The mechanical and metabolic stress (mitochondrial dysfunction and challenge by energy ADP/ATP ratio), induced by the repetitive contractions of muscle fibers, causes a disruption of the sarcolemma and extracellular matrix, swelling of mitochondria, dilation of the transverse tubule system, and fragmentation of the sarcoplasmic reticulum, thereby promoting the increased permeability of the membrane and efflux of myocellular proteins into the blood circulation (Peake et al., 2017; Hody et al., 2019). The loss of calcium homeostasis, oxidative stress, increased calpain activity, and inflammation contribute to muscle injury (Hody et al., 2019). An increase in the levels of systemic inflammatory mediators, such as interleukin (IL)-1 beta, IL-6, IL-8, IL-1ra, and IL-10, is induced during the muscle damage due to endurance exercise. However, the cellular sources of these mediators, such as endothelial cells, pericytes, fibroblasts, neutrophils and monocytes/macrophages, or skeletal muscle cells, utilize the inflammatory mediators in the process of muscle repair and regeneration (Peake et al., 2015).

In addition to the classical inflammatory mediators, the changes have been observed in more than 650 myokines in response to muscle contraction and differentiation, with autocrine, paracrine, and endocrine actions (Whitham and Febbraio, 2016; Safdar and Tarnopolsky, 2018; Piccirillo, 2019; Bay and Pedersen, 2020; Laurens et al., 2020). Despite the identification of hundreds of myokines, biological function has only been described in a small portion of myokines. Many of these myokines have paracrine biological functions in muscle protein synthesis or degradation, proliferation and differentiation of myoblasts, activation of satellite cells, organization and remodeling of the extracellular matrix, as well as modulation of muscle wasting, repair, and regeneration (Hoffmann and Weigert, 2017; Lee and Jun, 2019; Laurens et al., 2020). The cellular sources of myokines include myocytes, satellite cells, endothelial cells, residential macrophages, and fibroblasts (Hoffman and Weigert, 2017). The myokines primarily studied after aerobic exercise include myostatin, IL-6, irisin, IL-15, growth differentiation factor 15 (GDF-15), brain-derived neurotrophic factor (BDNF), fibroblast growth factor (FGF)-21, apelin, angiopoietin-like protein 4, and decorin (Safdar and Tarnopolsky, 2018; Piccirillo, 2019; Bay and Pedersen, 2020; Laurens et al., 2020). The levels of systemic hepatokines, such as follistatin, FGF-21, and angiopoietin-like protein 4, have also been studied during and after the acute exercise (Gonzalez-Gil and Elizondo-Montemayor, 2020). The release of exercise-induced peptides is dependent on the type of exercise and training protocol (Piccirillo, 2019; Domin et al., 2021).

Understanding the time course of a variety of different exercise-induced peptides can help highlight the circulatory markers that participate in the different phases of the muscle damage and repair, such as pro and anti-inflammatory phase, muscle and connective tissue remodeling as well as synthesis and protein degradation after long-distance exercise. The present study is the first study to determine and correlate a large number of muscle damage markers and inflammatory and tissue repair mediators in long-distance runners. Our hypothesis is that the extent of muscle damage may influence the inflammatory response, which in turn may affects the dynamics of muscle tissue repair during the recovery period.

The present study aimed to evaluate the time course and relationship of exercise-induced cytokines and muscle damage markers after a marathon race. An understanding of the course of the release of exercise-induced peptides on circulation and their association during muscle damage and repair may contribute to elucidate the endurance exercise muscle adaptations and may yield potential molecular therapeutic targets to treat the myopathies that involve mitochondrial dysfunctions.

Immediately after the race, we observed leukocytosis, neutrophilia, and an increase in the muscle damage markers, IL-6, IL-8, IL-10, TNF-α, MIP-1, decorin, GDF-15, BDNF, follistatin, and FGF-21, owing to a reduction in the myostatin, musclin, IL-15 and apelin levels which were maintained reduced 72 h after the race (Figure 9). The muscle damage marker, plasmatic LDH activity, correlated with the mediators of inflammation (IL-10 and TNF-α) but not with myokines response. The classical anti-inflammatory mediators induced by exercise (IL-6 and IL-10) and IL-8 seem to be associated with myokines that affect the muscle repair. IL-10 and IL-8 response was associated with the musclin response, while IL-6 response was positively correlated with the FGF-21 and follistatin response after the race and with myostatin, apelin, and irisin response in the recovery period. Moreover, BDNF had a negative correlation with the troponin levels and further studies should be carried out to verify the role of BDNF levels in myocardial damage-induced by the race.

