi & Vanhoutte, 1986; Marvar et al. 2007) or indirectly via impairments in mitochondrial function and therefore O₂ demand (Cleeter et al. 1994). Accordingly, redox modulation has the ability to impact and which, in turn, dictate microvascular O₂ partial pressures () and blood–muscle O₂ flux.

Little is known about the effects of redox modulation on the matching of and , either at rest or during contractions, in young healthy animals. We reported recently that skeletal muscle redox modulation via acute antioxidant supplementation (76 mg kg⁻¹ ascorbic acid and 52 mg kg⁻¹ tempol) changes markedly the relationship and thus , in contracting aged rat spinotrapezius muscle (Herspring et al. 2008). However, whether antioxidant supplementation can modulate in healthy skeletal muscle of young animals has not been determined.

The primary purpose of the present study was to evaluate the effects of acute antioxidant supplementation on the regulation of the ratio (i.e. ) at rest and during electrically induced contractions in unfatigued young healthy rat spinotrapezius muscle. Based on the potential for antioxidants to alter the muscle redox state acutely (Herspring et al. 2008), we tested the specific hypotheses that after acute antioxidant supplementation in young rats: (1) spinotrapezius muscle force production, , and blood flow (and therefore ) would be decreased during electrically induced contractions; and (2) any changes in and would, depending on their proportionality, result in significant modulation of at rest and during contractions.

Methods

Animals

Nineteen young male Fischer 344 × Brown Norway F1 hybrid rats were obtained from Charles River Laboratories (Boston, MA, USA). Thirteen rats (6–8 months old, body mass 404 ± 22 g) were used in the experiments examining P_O2mv whereas six rats (6 months old, body mass 339 ± 12 g) were used in the experiments examining force production. Upon arrival, all rats were housed, two per cage, and maintained on a 12 h–12 h light–dark cycle with food and water provided ad libitum. Animal housing facilities were approved by the Association for Assessment and Accreditation of Laboratory Animal Care, and all experimental protocols described presently were approved by the Institutional Animal Care and Use Committee of Kansas State University.

Surgical preparation

All rats were weighed and subsequently anaesthetized with 50 mg kg⁻¹ of pentobarbitone sodium administered i.p. to effect. The level of anaesthesia was monitored continuously throughout the experimental protocol via the toe-pinch and blink reflexes and was supplemented as necessary. Anaesthetized rats were initially placed on a heating pad to maintain core temperature, measured via rectal probe, at ∼37–38°C. The carotid and caudal (tail) arteries were cannulated (Polyethelene-50, Intra-Medic Tubing, Clay Adams Brand, Sparks, MD, USA) for continuous monitoring of heart rate and mean arterial pressure (MAP) and for infusion of the phosphorescent probe.

Overlying skin and fascia were surgically removed from the right side of the dorsal, mid-caudal region of each rat, thereby exposing the right spinotrapezius muscle. The exposed muscle and surrounding tissue were constantly moistened throughout the surgery and experimental protocol via superfusion of Krebs–Henseleit bicarbonate-buffered solution (equilibrated with 5% CO₂, 95% N₂, pH 7.4, warmed to 37°C), and surrounding tissue was covered with Saran wrap (Dow Brands, Indianapolis, IN, USA). Silver wire electrodes were sutured to the rostral (cathode) and caudal (anode) region of the muscle for delivery of the contraction protocol. In our laboratory the rat spinotrapezius muscle has been used routinely for measurements of microcirculatory function (Diederich et al. 2002; Behnke et al. 2005; Ferreira et al. 2006).

