Recurrent Exercise-Induced Muscle Damage and Overtraining Syndrome: Is There Any Relationship?

Academic dissertation submitted with the purpose of obtaining a master’s degree in Physical Activity and Health from the Faculty of Sport of University of Porto, according to the Law nº 74/2006 from March 24th, in the drafting given by Law nº 65/2018 from August 16th.

Supervisor: Professor Doutor José Alberto Ramos Duarte.

Francisco António da Silva Leite

Porto, 2019

Leite, F. A. S. (2019). Recurrent Exercise-Induced Muscle Damage and Overtraining Syndrome: Is There Any Relationship?. Porto: F. Leite. Academic dissertation submitted with the purpose of obtaining a Master´s degree in Physical Activity and Health. Faculty of Sport, University of Porto, Portugal.

Key words: TRAINING OVERLOAD; MUSCLE INJURY; ECCENTRIC EXERCISE; SOLEUS MUSCLE; TIBIALIS ANTERIOR MUSCLE;

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To my parents,

brother and sister

ACKNOWLEDGMENTS

I want to thank all the people who helped and supported me to accomplish this objective, but I want to thank you especially for all the concern and affection that you have expressed in this troubled phase of my life. Firstly, I want to thank Professor José Alberto Duarte for has instigated this love for Physiology and Biochemistry over the years, as well as for has received and guided me throughout this project, sharing with me new ideas and new knowledges. I also want to thank you for all the support, concern and understanding that you have shown throughout this phase of my life. To all my laboratory colleagues, Toni, Catarina, Edyla, Grace, Sara, Beshoy and Maurílio, I want to thank the great environment they provided and the magnificent moments they shared with me. Thanks for all the help and suggestions, opinions and advices that allowed me to make this work much richer and fuller. An appreciation for the effort you have made for me and for my work. Without you would not have been able to achieve this goal. I want to give special recognition to Toni, for the concern with me and my work. His role was like that of a co-supervisor. His constant guidance was undoubtedly a guiding line that did not let me deviate from the right way. À Dona Celeste não é possível demonstrar por palavras o quão agradecido estou por tudo aquilo que me ensinou e ajudou. De facto, demonstrou-me que a idade e a experiência também são um posto. Agradeço- lhe por me ter recebido tão bem no seu laboratório e por me ter ensinado a ser autónomo na realização das tarefas laboratoriais. Além disso, obrigado por ter orientado o meu trabalho quando eu não o podia fazer e obrigado por todo o carinho e preocupação que demonstrou comigo não só na minha ausência, mas também durante todo o processo de recuperação. O seu apoio e animação ajudaram-me a superar os problemas e os dias menos bons. De facto, a senhora foi uma segunda mãe para mim. Espero que continue por muitos mais anos com esse sorriso e boa disposição que tão bem a caracterizam. Muito obrigado Celestinha!

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I also want to recognize the role of my friends throughout this course. Thank you for the dinners, the coffees and the moments that allowed me to unwind a bit and helped to restore energies and focus. To Catarina I want to leave a huge thank you for all the support, understanding and patience over these years. Thank you for never given up on me and for shown me that I have the potential to do all the things that I want. Finally, I want to distinguish and thank my father Artur, my mother Luzia, and my brothers Daniel and Ana Rita, for everything they have done for me. Thank you for all the sacrifices you have made throughout your life. All the efforts allowed me to realize this dream, which I know is not only mine but also yours. I hope someday I'll be able to reciprocate. Thank you also for the support, concern, affection and patience you have had throughout this phase. I know it was not easy for you too, but in the end everything worked. I thank you with all my heart.

Thanks a lot, to all of you.

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TABLE OF CONTENTS

ACKNOWLEDGMENTS……………………………………………………………. VII

TABLE OF CONTENTS……………………………………………………………. IX

LIST OF FIGURES…………………………………………………………………... XI

LIST OF TABLES……………………………………………………………………. XIII

RESUMO……………………………………………………………………………… XV

ABSTRACT…………………………………………………………………………... XVII

LIST OF ABBREVIATIONS………………………………………………………… XIX

CHAPTER 1………………………………………………………………………….. 1

1. INTRODUCTION………………………………………………………………. 3

2. STATE-OF-ART……………………………………………………………….. 7

2.1. Physical exercise as a muscle damage inducer………………... 7

2.1.1. Microscopic changes induced by exercise………………….. 9

2.1.2. The muscle inflammatory response and repairing process. 10

2.2. Muscle adaptations to repeated exercise………………………... 12

2.3. Exercise training principles………………………………………… 14

2.4. Exercise training periodization……………………………………. 16

2.5. Overtraining syndrome……………………………………………… 16

2.5.1. Over-reaching and overtraining syndromes…………………. 17

2.5.2. Overtraining risk factos………………………………………….. 18

2.5.3. Overtraining syndrome symptomatology……………………. 19

2.5.4. Prevention and treatment of overtraining……………………. 21

2.5.4.1. Recovery period…………………………………………… 21

2.5.5. Sport and Overtraining…………………………………………... 23

2.5.5.1. Explosive sports versus endurance sports………….. 24

CHAPTER 2………………………………………………………………………….. 27

1. EXPERIMENTAL STUDY……………………………………………………. 29 Chronic skeletal muscle damage induced by a demanding physical training is not a major physiopathological factor for 29 overtraining development in Wistar rats………………………………. CHAPTER 3………………………………………………………………………….. 53

1. CONCLUSIONS……………………………………………………………….. 55

CHAPTER 4………………………………………………………………………….. 57

1. REFERENCES………………………………………………………………… 59

LIST OF FIGURES

CHAPTER 2 – EXPERIMENTAL STUDY

Figure 1. The mean value with standard deviation of body weight and the food intake for the CG and exercised group at short and medium-term are depicted in A and B, respectively……………………………………………………………… 38

Figure 2. Representative light micrographs of Soleus muscles from control group (CG), 1-week eccentric exercise (EE1), and 3-weeks eccentric exercise (EE3), stained with Hematoxylin & Eosin, are depicted in A. The boxplot graphic presents the median value with 25 and 75 percentiles of red stained area for the CG and each exercised subgroup, indicative of tissue cross- sectional area, are shown in B……………………………………………………… 40

Figure 3. Representative light micrographs of Tibialis Anterior muscles from control group (CG), 1-week eccentric exercise subgroup (EE1), and 3-weeks eccentric exercise subgroup (EE3), stained with Hematoxylin & Eosin, are depicted in A. The boxplot graphic presents the median value with 25 and 75 percentiles of red stained area for the CG and each exercised subgroup, indicative of tissue cross-sectional area, are shown in B………………………… 40

