ABSTRACT
Intense muscle activity can result in fatigue, a state where tetanic force remains depressed for a considerable period after the end of activity. At the level of interaction between myosin and actin, the force loss might reflect either a decrease in the number of force-generating crossbridges or a decrease in the mean force generated by single crossbridge. The cause of these changes is unclear but one recurrent suggestion is that free radicals or reactive oxygen species (ROS) have modified the contractile proteins. The present experiments investigated this point using single fibres or small fibre bundles isolated from the mouse flexor digitorum brevis muscle at 22-24°C. Fibres were repetitively stimulated to induce fatigue and then force and stiffness recovery were followed during
exposure to normal Tyrode solution or Tyrode solution to which 1 mM dithiothreitol (DTT) had been added. Force and fibre stiffness were measured before fatigue and during recovery from fatigue during 30 and 120 Hz test tetani. During the whole recovery from fatigue, force was slightly though significantly depressed in DTT with respect to Tyrode solution, whereas fibre stiffness remained unchanged. Our findings suggest that during recovery from fatigue, the impaired force production of crossbridges is not easily reversed or modified by a powerful reducing agent. Since force reduction by DTT occurred without alteration of fibre stiffness, our results suggest that force reduction is caused mainly by a mechanism which does not reduce crossbridge number, such as a reduction of the mean crossbridge force. 9.1 IntroductionFollowing periods of intense exercise, a state of fatigue persists and an individual cannot generate the force or power output that was possible before exercise started. It has long been known that this force loss is more marked at low compared to high fibre recruitment or stimulation frequencies (Edwards et al., 1977). Similar results were found in isolated skeletal muscles that were induced to contract repeatedly and where force was monitored and subsequently followed during recovery. The causes and the mechanisms underlying this force loss after fatigue are incompletely understood. Several hypotheses have been advanced and one that is currently receiving much attention suggests that the oxidation-reduction status of the muscle has been altered by increased production of reactive oxygen species (ROS), including the superoxide anion (Allen et al., 2008; Bruton et al., 2008; Lamb and Westerblad, 2011).Production of ROS is generally agreed to increase with exercise. ROS induces modifications of both actin and myosin filaments (Fedorova et al., 2010). Recently, it was demonstrated that a variety of anti-oxidant agents alone or in combination were able to restore tetanic calcium transients but were unable to reverse the force depression associated with fatigue (Cheng et al., 2015). However, in the presence of saturating [Ca2+], high concentrations of oxidising agents have been shown to markedly
alter force in intact muscle (Andrade et al., 1998) and in skinned muscle fibres (Plant et al., 2000). It has also been demonstrated that dithiothreitol (DTT) can radically affect the oxidation status of muscle and can reverse the force depression induced by H2O2 (Andrade at al., 1998; Dutka et al., 2012; Posterino et al., 2003). Thus, we were interested to determine the effect of DTT on crossbridge properties during recovery from fatigue. 9.2 Methods
9.2.1 Fibre Dissection and MeasurementsMale mice (C57BL/6 strain, 3-6 months old) were housed at controlled temperature (21-24°C) with a 12-12 h light-dark cycle. Food and water were provided ad libitum. Mice were killed by rapid cervical dislocation, according to the procedure suggested by the Ethical Committee for Animal Experiments of the University of Florence and the EEC guidelines for animal care of the European Community Council (Directive 86/609/EEC). All efforts were made to minimize animal suffering and to use only the number of mice necessary to obtain reliable data. Both flexor digitorum brevis (FDB) muscles were removed and placed in oxygenated Tyrode solution: one FDB was used for DTT treatment and the contralateral muscle was used as control. Single intact fibres or small bundles of 2 to 10 fibres were dissected as described previously (Colombini et al., 2009). Aluminium clips were attached to tendons as close as possible to the end of the fibre preparations and used to mount the fibres horizontally in an experimental chamber (capacity 0.38 ml) between the lever arms of a capacitance force transducer (resonance frequency, 16-20 kHz) and an electromagnetic motor that was used to change fibre length. Fibres were perfused continuously at a rate of about 0.35 ml min−1 with a normal Tyrode (NT) solution of the following composition (mM): NaCl, 121; KCl, 5; CaCl2, 1.8; MgCl2, 0.5; NaH2PO4, 0.4; NaHCO3, 24; glucose, 5.5; EDTA, 0.1 and bubbled with 5% CO2-95% O2 (pH of 7.4). Foetal calf serum (0.2%) was routinely added to the solution. Tyrode solution containing 1 mM dithiothreitol (DTT) was prepared fresh before each experiment following the procedure of Andrade et al. (1998).
