Differential α-adrenergic modulation of rapid onset vasodilatation along resistance networks of skeletal muscle in old versus young mice

Abstract

Key points: Rapid onset vasodilatation (ROV) initiates functional hyperaemia upon skeletal muscle contraction and is attenuated during ageing via α-adrenoreceptor (αAR) stimulation, but it is unknown where this effect predominates in resistance networks. In gluteus maximus muscles of young (4 months) and old (24 months) male C57BL/6 mice, tetanic contraction while observing feed arteries and arterioles initiated ROV, which increased with contraction duration, peaked later in upstream versus downstream vessel branches and was attenuated throughout networks with advanced age. With no effect on muscle force production, inhibiting αARs improved ROV in old mice while activating αARs attenuated ROV in young mice. Modulating ROV through αARs was greater in upstream feed arteries and arterioles compared to downstream arterioles, with α₂ ARs more effective than α₁ ARs. ROV is coordinated along resistance networks and modulated differentially between young and old mice via αARs; with advanced age, attenuated dilatation of upstream branches will restrict muscle blood flow.

Abstract: Rapid onset vasodilatation (ROV) in skeletal muscle is attenuated during advanced age via α-adrenoreceptor (αAR) activation, but it is unknown where such effects predominate in the resistance vasculature. Studying the gluteus maximus muscle (GM) of anaesthetized young (4 months) and old (24 months) male C57BL/6 mice, we tested the hypothesis that attenuation of ROV during advanced age is most effective in proximal branches of microvascular resistance networks. Diameters of a feed artery (FA) and first- (1A), second- (2A) and third- (3A) order arterioles were studied in response to single tetanic contractions (100 Hz, 100-1000 ms). ROV began within 1 s and peaked sooner in 2A and 3A (∼3 s) than in 1A or FA (∼4 s). Relative amplitudes of dilatation increased with contraction duration and with vessel branch order (FA<1A<2A<3A). In old mice, attenuation of ROV was greater in FA and 1A compared to 2A and 3A. With no effect on muscle force production, inhibiting αARs (phentolamine; 10^-6 m) improved ROV in FA and 1A of old mice while subthreshold stimulation of αARs in young mice (noradrenaline; 10^-9 m) depressed ROV most effectively in FA and 1A. In young mice, stimulating α₁ ARs (phenylephrine; 10^-7 m) and α₂ ARs (UK 14304; 10^-7 m) attenuated ROV primarily in FA. In old mice, inhibiting α₂ ARs (rauwolscine; 10^-7 m) restored ROV more effectively in FA and 1A than did inhibiting α₁ ARs (prazosin; 10^-8 m). We conclude that, with temporal and spatial coordination along resistance networks, attenuation of ROV with advanced age is most effective in proximal branches via constitutive activation of α₂ ARs.

Keywords: adrenoreceptors; ageing; blood flow; functional sympatholysis; microcirculation.

Figure 1. Internal diameters and spontaneous tone in GM feed arteries and arterioles are similar for young and old mice

Resting diameter (A) and maximal diameter (B) decreased as vessel branch order increased for both age groups (FA>1A>2A.3A). Spontaneous tone (C; see Methods) increased with vessel branch order. Summary data are means ± SEM, n = 6 per age group. ^# P < 0.05, main effect of age. ^Δ P < 0.05, main effect of vessel branch order; ^* P < 0.05, old vs. young mice for designated branch order.

Figure 2. With similar time course, ROV is depressed throughout resistance networks of old versus young mice

For single tetanic contractions of 100, 250, 500, or 1000 ms duration at 100 Hz (key applies to all panels), vasodilatation (expressed in relative terms as % max; see Methods) tended to increase with vessel branch order (FA<1A<2A<3A) and with contraction duration (100<250<500<1000 ms). Across branch orders, ROV was lower for GM in old compared to young mice. Summary data are means ± SEM, n = 6 per age group.

Figure 3. Initiation of ROV increases with contraction duration and branch order but is diminished with advanced age

At 1 s post‐contraction, vasodilatation increased with branch order (FA<1A<2A<3A), and contraction duration (all at 100 Hz) whether expressed as absolute diameter change (μm, left panels) or relative responses (% max, right panels). These initial responses were attenuated for all branch orders in GM of old mice compared to young mice. ^Δ P < 0.05, main effect of branch order; ^# P < 0.05, main effect of age. ^* P < 0.05, old vs. young mice within respective branch order. Summary data are means ± SEM, n = 6 per age group.

