Glycogen phosphorylase (GP) controls glycogenolysis which can be key for muscle contraction both during the initial phase and at high work rates (> 60 % VO2,max) (Parolin et al. 1999). In the present study we have investigated the effects on inhibition of GP by ZM399527 (5-chloro-N-[2-(4-fluorophenyl)-1-(4-hydroxypiperidino carbonyl) ethyl]-1H-indole-2-carboxamide) upon muscle fatigue.
Anaesthetised Wistar rats (alfaxalone/alfadalone, 12 mg kg-1 h-1, I.V., n = 16) had electrodes placed on the sciatic nerve to induce isometric contraction of the extensor digitorum longus muscle (EDL) (stimulated for 10 min at 2 Hz, 10 ms, supramaximal voltage). Muscle force (set initially at 5 g) was measured 30 min following either the GP inhibitor (ZM399527, 10 mg kg-1 I.V.) or vehicle (10 % hydroxypropyl β cyclodextrin/0.2 % saline). At 10 min of contraction both the contracting and contralateral muscles were removed, and frozen immediately in liquid nitrogen before storing at -80°C prior to analysis. The animals were killed humanely with an overdose of pentobarbitone whilst under anaesthesia, in accordance with UK regulations. Samples were digested in 1 M KOH for the measurement of tissue glycogen (amyloglucosidase method) and lactate (lactate oxidase method). Concentration of ZM399527 in ethyl acetate extracts of KOH digest of the gastrocnemius muscle was measured by HPLC analysis.
Muscle homogenate concentration of ZM399527 was 15.0 ± 1.3 µM (mean ± S.E.M.). IC50 for the compound on GP is 83 nM (Hoover et al. 1998). Peak EDL tension was not different in the two groups (28.8 ± 3.0 and 26.3 ± 0.8 g following vehicle and ZM399527, respectively; Student’s t test for paired data). In addition, the time to peak tension was unaltered (160 ± 30 vs. 113 ± 15 s). Femoral blood flow responses (measured by Doppler flow probe) both at rest (0.00 ± 0.08 vs. -0.02 ± 0.21 ml min-1) and during hindlimb contraction (1.58 ± 0.28 vs. 1.95 ± 0.16 ml min-1) were not different between vehicle and ZM399527-treated animals. However, EDL fatigue rate was lower (1.02 ± 0.18 and 0.54 ± 0.06 g min-1, P < 0.03) and lactate content of the contracting muscle lower in the ZM399527 group (233.2 ± 26.0 vs. 164.2 ± 16.9 nmol g-1, P < 0.05). Muscle glycogen content after contraction was unaltered by ZM399527 treatment (21 ± 3 vs. 19 ± 4 mmol g-1, n.s.).
These data show that at concentrations of GP inhibitor equivalent to 180-fold higher than required for inhibition of muscle GP isoform, lactate formation and muscle fatigue were reduced during muscle contraction. These data therefore suggest that muscle glycogen phosphorylase is not normally a rate-limiting factor in the ability of muscle to maintain work output in the present protocol.