Brad J. Behnke

Dynamics of microvascular oxygen pressure across the rest-exercise transition in rat skeletal muscle.

There exists substantial controversy as to whether muscle oxygen (O2) delivery (QO2) or muscle mitochondrial O2 demand determines the profile of pulmonary VO2 kinetics in the rest-exercise transition. To address this issue, we adapted intravascular phosphorescence quenching techniques for measurement of rat spinotrapezius microvascular O2 pressure (PO2m). The spinotrapezius muscle intravital microscopy preparation is used extensively for investigation of muscle microcirculatory control. The phosphor palladium-meso-tetra(4-carboxyphenyl)porphyrin dendrimer (R2) at 15 mg/kg was bound to albumin within the blood of female Sprague-Dawley rats ( approximately 250 g). Spinotrapezius blood flow (radioactive microspheres) and PO2m profiles were determined in situ across the transition from rest to 1 Hz twitch contractions. Stimulation increased muscle blood flow by 240% from 16.6 +/- 3.0 to 56.2 +/- 8.3 (SE) ml/min per 100 g (P < 0.05). Muscle contractions reduced PO2m from a baseline of 31.4 +/- 1.6 to a steady-state value of 21.0 +/- 1.7 mmHg (n = 24, P < 0.01). The response profile of PO2m was well fit by a time delay of 19.2+/-2.8 sec (P < 0.05) followed by a monoexponential decline (time constant, 21.7 +/- 2.1 sec) to its steady state level. The absence of either an immediate and precipitous fall in microvascular PO2 at exercise onset or any PO2m undershoot prior to achievement of steady-state values, provides compelling evidence that O(2) delivery is not limiting under these conditions.

Oxygen exchange profile in rat muscles of contrasting fibre types.

To determine whether fibre type affects the O2 exchange characteristics of skeletal muscle at the microcirculatory level we tested the hypothesis that, following the onset of contractions, muscle comprising predominately type I fibres (soleus, Sol, 86 % type I) would, based on demonstrated blood flow responses, exhibit a blunted microvascular PO2 (PO2,m, which is determined by the O2 delivery (Q̇O2) to O2 uptake (V̇O2) ratio) profile (assessed via phosphorescence quenching) compared to muscle of primarily type II fibres (peroneal, Per, 84 % type II). PO2,m was measured at rest, and following the rest‐contractions (twitch, 1 Hz, 2–4 V for 120 s) transition in Sol (n= 6) and Per (n= 6) muscles of Sprague‐Dawley rats. Both muscles exhibited a delay followed by a mono‐exponential decrease in PO2,m to the steady state. However, compared with Sol, Per demonstrated (1) a larger change in baseline minus steady state contracting PO2,m (ΔPO2,m) (Per, 13.4 ± 1.7 mmHg; Sol, 8.6 ± 0.9 mmHg, P < 0.05); (2) a faster mean response time (i.e. time delay (TD) plus time constant (τ); Per, 23.8 ± 1.5 s; Sol, 39.6 ± 4.3 s, P < 0.05); and therefore (3) a greater rate of PO2,m decline (ΔPO2,m/τ; Per, 0.92 ± 0.08 mmHg s−1; Sol, 0.42 ± 0.05 mmHg s−1, P < 0.05). These data demonstrate an increased microvascular pressure head of O2 at any given point after the initial time delay for Sol versus Per following the onset of contractions that is probably due to faster Q̇O2 dynamics relative to those of V̇O2.

Dynamics of oxygen uptake following exercise onset in rat skeletal muscle

Technical limitations have precluded measurement of the V(O(2)) profile within contracting muscle (mV(O(2))) and hence it is not known to what extent V(O(2)) dynamics measured across limbs in humans or muscles in the dog are influenced by transit delays between the muscle microvasculature and venous effluent. Measurements of capillary red blood cell flux and microvascular P(O(2)) (P(O(2)m)) were combined to resolve the time course of mV(O(2)) across the rest-stimulation transient (1 Hz, twitch contractions). mV(O(2)) began to rise at the onset of contractions in a close to monoexponential fashion (time constant, J = 23.2 +/- 1.0 sec) and reached its steady-state value at 4.5-fold above baseline. Using computer simulation in healthy and disease conditions (diabetes and chronic heart failure), our findings suggest that: (1) mV(O(2)) increases essentially immediately (< 2 sec) following exercise onset; (2) within healthy muscle the J blood flow (thus O(2) delivery, J Q(O(2)m)) is faster than JmV(O(2)) such that oxygen delivery is not limiting, and 3) a faster P(O(2)m) fall to a P(O(2)m) value below steady-state values within muscle from diseased animals is consistent with a relatively sluggish Q(O(2)m) response compared to that of mV(O(2)).