Experimental protocol

After surgery, the phosphorescent probe palladium meso-tetra (4-carboxyphenyl) porphyrin dendrimer (R2, Oxygen Enterprises, Philadelphia, PA, USA; 15–20 mg kg⁻¹ dissolved in saline) was infused via the carotid artery catheter. After a ∼10–15 min stabilization period, 1 Hz (∼7–9 V, 2 ms pulse duration) twitch contractions were elicited via a Grass stimulator (model S88; Quincy, MA, USA) for 180 s. Values of were measured at baseline and for the duration of the contraction protocol. Following a brief recovery from the contractions, rats were infused with a saline antioxidant mixture (76 mg kg⁻¹ of ascorbic acid and 52 mg kg⁻¹ of tempol; dissolved in 1.5 ml saline) via a Harvard pump (model 907, Cambridge, MA, USA) at a rate of 1.5 ml over 30 min. It has been demonstrated previously that this dose of ascorbic acid may restore microvascular function in sepsis (Armour et al. 2001; Tyml et al. 2005), and the dose of tempol selected for the present investigation has exhibited cardioprotective benefits via its actions on the sympathetic nervous system (Xu et al. 2004). Importantly, the rationale for the selection of the specific combination of antioxidants in the present study is supported by the observation that this mixture produces significant increases in antioxidant capacity (and thus alterations in redox state) in aged rats (Herspring et al. 2008). After the 30 min infusion, a second contraction bout was completed exactly as described above. Blood samples were taken after the contraction protocols to determine arterial O₂ content and serum antioxidant capacity. At the end of each experiment, rats were killed via an intra-arterial overdose of pentobarbitone (∼50 mg kg⁻¹).

Total serum antioxidant capacity

The sampled blood was placed in EDTA tubes with heparin (Elkins-Sinn, Inc., Cherry Hill, NJ, USA) as an anticoagulant, centrifuged for 7 min at 7245 g to obtain serum samples, and stored immediately at −80°C until analysed. The samples were aliquoted in duplicate, and the total serum antioxidant capacity was determined via a commercially available kit (no. K274-100, BioVision Total Antioxidant Capacity, Biovision, Mountain View, CA, USA). Trolox (Biovision) was used to standardize all antioxidants, and the total serum antioxidant capacity was measured in Trolox equivalents. The sample absorbance was analysed at 570 nm (Bio-Tek, Winooski, VT, USA). Antioxidant capacity was calculated from a standard curve that spanned the range of measurements.

Measurement of blood flow and calculation of

In nine of the 13 rats used in the present study, blood flow to the left and right spinotrapezius muscles and selected locomotor muscles of the hindlimb was determined using radiolabelled microspheres as described in detail previously (Musch & Terrell, 1992). Briefly, the caudal artery was connected to a Harvard pump, and blood withdrawal was initiated at 0.25 ml min⁻¹. Differentially labelled 15 μm diameter microspheres (⁴⁶Sc, ⁸⁵Sr; Perkin Elmer Life and Analytical Sciences, Boston, MA, USA) were infused, in random order, into the carotid artery catheter during the contracting steady state (i.e. the last 30 s) of both the control (before antioxidant supplementation) and experimental contraction bouts (after antioxidant supplementation). In each set of conditions, the stimulated right and non-stimulated left spinotrapezius muscles represented the contracting and resting blood flow measurements, respectively. Upon completion of each experiment, the right and left spinotrapezius muscles, right and left kidneys and selected hindlimb muscles were carefully dissected, removed and weighed. Tissue radioactivity was determined via a γ-scintillation counter (Packard Auto Gamma Spectrometer, Cobra model 5003, Downers Grove, IL, USA), and individual tissue flows were determined by the reference sample method (Ishise et al. 1980) and expressed in millilitres per minute per 100 grams of tissue. Vascular conductance was determined as blood flow/MAP. All rats exhibited a < 15% difference in blood flow between the left and right kidneys, which indicated an adequate mixing of microspheres during the blood flow measurements.

