Aging blunts the dynamics of vasodilation in isolated skeletal muscle resistance vessels

Abstract

Aging is associated with an altered ability to match oxygen delivery (Qo₂) to consumption (V̇o₂) in skeletal muscle and differences in the temporal profile of vasodilation may provide a mechanistic basis for the Qo₂-to-V̇o₂ mismatching during the rest-to-exercise transition. Therefore, we tested the hypothesis that the speed of vasodilation will be blunted in skeletal muscle first-order arterioles from old vs. young rats. Arterioles from the soleus and the red portion of the gastrocnemius (Gast_Red) muscles were isolated from young (Y, 6 mo; n = 9) and old (O, 24 mo; n = 9) Fischer 344 rats and studied in vitro. Vessels were exposed to acetylcholine (ACh; 10⁻⁶ M), sodium nitroprusside (SNP; 10⁻⁴ M), and increased intraluminal flow, and the subsequent vasodilation was recorded at 30 frames/s. The data were fit to a monoexponential model and the dynamics of vasodilation [i.e., time delay, time constant (tau), and rate of change (delta/tau)] were calculated. With old age, the rate of vasodilation was significantly blunted in resistance vessels from the soleus to ACh (Y, 27.9 ± 3.6; O, 8.8 ± 2.6 μm/s) and flow (Y, 12.8 ± 2.1; O, 3.1 ± 0.9 μm/s). In the Gast_Red the old age-associated impairment of endothelium-dependent vasodilator dynamics was even greater than that of the soleus. With SNP neither the magnitude nor time constant of vasodilation was affected by age in either muscle. The results indicate that aging impairs the dynamics of vasodilation in resistance vessels from the soleus and Gast_Red muscles mediated, in part, through the endothelium. Thus the old age-associated slower rate and magnitude of vasodilation could inhibit the delivery of O₂ during the critical transition from rest to exercise in moderate to highly oxidative skeletal muscle.

aging is associated with a decline in maximal aerobic capacity (V̇o_{2 max}) (41–43, 56) due, in part, to the reduced ability of the cardiovascular system to provide adequate blood flow to the active muscles. Specifically, with advancing age there is a lower bulk blood flow (21, 29, 47, 61) and an altered distribution of perfusion within muscle (39) during physical activity. Recently, Behnke et al. (6) have further demonstrated a temporal mismatching of oxygen delivery (Qo₂) to oxygen consumption (V̇o₂) across the rest-to-contractions transition in the spinotrapezius muscle of old rats, resulting in a transiently lowered microvascular Po₂. These altered microvascular Po₂ dynamics will, according to Fick's law, impair blood-myocyte O₂ transfer in the elderly. Using the modeling techniques of Barstow and Móle (3), the old age-related microvascular Po₂ response described by Behnke et al. (6) can arise only if blood flow dynamics are compromised (increased time constant) vs. the normal hyperemic time course observed in skeletal muscle from young subjects (31). There are several mechanisms responsible for the old age-associated perturbations of muscle blood flow, including a reduction in the number of feed arteries (5) and an impairment of endothelium-dependent vasodilation (20, 37, 55, 62) mediated by a reduced bioavailability of nitric oxide (NO) (18). These changes in resistance artery structure and function with advancing age culminate in significant impairments in capillary hemodynamics at rest (46) and during exercise (14). Despite the resolution of several mechanisms of the old age-related vasomotor dysfunction, the effects of aging on the dynamic responses of resistance arterioles (i.e., the speed of vasodilation) remain unknown.

Given the quintessential role of targeted arterial vasodilation in the matching of Qo₂ to V̇o₂ during the exercise on-transient, the purpose of this investigation was to quantify the effects of aging on skeletal muscle resistance artery vasomotor dynamics. We hypothesized that the speed of vasodilation will be blunted in arterioles from oxidative skeletal muscles from old vs. young animals. To test this hypothesis, resistance arterioles from the soleus and the red portion of the gastrocnemius (Gast_Red), two highly oxidative muscles (19), were studied since this muscle type demonstrates large old age-associated deficits in blood flow during exercise (39). In addition, with old age, blood flow is elevated to several low oxidative muscles of primarily fast glycolytic fiber type composition (39). Therefore, to further determine how aging affects vasomotor function, a second set of experiments was performed to quantify the speed of endothelium-dependent vasodilation in the low oxidative white portion of the gastrocnemius (Gast_White) muscle. Given the key role of NO in regulating resting vasomotor tone (28, 48, 49) and our interest in the dynamic transition from a resting state to a steady-state vasodilation, these studies focus primarily on NO-mediated vasodilation.