Dynamics of microvascular oxygen partial pressure in contracting skeletal muscle of rats with chronic heart failure.

OBJECTIVE This investigation tested the hypothesis that the dynamics of muscle microvascular O(2) pressure (PO(2)m, which reflects the ratio of O(2) utilization [V*O(2)] to O(2) delivery [Q*O(2)]) following the onset of contractions would be altered in chronic heart failure (CHF). METHODS Female Sprague-Dawley rats were subjected to a myocardial infarction (MI) or a sham operation (Sham). Six to 10 weeks post Sham (n=6) or MI (n=17), phosphorescence quenching techniques were utilized to determine PO(2)m dynamics at the onset of spinotrapezius muscle contractions (1 Hz). RESULTS MI rats were separated into groups with Moderate (n=10) and Severe (n=7) CHF based upon the degree of left ventricular (LV) dysfunction as indicated by structural abnormalities (increased right ventricle weight and lung weight normalized to body weight). LV end-diastolic pressure was elevated significantly in both CHF groups compared with Sham (Sham, 3+/-1; Moderate CHF, 9+/-2; Severe CHF, 27+/-4 mmHg, P<0.05). The PO(2)m response was modeled using time delay and exponential components to fit the PO(2)m response to the steady-state. Compared with Shams, the time constant (tau) of the primary PO(2)m response was significantly speeded in Moderate CHF (tau, Sham, 19.0+/-1.5; Moderate CHF, 13.2+/-1.9 s, P<0.05) and slowed in Severe CHF (tau, 28.2+/-3.4 s, P<0.05). Within the Severe CHF group, tau increased linearly with the product of right ventricular and lung weight (r=0.83, P<0.05). CONCLUSIONS These results suggest that CHF alters the dynamic matching of muscle V*O(2)-to-Q*O(2) across the transition from rest to contractions and that the nature of that perturbation is dependent upon the severity of cardiac dysfunction.

Measurement of muscle microvascular oxygen pressures: compartmentalization of phosphorescent probe.

Objective: To determine whether the phosphorescent probe Oxyphor R2 (a palladium porphyrin dendrimer) becomes extravasated within normotensive skeletal muscle, R2 perfusion and washout studies were performed using a perfused rat hindlimb preparation.

Aging reduces skeletal blood flow, endothelium-dependent vasodilation, and NO bioavailability in rats.

We determined whether aging diminishes bone blood flow and impairs endothelium‐dependent vasodilation. Femoral perfusion was lower in old animals, as well as endothelium‐dependent vasodilation and NO bioavailability. These effects could contribute to old age—related bone loss and the increased risk of fracture.

Effects of prior contractions on muscle microvascular oxygen pressure at onset of subsequent contractions