Figure 4. Representative light micrographs of Soleus muscles from control group (CG), 1-week eccentric exercise subgroup (EE1), and 3-weeks eccentric exercise subgroup (EE3), stained with Hematoxylin & Eosin, are depicted in A. The boxplot graphic presents the median value with 25 and 75 percentiles of central violet stained area for the CG and each exercised subgroup, indicative of tissue nuclei, are shown in B…………………………………………………….. 41

Figure 5. Representative light micrographs of Soleus muscles from control group (CG), 1-week eccentric exercise subgroup (EE1), and 3-weeks eccentric

exercise subgroup (EE3), stained with Sirius red, are depicted in A. The mean

value with standard deviation of red stained area for the CG and each exercised subgroup, indicative of tissue collagen content, are shown in B……... 43

The mean value with standard deviation of red stained area for the CG and each exercised subgroup, indicative of tissue collagen content, are shown in B

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LIST OF TABLES

CHAPTER 2 – EXPERIMENTAL STUDY Table I. Muscles weights ...... 39

Table II. Muscular damage in soleus muscle ...... 42

Table III. Muscular damage in tibialis anterior muscle ...... 43

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RESUMO

O exercício físico extenuante e inabitual, com predominância de contrações excêntricas, constitui um veículo indutor de dano muscular. Assume- se que a sucessiva acumulação deste dano esteja na origem de uma condição crónica designada de síndrome de sobretreino (OTS). Contudo, não existem evidencias científicas diretas que associem o dano estrutural crónico muscular com a ocorrência de OTS. Assim, o objetivo deste estudo é testar o pressuposto de que a sucessiva acumulação de dano muscular induzido pela sobrecarga do treino esta na origem da OTS, usando os músculos soleus (SOL) e tibialis anterior (TA) de ratos Wistar expostos a um protocolo exigente de treino. Os animais foram aleatoriamente distribuídos pelos grupos de controlo (CG, n=5) ou de treino (EE, n=10). O grupo EE realizou corrida em tapete rolante (-20º; a partir dos 25m/min, aumento progressivo de 1.25m/min/dia; 60 min) 6 vezes/semana, sendo os animais sacrificados 1 (EE1, n=5) e 3 semanas (EE3, n=5) após o início do programa de treino. O peso corporal, a ingestão de comida, a aparência do pelo, a capacidade de realizar trabalho e o comportamento dos animais foram mensurados durante o protocolo. O SOL e o TA foram coletados para analise histológica, após sacrifício. Ambos os músculos exibiram sinais de dano muscular nos subgrupos EE1 e EE3 pelo aumento da degeneração celular, da necrose tecidual e da atividade inflamatória. Porém, o protocolo de treino não foi capaz de induzir OTS. Paralelamente à ocorrência de lesão muscular, foram encontrados sinais adaptativos nos músculos exercitados, como melhorias no conteúdo de colagénio e na área de secção transversal. Em suma, a grande quantidade de dano muscular no SOL e no TA não está associada com a OTS a curto e médio-termo. Ademais, o dano muscular apresenta diferentes comportamentos de acordo com o tipo de trabalho desempenhado pelo músculo, questionando o uso de marcadores sistémicos de dano muscular como métodos fiáveis no estudo da relação ente o dano muscular e a OTS. Palavras-chave: SOBRECARGA DE TREINO; LESÃO MUSCULAR; EXERCÍCIO EXCÊNTRICO; MÚSCULO SOLEUS; MÚSCULO TIBIALIS ANTERIOR;

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ABSTRACT

Strenuous and unusual physical exercise, with predominance of eccentric contractions, constitutes a vehicle that potentially induces muscular damage. It is believed that this successive muscle damage accumulation due to the training overload is the origin of a chronic condition designated by overtraining syndrome (OTS). However, there is no scientific direct evidences associating the chronic structural muscle damage induced by training overload with the occurrence of OTS. Thus, this study aim is to test the assumption that successive muscle damage accumulation due to the training overload is in the origin of the OTS, using the soleus (SOL) and tibialis anterior (TA) muscles of Wistar rats exposed to a demanding exercise training protocol. Animals were randomized distributed to a control group (CG, n=5) or to an exercised training group (EE, n=10), which performed a treadmill running training (-20º; from 25m/min, with progressive increase of 1.25m/min per day; 60 min) for 6 times/week being animals sacrificed 1 (EE1, n=5) and 3 weeks (EE3, n=5) after the beginning of the training program. Body weight, food intake, hair appearance, ability to perform work, and animals’ behavior were measured during the protocol. After sacrifice, SOL and TA muscles were collected for histological analysis. Both showed muscle damage signs in EE1 and EE3 through the increase of cell degeneration, tissue necrosis, and inflammatory activity. However, the exercise training protocol was not able to induce OTS. In parallel to the occurrence of muscle injury, adaptative signs in the exercised muscles were found, such as enhanced collagen content and cross- sectional area were also observed. In conclusion, the great amount of chronic muscular damage in SOL and TA is not associated with the OTS in the short and medium-term. Further, muscle damage demonstrates different behaviors according to the type of work that each muscle performs, questioning the use of systemic markers of muscle damage as reliable methods to study the relationship between skeletal muscle damage and OTS.

Key words: TRAINING OVERLOAD; MUSCLE INJURY; ECCENTRIC EXERCISE; SOLEUS MUSCLE; TIBIALIS ANTERIOR MUSCLE;

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LIST OF ABBREVIATIONS

ATP Adenosine triphosphate

BW Body weight

Ca2+ Calcium ion

CD Cellular degeneration

CG Control group

CK Creatine Kinase

CSA Cross-sectional area

DPX Dibutyl xylene phthalate

EC Eccentric contractions

EE Exercise training group

EE1 1-week exercise subgroup

EE3 3-weeks exercise subgroup

EM Exercise myopathy

FI Food intake

GLUT-4 Glucose transporter type 4

H&E Hematoxylin-Eosin

IA Inflammatory activity

IL-1β Interleukin-1-beta

IL-6 Interleukin-6

IL-10 Interleukin-10

LDH Lactate dehydrogenase

MMPs Metalloproteinases

MPTP Mitochondrial permeability transition pore

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N Necrosis

NP Nuclei position

OTS Overtraining syndrome

PE Physical exercise

ROS Reactive oxygen species rSW Relative Soleus weight rTA Relative Tibialis Anterior weight