METHODS

All procedures performed in this study were approved by the University of Florida Institutional Animal Use and Care Committee. Twelve young (6 mo old; 382 ± 15 g) and 12 old (24 mo old; 421 ± 14 g) male Fischer 344 rats were obtained from the National Institute on Aging colony. The rats were housed in a temperature-controlled (23 ± 2°C) room with a 12:12-h light-dark cycle. Water and rat chow were provided ad libitum.

Arteriolar preparation.

Animals were anesthetized with pentobarbital sodium (85 mg/kg ip) and killed by exsanguination. The gastrocnemius-plantaris-soleus muscle complex was carefully excised from each leg and placed in cold (4°C) physiological saline solution (PSS). From the soleus and Gast_red muscles, 1A arterioles (i.e., resistance arterioles, intraluminal diameter 85–205 μm, length 0.5–1.0 mm), defined as the first branch off the feed artery that perforates the muscle, were isolated. The arterioles were cleared of surrounding muscle fibers, removed from the muscle, and cannulated on both ends to glass micropipettes. After cannulation, the arterioles were transilluminated by an inverted microscope (Olympus IX71) equipped with a video camera (Orca-HR, Hamamatsu) attached to a digital video recorder (Sony DSR-30 DVR). Resistance arterioles were initially pressurized to 60 cmH₂O with two independent hydrostatic pressure reservoirs. Leaks were detected by pressurizing the vessel and determining whether vessel diameter was maintained. Arterioles that exhibited leaks were discarded. Arterioles were warmed to 37°C and allowed to develop spontaneous tone during a 30- to 60-min equilibration period.

Assessment of vasomotor time courses followed similar methods as previously described (13, 63). Briefly, on displaying a steady level of spontaneous tone, vessels were exposed to either acetylcholine (ACh: 5 × 10⁻⁸ and 1 × 10⁻⁶ M), increased intraluminal flow, or sodium nitroprusside (SNP: 1 × 10⁻⁴ M), and images were time referenced by frame and stored on digital media (Maxell Mini DV, DVM60SE) for off-line analysis via digital video recorder (Sony DSR-30 DVR). Both ACh and SNP were administered via a microinjector placed near the vessel wall. The increase in intraluminal flow was accomplished by altering the heights of the independent fluid reservoirs in equal and opposite directions so that a pressure difference was created across the vessel without altering mean intraluminal pressure. In a preliminary set of experiments vasodilation dynamics to two rates of flow were investigated. Specifically, diameter measurements were determined in response to pressure differences from the static state to 10 (n = 5) or 60 (n = 5) cmH₂O to represent low and high intraluminal flow rates, respectively. No difference in the temporal profile of vasodilation was observed between the two flow rates. Therefore, flow data presented herein are from a pressure difference of 10 cmH₂O, which corresponds to an intraluminal flow rate of 13.4 ± 0.5 nl/s (37). On completion of the dynamics study, the vessel was incubated in calcium-free PSS solution for 60 min to determine maximal passive diameter (37).

Based on the results from the high oxidative skeletal muscle and the findings of Musch et. al. (39) of an old age-associated increased blood flow to low oxidative glycolytic muscles during exercise, an additional protocol was added to quantify the effects of aging on the dynamics of vasodilation in the low oxidative Gast_White muscle. Arterioles (young, n = 7; old, n = 6) were isolated from the superficial portion of the Gast_White and, on displaying a steady level of spontaneous tone, exposed to ACh (1 × 10⁻⁶ M); images were recorded as described above.

Off-line analysis.

To determine the rate of dispersion of a given compound in the buffer solution surrounding the vessel, India ink was initially infused through the microinjector during high-speed video recording. It was found that dispersion of the compound was complete within three frames or 1/10th of a second. Changes in arteriolar luminal diameter with exposure to ACh, increased intraluminal flow, and SNP were determined during frame-by-frame playback (30 frames/s) using a video caliper (Colorado Video, 307A, Boulder, CO). Subsequently, the time-diameter values were curve-fit to a monoexponential plus delay model (8) using an iterative least-squares technique by means of a commercial graphing/analysis package (KaleidaGraph 3.5).