Values of were estimated from blood flow and as described previously (Behnke et al. 2002a). Briefly, the arterial O₂ concentration () was calculated directly from arterial blood samples, and the venous muscle effluent blood O₂ concentration () was estimated from either the baseline (rest) or the contracting steady-state (contractions) using the rat dissociation curve (Hill coefficient of 2.6), the measured haemoglobin (Hb) concentration, a partial pressure of O₂ at which haemoglobin is 50% saturated (P₅₀) of 38 mmHg and an O₂ carrying capacity of 1.34 ml O₂ (g Hb)^–1 (Altman & Dittmer, 1974). The measures of the resting and contracting spinotrapezius blood flows were then used to calculate via the direct Fick calculation [i.e. ]. In four animals in which blood flow was not measured owing to technical limitations, the average spinotrapezius blood flow that was measured in the nine other rats was used to calculate .

Measurement of force production

Owing to differences in the surgical preparation required for measurement of muscle force production rather than , an additional group of rats (n= 6) was used to determine the effects of antioxidant supplementation on muscle contractile function. Rats underwent the procedure to expose the spinotrapezius muscle. The caudal end of the muscle was exteriorized and sutured to a thin, wire horseshoe manifold and attached to a swivel apparatus and a non-distensible light-weight (0.4 g) cable, which linked the muscle to a Grass force transducer (model FTO3, Quincy, MA, USA). The preload tension of the muscle was set at ∼4 g, which elicited the optimal length of the muscle for twitch force production. Muscle force production was measured throughout control and post-antioxidant supplementation contraction bouts, which were identical (i.e. 1 Hz, ∼7–9 V) to the contraction protocols described for the measurement of .

Measurement of and curve fitting

The principles of the phosphorescence quenching technique have been discussed previously (Behnke et al. 2001, 2005; McDonough et al. 2001). The Stern–Volmer relationship allows the calculation of through the direct measurement of a phosphorescence lifetime via the following equation (Rumsey et al. 1988):

• 

where k_Q is the quenching constant (expressed in mmHg s⁻¹) and τ° and τ are the phosphorescence lifetimes in the absence of O₂ and the ambient O₂ concentration, respectively. For R2, k_Q is 409 mmHg s⁻¹ and τ° is 601 μs (Lo et al. 1997), and these characteristics do not change over the physiological range of pH and temperature in the rat in vivo and, therefore, the phosphorescence lifetime is solely affected by the O₂ pressure (Rumsey et al. 1988; Lo et al. 1997).

The R2 phosphorescent probe binds to albumin and, consequently, is uniformly distributed throughout the plasma. This characteristic allows R2 to remain within the microvascular space and not filter into surrounding tissue, thereby ensuring a valid measurement of (Poole et al. 2004). The was determined with a PMOD 1000 Frequency Domain Phosphorometer (Oxygen Enterprises, Philadelphia, PA, USA). The common end of the light guide was placed ∼2–4 mm superficial to the dorsal surface of the exposed right spinotrapezius muscle. The randomly selected muscle field is comprised principally of capillary blood, and was measured continuously and reported at 2 s intervals throughout the duration of the contraction periods.

For the measured responses, curve fitting was performed with commercially available software (SigmaPlot 9.01), and the data were fitted with either a one- or two-component model as described below:

• 

• 

where represents the at any given time t, corresponds to the precontracting resting , Δ₁ and Δ₂ are the amplitudes for the first and second component, respectively, TD₁ and TD₂ are the time delays for each component, and τ₁ and τ₂ are the time constants (i.e. time to 63% of the final response value) for each component. Goodness of fit was determined using the following criteria: (1) the coefficient of determination (r²); (2) sum of the squared residuals; and (3) visual inspection and analysis of the model fits to the data and the residuals.

Time control experiments

The stability of the spinotrapezius muscle preparation has been addressed previously (Herspring et al. 2008) and was reconfirmed in the present study via time control saline-infusion experiments (n= 6). There were no differences qualitatively or quantitatively (P > 0.05) for any parameter between initial and subsequent contraction bouts separated by 30 min of saline infusion. Additionally, time-control force production measurements demonstrated only a 5% change (P > 0.05) between initial (11.5 ± 1 g) and subsequent contraction bouts (10.9 ± 1 g). Therefore, it is highly unlikely that fatigue and/or deterioration of the muscle preparation per se could account for any changes in measured variables after antioxidant supplementation.

