Aged skeletal muscles become smaller and weaker, which are also more susceptible to injury and take considerably longer to repair (Menshikova et al., 2006; Pasini et al., 2012). Previous studies have shown that the regular exercise leads to training-induced adaptation of aged muscles through contractile protein synthesis (Menshikova et al., 2006; Ogura et al., 2011). This adaptation includes structural remodeling and biochemical changes such as an upregulation of antioxidant enzymes, content of stress and heat shock proteins (HSPs) (McArdle and Jackson, 2000; Vasilaki et al., 2003).
HSPs play an important role in cellular processes, such as cell survival, proliferation and prevention of apoptosis against oxidative stress and aging (Koh and Escobedo, 2004; Vasilaki et al., 2003; Weber et al., 2012). HSPs are classified into hsp 27, 60, 70 and 90 according to the molecular weight. The expression of HSPs in skeletal muscles increases with exercise training, and it causes inhibition of apoptosis and cytoskeletal protection, which are needed for maintaining homeostasis and preventing myotube degeneration (Gabai and Sherman, 2002). Furthermore, HSPs correlates with high expression of SODs, which acts as a protective mechanism against oxidative stress in skeletal muscles after exercise (Murlasits et al., 2006; Zembron-Lacny et al., 2008). Among signaling pathway, MAPKs are general mediators of the stress response during exercise training (Nader and Esser, 2001). ERK1/2, p38 and JNK of MAPKs families are activated by various environmental stresses and related to activation of HSPs (Cargnello and Roux, 2011). Study about the exercise-induced production of HSPs in the skeletal muscle of aged rats is important as it may provide a valuable insight into the molecular mechanisms which can increase muscle mass against age-related stressors by regular exercise (Morton et al., 2009). Previous studies have demonstrated that HSP may differ in aged animal skeletal muscles following a period of duration and frequency exercise as well as numerous muscle structural and biochemical changes including increased antioxidant enzymes activity (McArdle and Jackson, 2000; Nader and Esser, 2001; Noble et al., 2008). Nevertheless, the effects of duration and frequency of exercise on the expression of HSPs in the skeletal muscle of aged animals are unknown. Therefore, the purpose of this study was to investigate whether different types of exercise training affect the expression of HSPs, SODs, and MAPKs in skeletal muscles of aged rats.
Male Sprague-Dawley rats [young (4 months old) and aged (21 months old) rats] were kept in a room that had an inverted 12h light/dark cycle under standard conditions of temperature and humidity. The animals were divided into the following 8 groups: young control (YC, n = 6) and old control (OC, n = 6); single long-duration exercise-trained old groups (OS1, n = 7): 1 x 30 min, 5 days x [week.sup.-1] for 6 weeks; second type of single long-duration exercise-trained old groups (OS2, n = 8): 1x30 min, 3 days x [week.sup.-1] for 6 weeks; multiple short-duration exercise-trained old groups (OM1, n = 8): 3 x 10 min x [day.sup.-1], 5 days x [week.sup.-1] for 6 weeks; second type of multiple shortduration exercise-trained old groups (OM2, n = 8): 3 x 10 min, 3 days/week for 6 weeks. The rats in the exercise groups were subjected to a treadmill physical training program. A standard laboratory animal chow (56.8% carbohydrates, 22.5% proteins, 3.5% lipids and 17.2% other nutrients; Korean animals) and water were given ad libitum. The experiment was approved by the Ethical Committee of the Chonbuk National Universityv (CBU: 2012-0046) for care and experimentation of laboratory animals.
Physical training protocol
The training protocol has been described previously (Ogura et al., 2011). In the first week of the preliminary experiment, the rats were adapted to the treadmill (Omnipacer model LC-4, Omnitech, Columbus, OH), and Exercise consisted of 5~10 min at a speed of 5~10 m x [min.sup.-1] with an incline of 0[degrees]. This program comprised of running on a treadmill for 30 (30 x 1) min, 5 days or 3 days per week for 6 weeks, and for 30 (10 x 3) min, 5 days or 3 days per week for 6 weeks at a maximal intensity or speed by aging rats (10 m x [min.sup.-1], 5[degrees] incline). Electric shocks were used sparingly to motivate the animals to run. Electrical shocks were applied to the metal grid behind the lane to stimulate the rats that failed to run spontaneously. The rats in the non-exercise group remained in their cages. Rats were sacrificed 48 h after their last exercise bout to minimize the influence of the last bout of exercise. Samples of blood and extensor digitorum longus (EDL; fast type) and soleus (SOL; slow type) skeletal muscles were collected. Separated serum and skeletal muscles were stored at -80[degrees]Cbefore determining cytokine levels and protein activity.
Western blot analysis
The total proteins were extracted from soleus and EDL muscle tissues using a lysis buffer containing 150 mmol x [L.sup.-1] NaCl, 5 mmol x [L.sup.-1] EDTA, 50 mmol x [L.sup.-1] Tri-HCl (pH 8.0), 1%-NP 40, 1 mmol x [L.sup.-1] aprotinin, 0.1 mmol leupeptin, and 1 mmol x...