Role of Heat Shock Proteins in Skeletal Muscle

The ability of the human organism to respond with rapid and appropriate modification during physiological challenges is an essential feature for its own survival. These modifications are inseparable from a satisfactory adjustment of the physiological processes of the whole body, where physiological systems tend to maintain relatively constant composition of the internal environment (milieu intérieur), despite the constant challenges that the body is submitted daily to, which is known as homeostatic control [1, 2].


Introduction
The ability of the human organism to respond with rapid and appropriate modification during physiological challenges is an essential feature for its own survival. These modifications are inseparable from a satisfactory adjustment of the physiological processes of the whole body, where physiological systems tend to maintain relatively constant composition of the internal environment (milieu intérieur), despite the constant challenges that the body is submitted daily to, which is known as homeostatic control [1,2].
The homeostatic regulation of any physiological variable depends on cooperative mechanisms which are activated simultaneously or in succession. Thus, intense or critical challenges to life require numerous and complex mechanisms to restore or to maintain the homeostasis. In this way, challenges generate a stress situation to the body and, depending on the severity, can cause harmful effects. Moreover, moderate challenges, which are also caused by stress, results in profitable physiological adjustments [3]. In this context, stress response may be considered a nonspecific response of the body to any demand. A stressor is an agent that produces stress at any time by different ways. The physiological adaptations of the body represent the chronologic development of the response to stressors when their action is prolonged [4].
Since the muscle represents about ¾ of the body mass, a healthy muscular system is associated with the status of the other system of the body. Sick muscle system has harmful effects on human healthy and to its the capacity of interaction in this world. Skeletal muscle is a high plastic tissue that may be modified by use or disuse. Muscle composition, at chemical and structural levels, can be altered and these modification are related to the specific type of exercise that the organism is submitted. For example, the well know effect of strenght training is the hypertrophy of the muscle, which is associated to an increase in force production capacity. Strenght training may induce hypertrophy (muscle fiber enlargement) and hyperplasia (increase in muscle fiber number) while endurance training promotes elevated muscle oxidative capacity (increase in mitochondrial number). All these adaptations are related to the health status of the body. On the other hand, the absence of exercise stimulus results in atrophy process and loss of functional capacity, marked by both impaired force production and metabolism of the muscle [5].
In this chapter, we focus on cell physiology and molecular biology of the muscle cells from a special point of view concerning to the stress response: the role of heat shock proteins (HSPs) in muscle.

Classical roles of heat shock proteins expression
Living organisms respond at cellular level to unfavorable conditions such as heat shock, and other stressful situations of many different origins, by a rapid, vigorous and transient acceleration in the rate of expression of specific genes: the heat shock genes. The products of these genes are commonly referred to as stress proteins or heat shock proteins (HSPs). Besides activation of heat shock genes, the expression of most other genes is inhibit as a result of stress. Thus, the stress intensity and duration leads to a perturbation of normal gene expression, which, if prolonged, can have drastic consequences for cells and system homeostasis [6,7].
HSPs are highly conserved proteins in both eukaryotic and prokaryotic organisms and are expressed in many cell types including striated skeletal muscle. The first report about HSPs was documented by Ritossa [8,9], after a serendipitous heat shock in salivary gland cells of Drosophila buskii, but heat shock proteins were only characterized later in 1974 [10]. Actually, HSPs are categorized in families according to their molecular sizes and include HSP110, HSP100, HSP90, HSP70, HSP60, HSP30 and HSP10 subclasses. In this chapter, the role of HSPs in muscle will be discussed in terms of the most studied (due to its evident high expression in mammalian cells under stress conditions) and conserved: the 70-kDa family (HSP70), which comprises a number of related proteins whose molecular weights range from 66 to 78 kDa. Many studies in human, rat and mice will be listed throughout the text, thus it is necessary to learn about the HSP70 isoforms that are encoded by a multigene family in each mammalian that will be listed below.
