Advanced age is associated with altered skeletal muscle hemodynamic control during the transition from rest to exercise. This study investigated the effects of aging on the functional role of nitric oxide (NO) in regulating total, inter-, and intramuscular hindlimb hemodynamic control at rest and during submaximal whole body exercise. We tested the hypothesis that NO synthase inhibition (NG-nitro-l-arginine methyl ester, l-NAME; 10 mg/kg) would result in attenuated reductions in vascular conductance (VC) primarily in oxidative muscles in old compared with young rats. Total and regional hindlimb muscle VCs were determined via radiolabeled microspheres at rest and during treadmill running (20 m/min, 5% grade) in nine young (6–8 mo) and seven old (27–29 mo) male Fisher 344 × Brown Norway rats. At rest, l-NAME increased mean arterial pressure (MAP) significantly by ∼17% and 21% in young and old rats, respectively. During exercise, l-NAME increased MAP significantly by ∼13% and 19% in young and old rats, respectively. Compared with young rats, l-NAME administration in old rats evoked attenuated reductions in 1) total hindlimb VC during exercise (i.e., down by ∼23% in old vs. 43% in young rats; P < 0.05), and 2) VC in predominantly oxidative muscles both at rest and during exercise (P < 0.05). Our results indicate that the dependency of highly oxidative muscles on NO-mediated vasodilation is markedly diminished, and therefore mechanisms other than NO-mediated vasodilation control the bulk of the increase in skeletal muscle VC during the transition from rest to exercise in old rats. Reduced NO contribution to vasomotor control with advanced age is associated with blood flow redistribution from highly oxidative to glycolytic muscles during exercise.
- blood flow
- endothelium-dependent vasodilation
- skeletal muscle fiber type
age-related alterations in cardiovascular structure and function impair oxygen transport to skeletal muscle and decrease exercise capacity (24, 25, 53). The peripheral circulation is particularly susceptible to the aging process so that alterations at the level of the skeletal muscle (5–7, 9, 18, 36, 40, 47, 48, 66) contribute significantly to the impairments in blood flow and its distribution during contractions (11, 26, 35, 49). The resultant mismatch between muscle oxygen delivery and utilization during metabolic transients in old individuals (8, 29) is considered to play a fundamental role in the decline in exercise performance with aging (54, 55).
Alterations in skeletal muscle vascular reactivity (i.e., the capacity to dilate or constrict) with advanced age are known to vary according to fiber type composition and oxidative capacity (7, 47, 48, 66). Accordingly, we demonstrated previously that aging is associated with a profound redistribution of blood flow away from highly oxidative (slow-twitch oxidative and fast-twitch oxidative glycolytic) toward glycolytic (fast-twitch glycolytic) muscles during submaximal dynamic exercise (19, 49). While likely to have complex and multifactorial causes, evidence from both human and animal studies suggests that age-induced endothelial dysfunction mediated by reduced nitric oxide (NO) bioavailability is a principal contributor to these responses (7, 21, 48, 56, 62, 66). This derives mainly from the fact that, in healthy young rats, highly oxidative muscles place a relatively greater reliance on NO-mediated vasodilation during the transition from rest to exercise compared with predominantly glycolytic muscles (31, 50).
However, it is still unknown whether age-related alterations to pharmacological (e.g., acetylcholine, bradykinin) and/or mechanical (e.g., flow-mediated vasodilation) stimuli in some studies translate to impairments in regional muscle blood flow delivery and distribution during more physiological conditions such as dynamic exercise. Reports that endothelial dysfunction with aging might be agonist-specific (16) cloud this issue. Although isolated blood vessel models provide an invaluable framework for mechanistic assessment of age-related cardiovascular impairments, fiber type differences in recruitment order (3), blood flow patterns (41), and contribution of NO to vasodilation (12, 31, 45, 50) at rest and during exercise require an integrated in vivo approach to investigate regional hemodynamic control. In addition, given that human studies often employ exercise of small muscle mass to limit the influence of cardiac output and the sympathetic nervous system on the modulation of muscle blood flow, the effects of aging on the contribution of NO to skeletal muscle vasomotor control during exercise using a large muscle mass have yet to be investigated systematically. Ethical and technical limitations also preclude determination of the role of NO in regional vascular responses (i.e., blood flow distribution within and among muscles) during exercise in aged humans.
