Programme Contents

Effect of S-nitrosoglutathione (GSNO) on excitation-contraction coupling in mechanically-skinned muscle fibres of the rat

S-nitrosoglutathione (GSNO) is a naturally-occurring compound found in fast-twitch muscle fibres, which is able to S-glutathionate and/or S-nitrosylate various target proteins involved in excitation-contraction (EC) coupling (Aracena et al., 2003). S-glutathionation or S-nitrosylation-induced changes to EC coupling proteins such as the Na⁺/K⁺ and SR Ca²⁺-ATPases might alter their efficacy and contribute to muscle fatigue under certain conditions. We sought to identify which EC coupling steps are more susceptible than others to GSNO-modulated changes and whether these changes might be important in understanding of muscle fatigue.

Male Long-Evans hooded rats were killed under deep anaesthesia (2% v:v isoflurane) and the extensor digitorum longus and soleus muscles swiftly excised and immersed in paraffin oil at resting length. Single fibres were mechanically-skinned with forceps and a segment connected to a force transducer (stretched to 120% resting length) and immersed in either a standard K-HDTA solution with weak Ca²⁺ buffering (50 μM EGTA, pCa 7) to examine transverse tubular (T-) system depolarization-induced force responses or into heavily Ca²⁺-buffered (50 mM EGTA) solutions to examine the contractile apparatus properties. All solutions contained as follows: 1 mM free Mg²⁺, 8 mM ATP; 10 mM creatine phosphate, at pH 7.1, and were equilibrated to room temperature (∼23°C) before use. GSNO was dissolved directly into solution immediately (∼1 min) before use.

Single fibres were electrically stimulated (75 V cm^-1, 1 ms pulse) to produce twitch or tetanic (50 and 100 Hz) force responses before and after GSNO exposure (2 mM for 2 min in all cases). Additionally, paired pulses with differing intervals (0-20 ms) were applied to determine the repriming period of sodium channels in the T-system membrane (an indirect but sensitive measure of T-system polarization, see Dutka & Lamb, 2007). The Ca²⁺-sensitivity of the contractile apparatus was determined by transferring the single skinned fibre segment from the 50 mM EGTA-based solution to solutions with progressively higher free [Ca²⁺] (made by mixing 50 mM EGTA solution with 50 mM Ca-EGTA solution as appropriate, pCa range ∼10-4.6) before and after GSNO treatment (2 mM, 2 min in 50 mM EGTA solution). In some other cases, 10 mM DTT (added from a 1M stock) was added to the solution. Maximum Ca²⁺-activated force produced by the contractile apparatus was virtually unchanged by exposure to GSNO. The Ca²⁺-sensitivity of the contractile apparatus was substantially potentiated (∼0.12 pCa) after exposure to fresh GSNO (n = 7), and this effect was fully reversed by 10 mM DTT. In two slow-twitch SOL fibres GSNO exposure virtually had no effect. Interesting in electrically-stimulated fast-twitch fibres, the equivalent exposure to GSNO initially caused a moderate decrease of 35% and 20% in peak twitch and tetanic force responses respectively (compared to the pre-exposure level, n = 11). Furthermore, subsequent twitch and tetanic force responses became progressively smaller and this was not reversed fully by 10 mM DTT but was substantially ameliorated by a small additional Ca²⁺ load (10 s at pCa 6.7 or equivalent to ≤50% of the endogenous SR Ca²⁺ level), suggesting that the cause of the decline in force involved loss of SR Ca²⁺. The progressive decrease in depolarization-induced force was not attributable to changes in T-system excitability because the repriming period for T-system Na⁺ channels was not significantly altered (4.3 ± 0.2 ms and 4.0 ± 0.3 ms before and after GSNO). These data indicate that the GSNO exposure glutathionated the contractile apparatus, causing a substantial increase in Ca²⁺-sensitivity (∼0.12 pCa). GSNO treatment had little effect on T-system excitability, thus implying Na⁺ channels, voltage-sensors, and Na⁺/K⁺ pumps remained functional unaltered. The progressive loss of depolarization-induced force after GSNO treatment was seemingly due to a loss of Ca²⁺ from the SR either through the SR Ca²⁺ pump itself or through the RyR. Alternatively, the uptake by SR Ca²⁺ pumps might be reduced at the resting Ca²⁺ level. Taken together, glutathionation in intact fibres might aid in maintaining force output when it might otherwise decline.

Aracena P, Sánchez G, Donoso P, Hamilton SL & Hidalgo C. (2003) Journal of Biological Chemistry, 278: 42927-35.

Dutka TL and Lamb GD. (2007) American Journal of Physiology, 292: C2112-21.