Hydrogen peroxide increases depolarization-induced contraction of mechanically skinned slow twitch fibres from rat skeletal muscles

Abstract

The effect of exogenous hydrogen peroxide (H(2)O(2)) on excitation-contraction (E-C) coupling and sarcoplasmic reticulum (SR) function was compared in mechanically skinned slow twitch fibres (prepared from the soleus muscles) and fast twitch fibres (prepared from the extensor digitorum longus; EDL muscles) of adult rats. Equilibration (5 min) with 1 mM H(2)O(2) diminished the ability of the Ca(2+)-depleted SR to reload Ca(2+) in both slow (P < 0.01) and fast twitch fibres (P < 0.05) compared to control. Under conditions when all Ca(2+) uptake was prevented, 1 mM H(2)O(2) increased SR Ca(2+) "leak" in fast twitch fibres by 24 +/- 5 % (P < 0.05), but leak was not altered in slow twitch fibres. Treatment with 1 mM H(2)O(2) also increased the peak force of low [caffeine] contracture by approximately 45% in both fibre types compared to control (P < 0.01), which could be partly reversed following treatment with 10 mM dithiothreitol (DTT). The changes in SR function caused by 1 mM H(2)O(2) were associated with an approximately 65% increase in the peak height of depolarization-induced contractile response (DICR) in slow twitch fibres, compared to control (no H(2)O(2); P < 0.05). In contrast, peak contractile force of fast twitch fibres was not altered by 1 mM H(2)O(2) treatment. Equilibration with 5 mM H(2)O(2) induced a spontaneous force response in both slow and fast twitch fibres, which could be partly reversed by 2 min treatment with 10 mM DTT. Peak DICR was also increased approximately 40% by 5 mM H(2)O(2) in slow twitch fibres compared to control (no H(2)O(2); P < 0.05). Our results indicate that exogenous H(2)O(2) increases depolarization-induced contraction of mechanically skinned slow but not fast twitch fibres. The increase in depolarization-induced contraction in slow twitch fibres might be mediated by an increased SR Ca(2+) release during contraction and/or an increase in Ca(2+) sensitivity.

Figure 1. The ability of Ca²⁺-depleted SR to reload Ca²⁺ is decreased by 1 mm H₂O₂ in both slow and fast twitch mechanically skinned muscle fibres

Relationship between the time in loading solution and SR Ca²⁺ content of mechanically skinned slow (circles, n = 5) and fast twitch (squares, n = 6) fibres, before (continuous line, filled symbols) and after (dashed line, open symbols) 1 mm H₂O₂ treatment. **P < 0.01, *P < 0.05; pre- vs. post-H₂O₂ treatment.

Figure 2. SR Ca²⁺‘leak'is increased by 1 mm H₂O₂ in fast but not slow twitch muscle fibres

Ratio of SR Ca²⁺ content following Ca²⁺‘leak’ in 1 mm H₂O₂ and control conditions (before H₂O₂ treatment) in slow (open bar) and fast twitch (hatched bar) mechanically skinned fibres (n = 5 fibres per group). Due to the suppressive effect of H₂O₂ on SR Ca²⁺ reloading (see Fig. 1), SR Ca²⁺ content was standardized (SR Ca²⁺ reloading under control conditions) before H₂O₂‘leak’ was assessed. When all Ca²⁺ uptake was prevented, H₂O₂ increased SR Ca²⁺ leak from fast but not slow twitch muscle fibres. *P < 0.05 compared to control; pre-H₂O₂ treatment; dashed line.

Figure 3. H₂O₂ increases the sensitivity of both slow and fast twitch mechanically skinned muscle fibres to caffeine-induced Ca²⁺ release

Peak force responses during low [caffeine] contracture under control conditions, 1 mm H₂O₂ equilibration (5 min), and 10 mm DTT equilibration (5 min) in slow (open bars) and fast twitch (hatched bars) mechanically skinned fibres (n = 5 fibres). The sensitivity to caffeine-induced Ca²⁺ release was increased after equilibration with 1 mm H₂O₂, indicating increased activity of the SR CRC. The effect of H₂O₂ was fully reversed in slow twitch fibres but only partly reversed in fast twitch fibres exposed to 10 mm DTT. *P < 0.05, **P < 0.01 pre- vs. post-H₂O₂ treatment for each fibre.

Figure 4. Depolarization-induced force responses of mechanically skinned slow and fast twitch muscle fibres

Original recordings from (A) a slow twitch fibre from the soleus muscle and (B) a fast twitch fibre from the EDL muscle repeatedly depolarized and repolarized (30s in K-HDTA) between successive depolarizations. Many fewer depolarizations could be elicited from the slow than fast twitch fibres before run-down. Incubation with a low [Mg²⁺] solution (0.1 mm free Mg²⁺) elicited a transient force response indicative of functional CRC, even at the point of fibre rundown. Soleus fibres were also incubated with an Sr-EGTA solution (pSr ∼5.50) to distinguish slow (type I) from fast (type II) fibres (see text). At the conclusion of all experiments, each fibre was bathed in a Ca-EGTA solution (pCa < 4.7) to determine maximum Ca²⁺-activated force. Here and in subsequent figures: Depol, depolarization with Na-HDTA solution; Low [Mg²⁺], 0.1 mm free Mg²⁺ in K-HDTA; Max, Ca-EGTA (pCa < 4.7); Sr²⁺, Sr-EGTA (pSr ∼5.50).

Figure 5. Peak depolarization force is increased by 1 mm H₂O₂ in mechanically skinned slow but not fast twitch muscle fibres

Original experimental trace recorded from (A) a slow and (B) a fast twitch fibre repeatedly depolarized (Na-HDTA) and repolarized (30 s in K-HDTA) during 1 mm H₂O₂ exposure. H₂O₂ treatment increased the peak height of DICR in slow but not fast twitch fibres.

Figure 6. Spontaneous force production is caused by 5 mm H₂O₂ in both slow and fast twitch mechanically skinned muscle fibres

Original experimental recording of a mechanically skinned (A) slow and (B) fast twitch muscle fibre during exposure to 5 mm H₂O₂. Spontaneous force production was observed soon after exposure to 5 mm H₂O₂. On average the peak height of DICR was increased following H₂O₂ treatment in slow but not fast twitch fibres. The spontaneous force production induced by H₂O₂ was reduced by treatment with 10 mm DTT.