Contraction-mediated glycogenolysis in mouse skeletal muscle lacking creatine kinase: the role of phosphorylase b activation

Abstract

Skeletal muscle that is deficient in creatine kinase (CK-/-) exhibits accelerated glycogenolysis during contraction. Understanding this phenomenon could provide insight into the control of glycogenolysis during contraction. Therefore, glycogen breakdown was investigated in isolated extensor digitorum longus CK-/- muscle. Muscles were stimulated to produce repeated tetani for 20 s in the presence of sodium cyanide to block mitochondrial respiration. Accumulation of lactate after stimulation was similar in wild-type (WT) and CK-/- muscles, whereas accumulation of glucose-6-phosphate was twofold higher in CK-/- muscles, indicating greater glycogenolysis in CK-/- muscles. Total phosphorylase activity was decreased by almost 30 % in CK-/- muscle (P < 0.001). Phosphorylase fractional activity (-/+ 3.3 mM AMP) was similar in both groups in the basal state (about 10 %), but increased to a smaller extent in CK-/- muscles after stimulation (39 +/- 4 % vs. 52 +/- 4 % in WT, P < 0.05). Inorganic phosphate, the substrate for phosphorylase, increased marginally in CK-/- muscles after stimulation (basal = 25.3 +/- 2.2 micromol (g dry muscle)-1; stimulated = 33.9 +/- 2.3 micromol (g dry muscle)-1), but substantially in WT muscles (basal = 11.4 +/- 0.7 micromol (g dry muscle)-1; stimulated = 54.2 +/- 4.5 micromol (g dry muscle)-1). Kinetic studies of phosphorylase b (dephosphorylated enzyme) from muscle extracts in vitro demonstrated higher relative activities in CK-/- muscles (60-135 %) in response to low AMP concentrations (up to 50 microM) in both the basal state and after stimulation (P < 0.05), whereas no differences in activity between CK-/- and WT muscles were observed at high AMP concentrations (> 100 microM). These data indicate that allosteric activation of phosphorylase b accounts for the accelerated glycogenolysis in CK-/- muscle during contraction.

Figure 1. Phosphorylase activation is blunted in CK^−/− muscle during contraction

EDL muscles from CK^−/− (^) and WT (•) mice were either not simulated (0 s) or stimulated with 330 ms tetani (1 s⁻¹) for 10 or 20 s in the absence or presence of 3 mm NaCN. Since NaCN did not significantly affect the results, they have been pooled. The 10 s stimulations were performed only in the absence of NaCN. Phosphorylase was measured in the absence and presence of 3.3 mm AMP and the fractional activity is the ratio of the two. Values are means ± s.e.m. for 8–18 muscles, except for WT at 10 s, where n = 2. CK^−/− values during contraction are significantly lower than for WT (P < 0.05).

Figure 2. Phosphorylase b sensitivity to AMP is increased in extracts from CK^−/− muscle

Extracts were prepared from CK^−/− (^) and WT (•) EDL muscles that were either not stimulated (A) or stimulated for 20 s (B) and analysed for phosphorylase activity with various concentrations of AMP, as described in the text. Activity in the absence of AMP was set to zero and the activity measured in the presence of AMP is considered to reflect phosphorylase b activity. Insets show activities at low AMP concentrations, where mean WT values (control, black bars) are set to 100 % and CK^−/− values (open bars) are given as a percentage of WT values. Values are means ± s.e.m. for 6–8 muscles. *P < 0.05; **P < 0.01; ***P < 0.001 between WT and CK^−/− at a given AMP concentration.

Figure 3. Fatigue is accelerated in CK^−/− muscle during repeated contractions in the presence of cyanide

EDL muscles were stimulated with 330 ms tetani at 1 s intervals. Values are means ± s.e.m. for CK^−/− (n = 6, unfilled circles) or WT (n = 8, filled circles) muscles. Other than the first tetanus, differences between groups are significant during all contractions (P < 0.01–0.001).