SOL Soleus

SOLCC Soleus collagen content

SOLCSA Soleus cross-sectional area

SOLMD Soleus damage

SOLNP Soleus nuclei position

SW Soleus weight

TA Tibialis Anterior

TACC Tibialis Anterior collagen content

TACSA Tibialis Anterior cross-sectional area

TAMD Tibialis Anterior damage

TANP Tibialis Anterior nuclei position

TAW Tibialis Anterior weight

TD Total muscle damage

TIMPs Tissue inhibitors of metalloproteinases

TNF-α Tumor necrosis factor alpha

TO Tissue organization

CHAPTER 1

INTRODUCTION AND STATE-OF-ART

1. INTRODUCTION

Strenuous and unusual physical exercise (PE), with predominance of eccentric contractions, constitutes a vehicle that potentially induces muscular damage (Hyldahl & Hubal, 2014; Peake et al., 2017). Indeed, the level of muscle damage is directly related to the type of contraction performed, with the eccentric contractions (EC) presenting a greater damage comparatively to the isometric and concentric contractions, based on the fact that, for the same workload, fewer motor units are recruited, resulting in a greater mechanical stress applied on each active motor unit (Douglas et al., 2017; Hyldahl & Hubal, 2014). The EC are characterized by specific muscular movements, in which the force generated is inferior to the load, resulting in the stretching of the muscle (Douglas et al., 2017). The isolated strenuous PE is responsible for an amount of acute changes in skeletal muscle with local and systemic repercussions, characterizing an anatomopathological condition called exercise myopathy (EM) (de Almeida et al., 2017; Duarte, 1993; Duarte et al., 2001). Specifically, this muscle damage is notorious at the micro-structural level, visible by the disturbances in the structural elements of muscle fibers, such as disorganization on the Z-lines, misalignment of the adjacent sarcomeres and damage in the contractile filaments of the cytoskeleton, which lead to the occurrence of muscle striated pattern disorganizations (Butterfield, 2010; Peake et al., 2017). On the other hand, it is possible to find necrotic areas and an inflammatory response in the affected muscles with sarcolemma and sarcoplasmic reticulum injuries, which favors the increased cytoplasmic Ca2+ contents within injured fibers (Peake et al., 2017; Torres et al., 2013). However, between two to three weeks after exercise, it’s possible to observe the return to normal muscle tissue structure (Duarte, 1993). Considering the repeated exercise practice, a required balance between loads and recovery periods provides favorable adaptations in the myofibrillar structure and in other cellular compartments, which enable the skeletal muscle to tolerate the mechanical, metabolic, thermic, and oxidative overload induced by subsequent training sessions (Friden et al., 1983; Lira et al., 2013). An example is the adaptation that occurs at the extracellular matrix level, through the increase

amount of connective tissue (Hyldahl et al., 2015; Mackey & Kjaer, 2016). The increase of the intramuscular connective tissue is assumed as a determinant factor in the muscular protection against the PE damage due to the greater capacity of this tissue to dissipate the mechanical stress imposed on the muscular fibers, which provide a greater tolerance against the acute exercise, especially when it is composed by EC (Hyldahl et al., 2015; Takagi et al., 2016). On the other hand, also skeletal muscle fibers experience several beneficial phenotype changes with physical training, allowing increased tolerance to further exercises, resulting from changes in gene expression in healthy fibers as well as from the regenerative process involved in injured fibers, through the activation of satellite cells with fusion to the existing fibers (Liu et al., 2013). The increased muscle fibers cross-sectional area, as a favorable adaptation to physical training, is the result of this regenerative process (Egner et al., 2016). This hypertrophy facilitates the dissipation and distribution of the mechanical stress exerted, improving the tolerance of muscular fibers to mechanic stress overload (Hedayatpour & Falla, 2015). However, training programs that combine elevated demands, great amount of eccentric contractions, and reduced rest periods provide to athletes a greater risk of exceeding the limits of their adaptative ability and, subsequently, evidencing progressively lower tolerance to training stimuli with the reduction of sport performance (Julian et al., 2018; Kentta & Hassmen, 1998; Kreher & Schwartz, 2012). A lower tolerance to the stimulus promotes the muscle damage accumulation with the repeated exercises, worsening progressively the damage induced by previous training sessions (Tiidus, 1998). Further, an acute inflammatory response amplification occurs, characterized by fluid, leukocytes and plasma proteins infiltration into injured tissues, reaching the level of chronic and pathological condition. Thus, the localized chronic inflammatory response may become a systemic inflammation, with adverse consequences to the whole organism (Kreher & Schwartz, 2012; Peake et al., 2017). The decreased sports performance associated with the clinical manifestations of the parallel organic maladaptations is usually referred as overtraining syndrome (OTS) (Kreher & Schwartz, 2012). However, terms as overtrained, burnout, overstrained,

stagnation, overused, overstressed, chronic fatigue syndrome, overreaching, overworked, staleness and staleness syndrome are often mistakenly used to define the same event, though without considering factors such as the recovery time required to return to previous levels of performance (Kreher, 2016). Therefore, influenced by daily life and training factors, such as the imbalance between training loads and the recovery periods, the OTS is defined as the progressive decrease of functional performance accompanied by clinical signs and symptoms resulting from modifications in the nervous, endocrine, and immune systems functionality (Cardoos, 2015; Carfagno & Hendrix, 2014; Kreher, 2016). Doubts remain regarding the exact etiology and pathogenesis of the OTS, as well as the existence of direct markers that allow the early and accurate diagnosis of this clinical condition, although some symptoms have been suggested in the literature as early markers (Carfagno & Hendrix, 2014; Kreher, 2016). Among the most commons, it is possible to highlight the appetite and weight loss, irritability and aggressiveness, and sleep disturbances, which are usually associated with laboratorial signs of a proinflammatory state (Gleeson, 2002; Kentta & Hassmen, 1998). Although the assumption that the successive muscle damage accumulation due to the training overload is the base of the OTS origin, there is not direct markers in the literature supporting this association. The literature just present indirect markers of muscle damage, such as the seric levels of creatine kinase (CK), proinflammatory cytokines or lactate dehydrogenase (LDH) to explain the relationship between muscle injury and the occurrence of OTS (Cardoos, 2015; da Rocha et al., 2017; Lira et al., 2010). However, the response of these markers during muscular damage shows a huge interindividual variability, which reducing the sensitivity and specificity of the parameters as indicators of damage, makes the association of OTS and chronic muscle injury less reliable (Magal et al., 2010). Thus, the aim of this dissertation was to verify in soleus (SOL) and tibialis anterior (TA) muscles of male Wistar rats, the relationship between chronic muscle damage induced by a demanding exercise training and the occurrence of the overtraining syndrome. For that purpose, a literature review and an

experimental study were elaborated in order to test the working hypothesis that the great amount of chronic muscle damage quantified by histology in the studied muscles is closely associated with OTS signs, in the short (1 week) and medium- term (3 weeks). Therefore, the present dissertation is divided into 4 main chapters. In the 1st chapter (Introduction and State-of-art) the problem, aims and hypothesis to be studied are presented as well as a synthesize with the most relevant themes associated with the experimental study, namely muscle damage, adaptive effects induced by PE, regenerative process, sports training and OTS. In the 2nd chapter, the Experimental Study was presented, with the aim to verify in SOL and TA muscles of Wistar rats, the relationship between chronic muscle damage induced by a demanding exercise training and the occurrence of the OTS. This dissertation ends with the Conclusions and References that correspond to 3rd and 4th chapter, respectively.