Leukocytosis after long-distance exercise is a result of hemodynamic and catecholamine induced demargination of vascular and pulmonary pools followed by a cortisol-induced release of neutrophils from the bone marrow. In the recovery period, these stress hormones also contribute to lymphocytopenia and both innate and acquire immune cell dysfunction (Nieman and Mitmesser, 2017; Jones and Davison, 2019; Suzuki et al., 2020). Muscle tissue infiltration of the innate immune cells is regulated by chemokines, such as IL-8 (Nieman and Mitmesser, 2017; Jones and Davison, 2019; Suzuki et al., 2020). The endurance exercise induces a pro inflammatory response mediated by the neutrophils and pro inflammatory macrophages, M1 in the early hours after exercise, follow an anti-inflammatory compensatory response mediated by M2 macrophages and T cells (Treg and CD8+) leading a period of immunosuppression called “open window” (Peake et al., 2017; Jones and Davison, 2019; Suzuki et al., 2020).

In this study, the levels of skeletal muscle damage markers were noted to be correlated with the immune changes and inflammatory mediators (IL-10 and TNF-α); however, no association was found between the variations in the levels of skeletal muscle damage markers and myokines. We suggest that regardless of the extent of skeletal muscle damage, other intrinsic muscle factors may modulate the release of myokines, as indicated by an association of free fat mass and/or training experience with the variations in BDNF, GDF-15, irisin, and myostatin levels. We observed an association between age and GDF-15, before and after the race, as proposed by Conte et al. (2020); however, we did not observe a correlation with the total number of leukocytes, neutrophils, and lymphocytes showed in the cyclists after exhaustive exercise (Conte et al., 2020). A GDF-15 is a stress-induced cytokine released from the TGF-β superfamily, in response to the mitochondrial stress and/or inflammatory stress. A GDF-15 has been suggested to induce reorganization and adaptation of systemic metabolism (Chung et al., 2017; Conte et al., 2020; Laurens et al., 2020). Although the contracting skeletal muscle releases GDF15, leading to an increase in the plasma levels, the source of systemic GDF-15 levels after acute exercise remains unclear (Kleinert et al., 2018; Conte et al., 2020).

IL-6, induced by exercise, has a well-known anti-inflammatory effect that modulates the release of IL-10 and IL-1ra. The other paracrine effects of IL-6 on the skeletal muscle include intramuscular lipolysis and improvement of insulin sensitivity and glucose intake (Carey et al., 2006; Wolsk et al., 2010; Laurens et al., 2020). Moreover, the previous studies have observed that an IL-6 induces the following: myogenic differentiation in the C2C12 myoblast cell line and primary human myoblasts, myotube protein synthesis in the C2C12 myoblast cell line, and murine and myoblast proliferation in the human satellite cell proliferation (Pedersen et al., 2001; Gao et al., 2017; Steyn et al., 2019). The studies in animals have elucidated the role of IL-6 in the activation of M2 macrophages, which promotes angiogenesis and damages tissue repair (Pilny et al., 2019), satellite cell proliferation, and myonuclear accretion (Serrano et al., 2008).

In this study, in response to the exercise, the IL-6 expression was positively correlated with that of FGF-21 and follistatin, which are considered hepatokines, rather than the myokines (Domin et al., 2021). Follistatin has myogenic properties that directly inhibit myostatin from binding to the activin IIb receptor and suppression of small mothers against decapentaplegic 3 (Smad3) phosphorylation, consequently increasing the protein synthesis by the mTOR/S6K/S6RP signaling cascade and contributing to skeletal muscle mass (Winbanks et al., 2012; Hoffmann and Weigert, 2017; Lee and Jun, 2019). In our study, the follistatin levels were correlated with those of BDNF, which are responsible for the activation and proliferation of satellite muscle cells after injury (Hoffmann and Weigert, 2017; Lee and Jun, 2019). BDNF had a negative correlation with a myocardial damage marker. A recent review highlighted that BDNF acts on myocardial tissue by decreasing the cardiomyocyte apoptosis and mitochondrial dysfunction and increasing angiogenesis, cardiomyocyte contraction, and calcium cycling via tropomyosin-related kinase receptor B (TrkB) signaling pathways (Hang et al., 2021).

Mitochondrial dysfunction and endoplasmic reticulum stress trigger the FGF-21 expression in the skeletal muscles (Tezze et al., 2019). FGF-21 modulates PI3K-AKT signaling, activates ATF4 in skeletal muscle, and is involved in the removal of damaged mitochondria via mitophagy and alteration of the type of muscle fiber; thus, it is involved in the regulation of both the muscle mass and function (Hoffmann and Weigert, 2017; Oost et al., 2019; Lee and Jun, 2019). In addition, FGF-21 seems to have metabolic effects similar to those of IL-6 in the skeletal muscle (Tanimura et al., 2016; Struik et al., 2019; Tezze et al., 2019). The FGF-21 response correlated negatively with the decorin and apelin response, indicating that better FGF-21 response may avoid decorin and apelin reduction after the race.