Statistical analysis

Data are presented as means ±s.e.m. All comparisons between before and after antioxidants were made using paired Student's t test. One-way ANOVAs were used to evaluate force production measurements over time within the control and antioxidant contraction bouts. When a directional hypothesis was tested, a one-tailed test was performed. Standard liner regression techniques were used to evaluate the change in blood flow (Δblood flow)/change in () ratio before and after antioxidants. The significance level was set at P < 0.05.

Results

Antioxidant capacity

Serum antioxidant capacity after antioxidant supplementation was increased by 72% (P < 0.05) above control values.

Muscle force production

Muscle force production did not decrease from the initial values throughout the duration of the contraction protocol, either before or after antioxidant supplementation (P > 0.05 for both). However, antioxidants reduced muscle force production by an average of 25% over the 180 s contraction protocol (before, 11.8 ± 0.8; after, 8.8 ± 1.1 g; P < 0.05; Fig. 1). The ratio of the average muscle force production-to- during contractions was not different (P > 0.05) before (0.70 ± 0.12 g (ml O₂)⁻¹) compared with after antioxidant supplementation (0.83 ± 0.12 g (ml O₂)⁻¹).

Figure 1. Spinotrapezius muscle force production before and after antioxidant supplementation, measured with identical stimulation parameters and over an identical time period as for themeasurement *P < 0.05 compared with before antioxidant supplementation.

Profile of

Similar to our previously established profile in young healthy rats (Behnke et al. 2001, 2005), the control contraction in the present investigation elicited a mono-exponential decrease to the steady-state value (Fig. 2) in the majority (9 of 13) of animals and this did not change after antioxidant supplementation (monoexponential fit in 8 of 13 rats). The values of the parameters before and after antioxidant supplementation are presented in Table 1. A principal effect of antioxidants on was to decrease (P < 0.05) the resting baseline from 29.9 ± 1.2 to 25.6 ± 1.3 mmHg. Following the onset of contractions, similar amplitudes were obtained before and after antioxidant supplementation (P > 0.05); therefore, as a result of the lower initial value, the steady-state was reduced after antioxidants (before, 16.4 ± 0.7 mmHg; after, 13.6 ± 0.9 mmHg; P < 0.05). Additionally, the temporal association between and during contractions was significantly altered such that the time constant (τ, time to reach 63% of the steady-state ) was reduced (P < 0.05) after antioxidant supplementation.

Figure 2. Microvascular() profiles along with the modelled fits from a representative young rat before (dashed line) and after antioxidant supplementation (continuous line) The inset shows the average modelled fits of the before and after antioxidant supplementation in young rats. Standard errors are omitted for clarity.

Table 1.  Microvascular() variables and model parameters before and after antioxidant supplementation

	Before antioxidant supplementation	After antioxidant supplementation
Values are means ±s.e.m. Abbreviations: , baseline ; τ, time constant, i.e. time taken to reach 63% of the final response value. *P < 0.05 compared with before antioxidant supplementation.
(mmHg)	29.9 ± 1.2	25.6 ± 1.3*
Time delay (s)	6.2 ± 1.1	9.5 ± 1.3*
amplitude (mmHg)	14.9 ± 1.2	13.2 ± 1.3
τ (s)	23.4 ± 4.3	15.6 ± 2.7*
Steady-state	16.4 ± 0.7	13.6 ± 0.9*
(mmHg)

Haemodynamic and O₂ transport variables

Antioxidant supplementation consistently reduced blood flow (P < 0.05) to both the resting left and contracting right spinotrapezius muscles (Table 2). This decreased blood flow occurred consequent to a reduced MAP and vascular conductance (P < 0.05 for both; Table 2). In addition the Δblood flow from rest to contractions was reduced after antioxidants (before, 116 ± 14 ml min⁻¹ (100 g)^–1; after, 74 ± 10 ml min⁻¹ (100 g)^–1; P < 0.05).