2. STATE-OF-ART 2.1. Physical exercise as a muscle damage inducer

Strenuous and unusual physical exercise (PE), particularly with predominance of eccentric contractions (EC), constitutes a vehicle of skeletal muscle damage (Hyldahl & Hubal, 2014; Peake et al., 2017). Indeed, the level of muscle damage caused is directly related to the type of contractions performed, with the EC presenting a greater damage compared to the isometric and concentric contractions, since, for the same workload, fewer motor units are recruited in EC, resulting in a greater mechanical stress applied on each active motor unit (Douglas et al., 2017; Hyldahl & Hubal, 2014). The eccentric contractions are present in daily activities, such as down stairs or land shopping bags, and they are characterized by specific muscular movements in which the force generated is inferior to the load, resulting in the stretching of the muscle (Douglas et al., 2017). Any acute PE session, depending of the muscle tolerance to that specific exercise, might induce damage by mechanical, metabolic, thermal and oxidative overload, consequent to the high functional demands imposed to the recruited muscles (Armstrong et al., 1991; Duarte, 1993; Formenti et al., 2013; Naughton et al., 2017; Qaisar et al., 2016). The mechanical overload is traduced by a high contractile tension imposed to the sarcolemma and sarcoplasmic reticulum, which provides their elongation and disruption with further disturbances in the calcium ion (Ca2+) homeostasis (Armstrong et al., 1991; Duarte, 1993). The metabolic overload, expressed by the occurrence of oxidative phosphorylation disturbances during energy production process, also causes the Ca2+ homeostasis loss due to energy deficits affecting Ca2+ pumps (Armstrong et al., 1991; Duarte, 1993). As a consequence of the enhanced metabolic rate, the resulting thermal overload potentiates the development of sarcolemma, lipid and protein abnormalities, compromising mitochondrial functionality and Ca2+ homeostasis (Armstrong et al., 1991; Formenti et al., 2013). On the other hand, the enhanced metabolic rate during exercise also produces a great amount of reactive oxygen species (ROS), affecting not only sarcolemma and sarcoplasmic

membranes, but also enzymes activity with Ca2+ homeostasis loss (Duarte, 1993; Qaisar et al., 2016). The Ca2+ homeostasis loss leads to the activation of Ca2+- activated neutral proteases, the calpains. In the skeletal muscle, three calpain isoforms emerge: μ-calpain, m-calpain and p94 (Gissel, 2006). The maintenance of elevated Ca2+ levels activate the calpains, promoting the degradation of cytoskeletal, myofibrillar and membrane proteins, namely titin, desmin and vimentin, whose functions are based on the structural sarcomeres integrity (Gissel, 2006; Pompeani et al., 2014). Thus, the degradation of these proteins by the calpains contributes to increase the sarcomeres vulnerability to exercise- induced damage (Pompeani et al., 2014). Furthermore, the increased Ca2+ also activates a specific cysteines protease called caspases (Sandri et al., 2001). The caspases present several functions, such as inflammatory response, tissue differentiation, cell proliferation, tumor formation and metastasis and DNA damage, acting in both necrosis and apoptosis processes (Fan et al., 2005; Shalini et al., 2015). Indeed, the caspases are directly associated with cell disassembly by the proteolytic cleavage as well as involved in upstream regulatory events (Sandri et al., 2001). Simultaneously, a mitochondrial Ca2+ overload can also induce necrosis and apoptosis processes (Lim et al., 2015). Elevated Ca2+ concentrations promote the opening of the mitochondrial permeability transition pore (MPTP), which allows free passage of molecules, affecting the ionic homeostasis and the uncoupling of oxidative phosphorylation (Paradies et al., 2009). Thus, a dysfunctional mitochondria development occurs with subsequent release of proapoptotic proteins, which increase the ROS production and the muscle damage (Lim et al., 2015; Paradies et al., 2009). In opposition to the traumatic injuries induced by PE, such as muscle ruptures and bruising, these non-traumatic damages associated with metabolic, mechanical, oxidative and thermal overload induced by unusual and exhaustive exercise are manifested at a muscular micro-structural level, accompanied by voluntary maximum strength decrease, and increase of muscle lysosomal enzymes and plasma muscle proteins (Duarte, 1993; Duarte et al., 2001). This anatomopathological condition induced by acute unusual and exhaustive exercise, composed by local morphological and functional alterations in recruited

skeletal muscles and attended by systemic repercussions, is usually known as exercise myopathy (EM) (de Almeida et al., 2017; Duarte, 1993; Duarte et al., 2001).

2.1.1. Microscopic changes induced by exercise

As above referred, the strenuous and unusual PE, namely with predominance of EC has been referred in literature as a major local damage inducer in the skeletal muscle. Immediately after PE is possible to find micro- structural changes within skeletal muscle fibers, such as striated pattern irregularities, with changes in isotropic and anisotropic bands dimensions, which results in disruptions, fleshes or extension of the material of the Z-lines to the adjacent I-bands (Duarte, 1993; Duarte et al., 2001). Moreover, it can be also noted sarcolemma and T-tubule disruptions, and a sarcoplasmic reticulum dilatation, which cause disturbances in the Ca2+ homeostasis, and the development of muscular lipid and protein structures self-destruction (Butterfield, 2010; Duarte, 1993; Peake et al., 2017). These cellular changes are part of an event called intrinsic muscle fiber degeneration (Duarte, 1993). Besides the cellular alterations presented, the PE may also causes extracellular changes, namely extracellular matrix problems, visible by the connective tissue disturbances (Hyldahl et al., 2015; Mackey & Kjaer, 2016). Muscle connective tissue is organized in three interconnected layers: epimysium, perimysium and endomysium. Epimysium is the outermost layer and covers the entire muscle, the perimysium is the layer that surrounds the muscle fascicles, and the endomysium is the innermost layer that lines each muscle fiber (Böl et al., 2014). Considering the connective tissue as the major extracellular matrix component, the connective tissue damages are usually referred as extracellular matrix problems (Hyldahl et al., 2015). The extracellular matrix is constituted by proteoglycans, glycoproteins, elastin, fibronectin and collagen, where type I and III isoforms predominate, being interconnected with the myofibrillar sarcolemma through a specialized basement membrane associated with an integrins network that potentiate not only the structural integrity of the muscle fibers, but also the