In humans, there are at least 13 distinct genes so far studied [11]. For the rationalization of the current nomenclature, human HSP70 genes (rat and mouse, also) have given the locus symbol HSPAx, where A defines members of HSP70 family and X designates the individual loci. In this sense, HSPA8 is the human gene that encodes a 73-kDa constitutive form of HSP70 (HSP73 or HSC70, the cognate form), while HSPA1A gene, located at the major histocompatibility complex (MHC) III region, encodes an inducible form (HSP72 or simply HSP70). In humans, but not in the rat or the mouse, there is an even higher inducible form (HSP70B') encoded by HSPA6 gene. Other representative members, besides mitochondrial (HSP75) and endoplasmic reticulum (HSP78) members of HSP70 family, are found in the intracellular space. While the constitutive form is expressed in a wide variety of cell types at basal levels (being only moderately inducible), the so-called inducible HSP70 forms (which are barely detectable under non-stressful conditions) could be promptly synthesized under a condition of 'homeostatic stress', this being any 'homeostasis threatening' condition, such as heat, glucose deprivation, lack of growth factors and so forth. Habitually, research groups indistinctly use HSP70 as a unified term for both constitutive and inducible form. However, HSP70 is the preferable form to be used when one refers to the inducible HSP72 protein encoded by HSPA1A gene [12,13] In rodents, the Hsp70 family consists of at least nine members that differ from each other by the intracellular localization and expression pattern. Two of them, Grp78 and Grp75, are localized in the lumen of the endoplasmic reticulum and in the mitochondrial matrix, respectively, whereas the remaining seven HSP70s reside mostly in the cytosol. The only cytosolic HSP70 abundantly and constitutively expressed in all cells is Hspa8 (related to Human constitutive form HSP73). The related form for HSP72 in rodents are two proteins encoded by almost identical genes, Hspa1a and Hspa1b, termed collectively Hsp70i. As in humans, the expression of Hsp70i in rodents is low or undetectable in most "resting" normal cells and tissues, but it increases rapidly in a variety of stress conditions [14][15][16].
The heat shock response is regulated by high conserved cis-acting regions of the DNA (heat shock elements -HSE) and also by high conserved DNA associated trans-acting proteins named Heat Shock Transcription Factors (HSF) (Morimoto et al., 1992). While more simple organisms as insects (Drosophila melanogaster) and yeasts (Saccharomyces cerevisiae) have only one HSF, rodents have at least 2 HSF (HSF1 and HSF2) and humans have 3 isoforms of HSF (HSF1, HSF2 e HSF4) [17][18][19][20]. Possibly, more complex organisms could have used multiple HSF against different challenges during evolution. Comparing the structure of each isoform of HSF in one single species, the sequence of amino acids may be 40% identical, as HSF1 and HSF2 in mice [21,22]. Comparing among species, the homology may be as great as 92%, as seen between HSF1 of human and rodents, or in HSF2 (95% homology between humans and rodents). Interestingly, HSF1 and HSF2 may be activated by distinct physiological phenomenon (Sistonen et al., 1992). While HSF1 and HSF2 are found in all kinds of cells, HSF4 is specifically for cells such as neurons or cardiac and skeletal muscle cells. It has also inhibitory function in heat shock response as negative regulator of HSPs expression [23,24]. Additionally, in experimental models and cell culture procedures, is possible to identify differences in the activation of HSF1 and HSF2. The former is activated seconds after stress and this response is attenuated quickly, while the latter appears to present a latency period between the event and the response, but it remains activated for 72 h. This HSF different latency for activation suggest a cooperative role of HSF isoforms in cell protection [19] and that these genes are high conserved in nature [7].
In unstressed cells, HSP70 may bind to the regulatory protein HSF for prevention of the trimmer formation of HSF that is required for HSPs transcription. Under stressful conditions, the free HSP70 captures the denatured proteins and then dissociates from the HSP70-HSF complex allowing the formation of HSF trimmer, thus triggering a HSP70 production. Once synthesized, new HSP70 molecules may be involved in a variety of cellular processes and exert different functions [25,26].