The purpose of this study was to determine the effects of aging on the contribution of NO to systemic [mean arterial pressure (MAP) and heart rate (HR)] and regional (total, inter- and intramuscular hindlimb, renal, and splanchnic) hemodynamic control at rest and during submaximal whole body exercise (treadmill running). Specifically, we examined whether reduced NO contribution to hemodynamic control with aging is involved mechanistically in the blood flow redistribution from highly oxidative to glycolytic muscles and muscle parts from old rats (19, 49). The hypothesis was tested that NO synthase (NOS) inhibition in old rats would result in attenuated reductions in vascular conductance (VC) in muscles composed predominantly of oxidative fibers compared with young rats, thus supporting a role for reduced NO contribution to vasomotor responses in blood flow redistribution within and among muscles of distinct fiber types with advanced age.
Animal selection and care.
Nine young (6–8 mo) and seven old (27–29 mo) male Fisher 344 × Brown Norway (F344 × BN) rats were used in this investigation. These ages represent young adult and senescent rats according to the lifespan of the F344 × BN rodent strain (40). Moreover, the F344 × BN rat has the distinct advantage of not acquiring many of the age-related pathologies observed in their highly inbred counterparts (42). Blood flow, MAP, and HR responses at rest and during exercise without NOS blockade were reported previously (49). The present investigation focuses on the effects of aging on the contribution of NO to regional hemodynamic control in the same animals used in the previous report (49). All experimental procedures were conducted under the guidelines established by the National Institutes of Health and were approved by the Institutional Animal Care and Use Committee of Kansas State University. Rats were maintained on a 12:12-h light-dark cycle with food and water provided ad libitum. Before the initiation of the experimental protocol, rats were familiarized with running on a custom-built motor-driven treadmill over the course of a 2- to 3-wk period (5–10 min/day at a speed of 20 m/min and 5–10% grade). This familiarization protocol has been demonstrated previously not to induce exercise training adaptations [e.g., no changes in peak oxygen consumption (V̇o2peak), endurance capacity, skeletal muscle citrate synthase, and/or succinate dehydrogenase activities] (2, 51).
On the day of data collection, rats were initially anesthetized with 5% halothane gas. Subsequently, while being maintained on a 2% halothane-oxygen mixture, one catheter (PE-10 connected to PE-50) was placed in the ascending aorta via the right carotid artery and another in the caudal (tail) artery as described previously (49, 52). Catheters were tunneled subcutaneously to the dorsal aspect of the cervical region and exteriorized through a puncture wound in the skin. Anesthesia was terminated after closure of the incisions and the animal was given ≥2 h to recover.
Each rat was placed on the treadmill after the surgical recovery period (∼3 h after instrumentation) and the tail artery catheter was connected to a 1-ml syringe and a Harvard pump (model 907). The speed of the treadmill was increased progressively during ∼30 s to a speed of 20 m/min up a 5% grade (49). The rat was required to exercise steadily for another 3 min. After 3.5–4 min of total exercise time, blood withdrawal from the tail artery catheter was initiated at a rate of 0.25 ml/min. Simultaneously, HR and MAP were measured (Gould Statham P23ID) and recorded via the carotid artery catheter. After 4 min of total exercise time, the carotid artery catheter was disconnected from the pressure transducer and ∼0.4 × 106 microspheres (46Sc, 85Sr, 113Sn, or 141Ce in random order; 15-μm diameter; Perkin Elmer) were injected into the aortic arch to determine regional blood flows. Blood withdrawal from the tail artery catheter was stopped ∼30 s after microsphere injection and exercise was terminated. Following a minimum 60-min recovery period, resting hemodynamic parameters were evaluated while each rat sat quietly on the treadmill. A second microsphere injection and blood withdrawal were performed as described above. This sampling strategy minimizes potential influences of the preexercise anticipatory response (1) on hemodynamic measurements.
