Danielle J. Padilla

Control of microvascular oxygen pressures in rat muscles comprised of different fibre types

In response to an elevated metabolic rate , increased microvascular blood–muscle O2 flux is the product of both augmented O2 delivery and fractional O2 extraction. Whole body and exercising limb measurements demonstrate that and fractional O2 extraction increase as linear and hyperbolic functions, respectively, of . Given the presence of disparate vascular control mechanisms among different muscle fibre types, we tested the hypothesis that, in response to muscle contractions, would be lower and fractional O2 extraction (as assessed via microvascular O2 pressure, P  mvO 2) higher in fast‐ versus slow‐twitch muscles. Radiolabelled microsphere and phosphorescence quenching techniques were used to measure and P  mvO 2, respectively at rest and across the transition to 1 Hz twitch contractions at low (Lo, 2.5 V) and high intensities (Hi, 4.5 V) in rat (n= 20) soleus (Sol, slow‐twitch, type I), mixed gastrocnemius (MG, fast‐twitch, type IIa) and white gastrocnemius (WG, fast‐twitch, type IIb) muscle. At rest and for Lo and Hi (steady‐state values) transitions, P  mvO 2 was lower (all P < 0.05) in MG (mmHg: rest, 22.5 ± 1.0; Lo, 15.3 ± 1.3; Hi, 10.2 ± 1.6) and WG (mmHg: rest, 19.0 ± 1.3; Lo, 12.2 ± 1.1; Hi, 9.9 ± 1.1) than in Sol (rest, 33.1 ± 3.2 mmHg; Lo, 19.0 ± 2.3 mmHg; Hi, 18.7 ± 1.8 mmHg), despite lower and in MG and WG under each set of conditions. These data suggest that during submaximal metabolic rates, the relationship between and O2 extraction is dependent on fibre type (at least in the muscles studied herein), such that muscles comprised of fast‐twitch fibres display a greater fractional O2 extraction (i.e. lower P  mvO 2) than their slow‐twitch counterparts. These results also indicate that the greater sustained P  mvO 2 in Sol may be important for ensuring high blood–myocyte O2 flux and therefore a greater oxidative contribution to energetic requirements.

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.

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.

Effects of altered nitric oxide availability on rat muscle microvascular oxygenation during contractions.

Aim:  To explore the role of nitric oxide (NO) in controlling microvascular O2 pressure (Po2mv) at rest and during contractions (1 Hz). We hypothesized that at the onset of contractions sodium nitroprusside (SNP) would raise Po2mv and slow the kinetics of Po2mv change whereas l‐nitro arginine methyl ester (l‐NAME) would decrease Po2mv and speed its kinetics.

Muscle microvascular oxygenation in chronic heart failure: role of nitric oxide availability

Aim:  To test the hypothesis that diminished vascular nitric oxide availability might explain the inability of individuals with chronic heart failure (CHF) to maintain the microvascular PO2’s (PO2mv ∝ O2 delivery‐to‐uptake ratio) seen in healthy animals.

Temporal profile of rat skeletal muscle capillary haemodynamics during recovery from contractions

In skeletal muscle capillaries, red blood cell (RBC) flux (FRBC), velocity (VRBC) and haematocrit (HctCAP) are key determinants of microvascular O2 exchange. However, the mechanisms leading to the changes in FRBC, VRBC and HctCAP during muscle contractions and recovery thereafter are not fully understood. To address this issue we used intravital microscopy to investigate the temporal profile of the rat spinotrapezius muscle (n= 5) capillary haemodynamics during recovery from 3 min of twitch muscle contractions (1 Hz, 4–6 V). Specifically, we hypothesized that (1) during early recovery FRBC and VRBC would decrease rapidly and FRBC would display a biphasic response (consistent with a muscle pump effect on capillary haemodynamics), and (2) there would be a dynamic relationship between changes (Δ) in VRBC and HctCAP. The values at rest (R) and end‐recovery (ER) were significantly lower (P < 0.05) than at end‐contraction (EC) for FRBC (in cells s−1, R = 30.1 ± 7.8, EC = 46.2 ± 7.3 and ER = 26.0 ± 6.1), VRBC (in μm s−1, R = 368 ± 83, EC = 497 ± 62 and ER = 334 ± 59) and HctCAP(R = 0.193 ± 0.016, EC = 0.214 ± 0.023 and ER = 0.185 ± 0.019). The first data point where a significant decrease in FRBC, HctCAP and VRBC occurred was at 5, 5 and 20 s post‐contraction, respectively. The decrease in FRBC approximated a monoexponential response (half‐time of ∼26 s). The relationship between ΔVRBC and ΔHctCAP was not significant (P > 0.05). Based on the early decrease in FRBC(within 5 s), overall dynamic profile of FRBC and the ∼20 s ‘delay’ to the decrease in VRBC we conclude that the muscle pump does not appear to contribute substantially to the steady‐state capillary haemodynamics in the contracting rat spinotrapezius muscle. Moreover, our findings suggest that alterations in VRBC do not obligate proportional changes in HctCAP within individual capillaries following muscle contractions.

