The effectiveness of physical training depends on physiological parameters of participants, applied workload as well as individual susceptibility to tolerate fatigue. Imbalance between the last two may leads to under or overtraining. Depending on the applied workload, different immunological responses to training can be induced. A practice, imposing an excessive stress, result in an inflammatory response robust and likely, sufficiently powerful, to modify subsequent responses. The long term consequences of such impact may occur via mechanisms of immune tolerance and/or training-associated reduction in the innate immune response to brief exercise (Cooper et al., 2007). Thus, there is only a fine line between improved performance and deterioration (Smith, 2000).
Exercise triggers simultaneous increase of various antagonistic mediators, yet also, elevates catabolic pro inflammatory cytokines such as interleukin 1[beta] (IL-1[beta]) and tumour necrosis factor alpha (TNF[alpha]). On the other hand, it also stimulates anabolic components such as interleukin 6 (IL-6), interleukin 10 (IL-10) and heat shock proteins (Hsps), which protect against stressors. If an anabolic response is stronger, training will probably, ultimately lead to an enhanced muscle mass and improved exercise adaptation (Noble et al., 2008; Pedersen, 2011; Roubenoff, 2007).
The role of pro-inflammatory cytokines in skeletal muscle growth still has not been fully explored. It was observed that after IL-1[beta] stimulation the total of protein synthesized does not increase, but rather synthesis of the acute phase proteins is favoured (Weissman, 1990). A study by Tayek (1996) showed that TNF[alpha] has significant short- and long-term effects on protein synthesis. It was also demonstrated to be able to reduce weight gain and enhance muscle catabolism (Tracey et al., 1988), yet, the suppression of TNF[alpha] synthesis with anti-inflammatory drug delays muscle restoration. At the same time, an excessive IL-1[beta] and TNF[alpha] release may be responsible for the overtraining (Mackey et al., 2007; Main et al., 2009).
The measurement of both pro- and anti-inflammatory cytokines IL-1[beta], TNF[alpha], IL-6 and IL-10 within a population of athletes during training has not been yet widely reported (Main et al., 2009; Marin et al., 2011; Reinke et al., 2009; Zembron-Lacny et al., 2010). Nowadays, it is known that pro- and anti-inflammatory cytokines concentrations alter as a result of physical activity in a way dependent on a discipline; yet we still lack information on the levels of inflammatory mediators appropriate and most beneficial for athletes training a particular sport. Research in this area, particularly in tennis, is challenging due to numerous factors that require analysis, including number of matches played, their intensity and duration time. Their unpredictable occurrence makes running an investigation during a tournament season very demanding. Nevertheless, such research is vital to allow trainings to be planned in a way to stimulate and emphasize the anti-inflammatory response.
Heat shock proteins (Hsps) represent cell-protective system that may be induced by reactive oxygen species, cytokines, and hyperthermia. Under physiologically balanced conditions, constitutively expressed Hsps function as molecular chaperones, whereas under stress conditions, Hsps protect proteins against misfolding, aggregation and denaturation. Non adequate Hsps biosynthesis may be deleterious to cells and make them more sensitive to stress. HSPs may also directly regulate specific stress-responsive signalling pathways and may antagonize signalling cascades that result in apoptosis (Madamanchi et al., 2001; Noble et al., 2008). Hsps increase the stress tolerance and participate in the cellular repair processes. Moreover Hsp are involved in a number of remodeling processes associated with exercise training, such as facilitating mitochondrial biogenesis (Hood et al., 2000), regulators of apoptotic pathways (Samali and Orrenius, 1998), and inducing improvements in insulin sensitivity (Chung et al., 2008). No data are available about role of Hsp in overreaching syndrome.