Subsequently, the l-arginine analog NG-nitro-l-arginine methyl ester (l-NAME, a non-isoform-specific NOS inhibitor; Sigma-Aldrich) was administered via the tail artery catheter at 10 mg/kg. We have demonstrated previously that this dose of l-NAME blunts the hypotensive response to acetylcholine and produces a maximal increase in MAP and decrease in HR without any noticeable alterations in behavior (including willingness to run on the treadmill) (12, 31, 50). The tail artery catheter was then reconnected (as above) and a second bout of exercise was initiated ∼3 min after l-NAME administration. The microsphere injections and blood withdrawal during exercise and at rest were performed exactly as for the control (i.e., without NOS blockade) condition.
Determination of blood flow and vascular conductance.
Following the final microsphere injection, each animal was euthanized with pentobarbital sodium overdose (>50 mg/kg ia). The thorax was opened, and placement of the carotid artery catheter into the aortic arch was confirmed by anatomical dissection. The right and left kidneys, organs of the splanchnic region and individual muscles, and muscles parts of both hindlimbs were identified and removed. The tissues were blotted, weighed, and placed immediately into counting vials.
The radioactivity of each tissue was determined on a gamma scintillation counter (Packard Auto Gamma Spectrometer, model 5230). Taking into account the cross-talk among isotopes, blood flows to each tissue were determined using the reference sample method (52) and expressed as milliters per minute per 100 g. Adequate mixing of the microspheres was verified for each injection by demonstrating a <15% difference between blood flow to the right and left kidneys and to the right and left hindquarter musculature. Blood flow data were normalized to MAP and expressed as vascular conductance (VC; ml·min−1·100 g−1·mmHg−1).
Data comparison was performed using paired or unpaired Student's t-tests, one-way analysis of variance (ANOVA), and two-way repeated-measures ANOVA. Post hoc analyses were conducted with the Student-Newman-Keuls test when a significant F-ratio was detected. Pearson's product-moment correlations and linear regression analyses were performed to examine relationships between variables. Linear regression slope comparisons were performed as described by Zar (70). Muscle fiber type composition was based on the percentage of type I, IIa, IIb, and IId/x fibers in the individual muscles and muscle parts of the rat hindlimb as described by Delp and Duan (14). Significance was accepted at P < 0.05. Results are presented as means ± SE.
As reported previously in these animals, old rats had greater body mass (young: 339 ± 8; old: 504 ± 18 g; P < 0.05) and total hindlimb muscle mass (young: 15.35 ± 0.36; old: 18.31 ± 0.54 g; P < 0.05) compared with their younger counterparts (Ref. 49; individual muscle masses are shown in Supplemental Table, available with the online version of this article). However, total hindlimb muscle mass normalized to body mass was reduced significantly in old (36.43 ± 0.61 mg/g) compared with young (45.32 ± 0.36 mg/g) rats.
The effects of l-NAME administration on HR and MAP at rest and during treadmill exercise in young and old rats are shown in Table 1. Resting MAP was not significantly different in old compared with young rats during the control condition (i.e., without NOS inhibition; Table 1; as reported previously in Ref. 49). At rest, l-NAME increased MAP significantly by ∼17% and 21% in young and old rats, respectively. During exercise, l-NAME increased MAP significantly by ∼13% and 19% in young and old rats, respectively. Given that l-NAME also elicited differences in systemic driving pressure between young and old rats (Table 1), hindlimb muscle and abdominal organ blood flow measurements made at rest and during exercise were normalized to MAP and presented below as VC (absolute blood flow data from individual hindlimb muscles are shown in Supplemental Table).