HSP70s are known to function as intracellular molecular chaperones that facilitate protein transport, prevent protein aggregation during folding and protect newly synthesized polypeptide chains against misfolding and protein denaturation. The molecular chaperones properties of such a protein allow them to assist the non-covalent assembly/disassembly of other macromolecular structures without being permanent components of such structures. Additionally, molecular chaperones assist the unfolded protein to achieve its single correct three-dimensional configuration (by still unknown mechanism it has evolved to generate this folded state), without becoming a constituent of the final folded protein [25,26].
Most proteins destined for cell organelles are synthesized in cytosol and must cross one or more organelle membranes to reach their functional destination. For example, in the mitochondria, 95% of the proteins are made as precursor proteins in the cytosol and are mainly post translationally imported into the mitochondrial sub-compartments. In this situation, cytosolic HSP70 play an important role for maintenance of a transport-competent conformation of precursor proteins. The precursor protein is translocated in an unfolded state and are refolded later, sorted to their final destination and assembled into functional complexes [27].
The chaperone function of HSP70 includes the inhibition of the formation of nascent polypeptides. This inhibition is dose-dependent over a range of 0.1-0.4 nmoles of HSP70 and this effect is greater for the larger polypeptides. All these data suggest that high concentration of HSP70 can perturb the normal folding of nascent proteins, block cell growth and impair the cell viability. Then, these are reasons that may explain why the cells of human body have to carefully auto-regulate the levels of HSP70. Many characteristics and functions of HSFP70 are listed in table 1 and are discussed in this text.
Since the skeletal muscle is one of the most adaptable tissues of the body, every structural aspect of the muscle that can change in response to the stimulus challenge (or to its lack) may require the chaperon action of HSP70 at the molecular level. For example, major adaptations to (dis)use muscle, such as fiber type distribution, fiber diameter, myosin heavy chain profile and mitochondrial distribution, are factors that are associated to the up-or down-regulation of HSP70.

HSP70 function in the muscle
The ability of muscle cells to express HSPs (mainly HSP70s) represents a cytoprotection mechanism because HSP70 proteins share the same overall structure. They are composed of an actin-like N-terminal nucleotide binding/ATPase domain of 45 kDa, a substrate-binding domain (SBD) of approximately 15 kDa and a C-terminal domain of approximately 10 kDa that is involved in co-chaperone binding (HU et al, 2006). It is of note that N-and C-terminal domains have expressive relevance to antigen presentation, an important way by which HSP70s participate in immune responses. With this structure, HSP70 may act as a molecular chaperone inside the muscle cell: they facilitate protein transport; prevent protein aggregation during folding; protect newly synthesized polypeptide chains against misfolding and protein denaturation [28,29]. Historically, studies about the effects of exercise on heat shock protein expression have been dedicated to HSP70 analysis in cardiac or skeletal muscle after exhaustive animal protocols (for review see Noble et al., 2008) [30]. It has been demonstrated that 30 minutes after an exercise bout there is an increase in mRNA expression of muscle inducible form of HSP70 (HSP72) and that is a later heat shock response related to mRNA of constitutive form of HSP70 (HSP73) [31]. Similar response may be observed in humans, and are related to glycogen depletion and the muscle heating [32]. This acute heat shock response (intracellular HSP70 content) remained increased 24 hours after an acute exercise session, according to exercise load [33].
In humans, repeated bouts of eccentric exercise showed an impressive result in terms of HSP70 expression. Sets of Eccentric contractions contractions of the elbow flexors promote ~2-fold increase in HSP70 expression in biceps brachii. Four weeks later, the basal levels of HSP70 was reduced but the muscle still presents the heat shock response to exercise in the same magnitude but in less HSP70 content [34,35]. Accompanying this effect, higher levels of both serum creatine kinase, soreness, lower levels of both relaxed arm angle and measured torque (indicators of muscle damage) occurs after the first bout, but the levels of these parameters are lower in the second bout. This may represents an association between muscular adaptations and the phenomenon called "acquired thermo tolerance" in terms of HSP70 expression. This study suggests that equivalent HSP70 response imply equivalent levels of stress in each bout and that may be an indicative that the heat shock response is a marker of muscle adaptation. Moreover, basal modification of HSP70 levels remains four weeks after the first bout of exercise and suggests that a single exercise challenge can promote deeper molecular adaptations in muscle cells [34].