The effect of herbal supplementation on the severity of exercise-induced pulmonary haemorrhage

Exercise-induced pulmonary haemorrhage (EIPH) is a serious condition that affects the health and possibly the performance of all racehorses. However, only two treatments, furosemide and the Flair™ equine nasal strip, both of which reduce capillary transmural pressure, have been successful in reducing EIPH. Alternatively, transient impairment of platelet function and coagulation during exercise has been considered an additional contributor to EIPH. Consequently, herbal formulations designed to enhance platelet function, and hence coagulation, are hypothesized to reduce EIPH. To investigate the validity of this hypothesis, five Thoroughbred horses completed three maximal incremental exercise tests on a 10% inclined treadmill in a randomized cross-over design experiment. Treatments included twice daily oral administration (for 3 days) of a placebo (PL; cornstarch) and two herbal formulas, Yunnan Paiyao (YP) or Single Immortal (SI). Blood samples for coagulation profiles, complete blood counts and biochemistry profiles were collected before each exercise test. During each test, pulmonary arterial pressure, oxygen uptake, arterial blood gases, plasma lactate and time-to-fatigue were measured. Severity of EIPH was quantified via bronchoalveolar lavage (BAL) at 30–60 min post-exercise. The herbal formulations were not effective in decreasing EIPH (×10 6 red blood cells ml −1 BAL fluid: PL, 27.1±11.6; YP, 33.2±23.4; SI, 35.3±15.4, P >0.05) or in changing any of the other variables measured with the exception of time-to-fatigue, which was slightly but significantly prolonged by Single Immortal compared with placebo and Yunnan Paiyao (PL, 670±9.6 s; YP, 665±5.5 s; SI, 685±7.9 s, P

Evidence supporting exercise-induced pulmonary haemorrhage in racing greyhounds

Exercise-induced pulmonary haemorrhage (EIPH) is a major health concern in performance horses, but the presence and severity of this condition in racing greyhounds has received little attention. While equids and greyhounds share many physiological attributes, there are important structural and functional differences that may help protect greyhounds from EIPH. We tested the hypothesis that greyhounds performing a simulated 503m race would experience EIPH and that the time course of recovery would be similar to the horse, even though the severity or relative extent as indexed by the concentration of red blood cells [RBCs] in bronchoalveolar lavage (BAL) fluid would be lower in comparison with that demonstrated previously in horses. Greyhound dogs (n 1⁄4 6) raced on two occasions (separated by 7 weeks) and BAL was performed 1 week before, 2 h after and each week for 4 weeks following each race to examine the [RBC], concentration of white blood cells [WBCs], WBC differentials and haemosiderophages in the lungs. Racing increased 10min post-exercise venous blood [lactate] to 18.6 ^ 0.4mmol l. No epistaxis or pink froth was observed at the nose or mouth of any of the dogs. The [RBC] in the BAL fluid was increased significantly 2 h post-race (baseline 1⁄4 109.6 ^ 11.7 £ 10; post-race 1⁄4 292.3 ^ 69.9 £ 10 RBCml BAL fluid, P , 0.05) and returned to baseline 1 week post-race (149.2 ^ 46.2 £ 10 RBCml BAL fluid, P . 0.05 versus baseline). The number of haemosiderophages was not different for any of the measurement periods. The [WBC] in the BAL fluid decreased from baseline and race values at 2, 3 and 4 weeks post-exercise (all P , 0.05). Alveolar neutrophil concentrations were also decreased from baseline and immediate post-race values for 4 weeks post-race. The increased [RBC] in the BAL fluid post-exercise is consistent with the presence of EIPH in these greyhounds. However, the relative extent of EIPH in greyhounds (as indexed by [RBC] in the BAL fluid), as compared with that in the horse, was mild, and the lack of elevation of WBC suggests that, unlike their equine counterparts, inflammatory airway disease was absent.