Decorin is a member of the small leucine-rich proteoglycan family of extracellular matrix proteins that interact with the collagen fibers. It has been reported to modulate the proliferation of human skeletal muscle cells and autophagy and the consequent promotion of muscle regeneration (Li et al., 2007); moreover, it directly inhibits myostatin by activating the SMAD-2/3 complex, thereby reducing the degradation of proteins in the skeletal muscle (El Shafey et al., 2016).

In the recovery period, IL-6 correlated with the myostatin, irisin, and apelin levels. Myostatin acts on the activin receptors (type I and II), promoting the phosphorylation and activation of SMAD proteins. SMAD-2 and SMAD-3 form a complex with SMAD-4, which induces the transcription of catabolic genes. Moreover, myostatin participates in the process of protein degradation through the ubiquitin-proteasome system and autophagy (Hoffmann and Weigert, 2017; Lee and Jun, 2019; Piccirillo, 2019).

The decrease in the levels of other well-known myokines that promote muscle regeneration and modulate autophagy, such as IL-15, apelin, and musclin, indicate that these molecules do not play decisive roles in the repair and regeneration after endurance exercise-mediated muscle damage. IL-15 is a myokine whose levels affect the differentiation of myoblasts, and this myokine modulates autophagy (Hoffmann and Weigert, 2017; Lee and Jun, 2019; Piccirillo, 2019; Trovato et al., 2019; Pesce et al., 2020); moreover, IL-15 is associated with the vascular smooth muscle cell proliferation, stem cell proliferation and differentiation, mitochondriogenesis, autophagy, and anti-inflammatory properties in skeletal muscle (Mughal and O'Rourke, 2018; Vinel et al., 2018).

The exercise-induced inflammatory mediators (IL-10 and IL-8) correlated with the changes in musclin which contains a region homologous to the members of the natriuretic peptide (NP) family. Musclin seems to promote the skeletal muscle oxidative capacity by mitochondrial biogenesis (Subbotina et al., 2015). In contrast to the studies in animals (Farrash et al., 2021), our data showed a decrease of musclin after acute exercise.

Daily dietary intake and food intake during the competition may affect the inflammatory response (Nieman and Mitmesser, 2017). A previous study of our group demonstrated the association of energy, electrolyte, and carbohydrate intakes and inflammatory response after endurance exercise (Passos et al., 2019). The limitation of this study was that it did not assess the role of food intake during the marathon race. Further studies are needed to evaluate the role of daily dietary intake, nutritional supplements, and food intake during the race on the exercise-induced cytokines.

Based on the results of the current study, we conclude that the endurance exercises, such as a marathon could alter the levels of inflammatory markers, and the interaction between the peptides shows that an increase in GDF-15, BDNF, follistatin, decorin, and FGF-21 expression, owing to the decrease in myostatin levels during the recovery period, appears to contribute to the muscle repair and regeneration after a long and high-intensity race. In addition, the regeneration of exercise-induced muscle damage involves the functioning of classical inflammatory mediators and myokines, whose functions include mediation of angiogenesis (IL-6), myogenesis (IL-6 and follistatin), mytophagia (FGF-21), and satellite cell activation (BDNF), in addition to the downregulation of protein (follistatin and decorin) degradative pathways. Additionally, further studies should be carried out to verify the role of BDNF levels in myocardial damage-induced by the race. Furthermore, IL-6 modulates the anti-inflammatory and metabolic responses, and it is associated with the levels of myokines (follistatin, FGF-21, myostatin, apelin, and irisin) that are involved in the muscle repair. The discovery of plasmatic myokines involved in the muscle damage and repair may help to elucidate the endurance exercise muscle adaptations and may yield potential molecular therapeutic targets to treat the myopathies that involve mitochondrial dysfunctions.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving human participants were reviewed and approved by Ethics Committee of Dante Pazzanese Institute of Cardiology, Brazil (Permit Number: 979/2010). The patients/participants provided their written informed consent to participate in this study.

CS carried out the data collection, acquisition analysis, and data interpretation. AS carried out the data collection, participated in its design, and helped draft the manuscript. BM performed acquisition analysis and data interpretation and helped draft the manuscript. JM and RM were responsible for the data collection. HB and HS participated in the experimental design, acquisition analysis, and data interpretation, as well as helped to draft the manuscript. MC-B conceived the study, participated in its design and coordination, helped to perform the statistical analysis, and drafted the manuscript. All authors have read, revised, and approved the final version of the manuscript and agree with the order of presentation of the authors.

This work was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) [grant number 2018/26269-0].

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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