Skeletal muscle microvascular and interstitial PO2 from rest to contractions

Abstract

Key points: Oxygen pressure gradients across the microvascular walls are essential for oxygen diffusion from blood to tissue cells. At any given flux, the magnitude of these transmural gradients is proportional to the local resistance. The greatest resistance to oxygen transport into skeletal muscle is considered to reside in the short distance between red blood cells and myocytes. Although crucial to oxygen transport, little is known about transmural pressure gradients within skeletal muscle during contractions. We evaluated oxygen pressures within both the skeletal muscle microvascular and interstitial spaces to determine transmural gradients during the rest-contraction transient in anaesthetized rats. The significant transmural gradient observed at rest was sustained during submaximal muscle contractions. Our findings support that the blood-myocyte interface provides substantial resistance to oxygen diffusion at rest and during contractions and suggest that modulations in microvascular haemodynamics and red blood cell distribution constitute primary mechanisms driving increased transmural oxygen flux with contractions.

Abstract: Oxygen pressure (PO2) gradients across the blood-myocyte interface are required for diffusive O₂ transport, thereby supporting oxidative metabolism. The greatest resistance to O₂ flux into skeletal muscle is considered to reside between the erythrocyte surface and adjacent sarcolemma, although this has not been measured during contractions. We tested the hypothesis that O₂ gradients between skeletal muscle microvascular (PO2 mv ) and interstitial (PO2 is ) spaces would be present at rest and maintained or increased during contractions. PO2 mv and PO2 is were determined via phosphorescence quenching (Oxyphor probes G2 and G4, respectively) in the exposed rat spinotrapezius during the rest-contraction transient (1 Hz, 6 V; n = 8). PO2 mv was higher than PO2 is in all instances from rest (34.9 ± 6.0 versus 15.7 ± 6.4) to contractions (28.4 ± 5.3 versus 10.6 ± 5.2 mmHg, respectively) such that the mean PO2 gradient throughout the transient was 16.9 ± 6.6 mmHg (P < 0.05 for all). No differences in the amplitude of PO2 fall with contractions were observed between the microvasculature and interstitium (10.9 ± 2.3 versus 9.0 ± 3.5 mmHg, respectively; P > 0.05). However, the speed of the PO2 is fall during contractions was slower than that of PO2 mv (time constant: 12.8 ± 4.7 versus 9.0 ± 5.1 s, respectively; P < 0.05). Consistent with our hypothesis, a significant transmural gradient was sustained (but not increased) from rest to contractions. This supports that the blood-myocyte interface is the site of a substantial PO2 gradient driving O₂ diffusion during metabolic transients. Based on Fick's law, elevated O₂ flux with contractions must thus rely primarily on modulations in effective diffusing capacity (mainly erythrocyte haemodynamics and distribution) as the PO2 gradient is not increased.

Keywords: diffusion; dynamics; kinetics; oxygen gradients.

Figure 1. MAP and HR from rest to contractions

MAP and HR from rest to contractions during spinotrapezius muscle microvascular and interstitial $P_{{\mathrm{O}}_2}$ measurement. Values are the mean ± SD.

Figure 2. Mean spinotrapezius PO₂ within the microvascular and interstitial spaces at rest

Mean spinotrapezius $P_{{\mathrm{O}}_2}$ within the microvascular and interstitial spaces during a resting period preceding contractions. No significant reductions in resting $P_{{\mathrm{O}}_2}$ were observed over time in either compartment (P > 0.05), refuting the possibility of accumulated oxygen photoconsumption by phosphorescence quenching in the current experimental protocols. Values are the mean ± SD. ^* P < 0.05 for microvascular versus interstitial $P_{{\mathrm{O}}_2}$ for all data points. For further details, see text.

Figure 3. Spinotrapezius muscle PO2 mv , PO2 is and ΔPO2 from rest to contractions

Top: mean spinotrapezius muscle $P_{{\mathrm{O}}_2\mathrm{mv}}$ and $P_{{\mathrm{O}}_2\mathrm{is}}$ during the rest–contraction transient. Bottom: mean difference between muscle $P_{{\mathrm{O}}_2\mathrm{mv}}$ and $P_{{\mathrm{O}}_2\mathrm{is}}$ $(\mathrm{\Delta }{P}_{{\mathrm{O}}_2})$ from rest to contractions. Inset: mean values for the area under the $P_{{\mathrm{O}}_2}$ curves ($P_{{\mathrm{O}}_2\mathrm{area}}$) in the microvascular and interstitial spaces (M and I, respectively). $P_{{\mathrm{O}}_2\mathrm{area}}$ was determined through integration of the area under the $P_{{\mathrm{O}}_2\mathrm{mv}}$ and $P_{{\mathrm{O}}_2\mathrm{is}}$ curves over the 3 min stimulation period. Note the pronounced $P_{{\mathrm{O}}_2}$ gradient between muscle microvascular and interstitial spaces from rest to contractions. Time zero depicts the onset of muscle contractions. Values are the mean ± SD. ^* P < 0.05 versus M.