correct distribution of forces throughout the muscular structure (Hyldahl et al., 2015; Song et al., 2015). The unusual PE practice is related with the changes in certain components of connective tissue (Hyldahl et al., 2015). Histological analyzes show a significant perimysium and endomysium areas enlargement, with a muscle fibers extracellular matrix withdrawal into an enlarged interstitial space (Stauber et al., 1990). Moreover, an increase of collagen expression and its degradation enzyme are also described associated to the degradation and synthesis process, characteristic of extracellular matrix damage (Mackey et al., 2004). On the other hand, an increase in the amount of metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) as well as in the enzymes responsible for the extracellular matrix degeneration and remodeling were observed too (Mackey et al., 2004).

2.1.2. The muscle inflammatory response and repairing process

The hormonal alterations associated with PE promotes the increase of circulating inflammatory cells (Chazaud, 2016; Cruzat et al., 2007). However, in the case of PE-induced muscle damage, a local inflammatory response in committed muscles appears 24-48 hours after the exercise session, characterized by a leukocyte infiltration in injured areas (Chazaud, 2016; Philpott et al., 2018). Neutrophils are the first leukocytes recruited to the damaged sites, reaching the cells peak approximately 24 hours after the injury, and maintaining a high concentration beyond 5 days (Smith et al., 2008). Posteriorly, the macrophages and lymphocytes recruitment occur, namely monocytes, B and T- lymphocytes, which causes a proteolytic enzymes and ROS release, aggravating the damage of intrinsic degeneration, promoting the protein catabolism and the cleaning of damaged tissue, and consequently, facilitating the muscular repair (Chazaud, 2016; Duarte et al., 2001; Smith et al., 2008). This process is called extrinsic degeneration (Duarte, 1993). Subsequently to the degenerative phases characteristics of muscle damage induced by the unusual and demanding PE occur a monocytes concentration increase in the injured muscle areas (Paulsen et al., 2012). Firstly,

there is a pro-inflammatory macrophages (M1 macrophages) growth, during approximately 3 days, responsible not only for the necrotized cell areas removal by the phagocytosis process, but also for the cytokines production (Paulsen et al., 2012; Peake et al., 2017). Posteriorly, a change in macrophages phenotype begins, where pro-inflammatory macrophages convert into anti-inflammatory macrophages (M2 macrophages). This transition appears as a result of some regulatory signals, such as the interleukin-10 (IL-10) or the phagocytosis process of necrotized areas, presenting a major importance in the inflammation resolution (Peake et al., 2017). The increased macrophages (M1 and M2) in the damaged areas promotes an amount of stem cells recruitment, namely satellite cells (Schiaffino et al., 2017). Their concentrations increase within the first 24 hours after the stimulus, remaining elevated during the following 8 days (Paulsen et al., 2012). In the skeletal muscle, the satellite cells are located between the sarcolemma of adjacent fibers and the basal lamina of those fibers (Snijders et al., 2015). When stimulated, these cells enter into cell cycle originating two newborn cells, one of them will further differentiate to a myoblast to fuse with adjacent fiber while the other one will stay undifferentiated for maintaining the satellite cell store (Smith et al., 2010). The quiescent satellite cells with the interaction of M1 and M2 macrophages become active, and consequently, occurs their proliferation and differentiation (Snijders et al., 2015). Hence, the satellite cells proliferate and differentiate into myoblasts, melting to adjacent fibers or form multinucleated myotubes (Peake et al., 2017; Snijders et al., 2015). Between 1 to 3 weeks after a maturation process, a complete muscular fibers regeneration is observed by the normal striated pattern presence, and the myonuclei peripheral location (Duarte, 1993). The repair process also occurs at the extracellular level, especially in the extracellular matrix. Right after the damaging stimulus, proteases are released, namely MMPs, which cause an extracellular matrix degradation, and subsequently, a cellular adhesion decreases (Hyldahl et al., 2015). Among the several MMPs reported in the literature, evidences suggest MMP-2 and MMP-9 play a significant role in the extracellular matrix repair process, being a major

agent in the extracellular matrix degradation, satellite cells activation and new muscle fibers formation (da Cunha Nascimento et al., 2015; Giganti et al., 2016). After the cellular adhesion decreases, the TIMPs present an opposite response to MMPs. The TIMPs promote the extracellular matrix adhesion and the deposition of the needed components to remodulation (da Cunha Nascimento et al., 2015). A balance between the MMPs and the TIMPs is central to avoid misaligned changes in the extracellular matrix, as well as in the remodeling process, because it potentiates an adequate environment creation to this event occurrence (Dastani et al., 2014). The creation of this favorable environment promotes an easier recruitment and proliferation of the extracellular matrix remodeling agents, namely leukocytes, satellite cells and fibroblasts (da Cunha Nascimento et al., 2015; Hyldahl et al., 2015). As the leukocytes and satellite cells, fibroblasts also play a crucial role in the extracellular matrix development, because they product essential structural components, such as fibronectin, proteoglycans, elastin and collagen (Tracy et al., 2016). Most of the injuries that occur in skeletal muscle are repaired by regeneration, without the formation of a functionally disabling fibrotic scar. However, sometimes due to a greater damage degree, the excessive fibroblasts proliferation results in a dense scar tissue formation in the injured muscle, creating a mechanical barrier that considerably delays or completely restricts the muscle fibers regeneration, affecting the ability and, consequently, their functionality (Järvinen et al., 2005; Novak et al., 2014; Xiao et al., 2016).