During the l-NAME condition, no significant differences in MAP were observed between resting measurements made immediately before exercise and those performed following the 60-min recovery period in young (142 ± 6 and 143 ± 6 mmHg, respectively) and old (155 ± 5 and 157 ± 6 mmHg, respectively) rats. This indicates that a similar degree of NOS inhibition was achieved during both exercise and rest protocols within each age group.
Consistent with previous reports (Ref. 15; net infusion of ∼1.5 × 106 microspheres), analyses of HR and MAP data pre- and post-microsphere injections in young and old rats both at rest (young: HR = +3 ± 2%, MAP = −1 ± 2%; old: HR = −3 ± 2%, MAP = +1 ± 1%; P > 0.05) and during exercise (young: HR = −2 ± 1%, MAP = −3 ± 1%; old: HR = +1 ± 1%, MAP = +1 ± 1%; P > 0.05) indicate that accumulation of microspheres with serial injections did not influence successive hemodynamic measurements in the present study.
Effects of l-NAME on hindlimb muscle VC.
Total hindlimb muscle VC was not significantly different between young and old rats at rest and increased to similar levels during exercise in the control condition (Fig. 1). At rest, l-NAME reduced total hindlimb muscle VC significantly by ∼64% and 55% in young and old rats, respectively (Fig. 1). During exercise, l-NAME decreased total hindlimb VC in both young and old rats, but the reductions were attenuated in the old compared with their young counterparts (i.e., down by ∼23% in old vs. 43% in young rats; P < 0.05; Fig. 1). Thus, although affecting the increase in total hindlimb VC (+ΔVC) during the transition from rest to exercise in both young and old rats, l-NAME impaired total hindlimb +ΔVC to a greater extent in young compared with old rats (Fig. 2).
At rest, young and old rats had similar individual hindlimb muscle VC (with the exception of the plantaris, young: 0.06 ± 0.02; old: 0.12 ± 0.02 ml·min−1·100 g−1·mmHg−1; and gracilis, young: 0.08 ± 0.02; old: 0.19 ± 0.05 ml·min−1·100 g−1·mmHg−1; P < 0.05 for both) during the control condition. The decrease in VC (−ΔVC) evoked by l-NAME at rest and during exercise in young and old rats correlated significantly with the percent sum of type I and IIa fibers (%type I + IIa) in the individual muscles or muscle parts of the hindlimb (Fig. 3). Advanced age attenuated the −ΔVC with l-NAME in predominantly oxidative muscles both at rest and during exercise (i.e., different linear regression slopes relative to young rats; P < 0.05).
As illustrated in Fig. 4, the age-dependent effects of l-NAME on VC of muscles of distinct fiber type were observed mainly during exercise. Without NOS blockade, old rats showed relatively lower VC in predominantly oxidative muscles (i.e., >85% type I + IIa fibers: soleus, vastus intermedius, gastrocnemius red) and higher VC in predominantly glycolytic muscles (i.e., 100% type IIb + IId/x fibers: biceps femoris anterior, semimembranosus white, gastrocnemius white; Fig. 4) during exercise compared with young rats, which reflects the profound blood flow redistribution in contracting skeletal muscle with advanced age similar to that reported previously by Musch et al. (49). During exercise, l-NAME administration in young rats reduced VC in muscles possessing a majority of oxidative fibers to similar levels found in the same muscles or muscle parts of old rats without NOS blockade (Fig. 4). In marked contrast to their younger counterparts, old rats showed no significant reductions in VC with l-NAME in these highly oxidative muscles during exercise. Predominantly glycolytic muscles were considerably less affected than highly oxidative muscles by l-NAME at rest and during exercise in young and old rats (Fig. 4, bottom).
