Impact of cell-free hemoglobin on contracting skeletal muscle microvascular oxygen pressure dynamics

Free hemoglobin (Hb) associated with hemolysis extravasates into vascular tissue and depletes nitric oxide (NO), which leads to impaired vascular function and could impair skeletal muscle metabolic control during exercise. We tested the hypothesis that: 1) free Hb would extravasate into skeletal muscle tissue, reducing the contracting skeletal muscle O2 delivery/O2 utilization ratio (microvascular PO2, PO2mv) to a similar extent as that observed following NO synthase (NOS) blockade, and 2) that the Hb scavenging protein haptoglobin (Hp) would prevent Hb extravasation and inhibit these skeletal muscle tissue effects. PO2mv was measured in eight rats (phosphorescence quenching) at rest and during 180 s of electrically induced (1-Hz) twitch spinotrapezius muscle contractions (experiment 1). A second group of seven rats was also used to investigate the effects of Hb + Hp (experiment 2). For both experiments, measurements were made: 1) during control conditions, 2) following a bolus infusion of either Hb (50 mg/kg) or Hb + Hp (50 mg/kg), and 3) following local superfusion of NG-nitro-l-arginine methyl ester (L-NAME; 10 mg/kg). Additional experiments were completed to visualize Hb extravasation into the muscular tissue using Click chemistry techniques. There were no significant differences in the PO2mv observed at rest for any condition in either experiment (p > 0.05 for all). In experiment 1, both Hb and L-NAME reduced the PO2mv significantly during the steady-state of muscle contractions when compared to control conditions with no differences between Hb and L-NAME (control: 24 ± 1, Hb: 21 ± 1, L-NAME: 20 ± 1 mmHg, p < 0.05). In experiment 2, only L-NAME resulted in a significantly lower PO2mv during the steady-state of muscle contractions (control: 25 ± 1, Hb + Hp: 22 ± 2, L-NAME: 18 ± 1 mmHg, p < 0.05). Free Hb lowered the blood-myocyte O2 driving force to a level not significantly different from L-NAME. However, infusing Hb bound to Hp resulted in no significant differences in steady-state PO2mv during muscle contractions when compared to control. Surprisingly, we did not observe Hb accumulation in skeletal muscle tissue. Taken together these data suggests that free Hb impairs O2 delivery/utilization via a NO scavenging effect. Furthermore, the unchanged PO2mv steady-state observed following Hb + Hp further indicates that vascular compartmentalization of Hb by the scavenger protein haptoglobin may improve skeletal muscle metabolic control and potentially exercise tolerance in those afflicted with hemolytic diseases.