Exercise-induced stress and muscle damage are considered two out of many stimuli, which induce Hsps synthesis (Steinacker et al., 2004). The sustaining high Hsps synthesis may indicate a state of inadequate regeneration even after a couple of weeks of recovery from exhaustive exercise (Lehmann et al., 1997). The elevated blood level of Hsp70 was observed in rowers, soccer players and endurance runners (Banfi et al., 2006; Fehrenbach et al., 2000; Liu et al., 2000). Among the subset of stress-responsive proteins, Hsp27 and Hsp70 are considered to be a new approach to monitoring exercise training and adaptive mechanisms (Banfi et al., 2006). The regulation of Hsp within intracellular environment is well understood, but extracellular Hsp can also exert important biological functions (Lancaster and Febbraio, 2005). For example Hsp27 seems to both directly scavenge the free radicals and protect against the toxicity of reactive oxygen species (ROS) (Wyttenbach et al., 2002).
One of the factors, which may induce synthesis of Hsp is hydrogen peroxide ([H.sub.2][O.sub.2]). It is an important signalling molecule, generated during muscle contraction, involved in regeneration and adaptation of skeletal muscle to physical exercise. [H.sub.2][O.sub.2] is produced by the enzymes superoxide dismutase (isoforms CuZnSOD and MnSOD), which are localized in the muscle sarcolemma and mitochondria (Jackson et al., 2007). The studies in human isolated muscle and myotube culture demonstrated that [H.sub.2][O.sub.2] produced within contracting skeletal muscle is the key regulator of signalling pathways, leading to skeletal muscle adaptation (Powers et al., 2010).
Basing on the gathered data on immunological response indicators, the study was designed to evaluate the blood level of Hsp27 and Hsp70, as well as their relation to skeletal muscle damage and inflammation in tennis players. We hypothesized that our young tennis players experienced overreaching after a tournament season--a syndrome characterised by an increase in blood pro-inflammatory and lower anti-inflammatory cytokines. Consequently, we set our goal to verify the influence of Hsp70 and Hsp27 levels on restoring an immune balance.
Data collection and subjects
Our investigation was held during the sport camp (beginning of October 2011), organized annually by the Polish Tennis Association at the National Olympic Sport Centre in Cetniewo (Poland). All subjects occupied the same accommodations and followed the same training and diet schedules. Daily, energetic value of food offered in the menu did not exceed 4000 kcal. The proposed protein dose varied from 1.2-1.4 g x [kg.sup.-1] of body mass.
The main purpose of the camp is to support development of the best young tennis players. Participants (n = 15, age 16 years old) are selected by the national coaches according to tennis players' annual achievements and rankings. The examination is officially approved by the Bioethical Committee of the Regional Medical Society in Gdansk NKEBN/39/2009 according to the Helsinki Declaration. Participation must be approved with written consents from the players' parents.
Blood was collected three times: directly after arrival at the camp (I), after a 3-day active rest (II) and at the end of the camp (III). The schedule of the training program was planed basing on our previous experiences, which had revealed that directly after arriving at the camp, low grade inflammation was noted. It suggested that players had been taking part in many different tournaments till the very end of the season to improve their rankings. In fact they did not experience sufficient recovery afterwards. Therefore, three days of an active rest, after arrival at the camp, were introduced, aimed to familiarize participants with stretching exercises and low-intensity training. After this period, body composition and aerobic assessment were held.
The presented training structure was applied in the first part of preparatory season, encountering for a half-year macrocycle. The main goal of the practice was to improve players' physical abilities via focusing on the main training components--strength, endurance and flexibility training. Consequently, 70% of training hours was assigned to strength, endurance and flexibility training, while the remaining 30% was used to develop other training components, vital in tennis: speed, agility, coordination as well as strokes timing. The strength practice based on developing local strength capability, as it is the first step to achieve a long-term strength level increase (dynamics of strokes and court movement). At the same time, endurance training implemented methods improving energy metabolism mechanisms, whereas flexibility was practiced through systematic exercises aimed to normalize muscle tension. Details of the training program are presented in Table 1, whereas it summary is included in Table 2.
Body composition assessment
Body mass (BM) and body composition were estimated using a multi-frequency impedance plethysmograph body composition analyser (InBody 720, Biospace Analyzer,...