During the transition from rest to exercise, l-NAME administration attenuated the +ΔVC in ∼68% (19 of 28) of locomotory muscles analyzed in young rats and 25% (7 of 28) of muscles in old rats (Table 2). NOS blockade attenuated the +ΔVC from rest to exercise in the majority of highly oxidative muscles (i.e., >85% type I + IIa fibers: soleus, gastrocnemius red, and vastus intermedius) in young but not old rats (Table 2). More specifically, l-NAME had no effects on +ΔVC from rest to exercise in muscles containing ≥65% type I + IIa fibers (i.e., soleus, gastrocnemius red, vastus intermedius, vastus lateralis red, adductor longus) from old rats (Table 2). Moreover, the majority of muscles or muscle parts possessing a low percentage of oxidative fibers (i.e., 100% type IIb + IId/x fibers: gastrocnemius white, vastus lateralis white, biceps femoris anterior and semimembranosus white) showed greater increases in VC from rest to exercise in old compared with young rats during both control and l-NAME conditions (Table 2). The relationships between the effects of l-NAME on +ΔVC during the transition from rest to exercise in young and old rats and %type I + IIa fibers associated with the individual muscles or muscle parts of the hindlimb are depicted in Fig. 5. Accordingly, NOS blockade attenuated the +ΔVC of highly oxidative muscles from rest to exercise in young rats but did not change the relationship in old rats (i.e., similar linear regression slopes pre- and post-l-NAME administration; P > 0.05).
Effects of l-NAME on abdominal organ VC.
Old rats had lower total abdominal organ (i.e., kidneys, stomach, adrenals, spleen, pancreas, small and large intestines) VC during the control condition compared with young rats both at rest (young: 34 ± 3; old: 20 ± 1 ml·min−1·100 g−1·mmHg−1; P < 0.05) and during exercise (young: 23 ± 2; old: 9 ± 1 ml·min−1·100 g−1·mmHg−1; P < 0.05).
Absolute decreases in kidney and splanchnic organ VC (−ΔVC) evoked by l-NAME at rest and during exercise in young and old rats are shown in Fig. 6. Advanced age attenuated the −ΔVC with l-NAME in the majority of the organs examined from the abdominal region both at rest and during exercise. This resulted in attenuated total abdominal organ −ΔVC with l-NAME in old compared with young rats both at rest (young: −26 ± 3; old: −13 ± 1 ml·min−1·100 g−1·mmHg−1; P < 0.05) and during exercise (young: −15 ± 1; old: −4 ± 1 ml·min−1·100 g−1·mmHg−1; P < 0.05).
Because old rats demonstrated lower VC in a number of these organs at rest during the control condition compared with young rats (i.e., kidneys, stomach, adrenals, spleen; see also Fig. 3 in Ref. 49), it is important to consider the relative decrease in VC (i.e., −%ΔVC) induced by l-NAME administration. At rest, l-NAME resulted in similar −%ΔVC in all abdominal organs from young and old rats except the stomach (young: −87.2 ± 2.5; old: −68.7 ± 5.1%; P < 0.05). During exercise, old rats displayed attenuated −%ΔVC with l-NAME in the kidneys, stomach, spleen, and small intestine relative to their younger counterparts (data not shown). Similar to when the data are expressed in absolute terms, old rats showed attenuated total abdominal organ −%ΔVC with l-NAME relative to young rats both at rest (young: −74 ± 3; old: −62 ± 4%; P < 0.05) and during exercise (young: −63 ± 3; old: −48 ± 7%; P < 0.05).
The present study is the first to determine the effects of aging on the contribution of NO to systemic and regional hemodynamic control at rest and during submaximal whole body exercise. The main novel findings were that, relative to young rats, NOS inhibition with l-NAME in old rats induced attenuated reductions in 1) total hindlimb VC during exercise but not at rest, 2) VC in individual muscles or muscle parts composed primarily of oxidative fibers both at rest and during exercise, and 3) VC in the kidneys and the majority of the splanchnic organs examined both at rest and during exercise. These results indicate that the contribution of NO to the increase in VC during the transition from rest to exercise is markedly reduced in highly oxidative muscles of old compared with young rats. Furthermore, age-related blood flow redistribution within and among muscles comprised of different fiber types during exercise may be ascribed, at least in part, to reduced NO contribution to hemodynamic control.