Vasodilation was expressed as a percentage of maximal dilation using the following equation:

$$\%\mathrm{Relaxation}=(D_{\mathrm{t}}-D_{\mathrm{i}}/D_{\mathrm{m}}-D_{\mathrm{i}})\times 100$$

where D_t is the diameter recorded at a given time point, D_i is the diameter recorded immediately before addition of the vasoactive agent (i.e., initial diameter), and D_m is the maximal diameter recorded.

Statistics.

For the KaleidaGraph analysis program a user-defined function to the data was fit using the following equation:

$${\mathrm{Diameter}}_{\mathrm{t}}={\mathrm{Diameter}}_{\mathrm{b}}+\Delta {\mathrm{Diameter}}_{\mathrm{ss}}(1-e^{-(t-\mathrm{TD})\mathrm{/}\tau })$$

where Diameter_t is the change in diameter at time t, Diameter_b is baseline diameter, ΔDiameter_ss is the change in diameter from baseline (preintervention) to the steady-state value, TD is the time delay, and τ is the time constant of the response, which estimates the time taken to reach 63% of the final exponential response. The same model was applied to diameter responses of arterioles from the soleus, Gast_Red, and Gast_White of young and old animals. From the mathematical modeling results the rate of vasodilation [change in diameter (Δ)/τ] and the time to reach a steady-state diameter (4τ) were calculated.

A two-way ANOVA was used to determined differences in vessel characteristics and vessel dynamics data between the young and old groups and between the soleus and gastrocnemius muscles. When a significant F value was demonstrated a Student-Newman-Keuls (SNK) post hoc test was performed to determine differences among mean values. Significance was accepted at P ≤ 0.05. Values are presented as means ± SE.

RESULTS

Vessel characteristics.

Vessel characteristics are shown in Table 1. There was no difference in maximal diameter between age groups for the soleus and a tendency (P = 0.09) for a greater maximal diameter in the Gast_Red from the old age group. The level of spontaneous tone before each intervention is shown in Table 1. Consistent with previous findings in soleus muscle arterioles (37), there was no significant difference in the level of spontaneous tone between the young and old groups. In the Gast_Red the initial diameter after the equilibration period was significantly smaller in young; however, spontaneous tone was not different between the two groups (Table 1).

Table 1.

Characteristics of resistance arterioles from the soleus and red portion of the gastrocnemius muscles of young and old animals

	Soleus Muscle		GastRed Muscle
	Young (n = 9)	Old (n = 9)	Young (n = 9)	Old (n = 9)
Maximal diameter, μm	135±9	139±7	158±14	176±18*
Spontaneous tone, %
Flow	40±4	39±3	36±5	37±4
ACh	41±5	38±6	38±3	34±4
Nitroprusside	43±4	41±5	39±5	39±6

Values are means ± SE. Gast_Red, red portion of the gastrocnemius; maximal diameter, diameter measured in Ca²⁺-free solution.

^*P < 0.1 vs. corresponding young diameter.

Vasodilator dynamics to increased intraluminal flow.

The magnitude of vasodilation to an increase in intraluminal flow of 13.4 ± 0.5 nl/s was reduced in resistance arterioles of both the soleus (Fig. 1A) and Gast_Red (Fig. 2A) muscles from old vs. young animals. After the increase in intraluminal flow, the time to reach a steady-state diameter was over twice as long in arterioles from the soleus muscle, and almost three times as long in those from the Gast_Red muscle from old animals (Table 2). In resistance arterioles from both muscles of the old animals, the time delay before a statistically significant change in intraluminal diameter occurred was increased ∼170% (Table 2). The time constant of vasodilation to flow was significantly longer in the Gast_Red vs. the soleus in the old group (Table 2). The slower dynamics and smaller magnitude of vasodilation culminated in a reduced rate of vasodilation (i.e., delta/time constant) with old age in both the soleus (old 3.1 ± 0.9 vs. young 12.8 ± 2.1 μm/s) and Gast_Red (old 1.9 ± 0.7 vs. young 15.7 ± 1.9 μm/s) muscles.

Fig. 1.

Vasodilator dynamics of arterioles from the soleus muscle of young and old rats to flow (A), ACh (B), and sodium nitroprusside (SNP; C). Exposure to a given condition began at time 0. Dashed lines represent the 95% confidence intervals. *P < 0.05.