Table 2.  Spinotrapezius haemodynamic and oxygen transport variables before and after antioxidant supplementation at rest and during contractions

	Before antioxidant supplementation	After antioxidant supplementation
Values are means ±s.e.m. Abbreviations: , venous O2 saturation; , venous O2 content; and , muscle O2 utilization. † Arterial O2 content (17.3 ± 0.3 ml (100 ml)^–1) remained unchanged (P < 0.05) after antioxidant supplementation and was assumed to be unchanged from rest to contractions in both conditions. *P < 0.05 compared with before antioxidant supplementation.
Resting spinotrapezius
Blood flow (ml min^−1 (100 g)^−1)	23 ± 4	9 ± 1*
Mean arterial pressure (mmHg)	136 ± 4	126 ± 4*
Conductance (ml min^−1 (100 g)^−1 mmHg^−1)	0.16 ± 0.02	0.07 ± 0.01*
Estimated (%)	36 ± 0.1	26 ± 0.1*
Estimated (ml dl^−1)	6.5 ± 0.1	4.7 ± 0.1*
Estimated arteriovenous O2 difference (ml dl^−1)†	10.8 ± 0.3	12.6 ± 0.6*
Calculated (ml min^−1 (100 g)^−1)	2.4 ± 0.4	1.11 ± 0.1*
Contracting spinotrapezius
Blood flow (ml min^−1 (100 g)^−1)	139 ± 15	83 ± 11*
Mean arterial pressure (mmHg)	136 ± 3	125 ± 4*
Conductance (ml min^−1 (100 g)^−1 mmHg^−1)	1.0 ± 0.1	0.7 ± 0.1*
Estimated (%)	10.0 ± 0.1	7.0 ± 0.1
Estimated (ml dl^−1)	1.9 ± 0.2	1.2 ± 0.2*
Estimated arteriovenous O2 difference (ml dl^−1)†	15.4 ± 0.3	16.1 ± 0.3*
Calculated (ml min^−1 (100 g)^−1)	21.3 ± 2.3	13.4 ± 1.8*

Oxygen transport variables before and after antioxidant supplementation are displayed in Table 2. Antioxidant supplementation decreased both the resting and contracting spinotrapezius (P < 0.05). Similar to blood flow, the from rest to contractions was reduced after antioxidants (before, 18.9 ± 2.2 ml min⁻¹ (100 g)^–1; after, 12.2 ± 1.8 ml min⁻¹ (100 g)^–1; P < 0.05). However, from rest to contractions the spinotrapezius Δblood flow/ ratio was not different (P < 0.05) before (6.1 ± 0.1) compared with after antioxidant supplementation (6.1 ± 0.1; Fig. 3). Despite the substantial decreases in resting spinotrapezius blood flow after antioxidant supplementation, there was no consistent effect of antioxidant supplementation on resting hindlimb muscle blood flows, with select muscles showing either an increase or no change after antioxidant supplementation (Table 3).

Figure 3. Individual plots of the relationship between the change from rest to contractions in spinotrapezius muscle blood flow (Δblood flow) and O₂ utilization () before and after antioxidant supplementation The slope of the relationship before and after antioxidants was identical (i.e. 6.1 ± 0.1 for both). Inset shows average Δblood flow/ before and after antioxidants. *P < 0.05 for Δblood flow and compared with before antioxidant supplementation.