Since the heat shock response is a prompt molecular adaptation to the stress condition, to localize the elements that contemplates this phenomenon in muscle is a interesting point of research. The sarcoplasmic reticulum contains microdomains that are involved in translation and processing of transcripts which encode proteins requiring compartmentalization to specific organelles within the myofiber [5]. In skeletal myofibers, ribosomes are localized to both the subsarcolemmal and intermyofibrillar cytoplasm. HSP70 has been shown to be concentrated in a subsarcolemmal fashion and it localizes to the nucleolus and myofibrils in response to stress condition. Although various modes of exercise can induce HSP70 expression, it is clear that it has a different pattern of heat shock response between slow and fast fibers. Slow and more oxidative fibers express greater HSP70 content in response to exercise possibly by preferential recruitment or a more sensitivity to temperature challenges. In this way, the muscle that have different localization of HSP70 mRNA in according to the type of the stimulus: exercise is different to heat treatment [36]. Exercise promotes a concentrated and punctuated perinuclear localization of HSP70 mRNA near the periphery of skeletal myofibers after exercise session (1 to 24h). This result represents that HSP70 proteins are prompt to response because HSP70 mRNA appears rapidly close to the nuclei that transcribes this gene. Diffuse HSP70 mRNA was also observed any time after exercise bout that represents a quickly cytoplasmic distribution of heat shock response proteins [36,37]. Corroborating this discussion, Paulsen et al [38] showed that maximal eccentric exercise induces a 20 fold increase in HSP70 mRNA 24h after exercise and an increase about 200% in cytosolic HSP70 content.

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The chaperone function of HSP70 is more than microscopic laboratory measurements research field. Muscle disuse results in muscular atrophy that is represented by decrease in muscle mass, fiber cross sectional area and total myofibrillar protein content. In this situation contractile protein breakdown exceeds protein synthesis. Moreover, in atrophied muscle there occurs an increase in the proportion of fibers containing the fast myosin heavy chain by transformation from the slow myosin heavy chain (MyHC-I/β) to the fast myosin heavy chain (MyHC-IId/x). As early as 18 h after muscle disuse and persisting for as long as for 18 days, it is possible it is possible to measure a decrease in HSP72 in soleus muscle [39]. Interestingly, previous heat treatment is a strategy to induce HSP70 expression in muscle and this molecular adaptation results in maintenance of muscle mass during 7 days period of immobilization [40]. In this way, HSP70 expression appears to have, not a full protective effect on muscle mass, fiber cross sectional area and total myofibrillar protein content, but a preventive effect on the decrease in MyHC-I/β and the increase in MyHC-IId/x induced during the atrophy process [41]. These evidences suggest that HSP70 can inhibit a key signaling pathway for atrophy in muscle cell preventing the muscular atrophy.
Heat treatment has also been tested in humans. Short wave diathermy therapy is a clinical strategy that means to increase deep heating of tissues with higher water content. This strategy may promote a 58%-increase in HSP70 expression in vastus lateralis [42]. It is possible that the previous heat treatment cannot reduce markers of muscle damage but it is able to reduce muscular pain, to preserve strength and to improve range of motion following eccentric contractions. Curiously, there is a gender difference in heat shock response in both basal and exercise-induced HSP70 levels, with men showing lower pre-exercise levels and an attenuated HSP70 response as compared to women's values. The gender difference may be explained by the effects of estrogen modulation on heat shock response [42].
If muscle disuse is a trouble, the reuse of the musculature may represent many stages of soreness. After immobilization, the reload process to the muscle implies in newest molecular adaptations. If a less-required muscle is submitted to a challenge, the HSP70 expression increases greatly (~200%) in the first two weeks of reload process and return to basal levels (above disuse levels) as early as in 8 weeks [39]. This effect is accompanied by increase in percentage of slow type I MyHC fibers (MyHC-I/β). Although many factors appear to be related to the down-and up-regulation of HSP70 function, the expression of this protein is closely related with the morphological and functional changes of muscle cells.
