Elevated plasma creatine kinase (CK) activity is often used as a marker of muscle injury in myopathies, cardiomyopathies and encephalopathies (Brancaccio et al., 2007). Muscle exercise can also trigger an efflux of various molecules, including CK, from skeletal muscles (Reihmane et al., 2013). Muscle CK efflux occurs after various types of exercise, but is particularly large after eccentric contractions (Newham et al., 1986). However, the relationship between muscle CK efflux and exercise characteristics is often complicated by the inflammatory response which can lead to secondary muscle damage after exercise (McHugh, 2003: Tidball, 1995). Indeed, plasma CK activity shows no or only a small increase after exercise and peaks after 1-5 days of recovery which often coincides with the time of peak muscle soreness (Ahmadi et al., 2007; Armstrong et al., 1984). Interestingly, no evidence of damage was detected in skeletal muscle fibres showing significant swelling after eccentric exercise (Yu et al. 2013). It appears that plasma CK activity is influenced by the interplay between muscle damage, osmotic factors and CK clearance from the body fluids after exercise (McHugh, 2003; Tidball, 1995). It would be beneficial to separate these factors in order to gain a better understanding of the physiological mechanisms responsible for muscle CK efflux. Isolated muscles might provide a good model for such studies as the effects of secondary muscle damage and CK clearance can be minimized. CK efflux from the isolated muscles increases after muscle injury induced by chemical agents, such as calcium ionophore A23187 and dinitrophenol, supporting the feasibility of this experimental approach (Jackson et al., 1987).
Sex and age might affect susceptibility to muscle damage and loss of muscle proteins after exercise (Amelink et al., 1990; Lynch et al., 2008). However, the findings from the human studies are conflicting. When compared to women, men showed higher plasma CK activity after marathon running (Rogers et al., 1985), but the opposite was true for 50 maximal eccentric arm flexions (Miles et al., 1994). It appears that susceptibility to exercise-induced muscle damage increases from the preadolescent age to adulthood (Chen et al., 2014). However, children show greater impairment in muscle voluntary activation and this could affect comparison of muscle damage indicators in children and adult after exercise (Streckis et al. 2007). Most of the mouse studies of muscle damage focused on the very old animals (Brooks et al., 2001; Lynch et al., 2008). Old age is associated with impairments in motor coordination, reduced levels of physical activity and significant loss of muscle mass (Brooks et al., 2001; Wolfe et al., 2006). It is unclear if muscle susceptibility to damage changes from young age to adulthood when animals reach high levels of muscle strength.
The first aim of the present study was to examine if CK efflux from skeletal muscles is indeed affected by the type of muscle exercise. Thus we assessed CK efflux from the isolated soleus muscle (SOL) of adult female mice after either passive stretching, isometric contractions or eccentric contractions in vitro. The second aim of the study was to investigate if muscle CK efflux is dependent on age and sex of animals. We compared muscle CK efflux in young animals of both sexes and female adults at rest and after eccentric exercise.
Animals and experiments
All procedures involving mice were approved by the Lithuanian Republic Alimentary and Veterinary Public Office (Nr. 0223). As in our previous studies (Baltusnikas et al., 2015; Kilikevicius et al., 2013; Ratkevicius et al., 2010), C57BL/6J mice were housed in standard cages, one to three mice per cage at a temperature of 22-24[degrees]C and 40-60 % humidity with the normal 12/12-h light/dark cycle. Animals were fed standard chow diet and received tap water ad libitum. We assessed CK efflux from SOL in adult (7.5 month old) females after 100 non-stimulated stretches (n = 8), 100 isometric contractions (n = 9), 100 lengthening contractions (n = 10) and after passive incubation (n = 10). We have also studied CK efflux from SOL of young (3-month old) females and males after 100 eccentric contractions (n = 11 and n = 8, respectively) and without any exercise (n = 9 and n = 6, respectively).
Mice were euthanized by cervical dislocation. Assessment of contractile properties and CK efflux from SOL was performed using similar methods as in our recent study (Baltusnikas et al., 2015). Silk sutures were attached to the proximal and distal tendons of SOL from left leg. The muscle was then excised and fixed between two platinum plate electrodes in 50 ml Radnotti tissue bath filled with Tyrode solution (121 mM NaCl, 5 mM KCl, 0.5 mM Mg[Cl.sub.2], 1.8 mM Ca[Cl.sub.2], 0.4 mM Na[H.sub.2]P[O.sub.4], 0.1 mM NaEDTA, 24 mM NaHC[O.sub.3], 5.5 mM glucose, pH adjusted to 7.4) bubbled with 95% [O.sub.2] and 5% C[O.sub.2] at room (~25 [degrees]C) temperature. The distal tendon of the muscle was attached to a hook and the proximal end was tied directly to the lever of muscle test system (1200A-LR Muscle Test System, Aurora Scientific Inc., Aurora, Canada). The muscle was then left to equilibrate in the Tyrode solution at a slight pre-tension (~20-30 mN) for 7 min in order to minimize heterogeneity of sarcomere lengths that might have occurred as a result of muscle manipulations during the dissection. Afterwards, muscle length was increased in steps every 2 min and the muscle was stimulated supramaximally at 150 Hz for 3 s. This procedure was continued until no further increase in muscle force was seen with the increase in muscle length. Our measurements showed that SOL reaches its peak isometric force at 150 Hz of electrical stimulation. Therefore we used this stimulation frequency to set optimal muscle length ([L.sub.0]), and muscles were kept at this length during the subsequent procedures.
Afterwards, tetanus contraction time was assessed as the time from the beginning of the force development to 90% of the peak force. Then SOL was subjected to one of the three protocols of muscle manipulations which were repeated every 10 s. The typical force recordings from these experiments are presented in Figure 1. For the protocol of passive stretches, the muscle was subjected to a ramp 3.5-mm stretch corresponding to approximately 30% of muscle length over 200 ms followed by return to L0 in 200 ms. For isometric contraction protocol, SOL was stimulated at 150 Hz for 700 ms. For the eccentric exercise, SOL was also stimulated at 150 Hz for 700 ms, but subjected to 3.5 mm ramp stretch during the last 200 ms of this stimulation which was followed by return to the optimal length in another 200 ms. In less than 10 s after completion of all three protocols, SOL was removed from the tissue bath and incubated in 2 ml of Tyrode solution for 2 h at room temperature. In...