Effects of aging on the contribution of NO to vasomotor control.
Contrary to our hypothesis, NOS inhibition with l-NAME resulted in similar decreases in total hindlimb VC in young and old rats at rest (Fig. 1). Although these findings suggest that NO subserves a similar role in total hindlimb VC control in young and old rats at rest, it is important to note that aging altered the relationship between −ΔVC evoked by l-NAME at rest and the %type I + IIa fibers in the individual muscles or muscle parts of the hindlimb (Fig. 3, top). This indicates a reduced contribution of NO to resting vasomotor control in predominantly oxidative muscles from old rats relative to their younger counterparts and highlights the importance of examining regional hemodynamic responses.
During exercise, attenuated reductions in total hindlimb VC following l-NAME administration in old compared with young rats (Fig. 1) are consistent with our hypothesis and demonstrate that aging reduces the contribution of NO to functional hyperemia (see also Refs. 13, 56). Combined with similar total hindlimb VC levels reached during exercise in young and old rats without NOS blockade (Fig. 1), these results demonstrate that the relative importance of distinct vasodilatory pathways varies as a function of age. Accordingly, attenuated −ΔVC with l-NAME in predominantly oxidative muscles from old compared with young rats during treadmill running (Fig. 3, bottom) reveals that the contribution of NO to vasomotor responses to exercise in these muscles is severely reduced with aging.
The age-induced redistribution of blood flow among muscles of different fiber type reported previously by Musch et al. (49) is illustrated in Fig. 4 for representative muscles spanning the range of fiber type composition. Old rats exercising without NOS inhibition presented lower VC in predominantly oxidative muscles (i.e., >85% type I + IIa fibers: soleus, vastus intermedius, gastrocnemius red) and higher VC in predominantly glycolytic muscles (i.e., 100% type IIb + IId/x fibers: biceps femoris anterior, semimembranosus white, gastrocnemius white) compared with young rats. As expected, NOS inhibition had strikingly different effects on highly oxidative and glycolytic muscles of young and old rats. Consistent with the relatively greater reliance on endothelium-dependent vasodilation to power oxidative metabolism by oxidative fibers (31, 45), l-NAME administration in young rats induced greater reductions in VC in highly oxidative compared with glycolytic muscles during exercise (Fig. 4). Interestingly, NOS inhibition during exercise 1) reduced VC in highly oxidative muscles from young rats to similar levels found in old rats without l-NAME, 2) had no effect on VC of highly oxidative muscles from old rats, and 3) had either no effect (e.g., semimembranosus white, gastrocnemius white) or decreased VC of highly glycolytic muscles from old rats to similar levels found in young rats without l-NAME (e.g., biceps femoris anterior; Fig. 4). These data suggest that reduced NO contribution to hemodynamic control is implicated in age-related blood flow redistribution within and among muscles of distinct fiber types during exercise.
Analyses of the relationships between the effects of l-NAME on +ΔVC during the transition from rest to exercise and the %type I + IIa fibers in the hindlimb muscles of young and old rats (Fig. 5) provide useful information pertaining to the contribution of NO to functional hyperemia in the individual muscles of the hindlimb. In marked contrast with their younger counterparts, the unaltered slope of this relationship in old rats with l-NAME indicates that 1) the dependency of predominantly oxidative muscles on NO-mediated vasodilation during exercise is practically abolished; and 2) that mechanisms other than NO are responsible for the bulk of augmented skeletal muscle VC during the transition from rest to exercise with advanced age (e.g., endothelium-derived hyperpolarizing factor, EDHF; Refs. 33, 58). In fact, NOS inhibition in old rats impaired the +ΔVC from rest to exercise in only 7 of 28 muscles (notably none of these muscles affected by l-NAME contained ≥65% type I + IIa fibers; Table 2). On the other hand, NOS inhibition in young rats attenuated the +ΔVC from rest to exercise in 19 of 28 muscles (including highly oxidative muscles or muscle parts such as soleus, gastrocnemius red, and vastus intermedius, which contain >85% type I + IIa fibers; Table 2).