Although initially the HSP70s have been described essentially in studies that addressed molecular chaperone action of such proteins, HSP70s have also been studied as limiting of protein aggregation, facilitating protein refolding and maintaining structural function of proteins [43]. Intracellular HSP70s have further been demonstrated to be anti-inflammatory [44,45], providing cytoprotection through anti-apoptotic mechanisms, inhibiting gene expression and regulating cell cycle progression [46].
Besides the now classical molecular chaperone action, the most remarkable intracellular effect of HSP70 is the inhibition of nuclear factor κB (NF-κB) activation, which has profound implications for immunity, inflammation, cell survival and apoptosis. Indeed, HSP70 blocks NF-κB activation at different levels. For instance, HSP70 inhibits the phosphorylation of inhibitor of κB (IκBs), while heat-induced HSP70 protein molecules are able to directly bind to IκB kinase gamma (IKKγ) thus inhibiting tumour necrosis factor-α (TNFα)-induced apoptosis [47,48] . In fact, the supposition that HSP70 might act intracellularly as a suppressor of NF-κB pathways has been raised after a number of discoveries in which HSP70 was intentionally induced, such as the inhibition of TNFα-induced activation of phospholipase A2, the suppression of inducible nitric oxide (NO) synthase (iNOS, encoded by NOS-2 gene) expression paralleled by decreased NF-κB activation. Hence, HSP70 is antiinflammatory per se, when intracellularly located, which also explains why cyclopentenone prostaglandins (cp-PGs) are powerful anti-inflammatory autacoids [49,50].
Another striking intracellular effect of HSP70 is the inhibition of apoptosis. Caspases form an apoptotic cascade by the intrinsic pathway, characterized by the release of mitochondrial proapoptotic factors into the cytosol, while stimulation of cell surface receptors triggers the extrinsic pathway by external signaling factors that may induce the apoptotic process. The inhibitory potential of HSP70 over apoptosis occurs via many intracellular downstream pathways (e.g. JNK, NF-κB and Akt), which are both directly and indirectly blocked by HSP70 either, besides the inhibition of Bcl-2 release from mitochondria. Together, these mechanisms are responsible for HSP70 anti-apoptotic function in cells under stress conditions [51][52][53].
These intracellular effects of HSP70 are closely related to aging and disuse (or both, in a synergic way) effects on muscle wasting, because there is comprehensive evidence that NF-κB activity is increased during disuse and is required for muscle atrophy. NF-κB activation is actually decrease in the first week of immobilization but it is increased in longer atrophy process (by 3-fold) and aged disused muscle (5-fold increase), both reversible effects with HSP70 overexpression that inhibit NF-κB activity owing to increasing the levels of IκBα that are available to bind and to retain NF-κB proteins in cytosol [40].
In terms of metabolic function, increased HSP70 protein expression (~50%) by heat treatment, muscle-specific transgenic over expression, or pharmacological means can protect against diet-or obesity-induced hyperglycemia, hyperinsulinemia, glucose intolerance, and insulin resistance. This protection was tightly associated with the prevention of JNK phosphorylation, another role for HSP70 in the blocking of inflammation [54].

Muscle activity and HSP70, eHSP70 and cytokines
Cytokines are intracellular signaling molecules, typically proteins or glycoproteins, that mediate various aspects of cell function, including proliferative and adaptive responses. Cytokine signaling is essential for a coordinated inflammatory response. Diseases related to inflammatory processes as cancer, congestive heart failure, AIDS, sepsis and arthritis often lead to muscle catabolism and loss of muscle function, and this effects are attributed to circulating cytokines. On the other hand, exercise is known to alter immunological function in health individuals and this adaptation also is related to altered cytokine levels [55].