Table 3.  Resting blood flows to various hindlimb locomotor muscles

	Before antioxidant supplementation	After antioxidant supplementation
Values are means ±s.e.m. in ml min^−1 (100 g)^–1. *P < 0.05 compared with before antioxidant supplementation.
Soleus	8 ± 1	15 ± 2*
Plantaris	9 ± 3	7 ± 1
Red gastrocnemius	9 ± 2	13 ± 2
White gastrocnemius	4 ± 1	9 ± 1*
Mixed gastrocnemius	6 ± 1	5 ± 1
Extensor digitorum longus	6 ± 1	9 ± 1

Discussion

These results demonstrate, for the first time, that redox modulation via antioxidant supplementation alters the O₂ delivery–O₂ utilization balance in young healthy skeletal muscle. In the present investigation, antioxidant supplementation reduced spinotrapezius blood flow and at rest and during contractions and significantly depressed the ability of the muscles to generate force. These alterations led to a reduction in the resting baseline after antioxidants, and this reduction was maintained throughout the contraction protocol (Table 1 and Fig. 2). This demonstration of the ability of an antioxidant treatment to alter is significant because the decreased represents a reduction of the pressure head responsible for driving O₂ flux across the capillary-to-myocyte interface. These results lend important insights into the redox regulation of matching.

The sensitivity of redox regulation – relationship with the literature

A principal effect of antioxidant supplementation was a reduction in resting spinotrapezius muscle blood flow and . These reductions were disproportional such that the resting baseline ratio (i.e. ) was reduced. During contractions, antioxidants resulted in a 25% reduction in force production, which may account, at least in part, for the lower Δblood flow and from rest to contractions (and therefore the lower absolute contracting blood flow and values) after antioxidants. However, the Δblood flow/ of 6.1/1, which matches closely the value established for humans (i.e. 6/1; cf. Poole, 1997), was unaffected by antioxidants. The maintenance of this ratio, calculated from values at rest and during the contracting steady state, suggests that the mechanistic determinants of the ΔO₂ supply–O₂ utilization relationship were unaffected by antioxidant supplementation. However, it is important to consider that the mechanisms controlling the dynamic rate of adjustment of relationship were altered such that the fall in was speeded (i.e. shorter time constant) upon the initiation of contractions. It is worth noting that in other investigations, pathological states, such as diabetes (Behnke et al. 2002b; Padilla et al. 2007) and moderate heart failure (Diederich et al. 2002), are also associated with faster kinetics at the onset of contractions. As expected, the amplitude of the decrease was similar before and after antioxidants; therefore, the lower steady-state was a result of the reduced baseline (i.e. similar amplitudes).

The integration of the present results with existing studies of the effects of antioxidants on muscle and vascular function is challenging owing to potential variations in the actions of specific antioxidants, as well as differences in selected doses, administration methods and limitations in the scope of measurements made. Notwithstanding these potential confounding factors, such comparisons may lend important insights into the sensitivity of redox balance in health and disease. The observation of a 25% decrease in force production after antioxidants in the present investigation corroborates previous antioxidant interventions in animal muscles (Reid et al. 1993; Coombes et al. 2001; Herspring et al. 2008) and is consistent with the biphasic model of the redox regulation of contractile function (Reid, 2001). However, it is also pertinent to note that antioxidants have been efficacious in reducing skeletal muscle fatigue in certain experimental conditions (Reid et al. 1994; Travaline et al. 1997; Kelly et al. 2009). In the present study, it is logical that the decreased force production would be concurrent with a reduced contracting spinotrapezius and blood flow after antioxidant supplementation. It is also possible that antioxidants may have had direct effects on vascular function. For example, the redox control of vascular function appears to follow a biphasic response curve similar to muscle contractile function. Specifically, antioxidants have detrimental effects of on vascular function (reduced vasodilatation) in isolated vessel preparations (Rubanyi & Vanhoutte, 1986; Samora et al. 2008) and in vivo animal (Marvar et al. 2005, 2007) and human models (Richardson et al. 2007; Wray et al. 2009), whereas low concentrations of exogenous oxidizing agents may, in contrast, promote vascular plasticity and function (Marvar et al. 2007).