Figure 4. Spinotrapezius muscle PO2 within the microvascular and interstitial spaces

Mean and individual spinotrapezius muscle $P_{{\mathrm{O}}_2}$ within the microvascular and interstitial spaces at rest ($P_{{\mathrm{O}}_2(\mathrm{BL})}$; top) and during the steady‐state of contractions ($P_{{\mathrm{O}}_2(\mathrm{SS})}$; bottom). Microvascular $P_{{\mathrm{O}}_2}$ was higher than interstitial $P_{{\mathrm{O}}_2}$ in all instances irrespective of metabolic status. Values are the mean ± SD. ^* P < 0.05 versus microvascular.

Figure 5. TEM of the rat spinotrapezius showing the oxygen transport pathway within the muscle microcirculation

Left: cross‐sectional TEM image of the rat spinotrapezius showing the oxygen transport pathway from the red blood cell (RBC) to skeletal muscle. Note the thin plasma layer (p) between the RBC and capillary wall (w). The arrow illustrates the short distance between the RBC surface and the sarcolemma known as the carrier‐free region (CFR). i, interstitial space; s, sarcolemma; f, muscle fibre; m, mitochondrion; pe, pericyte. TEM magnification of 8000×. Scale bar = 1 μm. Right: quantitative analysis of TEM images showing the shortest distance between the RBC surface and adjacent sarcolemma (i.e. CFR). The boxplot depicts the median, interquartile range and individual values (n = 16).

Figure 6. TEM images of the rat spinotrapezius muscle showing the intracellular, interstitial and microvascular (lumen) compartments

Left (A–D): cross‐sectional TEM images of the rat spinotrapezius muscle showing the intracellular, interstitial and microvascular (lumen) compartments. The sequence from (A) to (D) shows increasingly higher magnification images of the same field. Note that no red blood cell was sectioned on the left vessel in (A). TEM magnifications of 700×, 2000×, 4000× and 8000×, respectively. Scale bars = 10, 2, 2 and 1 μm, respectively. Right: quantitative analysis of TEM images showing the relative volume occupied by each compartment. It should be noted that microvascular volume calculations based on cross‐sectional TEM images reflect total (as opposed to functional) values therefore overestimating the volume fraction participating effectively in blood–myocyte O₂ transport. The boxplots depict the median, interquartile range and individual values (n = 11). ^* P < 0.05 versus interstitial volume; †P < 0.05 versus microvascular volume.

Figure 7. Simulated profiles illustrating the effects of altered PO2 mv dynamics on transcapillary ΔPO2 during the transition from rest to contractions

Top: simulated $P_{{\mathrm{O}}_2\mathrm{mv}}$ profiles with varying time constants for the first component (τ₁) and fixed baseline, amplitudes and time delays. $P_{{\mathrm{O}}_2\mathrm{mv}}$ and $P_{{\mathrm{O}}_2\mathrm{is}}$ profiles were generated based on mean values reported in Table 1. Bottom: effects of $P_{{\mathrm{O}}_2\mathrm{mv}}$ τ₁ manipulations on transcapillary $P_{{\mathrm{O}}_2}$ (i.e. $\mathrm{\Delta }{P}_{{\mathrm{O}}_2(\mathrm{t})}=P_{{\mathrm{O}}_2\mathrm{mv}}-P_{{\mathrm{O}}_2\mathrm{is}}$). Fixed $P_{{\mathrm{O}}_2\mathrm{is}}$ kinetics parameters were used in these calculations. Note that progressively faster $P_{{\mathrm{O}}_2\mathrm{mv}}$ kinetics (i.e. smaller τ₁; characteristic of aged and diseased states) lowers $\mathrm{\Delta }{P}_{{\mathrm{O}}_2}$ and thus the exclusive driving pressure for transmural O₂ flux during the metabolic transient. Time zero depicts the onset of muscle contractions. ^*Mean $P_{{\mathrm{O}}_2\mathrm{mv}}$ response observed in the current study (Table 1). For further details, see text.