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Some cytokines are more closely related to exercise challenge. Circulating TNF-α (tumor necrosis factor -α) may promote cellular responses mediated by two receptors located on cell surface, the 55 kDa TNF-receptor 1 and the TNF-receptor 2. The chronic interaction of this cytokine with its receptors resulted in catabolic response, as loss of muscle mass and contractile dysfunction. TNF-α promotes loss of muscle protein associated to oxidative stress signaling that culminates in muscle wasting mediated by the NF-κB activation, a redox sensitive transcription factor. The impaired muscle function induced by TNF-α also may occur without changes in muscle mass [56] Exercise-associated muscle damage initiates the inflammatory cytokine cascade. Strenuous exercise increases plasma levels of TNFα, IL-1, IL-6, IL-1 receptor antagonist (IL-1ra), TNF receptors (TNFR), IL-10, IL-8, and macrophage inflammatory protein-1. Exercise induces immune changes and also alters neuroendocrinological factors including catecholamines, growth hormone, cortisol, β-endorphin, and sex steroids. It is generally assumed that the "brain-immune" axis also exists during stress. Release and/or expression of enkephalins can be regulated by different factors such as stress, exercise and cytokines [55,57]. IL-6 is generally considered a pro-inflammatory cytokine released from immune cells and reaching higher levels in the circulation and inside the muscle. However, muscle contraction during exercise is a signal for IL-6 release from the muscle. IL-6 increases ~100-fold after a marathon race and the increase was tightly related to the duration and intensity of the exercise. IL-6 is produced in the skeletal muscles in response to exercise and it has growth factor abilities and contributes to the anti-inflammatory effect of exercise. Interestingly, exercise, IL-6 and HSP70 have particular relationship: exercise training increases IL-6 response to immune related challenge (LPS treatment) and IL-10 plasma concentration; IL-6 can induce HSP70 expression but the absence of IL-6 during exercise do not attenuate the increase of HSP70 expression by exercise; in sedentary, the absence of IL-6 blunted HSP70 response in skeletal muscle after a immune challenge (LPS treatment); and the absence of IL-10 (an anti-inflammatory cytokine produced during exercise) increased the levels of IL-6 after the same immune challenge These data suggest that there are different pathways that leads to IL-6 and HSP70 up-regulation, with and without exercise stimulus [44,45,[58][59][60][61]. These cytokine signaling and HSP70 expression effects on muscle are summarizing in Figure 1.
Physical exercise has many effects on the Central Nervous System (CNS), much more than mood influence. Peripheral signals generated during and after an exercise session, such as IL-6 and IL-10, decrease endoplasmic reticulum stress markers at hypothalamic level, an effect related to the decrease in NF-κB activation. The processes of building certain behaviors and control of them can be analyzed under the optics of neuroimmunomodulation. The expression of 'sickness behavior' can be induced by immune modifications and immune capacities that are associated with distinct behavior in mammals. In this sense, it is clear the participation of mediators including TNF-α, interleukin-1β (IL-1 β), and IL-6 in the CNS. For instance, the release of skeletal musclederived IL-6 into the blood is the most remarkable alteration in cytokine pattern observed during exercise so that IL-6 is now considered as an exercise factor, a 'myokine' [60], not just an inflammatory mediator. Additionally, as previously hypothesized, [61] the exercise-evoked IL-6 may also act on the CNS to induce the fatigue sensation. In other words, the skeletal muscle must be considered as an auxiliary endocrine organ that interacts with the immune system and CNS, so that IL-6 is a robust exercise marker. Myokine signals are correlated with sensation of fatigue, and may be inducer of sleep or illness response and pyrogenic behavior. [62][63][64][65][66][67]. More recently, however, it has been demonstrated that the presence of HSP70s in the circulation (extracellular HSP70, eHSP70) in response to exercise [32,44,[68][69][70]. Since exercise is able to induce high concentrations of HSP70s in both muscle and plasma, the most obvious hypothesis was, primarily, that skeletal muscle should be the releaser of HSP70 during exercise. However, further studies have revealed that this is not the case, at all. The lack of evidence supporting the proposition that the muscle could be the major source of circulatory eHSP70 precluded the 'muscle hypothesis' and suggested that other tissues/cells should be responsible for the increase of eHSP70 in the circulation. In the early phase after high-intensity exercise, eHSP70 is elevated in peripheral blood.
