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. 1998 Jun 1;509 ( Pt 2)(Pt 2):565-75.
doi: 10.1111/j.1469-7793.1998.565bn.x.

Effect of hydrogen peroxide and dithiothreitol on contractile function of single skeletal muscle fibres from the mouse

F H Andrade  1 , M B Reid, D G Allen, H Westerblad
Affiliations

Effect of hydrogen peroxide and dithiothreitol on contractile function of single skeletal muscle fibres from the mouse

F H Andrade et al. J Physiol. .

Abstract

1. We used intact single fibres from a mouse foot muscle to study the role of oxidation-reduction in the modulation of contractile function. 2. The oxidant hydrogen peroxide (H2O2, 100-300 microM) for brief periods did not change myoplasmic Ca2+ concentrations ([Ca2+]i) during submaximal tetani. However, force increased by 27 % during the same contractions. 3. The effects of H2O2 were time dependent. Prolonged exposures resulted in increased resting and tetanic [Ca2+]i, while force was significantly diminished. The force decline was mainly due to reduced myofibrillar Ca2+ sensitivity. There was also evidence of altered sarcoplasmic reticulum (SR) function: passive Ca2+ leak was increased and Ca2+ uptake was decreased. 4. The reductant dithiothreitol (DTT, 0.5-1 mM) did not change tetanic [Ca2+]i, but decreased force by over 40 %. This was completely reversed by subsequent incubations with H2O2. The force decline induced by prolonged exposure to H2O2 was reversed by subsequent exposure to DTT. 5. These results show that the elements of the contractile machinery are differentially responsive to changes in the oxidation-reduction balance of the muscle fibres. Myofibrillar Ca2+ sensitivity appears to be especially susceptible, while the SR functions (Ca2+ leak and uptake) are less so.

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Figures

Figure 1
Figure 1. Brief exposure to H2O2 increases force in single skeletal muscle fibres
A, original [Ca2+]i (top) and force (bottom) records from a single skeletal muscle fibre during 40 Hz tetani. The bars under the force tracings represent the 350 ms stimulation periods. Shown are 2 responses before (Control, 4 min apart) and after being exposed to 150 μM H2O2 for 3 min. Observe that the control responses are virtually identical. H2O2 increased force whereas [Ca2+]i was almost unchanged. B, mean data from 10 single fibres show that tetanic [Ca2+]i in single skeletal muscle fibres remained unaffected during short exposures to H2O2 (100-300 μM) whereas force increased. * Significant difference from control (P < 0.05).
Figure 2
Figure 2. Prolonged exposure to H2O2 decreases force
A, original [Ca2+]i (top) and force (bottom) records from a single fibre, presenting 50 Hz tetani before (Control), and at 4 and 8 min in 300 μM H2O2. The bars under the force tracings represent the stimulation periods. H2O2 for 4 min increased [Ca2+]i by only 1 % and force by 22 %. After 8 min in H2O2, [Ca2+]i increased 6 %, and force decreased to 74 % of control. B, biphasic effects of H2O2 on single fibre function. During the first 2-4 min, H2O2 (300 μM) increased force (•) during submaximal tetani without altering [Ca2+]i (▴; n= 8). As exposure extended to 6-16 min, [Ca2+]i increased, while force declined by over 40 %. These trends continued during the wash-out period.
Figure 3
Figure 3. H2O2 alters myofibrillar Ca2+ sensitivity and SR function
A, force-[Ca2+]i relationship obtained from a single fibre under control conditions (○). The continuous line represents the Hill equation fitted to these data points (Ca50= 0.51 μM, N= 7.2). Open triangles show [Ca2+]i and force during 40 Hz tetani 3 min before (▵) and 8 min after (▿) producing the force-[Ca2+]i curve. Filled symbols represent [Ca2+]i and force during 40 Hz tetani at selected time points during incubation with 100 μM H2O2 (2 min, •; 6 min, ▾; 7 min, ▪; 8 min, ♦; 10 min, ▴). The arrow shows the step change from the control 40 Hz tetanus (▿) to the first time point during H2O2 exposure. B, averaged records from 8 fibres of [Ca2+]i after the end of stimulation. The dashed curves represent the double exponential functions fitted to them. C, plots of the rate of decline in [Ca2+]i (d[Ca2+]i/dt) vs.[Ca2+]i taken from the double exponential fits shown in B (control, □; H2O2, ▪). The lines represent the curve fitting of the data points to eqn (4).
Figure 4
Figure 4. The effects of H2O2 are reversible
A, original [Ca2+]i (top) and force (bottom) records from a single fibre, presenting 50 Hz tetani before (Control), after 6 min in 300 μM H2O2, and after 10 min exposure to 1 mM DTT. H2O2 increased [Ca2+]i by 10 % and decreased force by 78 %. DTT restored [Ca2+]i and force to 105 and 107 % of control, respectively. B, decline of [Ca2+]i to resting levels, after the end of stimulation, for the three experimental conditions. The prolonged exposure to H2O2 displaced the [Ca2+]i tail upwards and to the right. Incubation with DTT completely reversed this change.
Figure 5
Figure 5. DTT reverses the effects of H2O2
Incubation with 300 μM H2O2 for 6-16 min markedly decreased force (•), while tetanic [Ca2+]i (▴) was not significantly affected during submaximal tetani (n= 4). DTT (0.5-1 mM for 6-10 min) applied immediately following the exposure to H2O2 almost restored force to the initial control value, whereas [Ca2+]i remained virtually unaffected.
Figure 6
Figure 6. Typical effects of DTT on [Ca2+]i and force transients
Original records presenting [Ca2+]i (top traces) and force (bottom traces) from a single fibre during 40 Hz tetani. The bars under the force tracings represent the stimulation periods. Four sequential conditions are shown: control, incubation with 1 mM DTT for 6 min, wash-out for 8 min, and incubation with 150 μM H2O2 for 6 min. DTT barely decreased [Ca2+]i by 4 %, while force was reduced by 35 %. Force dropped a further 25 % in the subsequent wash-out period. Finally, a short incubation with 150 μM H2O2 partially restored force, to 79 % of the control level.
Figure 7
Figure 7. DTT does not change [Ca2+]i but decreases force
[Ca2+]i (▴) and force (•) during submaximal tetani as percentage of control values and under the same conditions as in Fig. 6. [Ca2+]i was not significantly affected during incubation with DTT. The wash-out period and subsequent exposure to H2O2 did not significantly alter [Ca2+]i either. On the other hand, DTT decreased force by over 40 % (P < 0.05). The wash-out period did not reverse this change, but incubation with H2O2 restored force to within 6 % of the control level.
Figure 8
Figure 8. Force as a function of cellular redox balance
Model of force production as a function of cellular redox balance. The baseline redox balance (○) is to the left of the peak, into the reduction range. DTT shifts the reduction state further to the left and decreases force (▪). A short exposure to H2O2 changes the baseline redox balance to the right, into the oxidation range and increases force during submaximal tetani (▾). As exposure to H2O2 continues, the redox balance moves further into the oxidation state, and force decreases (▴). The positioning of ‘reduction’ and ‘oxidation’ is arbitrary. For further explanation, see Discussion.
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