Attenuated reductions in total abdominal organ VC (expressed in absolute and relative terms) evoked by l-NAME at rest and during exercise in old compared with young rats is consistent with age-related diminished NO bioavailability in the renal (20, 32, 43, cf. 28, 63) and mesenteric (61, 69, cf. 71) circulations. Decreased contribution of NO to splanchnic vasomotor control with advanced age could partly account for lower total abdominal organ VC at rest and during exercise in old rats (Fig. 6; see also Fig. 3 in Ref. 49). This likely reduces the capacity to divert blood flow away from those tissues toward the contracting skeletal muscle and could impair exercise performance (2). Nevertheless, it is possible that potential differences in relative work rate may have influenced the splanchnic hemodynamic responses to exercise in old animals (see discussion below concerning the skeletal muscle).
Taken together, our data suggest that reduced NO contribution to hemodynamic control constitutes an integral component of age-related muscle blood flow redistribution during exercise. Specifically, reduced VC in predominantly oxidative muscles during exercise in aged rats might be attributed mainly to reduced NO participation in the hyperemic response (Figs. 3 and 4, Table 2). That l-NAME administration in young rats does not produce the redistribution pattern characteristic of old rats (i.e., ↓VC in oxidative muscles concomitant with ↑VC in glycolytic muscles; Fig. 4) suggests the involvement of multiple mechanisms in this process. Potential candidates include alterations in vascular control in predominantly glycolytic muscles (e.g., impaired myogenic vasoconstriction; Ref. 47) and changes in recruitment pattern of locomotor muscles during exercise (19, 49). The latter is presumably the result of rapid fatigue in highly oxidative fibers due to reduced oxygen delivery (mediated mainly via diminished NO contribution; present study) that requires recruitment of additional glycolytic fibers to sustain exercise (3, 4).
In contrast to these altered hemodynamic responses observed in old rats, Armstrong and Laughlin (2) reported that exercise training in young rats induces a preferential redistribution of blood flow away from highly glycolytic toward oxidative muscles during treadmill running. This adaptation is thought to derive, at least in part, from upregulation of endothelial and/or NOS function (54). Consequently, pharmacological (e.g., antioxidants, tetrahydrobiopterin; Refs. 21, 22, 27, 30, 58, 65, 67) and/or nonpharmacological (e.g., exercise training; Refs. 13, 17, 46, 58–60, 64) interventions aiming at improving endothelial function and/or NO bioavailability could mitigate age-related blood flow redistribution and increase exercise capacity.
We and others have employed the microsphere technique to assess the control of vascular tone in vivo in a variety of experimental conditions (1, 2, 12, 19, 25, 30, 31, 35, 41, 45, 46, 49–52, 63). The present protocol involved a total of four microsphere injections per animal to measure regional hemodynamic responses to exercise and l-NAME administration. Importantly, as mentioned above and in agreement with Delp et al. (15), accumulation of microspheres did not induce hemodynamic disturbances that could influence our results. This strategy thus provides the unique opportunity to determine effectively individual changes in muscle VC (Figs. 2, 3, 5, and 6, Table 2) and gain insights into the effects of aging on the contribution of NO to regional muscle hemodynamic control at rest and during exercise.
Histochemical data describing the fiber type composition of young Sprague-Dawley rat muscles (14) were used herein to characterize the %type I + IIa fibers in individual muscles or muscle parts of both young and old F344 × BN rats. To the best of our knowledge, no previous studies have evaluated systematically the effects of aging on fiber type composition of all hindlimb muscles from F344 × BN rats. Notwithstanding possible strain differences, comparison between representative hindlimb muscles of young and old F344 × BN rats within the age range used in the present study revealed no significant differences in fiber type composition (44; see also discussion in Ref. 10).