Fig. 2.

Vasodilator dynamics of arterioles from the red portion of the gastrocnemius muscle (Gast_Red) of young and old rats to flow (A), ACh (B), and SNP (C). Exposure to a given condition began at time 0. Dashed lines represent the 95% confidence intervals. *P < 0.05.

Table 2.

Temporal characteristics of resistance arterioles from the soleus and red portion of the gastrocnemius (Gast_Red) muscles from young and old animals

	Soleus Muscle		GastRed Muscle
	Young	Old	Young	Old
Flow	n = 9	n = 8	n = 8	n = 8
Vasodilation, %	42±9	18±8*‡	48±7	12±7*‡
Time to S-S, s	8.6±1.6	17.5±2.7*‡	8.8±1.5	22.1±3.4*‡
Time delay, s	1.9±0.4	5.1±1.1*	2.0±0.4	5.5±0.9*‡
Tau, s	1.7±0.3	3.1±0.8*‡	1.7±0.4	4.2±1.1*‡
Delta, μm	22±6‡	9±4*‡	27±6‡	8±3*‡
ACh	n = 9	n = 9	n = 9	n = 9
Vasodilation, %	81±6	54±8*‡	67±7	25±8*‡
Time to S-S, s	9.5±1.2	17.9±2.6*‡	8.4±0.5	20.0±3.0*‡
Time delay, s	3.1±0.4	4.4±1.6	2.5±0.2	4.5±1.6*
Tau, s	1.6±0.4	3.4±0.7*‡	1.5±0.3	3.9±0.6*‡
Delta, μm	45±6	30±7*‡	40±6	15±5*‡
Nitroprusside	n = 9	n = 9	n = 9	n = 9
Vasodilation, %	74±9	70±6	72±6	68±7
Time to S-S, s	7.8±1.1	11.2±2.0*	8.2±1.3	11.3±2.4*
Time delay, s	2.0±0.6	3.2±1.1†	2.0±0.5	3.8±1.1*
Tau, s	1.5±0.5	2.0±1.1	1.6±0.2	1.9±0.7
Delta, μm	43±5	40±4	44±6	47±8

Values are means ± SE. Vasodilation, percent difference between the steady-state (postintervention) and initial diameter; time to S-S, time taken to reach a stable (i.e., steady-state) diameter after intervention; time delay, initial delay before a significant change in diameter; tau, time taken to reach 63% of the final steady-state diameter

^*P < 0.05 vs. corresponding young value.

^†P < 0.1 vs. corresponding young value.

^‡P < 0.05 vs. value from sodium nitroprusside.

Vasodilator dynamics to ACh.

In response to 5 × 10⁻⁸ M ACh there was no significant change in intraluminal diameter vs. initial diameter in either group (data not shown). Therefore, dynamics data are presented only for the 1 × 10⁻⁶ M dose of ACh, which elicited a significant vasodilation in arterioles from both muscles in young and old animals (Figs. 1B and 2B). The magnitude of vasodilation to ACh was reduced in resistance arterioles from both the soleus (Fig. 1B) and Gast_Red (Fig. 2B) muscles of old vs. young animals. The temporal responses of vasodilation to ACh are demonstrated in Figs. 1B and 2B and reported in Table 2. In the soleus muscle, the time delay before the onset of vasodilation to ACh was not significant between young (3.1 ± 0.4 s) and old (4.4 ± 1.6 s; P = 0.16), whereas the time delay was less in the Gast_Red of young (2.5 ± 0.2 s) vs. old (4.5 ± 1.6 s; P < 0.05) animals. The time constant of vasodilation from both muscles was over twice as long in the old vs. young group (Table 2). The blunted dynamics in the resistance arterioles of the old group resulted in a longer time to attain a steady-state luminal diameter in both the soleus (old 17.1 ± 2.8 vs. young 9.4 ± 1.1 s; P < 0.05) and Gast_Red (old 20.1 ± 3.2 vs. young 7.8 ± 0.5 s; P < 0.05) muscles. The rate of vasodilation as a function of the exponential response was reduced in old compared with young in both the soleus (old 8.8 ± 2.6 vs. young 27.9 ± 3.6; μm/s; P < 0.05) and Gast_Red (old 3.8 ± 1.3 vs. young 26.9 ± 4.3; μm/s; P < 0.05) muscles.