2.2. Muscle adaptations to repeated exercise

As seen, the unusual, strenuous or predominantly eccentric PE practice is responsible for a set of events associated with the muscle damage. Considering the repeated exercise practice, a required balance between loads and recovery periods provides favorable adaptations in the myofibrillar structure and in other cellular compartments, which enable the skeletal muscle to tolerate the mechanical, metabolic, thermic, and oxidative overload induced by subsequent training sessions (Friden et al., 1983; Lira et al., 2013). Specifically, the literature

presents the assumption that from the first acute eccentric exercise session is possible to find adaptations that protect the muscle against injuries from subsequent eccentric exercise sessions, reducing initial damage symptoms. This event is called Repeated Bout Effect (McHugh, 2003). Among the several adaptations described in the literature, the mostly highlighted are: the lower acute inflammatory response, slower delayed sensation of muscle discomfort, lower circulating CK concentration, and a faster force indices recovery (Hyldahl et al., 2017). Muscle fibers hypertrophy is a potential favorable adaptation induced by the continuous and demanding PE practice (Tsumiyama et al., 2014; Vogt & Hoppeler, 2014). According to literature, eccentric exercise induces higher hypertrophy levels than concentric and isometric exercises (Hedayatpour & Falla, 2015; Margaritelis et al., 2015). Skeletal muscle hypertrophy is characterized by muscle cross-sectional area increase through muscle mass growth due to myoblasts fusion with protein synthesis and other growth factors increase (Egner et al., 2016; Tsumiyama et al., 2014; Vogt & Hoppeler, 2014). As the EC induce a greater mechanical tension in the muscular fibers in comparison to the concentric and isometric contractions, it is possible to chronically observe a greater addition of sarcomeres in parallel (sarcomerogenesis), which contribute for a higher cross-sectional area increase (Hedayatpour & Falla, 2015; Margaritelis et al., 2015). Therefore, the increase in the number of the muscular fibers sarcomeres facilitates the mechanical tension dissipation and distribution, which lead each muscle fiber to perform a smaller effort and, consequently, decreasing the damage induced. On the other hand, the higher mechanical stress during the EC stimulate the signaling pathways responsible for muscle growth, with fusion of myoblasts to adjacent recruited fibers (Egner et al., 2016; Hedayatpour & Falla, 2015). The PE is a physical activity subcategory characterized by being structured and organized. When it is done regularly with a purpose to improve or maintain one or more physical fitness components in a planned way, it is classified as exercise training (Caspersen et al., 1985). Exercise training appears as a vehicle for the development of local and systemic adaptations, such as

neuromotor, biochemical, physiological, behavioral and structural adaptations, in order to improve in the individual performance and the tolerance to acute exercise (Cunha, Minderico, et al., 2016).

2.3. Exercise training principles

In order to achieve the optimal individual performance, a correct training planning is needed, considering a short-term and long-term systematic coordination and scientific support of all programming, periodization, achievement, control, analysis and correction training measures (Grosser et al., 1989). Thus, to meet the proposed aims, as fitness improvement or prevention/treatment of diseases, training planning should consider a set of guiding principles, among which are the overload, specificity, individuality and reversibility principles (Ammann et al., 2014; Xu, 2015). The overload principle is based on the fact that an organ or an organic system should be exercised at a level beyond which it is accustomed in order to promote changes (adaptations), and consequently the training effect (Ammann et al., 2014). Moreover, it states that the stimulus (load) must be diversified when an adaptation to this overload is observed, through changing of the variables, such as intensity, duration, frequency, volume and density (Ciolac & Guimarães, 2004). The load intensity is a volitional training variable, which tries to measure the effort that a subject develops in the response to a certain load, i.e. to observe the requirement in relation to the maximal capacity. The load duration is a variable which demonstrates the exercise time execution. On the other hand, the frequency is associated with the number of repetitions and sets performed by the subject, and the volume is directly related to the amount of work performed. Finally, the load density is a variable that demonstrates the temporal relationship between the effort and the recovery (Cunha, Minderico, et al., 2016). Besides the overload principle, the literature presents the specificity principle. This principle exhibits the idea that adaptations to the training load just occur in the capacities, conditions and functional systems in which they are sustained, i.e. the adaptations obtained are specific to the exercises performed, which promotes

specific physiological responses (Ammann et al., 2014; Ciolac & Guimarães, 2004). On the other hand, the training loads induce adaptations which have a huge inter-individual variability, i.e., the load responses vary between individuals, suggesting that the training program must be individualized to each subject. This principle is named as individuality principle (Ciolac & Guimarães, 2004; Xu, 2015). Finally, the reversibility principle refers that all adaptations cease or regress to a pre-training state if the training program is discontinued (Ammann et al., 2014; Ciolac & Guimarães, 2004).

2.4. Exercise training periodization

Besides the training principles above, it is also important a careful attention to the aspects associated with the training program periodization (Kreher & Schwartz, 2012; Vogt & Hoppeler, 2014). The training periodization is a concept related to time management. It is a planning technique whose aim is the precise temporal limits definition, such as periods, stages and cycles, which allow to structure objectively, at each moment, the variables related to the training, in order to reach the training state and the results desired (Cunha, Louro, et al., 2016). The training periodization considers the periodic variation of the training components, such as volume and intensity as well as the training contents, trying to obtain a balance between work and recovery (Cunha, Louro, et al., 2016). The correct training periodization is based on the adequate time periods management. These plans try to organize the variation of training components over time and can be classified according to their duration (Cunha, Louro, et al., 2016; Oliveira et al., 2005). The annual plan determines the training process during a sporting season. It is the plan that directly influences the training program elaboration during an annual cycle and considers the number of moments of optimal performance (Cunha, Louro, et al., 2016). The annual plans can be divided into shorter time blocks, in order to become easier and more precise the training programs planning. These shorter time blocks can be distinguished in macrocycles, mesocycles and microcycles (Cunha, Louro, et al., 2016; Oliveira et al., 2005). The macrocycles are the most prolonged temporal blocks, usually

between 8-12 weeks and until 1 year, being responsible for the training organization within each sport season (Cunha, Louro, et al., 2016; Oliveira et al., 2005). Each annual plan can integrate one or more macrocycles, which are subdivided into training periods (preparatory, competition and transition) (Oliveira et al., 2005). The preparatory or preparation period allows the athlete to reach the desire competitive level. This period is composed by two phases: the basic phase, where there is a greater concern with the physical preparation and with general training components, and the specific phase, where the technical-tactical training is emphasized (Oliveira et al., 2005). The competition period is the time block where the athlete must reach his optimal performance, and where predominates the specific formation (Oliveira et al., 2005). The transition period is a phase with a duration of approximately 1 month, and whose athletes can recover physically and psychologically from the effort made during the competitive period (Oliveira et al., 2005). On the other hand, the mesocycles are time blocks between 3 and 5 weeks, which aim is to develop adaptations in the abilities and conditions related to the performance (Cunha, Louro, et al., 2016; Oliveira et al., 2005). According to the goals, mesocycles can be classified in: basic, control, recuperative, incorporation, stabilizer, pre-competitive and competitive (Oliveira et al., 2005). The microcycles are the shortest sections of the plan, with a duration of 1 week (Cunha, Louro, et al., 2016). These temporal blocks present as the most important reference for training planning, because they show detailly the training contents and the work and recovery phases (Oliveira et al., 2005). Such as mesocycles, the microcycles can be labeled in ordinary, recovery, incorporation, shock, pre-competitive and competitive (Oliveira et al., 2005).