In humans, pulmonary oxygen uptake (V̇O2) kinetics may be speeded by prior exercise in the heavy domain. This ‘speeding’ arises potentially as the result of an increased muscle O2 delivery (Q̇O2) and/or a more rapid elevation of oxidative phosphorylation. We adapted phosphorescence quenching techniques to determine the QO2‐to‐O2 utilization (Q̇O2/V̇O2) characteristics via microvascular O2 pressure (PO2,m) measurements across sequential bouts of contractions in rat spinotrapezius muscle. Spinotrapezius muscles from female Sprague‐Dawley rats (n= 6) were electrically stimulated (1 Hz twitch, 3–5 V) for two 3 min bouts (ST1 and ST2) separated by 10 min rest. PO2,m responses were analysed using an exponential + time delay (TD) model. There was no significant difference in baseline and ΔPO2,m between ST1 and ST2 (28.5 ± 2.6 vs. 27.9 ± 2.4 mmHg, and 13.9 ± 1.8 vs. 14.1 ± 1.3 mmHg, respectively). The TD was reduced significantly in the second contraction bout (ST1, 12.2 ± 1.9; ST2, 5.7 ± 2.2 s, P < 0.05), whereas the time constant of the exponential PO2,m decrease was unchanged (ST1, 16.3 ± 2.6; ST2, 17.6 ± 2.7 s, P > 0.1). The shortened TD found in ST2 led to a reduced time to reach 63 % of the final response of ST2 compared to ST1 (ST1, 28.3 ± 3.0; ST2, 20.2 ± 1.8 s, P < 0.05). The speeding of the overall response in the absence of an elevated PO2,m baseline (which had it occurred would indicate an elevated QO2/V̇O2) or muscle blood flow suggests that some intracellular process(es) (e.g. more rapid increase in oxidative phosphorylation) may be responsible for the increased speed of PO2,m kinetics after prior contractions under these conditions.

Blood flow and O2 extraction as a function of O2 uptake in muscles composed of different fiber types.

We examined how the greater vasodilatory capacity of slow--(ST) versus fast-twitch (FT) muscles impacts the relationship between blood flow (Q ) and O2 uptake (VO2) and, consequently, the O2 extraction (a-vO2 diff.)-to-VO2 relationship. Q was measured with radiolabelled microspheres, while VO2 was calculated by the Fick principle using measurements of microvascular O2 pressure (phosphorescence quenching) at rest, low--(2.5 V) and high-intensity contractions (4.5 V) for soleus (Sol; ST, n=5), mixed-gastrocnemius (MG; FT, n=7) and white-gastrocnemius (WG; FT, n=7). The slope of the Q-to-VO2 relationship (delta Q/delta VO2] ) was not different among muscles (Sol = 5.5 +/- 0.2, MG = 6.0 +/- 0.11 and WG = 5.8 +/- 0.06; P > 0.05). In contrast, the intercept was greater (P < 0.05) for Sol (16.3 +/- 2.7 ml min(-1) 100 g(-1)) versus MG and WG (in ml min(-1) 100 g(-1): 1.39 +/- 0.26 and 1.45 +/- 0.23, respectively; MG and WG, P > 0.05). In addition, the a-vO2 diff.-to-VO2] relationship for Sol was shifted rightward compared to MG and WG. These data suggest that the increase in Q for a given change in VO2 is similar for slow- and fast-twitch muscles, at least for the range of metabolic rates and muscles studied herein and that a-vO2 diff. differences result from the lower resting Q in FT muscles.

Effects of Type II diabetes on muscle microvascular oxygen pressures

We tested the hypothesis that muscle microvascular O2 pressure (PmvO2; reflecting the O2 delivery (QO2) to O2 uptake (VO2) ratio) would be lowered in the spinotrapezius muscle of Goto-Kakizaki (GK) Type II diabetic rats (n=7) at rest and during twitch contractions when compared to control (CON; n=5) rats. At rest, PmvO2 was lower in GK versus CON rats (CON: 29+/-2; GK: 18+/-2Torr; P<0.05). At the onset of contractions, GK rats evidenced a faster change in PmvO2 than CON (i.e., time constant (tau); CON: 16+/-4; GK: 6+/-2s; P<0.05). In contrast to the monoexponential fall in PmvO2 to the steady-state level seen in CON, GK rats exhibited a biphasic PmvO2 response that included a blunted (or non-existent) PmvO2 decrease followed by recovery to a steady-state PmvO2 that was at, or slightly above, resting values. Compared with CON, this decreased PmvO2 across the transition to a higher metabolic rate in Type II diabetes would be expected to impair blood-muscle O2 exchange and contractile function.

The final frontier: oxygen flux into muscle at exercise onset.

In humans at exercise onset, intramuscular phosphocreatine decreases immediately, whereas muscle oxygen (O2) uptake seems to rise after a delay of up to 15 s which is inconsistent with models of metabolic control. Novel microcirculatory investigations reveal that elevated capillary-to-myocyte O2 flux in rat muscle is, in fact, initiated simultaneously with contractions.