Once HSP70 protein release from muscle to extracellular fluid could eventually happen by lysis process, and considering that the lysis of muscle fiber occurs only under severe cellular stress condition, the presence of eHSP70 during moderate exercise was found to be unfeasible. Though it had been shown that both the intensity and duration of exercise have effects in plasma [71] and muscle [33] HSP70 concentration, this rise in circulating levels of HSP70 precedes, however, any gene or protein expression of HSP70 in skeletal muscle, [32,72] which is another strong argument against the 'muscle hypothesis'. Afterwards, eHSP70 blood concentration returns to the lower basal levels as soon as 2 h after the end of the physical effort, remaining practically undetectable for 24h. Similarly to the cytokines released by immune cells during exercise, serum eHSP70 concentration does rise after exercise sessions, mainly because of the contribution of lymphocytes [73]. As a corollary, lymphocyte-derived HSP70s may interplay with CNS to induce the state of 'fatigue behavior' activation [45]. Then, the equilibrium of immune signals during exercise is required to maintenance of the homeostasis and this equilibrium may be observed by several markers, listed in the Figure 2 in relation to the degree of exercise or disease challenge. Relation between the degree of exercise or disease challenge and inflammatory markers: The proposed markers may represent both the inflammatory status and muscle status of health. In this way, the figure represents the hypothesis that there is an equilibrium state of many markers in health and a disequilibrium in sedentary, disease and overtraining situations. These markers include extracellular/intracellular HSP70 ratio hypothesis and many others cytokines.
There are many diseases related with higher levels of eHSP70, suggesting that serum levels of this proteins may be considered a novel important biomarker. Whereby health people have low plasmatic levels of eHSP70, the association of these proteins with illness, disease progression and mortality was hypothesized, as well as longevity and health parameter status were attributed to this lower concentration. On the other hand, a rise 3.7-fold eHSP70 circulating levels in critically ill patients was correlated with less hospital treatment period [74] and death [75].
The increase in eHSP70 during the exposure to stresses has also been demonstrated to be the result of the activation of the sympathetic nervous system via alpha-adrenergic receptors leading to eHSP70 export and increased eHSP70 serum concentration [76,77]. Thus, even though the necrotic cell death might result in the appearance of HSP70 within the extracellular milieu, an increasing number of studies suggest that this is not the major rule but, on the contrary, physiological effectors (e.g. fever, hypoglycemia and sympathetic stimulation) are the true excitatory signals for the eHSP70 exocytotic pathway, which suggests that highly conserved evolutionary responses are tightened to eHSP70 production, meaning that extracellular HSP70 response may have had an important evolutive role.
The interaction of cytokines or eHSP70 with the complexes of toll-like receptor (TLR2 and/or T L R 4 ) a c t s a s i n f l a m m a t o r y s i g n a l t o c e l l s o f t h e i n n a t e i m m u n e r e s p o n s e (macrophage/dendritic cells/neutrophils). Under stimulation of TLRs, eHSP70 signalizes to the increase of the signal transduction of NF-κB dowstream pathways. Asea and co-workers have shown that eHSP70 induces NF-κB activation and the production of inflammatory cytokines in a process that requires CD14, in addition to TLR2 and TLR4 that are expressed in muscle cell surface [78][79][80][81].
By definition, cytokines are proteins secreted by cells with regulatory effects on other cells. Therefore, in addition to its function as an intracellular molecular chaperone, HSP70 in the extracellular milieu acts as a powerful cytokine, affecting the functional properties of immunocompetent cells. This dual role, as both a chaperone and cytokine, helps to elucidate recent findings indicating that heat-shock proteins can be potent adjuvant for many inflammatory related diseases [79].

Conclusion
In summary, HSP70s have physiological proprieties that are involved in maintenance of muscle muscle function by the interaction with molecular entities inside the skeletal muscle cell and also by via cell surface receptor. Exercise-induced increase in HSP70 expression and eHSP70 concentration have important role in the regulation of the inflammatory pathways that can be activated during high intensity exercise as well as in the course of atrophy process.