2.5. Overtraining syndrome

Nowadays, there is an increased competitiveness among athletes, namely among elite athletes (Carfagno & Hendrix, 2014; Kentta & Hassmen, 1998). The willing to achieve the desired results enhances the development of risk behaviors, particularly in the training programs planning, which can put at risk not only the

athletes’ career but also their own health (Borin et al., 2007; Carfagno & Hendrix, 2014; Kentta & Hassmen, 1998). As previously seen, the overload principle presents the assumption that an organ or an organic system should be stimulated beyond the levels in which it is accustomed to be able to observe changes (adaptations) and, consequently, the training effect. However, when the training programs are characterized by high intensity loads and short recovery periods, the athletes present a greater risk of exceeding the limits of their adaptive abilities and, subsequently, evidencing progressively a lesser muscle adaptative ability to training stimuli (Julian et al., 2018; Kentta & Hassmen, 1998; Kreher & Schwartz, 2012). Thus, the subjects do not express beneficial physiological adaptations, but instead their sports performance decrease as well as the occurrence of chronic maladaptations. This decreased sports performance associated with the clinical manifestations of the parallel organic maladaptations is usually referred as overtraining syndrome (OTS) (Kreher & Schwartz, 2012). According to the literature, several terms are used to describe the OTS, such as burnout, overstrained, stagnation, overused, overstressed, chronic fatigue syndrome, and overreaching, but they are often mistakenly used, because they do not consider factors such as the recovery time required to return to previous levels of performance (Kreher, 2016). Therefore, overtraining syndrome is the result of prolonged and chronic stress imposed by regular physical exercise, being influenced by daily life and training factors, such as the imbalance between training loads and the recovery periods, which results in chronic decreases in functional performance capacity and intolerance to exercise, accompanied by modifications in the nervous, endocrine, and immune systems functionality (Cardoos, 2015; Carfagno & Hendrix, 2014; Kreher, 2016).

2.5.1. Over-reaching and overtraining syndromes

According to literature, the OTS and over-reaching are part of the same training continuum. This continuum is characterized by four phases: undertraining, acute overload, overreaching and overtraining (Carfagno & Hendrix, 2014). The undertraining phase shows minimal physiological

adaptations, however no performances changes are observed. Contrary, the acute overload is characterized by the presence of positive physiological adaptations, which result in small performance improvements. On the other hand, the overreaching phase exhibits physiological adaptations and optimal performance but only after a required recovery period. Finally, the overtraining phase presents a stress increase induced by training load, which causes irreversible physiological maladaptations and performance decreases (Carfagno & Hendrix, 2014). The overreaching phase can be divided into two distinct states: the functional overreaching and nonfunctional overreaching. The functional overreaching state occurs when the training stress accumulation results in a temporary performance decline, between a few days and a few weeks. However, a performance improvement is obtained when adequate recovery periods are given to athletes. In contrast, the nonfunctional overreaching state demonstrates physiological and psychological problems, and a regression in the capacity to restore the performance due to a successive training loads accumulation. These problems can persist among few weeks to a few months (Cardoos, 2015; Kreher, 2016; Kreher & Schwartz, 2012). The nonfunctional overreaching and overtraining have many similarities, but the overtraining phase differs from the nonfunctional overreaching state because the performance declines and disturbances in the psychological, neurological, endocrine and immunological systems adaptations are observed over a longer period, usually more than 2 months, but may never return to normal, i.e. the physiological maladaptations presents a chronic character (Cardoos, 2015; Kreher, 2016; Kreher & Schwartz, 2012).

2.5.2. Overtraining risk factors

As above referred, inappropriate training programs planning appears as a major factor of OTS development. However, other factors are referred in the literature as inductors of this condition, namely the high competitions density; the low physical fitness level; the high injuries or infections frequency; the high

motivation levels; the high emotional instability and the adoption of unbalanced diets, with insufficient caloric intake (Kentta & Hassmen, 1998).

2.5.3. Overtraining syndrome symptomatology

There are several symptoms associated with the established OTS (Carfagno & Hendrix, 2014; Carter et al., 2014; Kreher, 2016). However, it is a problem to define exclusively the overtraining symptoms due to the tenuous threshold between the continuum phases (Kreher, 2016). Nevertheless, according to literature, the major overtraining symptoms are the irritability states, apathy and discouragement, abrupt and persistent performance decline, increased heart rate and resting blood pression, persistent muscle discomfort feeling, increased number of injuries and infections, decreased appetite and weight, sleep disorders, reflecting changes in the nervous, muscular, endocrine and immune systems activity (Carfagno & Hendrix, 2014; Carter et al., 2014; Kentta & Hassmen, 1998; Kreher, 2016). Strenuous and prolonged PE is responsible by ROS production. Elevated ROS concentrations promote cellular disorders at the muscular level, which develop asthenia and muscle inflammation, compromising the normal skeletal muscle function and, consequently, the optimal individual performance (Duarte, 1993; Kreher & Schwartz, 2012). Moreover, the asthenia development is directly related to decreased proprioceptive capacity, which helps the development of muscular injuries (Ribeiro & Oliveira, 2017). On the other hand, according to literature, overtraining athletes show lower citrate synthase levels during the endurance trainings, antagonistically to what is expected for athletes under normal conditions, supporting the assumption that overtraining subjects are less responsive to the oxidative stress induced by exercise, being more susceptible to oxidative damage (Kreher & Schwartz, 2012). In contrast, the strenuous PE practice, namely eccentric ones, promotes a higher number of mechanical injuries and lesions related to movement repetition, causing a tissue microtrauma which, subsequently, activate the cytokines recruitment, beginning a local inflammatory response. The continuous PE