Vasodilator dynamics to SNP.

In response to the NO donor SNP (1 × 10⁻⁴ M), the magnitude of vasodilation was not different between young and old in either muscle (Figs. 1C and 2C; Table 2). In the soleus muscle, there was a trend (P < 0.1) for an increased time delay before the onset of vasodilation to SNP in the old vs. young group, whereas the time delay was significantly longer in the Gast_Red with old age (Table 2). There was no age-associated difference in the time constant of vasodilation in either muscle. The rate of change was not different depending on age in either the soleus (old 22.3 ± 4.6 vs. young 27.5 ± 4.5 μm/s) or the Gast_Red (old 25.1 ± 4.7 vs. young 27.4 ± 5.1 μm/s) muscles.

Vasodilation dynamics in the Gast_White.

From the second series of investigations the maximal diameter of arterioles from the Gast_White was significantly greater in old (192 ± 18 μm) vs. young (158 ± 12 μm), but consistent with previous findings (37) the percent relaxation to ACh was not different between age groups (Fig. 3). With exposure to ACh, there were no age-associated differences in the time delay (old 3.2 ± 0.7; young 3.0 ± 0.6 s), time constant (old 2.4 ± 0.7; young 2.2 ± 0.6 s), or rate of vasodilation (old 28.6 ± 9.3; young 27.1 ± 8.2 μm/s).

Fig. 3.

Vasodilator dynamics of arterioles from the low oxidative white portion of the gastrocnemius muscle (Gast_White) of young and old rats to the endothelium-dependent vasodilator ACh. No age-related differences in the dynamics of vasodilation or the percent relaxation were observed.

DISCUSSION

Previous work has demonstrated that aging results in a temporal mismatching of Qo₂ to V̇o₂ in skeletal muscle during the rest-to-exercise transition that results in a low microvascular Po₂ (6). Modeling analysis indicates that this old age-induced transient reduction in microvascular Po₂ can only arise through a delay in the muscle hyperemia at the onset of contractions. However, to date there have been no studies describing whether aging alters vasodilation dynamics in isolated resistance arterioles. Therefore, the primary purpose of this study was to determine whether there is an old age-related blunting of the dynamics of vasodilation in resistance arterioles from highly oxidative skeletal muscle. The results demonstrate that with advancing age there is a prolonged time taken to reach a steady-state diameter and a reduced magnitude of endothelium-dependent vasodilation in resistance arterioles from both the soleus and Gast_Red muscles. The key finding of this study is a decrease of >70% and >85% in the rate of endothelium-dependent vasodilation in resistance arterioles from the soleus and Gast_Red muscles of old animals, respectively. Conversely, there were no age-related differences in the rate or magnitude of vasodilation from either muscle to the endothelium-independent NO donor SNP. The latter suggests that neither vascular structural nor mechanical properties can account for the slower rate of endothelium-dependent vasodilation in arterioles from old rats. The finding of an old age-associated blunting of endothelium-dependent vasodilator dynamics provides a plausible mechanism for the rapid decline in microvascular Po₂ across the exercise on-transient (6) and the greater depletion of high-energy phosphates (57) in older individuals. An additional study utilizing the low oxidative Gast_White muscle was performed to investigate whether aging blunts the dynamics of vasodilation uniformly across muscle fiber types. Neither the speed nor magnitude of endothelium-dependent vasodilation was affected by age in the Gast_White (Fig. 3). Compared with the Gast_White, the speed of vasodilation in arterioles from the soleus and Gast_Red muscles was faster in the young and slower in the old groups, suggesting that old age-related alterations in the speed of vasodilation are fiber type specific.

Time course of vasodilation.

The time course of exercise hyperemia has been of interest for well over a century since Gaskell (23) postulated a metabolite-associated vasodilator mechanism. However, the mechanistic bases for the rapid increase in blood flow with contractions remains unknown and is a matter of some debate, although it is likely a combination of the mechanical augmentation of blood flow (i.e., muscle pump) in skeletal muscle and vasodilation (for review, see Refs. 11, 17, 58). However, to our knowledge the literature contains no previous reports of the dynamics of isolated resistance arteriolar vasodilation from aged skeletal muscle.