There is a distinct lack of current literature that examines the redox regulation of matching. However, when the present results are considered in conjunction with those from our recent investigation in aged rats (Herspring et al. 2008) important insights can be gained regarding the effects of age and associated differences of the initial basal redox state on the redox regulation of muscle oxidative function and . Specifically, advanced age has been associated with increases in ROS generation, both at rest and during exercise (Bejma & Ji, 1999). This occurrence would promote a more oxidized basal state of aged versus young skeletal muscle, suggesting that identical antioxidant doses might affect young and old subjects differently. Indeed, an identical relative antioxidant dose in young (present investigation) and aged rats (Herspring et al. 2008) produced markedly dissimilar effects, most notably on (Table 4), suggesting that the initial basal redox state of the skeletal muscle exerts an important influence on the impact of antioxidants on the regulation of matching.

Table 4.  Comparison of the systemic and muscular (spinotrapezius) effects of antioxidants on young and aged rats

Variable	Young	Aged
The table shows the average percentage change from before to after identical antioxidant supplementation protocols in selected measured variables from young (present investigation) and aged rats (Herspring et al. 2008). *P < 0.05 between before and after antioxidant supplementation within the given age group.
Resting spinotrapezius
Baseline	−14%*	20%*
Mean arterial pressure	−8%*	−18%*
Blood flow	−61%*	−11%
Vascular conductance	−56%*	9%
Calculated	−54%*	−25%*
Contracting spinotrapezius
Time constant	−33%*	20%
Steady-state	−17%*	−10%
Mean arterial pressure	−8%*	−17%*
Blood flow	−40%*	−42%*
Vascular conductance	−30%*	−29%*
Calculated	−37%*	−26%*
Muscle force production	−25%*	−25%*

Potential mechanisms of antioxidant action

The present investigation was designed to evaluate the effects of redox modulation via antioxidant supplementation on the regulation of matching in young healthy muscle. Thus, our design makes it impossible to determine the specific mechanisms of the actions of the antioxidants. However, a discussion of the possible mechanisms sheds light on the specific nature of redox control in skeletal muscle and vascular tissues. The mechanisms responsible for the changes in resting spinotrapezius blood flow and estimated are presently unclear. Regarding the effects of antioxidants on blood flow, there is a litany of evidence in support of a vasodilatory role of ROS (Rubanyi & Vanhoutte, 1986; Cseko et al. 2004; Marvar et al. 2005, 2007; Richardson et al. 2007), either through direct action on vascular smooth muscle tone or through mobilization of vascular endothelial calcium stores (Edwards et al. 2008). Thus, we cannot discount the possibility that ROS make an important contribution to the regulation of basal (as well as contracting) skeletal muscle arteriolar tone, and therefore antioxidant supplementation may have reduced spinotrapezius blood flow directly (and thereby decreased resting ) by scavenging important ROS vasodilators. Additionally, changes in the autonomic nervous system impact local blood flow regulation significantly, and the antioxidant-induced reduction of resting MAP on spinotrapezius blood flow also should not be discounted. Antioxidants may also have elicited direct effects on by scavenging superoxide, thereby increasing intramyocyte nitric oxide bioavailability, which is known to competitively inhibit cytochrome c oxidase in the mitochondrial electron transport chain (Cleeter et al. 1994). An intriguing finding in the present investigation was the difference between the antioxidant-induced decrease in resting spinotrapezius blood flow compared with the effects on hindlimb muscle blood flow. This discrepancy may be due to functional differences between the predominantly postural spinotrapezius muscle compared with the locomotor muscles of the hindlimb. More importantly, the spinotrapezius muscle has a similar fibre-type composition (Delp & Duan, 1996) and oxidative capacity to the human quadriceps (Leek et al. 2001) and therefore, it may be argued, represents a more relevant model for the study of skeletal muscle function. Further work will be needed to examine the potential confounding influences of muscle function, fibre-type composition and oxidative capacities on specific antioxidant actions.