Control of microvascular oxygen pressures during recovery in rat fast-twitch muscle of differing oxidative capacity.

Whether the speed of recovery of microvascular O2 pressures ( ) differs within muscles composed primarily of type II fibres with contrasting oxidative capacity has not been determined. We tested the hypothesis that, following contractions, the recovery of would be slower in the white (WG; low oxidative capacity) versus the mixed gastrocnemius (MG; comparatively high oxidative capacity). Radiolabelled microsphere and phosphorescence quenching techniques were used to measure muscle blood flow ( , hence O2 delivery, ) and during contractions (1 Hz twitch) at low (LO, 2.5 V) and high intensities (HI, 4.5 V) in rat (n= 15) MG and WG muscle and during subsequent recovery. Following the LO protocol, end‐contraction was lower in WG (11.6 ± 0.5 mmHg) than in MG (16.2 ± 0.6 mmHg; P < 0.05) while, contrary to our hypothesis, the initial rate of change in during recovery ( /dt; MG 0.11 ± 0.01 mmHg s−1 and WG 0.06 ± 0.03 mmHg s−1) and mean response time (MRT; MG 110.3 ± 5.1 s and WG 113.5 ± 8.4 s, P > 0.05) were not different. In contrast, end‐contraction baseline was not different following the HI protocol (MG 10.3 ± 0.6 mmHg and WG 9.2 ± 0.6 mmHg; P > 0.05) but, in agreement with our hypothesis, /dt was slower (MG 0.07 ± 0.01 mmHg s−1 and WG 0.03 ± 0.003 mmHg s−1; P < 0.05) and MRT longer (WG 180.8 ± 4.5 s and MG 115.4 ± 6.7 s; P < 0.05) in WG versus MG following the HI protocol. These data suggest that following high‐intensity, though submaximal, muscle contractions, recovers much faster in the more oxidative mixed gastrocnemius than in the less oxidative white gastrocnemius.

Effects of Altered Nitric Oxide Availability on Rat Muscle Microvascular Oxygenation During Contractions: 1628

AIM To explore the role of nitric oxide (NO) in controlling microvascular O2 pressure (P(O2)mv) at rest and during contractions (1 Hz). We hypothesized that at the onset of contractions sodium nitroprusside (SNP) would raise P(O2)mv and slow the kinetics of P(O2)mv change whereas l-nitro arginine methyl ester (L-NAME) would decrease P(O2)mv and speed its kinetics. METHODS We superfused the spinotrapezius muscle of female Sprague-Dawley rats (n = 7, body mass = 298 +/- 10 g) with SNP (300 microM) and L-NAME (1.5 mm) and measured P(O2)mv (phosphorescence quenching) during contractions. RESULTS SNP decreased mean arterial pressure (92 +/- 5 mmHg) below that of control (CON, 124 +/- 4 mmHg) and L-NAME (120 +/- 4 mmHg) conditions. SNP did not raise P(O2)mv at rest but it did elevate the P(O2)mv-to-MAP ratio (50% increase, P < 0.05) and slow the kinetics by lengthening the time-delay (TD, 14.0 +/- 5.0 s) and time constant (tau, 24.0 +/- 10.0 s) of the response compared with CON (TD, 8.4 +/- 3.3 s; tau, 16.0 +/- 4.5 s, P < 0.05 vs. SNP). L-NAME decreased P(O2)mv at rest and tended to speed tau (10.1 +/- 3.8 s, P = 0.1), while TD (8.1 +/- 1.0 s) was not significantly different. L-NAME also caused P(O2)mv to fall transiently below steady-state contracting values. CONCLUSIONS These results indicate that NO availability can significantly affect P(O2)mv at rest and during contractions and suggests that P(O2)mv derangements in ageing and chronic disease conditions may potentially result from impairments in NO availability.