Figure 4. Peak ROV increases with contraction duration and branch order but is diminished with advanced age

Following each contraction duration at 100 Hz, peak vasodilatation expressed as diameter change (μm, left panels) was similar across branch orders while relative responses (% max, right panels) increased with branch order (FA<1A<2A<3A). Both absolute and relative peak ROV increased with contraction duration for all branch orders in both age groups, but peak ROV was diminished consistently in all branch orders for old mice compared to young mice, with the greatest effect of ageing in FA. ^Δ P < 0.05, main effect of branch order; ^# P < 0.05, main effect of age, ^* P < 0.05, old vs. young within respective branch order. Summary data are means ± SEM, n = 6 per age group.

Figure 5. ROV peaks earlier in downstream branches of the resistance network

The time‐to‐peak for ROV decreased as branch order increased (FA>1A>2A>3A) in GM of both young mice (R ^2 = 0.950) and old mice (R ^2 = 0.986). For each branch order, the time‐to‐peak ROV was consistent across contraction durations for both age groups. Note temporal lag between downstream 3A and upstream FA, particularly in old mice for 100 ms contraction. Each data point represents the mean value for given duration from n = 6; horizontal bar for each branch indicates mean time‐to‐peak ± SEM across contraction durations.

Figure 6. Sympathetic innervation of GM feed arteries for young and old mice

A, representative immunofluorescent staining (maximum Z‐stack projection of confocal image slices) of all perivascular nerves (protein gene product 9.5, PGP9.5) and of sympathetic nerves (tyrosine hydroxylase, TH) in GM feed arteries of young and old mice. Vessel edges indicated by dotted lines. B, innervation per vessel surface area (% fluorescence area; see Methods) for PGP9.5 and TH was not different between FAs of young and old mice. Summary data are means ± SEM, n = 4 per age group.

Figure 7. Differential modulation of ROV in GM branch orders by αARs in young versus old mice

Peak ROV (% max) in FA, 1A, 2A and 3A following 100, 250, 500 and 1000 ms contraction at 100 Hz in the GM of young and old mice. Peak ROV increased as contraction duration increased in both age groups. Stimulation of αARs with NA (10⁻⁹  m) attenuated peak ROV in young mice but not in old mice. In contrast, inhibition of αARs with phentolamine (Phentol, 10⁻⁶  m) enhanced peak ROV in old mice but not in young mice. The effect of αAR modulation on peak ROV tended to be greater in FA and 1A than in 2A and 3A. ^Δ P < 0.05, main effect of branch order. ^# P < 0.05, main effect of Phentol or NA. ^ψ P < 0.05, NA vs. control in designated branch order. ^Φ P < 0.05, Phentol vs. control in designated branch order. Summary data are means ± SEM, n = 6 per age group.

Figure 8. Muscle force production is unaffected during manipulation of adrenoreceptor activation

During single tetanic contractions at 100 Hz, active force produced by GM in young (n = 6; open symbols, means ± SEM) and old (n = 2, individual solid symbols) mice was similar across contraction durations as well as between control conditions and superfusion with either NA (10⁻⁹  m) or phentolamine (10⁻⁶  m).

Figure 9. Selective stimulation of α₁ARs or α₂ARs attenuates ROV in upstream branches of GM in young mice

Peak ROV (% max) in FA, 1A, 2A and 3A following 100, 250, 500 and 1000 ms contraction in the GM of young mice. Across contraction durations, selective activation of α₁ARs (PE, 10⁻⁷  m) or of α₂ARs (UK, 10⁻⁷  m) attenuated ROV (% max) in FA, 1A and 2A but had no effect on ROV in 3A. ^Δ P < 0.05 main effect of branch order; ^# P < 0.05 main effect of treatment; ^Φ P < 0.05, PE vs. control, ^ψ P < 0.05, UK vs. control. Summary data are means ± SEM for n = 6 young mice.

Figure 10. Selective inhibition of α₁ARs or α₂ARs improves ROV in upstream branch orders of GM in old mice

Peak ROV (% max) in FA, 1A, 2A and 3A following 100, 250, 500 and 1000 ms contraction in the GM of old mice. Across contraction durations, selective inhibition of α₁ARs (PZ, 10⁻⁸  m) or of α₂ARs (RW, 10⁻⁷  m) improved ROV in FA while inhibition of α₂ARs improved ROV in 1A. Nevertheless, inhibition of either αAR subtype alone had no effect in 2A or 3A. ^Δ P < 0.05 main effect of branch order; ^# P < 0.05, main effect of treatment; ^Φ P < 0.05, PZ vs. control, ^ψ P < 0.05, RW vs. control. Summary data are means ± SEM for n = 6 old mice.