Contrary to our hypothesis, we found that antioxidant supplementation produced a significant reduction in the resting baseline , which was sustained throughout the rest-to-contractions transition. The effects of antioxidants on resting and steady-state were consequent to reductions in resting and contracting spinotrapezius blood flow and (but to a lesser extent). The reductions in contracting blood flow and are likely to be coupled, at least in part, to the observed reduction in muscle force production and therefore, energetic demand. As discussed above, the ability of a muscle to produce force for a given contractile stimulus is related to the redox state of the muscle cells according to a bell-shaped response profile. The measured increase in total antioxidant capacity in the present study implies that a moderate leftward shift along this response curve was achieved (Fig. 4), which resulted in the depressed spinotrapezius force generation (Fig. 1). The observation that antioxidant supplementation resulted in muscle contractile dysfunction at the very start of contractions provides strong evidence that the dysfunction resulted from alterations in intracellular processes rather than from fatigue development consequent to reduced O₂ delivery. The specific mechanism responsible for the antioxidant-induced decrease in force production probably involves alterations in intracellular calcium regulation. For example, potential sites of redox regulation may include modulation of the open probability of the redox-sensitive ryanodine calcium channels (Anzai et al. 2000), alterations in SERCA pump function (Daiho & Kanazawa, 1994; Tupling et al. 2007) and/or changes in myofibrillar protein sensitivity (Haycock et al. 1996; Andrade et al. 1998).

Figure 4. The biphasic response profile of muscle force production as a function of the redox state of the skeletal muscle as originally described by Reid (2001) The theoretical basal skeletal muscle redox states of young, healthy unfatigued skeletal muscles in the present investigation are shown before and after antioxidant supplementation.

Methodological considerations

First, the present investigation used a combined antioxidant mixture consisting of intra-arterially administered doses of ascorbic acid and tempol. The nature of this combined antioxidant therapy makes it impossible to distinguish between potential individual or synergistic effects of these compounds. Future studies using both individual and combined supplementation regimes of these antioxidants will be needed to address this issue.

Second, it is difficult to speculate about the specific magnitude of change in the redox state of the skeletal muscle in the present investigation given that no specific units are associated with redox balance (cf. Reid, 2001). Future investigations using ranges of various reducing and oxidizing agents will contribute significantly to our understanding of the degree of redox control of skeletal muscle function across the redox spectrum.

Third, exercise performance is controlled by many different processes, and studies investigating only a few physiological variables may not comprehensively test the broad range of potential actions of antioxidants (Atalay et al. 2006). A strength of the present investigation was the measurement or calculation of multiple physiological variables, including , blood flow, and muscle force production within a single muscle.

Finally, the effects of spinotrapezius muscle exposure and exteriorization, the electrically stimulated contraction protocol, potential physicochemical effects of the antioxidants on and the validity of the calculation have been discussed in detail previously (Bailey et al. 2000; Behnke et al. 2001; Herspring et al. 2008).

Conclusions

The acute antioxidant supplementation protocol in the present investigation significantly reduced skeletal muscle blood flow and , both at rest and during contractions, in young rat spinotrapezius muscle. The alterations in blood flow and modified the microvascular O₂ delivery–utilization balance such that was reduced at rest and during contractions. These effects occurred concurrent with a significant decrease in spinotrapezius muscle force production. These results lend important insights into the sensitivity of at rest and during muscle contractions to the basal skeletal muscle redox state.

Appendix

Acknowledgements

The authors would like to thank K. Sue Hageman, Scott A. Hahn, Peter J. Schwagerl and Dr Mark D. Haub for their significant contributions to this study. This work was supported by the American Heart Association Heartland Affiliate Grant 070090Z.