In the present experimental protocol, young and old rats exercised at the same absolute work rate (i.e., treadmill running at 20 m/min up a 5% grade) based on the fact that both young and aged individuals usually perform daily work tasks that are equivalent to one another in absolute terms (e.g., walking on the street, climbing a flight of stairs). As discussed in detail previously (19, 49), potential differences in relative work rates, body mass, and muscle mass cannot explain the redistribution of blood flow away from highly oxidative to glycolytic muscles in aged rats. Specifically, given that rat hindlimb muscle blood flows do not increase markedly (and not at all in some instances) with running speeds from 15 to 45 m/min (a range that comprises both absolute and relative exercise intensities of young and old rats in this study) (41, 49), it is unlikely that distinct relative work rates induced the redistribution pattern observed in aged rats. Furthermore, a higher relative intensity would be associated with greater VC in predominantly oxidative muscles, which is essentially the opposite of the effect seen herein. Although greater VC in predominantly glycolytic fibers from old rats is consistent with higher relative work rates, data from Laughlin and Armstrong (41) indicate that these muscles do not exhibit major increases in blood flow (and by extension VC) until the rat is running at speeds greater than 45 m/min.
Considering that the contribution of NO to the hyperemic response to treadmill running appears to be progressively greater with increasing work rates (e.g., rats, Ref. 50; dogs, Ref. 57), greater absolute reductions in muscle VC with l-NAME are expected during higher exercise intensities. Therefore, attenuated reductions in skeletal muscle VC following l-NAME administration in aged rats are also unlikely to derive from potential differences in relative work rate.
We have reported previously that varying the timing of NOS inhibition (i.e., l-NAME administration prior vs. during exercise) alters the fiber type dependency of NO-mediated vasomotor control in healthy young rats (12). This suggests that the present experimental protocol could overestimate the dependency of highly oxidative muscles on NO-mediated vasodilation during exercise. However, as noted in that study, performing NOS inhibition prior to exercise (as done herein) may be more appropriate when examining vasomotor control in conditions associated with chronic reductions in NO bioavailability such as aging.
In addition to the modulation of peripheral vascular tone, NOS inhibition could influence skeletal muscle hemodynamic control via modulations in cardiac function. Accordingly, l-NAME administration is reported to reduce cardiac output in a variety of in vivo animal models (e.g., rats, Ref. 34; horses, Ref. 37; dogs, Ref. 38). Although evidence in the isolated rat heart preparation suggests that cardiac contractility is sensitive to altered NO levels (23, 39), reduced cardiac output during whole body measurements following l-NAME administration might result in part from increases in afterload and/or activation of baroreflex mechanisms. Nonetheless, irrespective of putative changes in cardiac function with l-NAME, it is difficult to conceive how altered cardiac output would affect preferentially oxidative muscles and produce the redistribution pattern observed in aged rats herein.
Given the oxidative stress that develops with advanced age, it is important to consider that l-NAME administration in old rats could impact vascular control not only via reductions in NO production (i.e., inhibition of NOS) but also via reductions in superoxide radical generation (i.e., inhibition of uncoupled NOS) (68).
This study demonstrates that the contribution of NO to regional (total, inter-, and intramuscular hindlimb) hemodynamic control from rest to exercise is severely compromised with advanced age. Specifically, old rats had significantly attenuated reductions in VC with NOS inhibition in highly oxidative muscles during exercise compared with their younger counterparts. Our data provide important mechanistic insights into age-related alterations in skeletal muscle hemodynamic control by indicating that reduced NO contribution to vascular responses in aged rats partly underlies blood flow redistribution from highly oxidative to glycolytic muscles during exercise (19, 49).
This project was supported in part by a Fellowship from the Brazilian Ministry of Education/CAPES-Fulbright to D. M. Hirai; a Kansas State University SMILE Grant to D. C. Poole; and American Heart Association Heartland Affiliate Grant 0750090Z to T. I. Musch and 10GRNT4350011 to D. C. Poole.
No conflicts of interest, financial or otherwise, are declared by the author(s).
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