At the onset of muscular contractions there is an immediate increase in red blood cell (RBC) velocity through the capillary bed (31) and feed arteries (60), whereas RBC flux does not increase quite as rapidly (31). There is disagreement regarding the latency before the onset of arteriolar vasodilation, with values ranging from 1–2 s (4), 3–6 s (36), and 2–18 s (35), up to >20 s (25). In isolated arterioles we have consistently observed a time delay of ∼3–4 s before the onset of vasodilation (present study and 63). In contrast, in isolated soleus muscle feed arteries from young animals, Clifford et al. (12) have demonstrated that mechanical deformation of the vessel by external compression elicits an immediate vasodilation that is complete within 3–4 s. The differences between these studies are likely due, in part, to dissimilar vasodilator stimuli (i.e., mechanical compression vs. pharmacological agents) and the spatially dependent physiochemical milieu of feed arteries (extramuscular) vs. resistance arterioles (i.e., intramuscular). For example, in smaller arterioles (i.e., 20- to 30-μm luminal diameter) located within the gluteus maximus muscle (and therefore subject to large changes in intramuscular pressure), after the onset of muscular contractions there is a clear delay before the onset of vasodilation (4).

With advancing age much less is known regarding the time course of exercise hyperemia. There is an old age-related deficit in steady-state blood flow (44) and vascular conductance (33) during dynamic leg exercise and electrically induced muscular contractions (27). Recently, Copp et al. (14) have investigated the effects of aging on the capillary hemodynamic response to muscular contractions. With advancing age these researchers found virtually no increase in RBC flux (index of O₂ delivery) over time with muscular contractions, indicating impaired peripheral circulatory control. In highly oxidative muscle such as the soleus and Gast_Red NO plays a significant role in the regulation of vascular tone in vivo (28). Therefore, the slowed endothelium-dependent vasodilator dynamics with advancing age (Figs. 1, 2, and 4; Table 2) and the results of previous work demonstrating reduced bioavailability of NO in arterioles during endothelium-dependent vasodilation (18, 51) provide a plausible mechanism for the blunted capillary hemodynamics (14) and the lower microvascular Po₂ (6) following the onset of muscle contractions with aging. Indeed, Ferreira et al. (22) have demonstrated that NO bioavailability is crucial in ensuring adequate Qo₂-to-V̇o₂ matching in skeletal muscle across the rest-to-exercise transition. In addition, the slower dynamics and reduced magnitude of endothelium-dependent vasodilation in the soleus and Gast_Red from old animals are consistent with the diminished blood flow to these muscles during exercise in conscious animals (39). However, the lack of change in the dynamics of vasodilation in the Gast_White (Fig. 3) is unlikely responsible for the enhanced exercise hyperemic response in glycolytic muscle with aging (39). The old age-associated elevation of blood flow during exercise in low oxidative muscle may be the result of 1) an increased recruitment of white glycolytic fibers due to an inadequate perfusion of high oxidative motor units and/or 2) an impaired myogenic autoregulation of vasomotor tone observed in resistance vessels from this muscle fiber type (38). Prospective studies are required to address this issue mechanistically.

Fig. 4.

Time course of vasodilation to endothelium-dependent stimuli increased intraluminal flow (A) and ACh (B) in arterioles from the soleus and Gast_Red of young and old rats. Data are plotted as a function of the delta (i.e., change in diameter between pre- and postintervention) to normalize the percent vasodilation between the means of the two groups.

Mechanistic bases for aging-induced alterations in the speed of vasodilation.

Many of the investigations focusing on vasomotor dysfunction with advancing age have demonstrated impaired endothelium-dependent vasodilation (20, 24, 37, 55, 62). Muller-Delp and colleagues (37, 52) have further determined that a large component of the old age-associated endothelial dysfunction originates from the NO signaling pathway. In the present study, a degree of old age-associated endothelial dysfunction on the dynamics of vasodilation is also evident (Fig. 4, A and B). In the soleus muscle, for example, the normalized vasodilator responses through endothelium-dependent mechanisms (i.e., flow and ACh) are significantly blunted with old age (Fig. 4, A and B). Interestingly, advancing age impaired the speed of vasodilation to a greater extent in arterioles from the Gast_Red vs. that from the soleus (Fig. 4A) muscle, although the mechanistic basis for this difference is not entirely clear. Given the Gast_Red is a locomotory muscle and the soleus is primarily a postural muscle (19, 32), it is likely that the reduced spontaneous physical activity with advancing age (5) may affect vessels from the Gast_Red to a greater extent than those from the soleus muscle.

Although there was no change in the rate of vasodilation to SNP, there was a tendency in the soleus muscle (Figs. 1C and 5) and a statistically longer delay in arterioles from the Gast_Red muscle (Fig. 2C and 5) before the onset of vasodilation to SNP with aging. The mechanism(s) responsible for this increased delay is currently unclear; however, an impaired diffusion of NO into the vascular smooth muscle or an inertia within the cGMP signaling pathway may be contributing factors. However, NO freely diffuses across cell membranes due to its small size and lack of charge making an impaired diffusion of NO into the vessel unlikely. The intransigent rate of vasodilation to SNP between age groups suggests the prolonged time delay of vasodilation with old age is manifest in the initial steps of the NO/cGMP pathway. To our knowledge, there are no studies reporting old age-associated changes in enzyme kinetics that would explain a slower onset of vasodilation. However, advancing age affects several key enzymes in the NO/cGMP signaling pathway, including 1) loss of expression of the β-subunit of soluble guanylyl cyclase (sGC) (10) and 2) decreased activation of cGMP-dependent protein kinase by cGMP (34), both of which have the potential to affect smooth muscle relaxation. In addition, with increasing age there is an elevated superoxide (O₂⁻) production (15) due, in part, to a loss of extracellular superoxide dismutase (SOD) (54) located within the vascular intima. Superoxide can exhibit a modulatory effect of NO concentration by scavenging NO to form the oxidant peroxynitrite (26), which likely contributes to the reduced bioavailability of the former with aging (30, 51, 59). Therefore, with exogenous NO administration to a milieu of increased O₂⁻ production, the time taken to reach the critical concentration of NO sufficient to activate cGMP may be prolonged, resulting in a delay in the onset of vasodilation (Figs. 1C, 2C, and 5).

Fig. 5.

Time course of vasodilation to endothelium-independent vasodilator SNP in arterioles from the soleus and Gast_Red of young and old rats. Data are plotted as a function of the delta (i.e., change in diameter between pre- and postintervention) to normalize the percent vasodilation between the means of the two groups.

Ramifications of slowed vasodilatory dynamics.

Skeletal muscle possesses a tremendous ability to augment blood flow from rest to exercise (1, 2, 9, 31, 40, 45, 50). Therefore, alterations in the fidelity in which the resistance vasculature can modulate vascular conductance can have severe ramifications on exercise tolerance. Across the rest-to-exercise transition, the time course of the whole muscle hyperemic response reflects the summation of the individual resistance arterioles vasodilator dynamic profiles. Blunted arteriolar vasodilator (and therefore Qo₂) dynamics (Figs. 1, A and B, and 2, A and B) would have a downstream affect on the individual microvascular units they perfuse. Specifically, a slower rate of Qo₂ would force a compensatory increase in fractional O₂ extraction at the capillary level to maintain O₂ flux and cellular energetics. Indeed, in both aged humans (16) and animals (6) the muscle oxygenation kinetic profile across the exercise on-transient supports a slower adaptation of muscle blood flow vs. that of V̇o₂. The blunted vasodilator dynamics with advancing age will likely potentiate the vascular dysfunction elicited by pathological conditions such as chronic heart failure (CHF) and diabetes. Indeed, an even slower skeletal muscle hyperemic response with contractions is predicted in aged subjects with CHF based on microvascular O₂ exchange profiles (7).

Conclusion.

In summary, data from the present study demonstrate that the time course of endothelium-dependent vasodilation in isolated arterioles is significantly slower in skeletal muscle from old animals. These blunted dynamics concurrent with a reduced magnitude of vasodilation would have severe ramifications on the ability to rapidly augment blood flow to exercising muscle in the elderly. Therefore, the ability to match Qo₂ to V̇o₂, which is crucial to maintain cellular energetics and exercise tolerance, is compromised in the aged muscle. It is known that chronic exercise training can mitigate the old-age associated vascular endothelial dysfunction (52, 53); however, whether exercise training can speed the dynamics of vasodilation in skeletal muscle from aged individuals remains to be determined.

GRANTS

This study was supported by National Institutes of Health Grant KO1-AG-031327-01 (B. J. Behnke).

DISCLOSURES

No conflicts of interest are declared by the authors.