practice causes an inflammatory response amplification, reaching the level of chronic and pathological condition (Carfagno & Hendrix, 2014; Kreher & Schwartz, 2012). The exacerbated cytokines increase, such as interleukin-1-beta (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α), characteristic of an overtraining state, are associated with the low muscle glycogen levels in overtrained athletes. Indeed, these cytokines action in the muscle glucose transporters, namely glucose transporter type 4 (GLUT-4), diminish the glycogen synthesis (Carfagno & Hendrix, 2014). Further, these cytokines act in the hypothalamic centers associated with hunger, inducing anorexia, and contributing to the glycogen stores decrease (Kreher & Schwartz, 2012). On the other hand, reduced muscle and seric glycogen levels are directly related to higher apathy, irritability and discouragement rates as well as sleep disturbances (Weiss et al., 2010; Zhou et al., 2015). The increased seric cytokines concentrations promotes the tryptophan levels decrease due to the higher brain absorption in order to occur the inflammatory proteins synthesis. Lower tryptophan levels are found in overtrained subjects and they are related to elevated asthenia and depression rates (Kreher & Schwartz, 2012). Furthermore, the proinflammatory cytokines IL-1β and TNF-α also cause behavioral and psychological effects, visible by the sleep disturbances, appetite loss, and psychological disorders such as depression (Kreher & Schwartz, 2012). Moreover, these cytokines also influence the immune system. Specifically, IL-1β and TNF-α stimulate the glutamine uptake into hepatocytes. Since glutamine is an important precursor of the inflammatory proteins’ synthesis as well as a major gluconeogenesis element, it is normal to observe reduced concentrations of plasma glutamine and an increase in glycolytic and protein metabolism in overstrained subjects (Kreher & Schwartz, 2012; Smith, 2000). The reduced glutamine concentrations induce an increased sensitivity to infections, and an increased catabolic state causes a weight loss (Kreher & Schwartz, 2012; Loureiro et al., 2014; Smith, 2000).

2.5.4. Prevention and treatment of overtraining

Based on the overtraining or over-reaching diagnosis, an amount of symptoms are observed influencing the treatment time needed (Cardoos, 2015; Kreher, 2016). According to the literature, the methods most used in the overtraining treatment is the increased recovery time with the training loads reduction or even the complete training cessation (Myrick, 2015). If the subject presents nonfunctional over-reaching, at least few weeks will be needed to treat the pathology, which will affect negatively the athlete goals for the season (Kreher, 2016). So, the OTS prevention has an important role in the training programs planning. The correct workloads and recovery planning, namely, the regular control of the training state, through medical-sports and performance evaluation, as well as through the early detection of weaknesses in motor behavior are the main measures adopted in the overtraining prevention (Cardoos, 2015; Myrick, 2015). Further, Aubry et al. (2014) present the tapering as a decisive factor in the overtraining prevention. According to these authors, tapering is characterized by the workload reduction before competitions or between training cycles, allowing the active recovery and the achievement of the overcompensation effect. This effect is obtained by the performance components and training variables diversification (Aubry et al., 2014). On the other hand, the diet control is also evidenced as an effective measure in the overtraining prevention, since an ineffective repletion of energetic substrates, such as glycogen, causes disturbances in the performance (Kreher, 2016; Kreher & Schwartz, 2012).

2.5.4.1. Recovery period

The recovery period presents as a major factor in the OTS prevention and treatment process (Carfagno & Hendrix, 2014). When an unusual or demanding workload is applied, the functional capacity to respond to the same stimulus is initially reduced. However, when appropriate recovery periods are given, a set of adaptations will increase the functional capacity and to develop the

overcompensation effect (Cardoos, 2015; Kreher, 2016; Kreher & Schwartz, 2012). The recovery can be defined as the process of returning to homeostasis after performing PE, which comprises the metabolic and structural reparation and regeneration as well as the restoration of the organism functional capacity (Tomlin & Wenger, 2001). According to Borin et al. (2007), initially occurs a work capacity recovery, responsible for the restoration of the energy deposits used and the basal levels in the cardiorespiratory, endocrine, nervous and muscular systems, as well as for the elimination of the metabolites produced. In the following phase an overcompensation effect is observed, with the restoration and improvement of the individual performance, through the physiological adaptations development. Finally, it is possible to observe a stabilization of the adaptations developed, i.e. the organism is adapted to the new performance level. Regarding to recovery methods, Gutiérrez & Castillo (2001) show four methods with different typologies. The synchronous method presents the recovery performed during the training session, through the hydration between work series. The primary restitution method shows the recovery between training sessions, over the energy substrates restitution and the protein drinks ingestion after training. Moreover, the secondary restitution method exhibits the proprioceptive and stretching strategies adoption as recovery process. Lastly, the authors present the overtraining restitution method, characterized by the immediate workloads decrease, associated with tapering strategies. As in the OTS, some factors are responsible for influence the recovery process. Namely, the exercise intensity and duration, where longer and strenuous exercises require higher recovery periods; muscular action type, whose EC predominance needed greater recovery periods compared to concentric and isometric contractions; metabolic pathways and substrates used in the exercise, where the glycolytic metabolic pathways use demand longer recovery periods, and the training state or training level, where overtraining and unconditioned athletes require longer recovery periods (Bernardes et al., 2004; Bigland-Ritchie & Woods, 1976; Kreher & Schwartz, 2012; Morgan et al., 1987; Naughton et al., 2017). Moreover, the recovery type performed (active recovery

vs passive recovery), diet, supplementation use, gender and psychological condition also influence the recovery periods needed (Davies et al., 2018; Garcia et al., 2018; Pastre et al., 2009; Tomlin & Wenger, 2001; Wooller et al., 2018). As referred above, recovery is a major factor in the OTS prevention as well as an important key in the construction of appropriate individual training programs. Thus, there is a need to adopt strategies and ways to promote an adequate recovery (Cardoos, 2015; Kentta & Hassmen, 1998). The coaches and athletes must search for the use of measures to organize and to program adequate the training, through the change of the load parameters, and the balance the between load and recovery (Cardoos, 2015; Kentta & Hassmen, 1998). Further, it is crucial the adoption of a balanced diet which proportionate an adequate caloric intake to the demand (Garcia et al., 2018). On the other hand, it is important to use auxiliary ways, such as massages, Turkish bath, ultrasounds and supplements as L-carnitine, creatine and antioxidants (Goto & Morishima, 2014; Pastre et al., 2009).

2.5.5. Sport and Overtraining

Nowadays, the sports world presents a very close connection with the competition. The competition is easily observed in high-performance sports, at elite levels (Kentta & Hassmen, 1998). However, it becomes common to detect the competition in different contexts, such as young categories and recreative environments (Vieira et al., 2010; Walters et al., 2018). The willing to achieve the desired results enhances the development of risk behaviors, which can potentiate the increase of overtraining cases number. For example, approximately 60% of elite and 33% of recreational runners will can to present a nonfunctional overreaching state during their careers (Myrick, 2015). As already referred, OTS is induced by imbalance between workloads and recovery periods. Regarding to the high competition athletes, the overtraining cases are developed as a result of the high pressure and stress conditions by coaches and the own athletes in order to obtain the desired performances and results (Borin et al., 2007; Gustafsson et al., 2017). On the other hand, the young

athletes present a pressure increase in order to improve their performance and participate quickly in high-performance context. This increased emphasis in high- performance competition is associated with overuse injuries and overtraining cases enhancement (Brenner et al., 2016). In the recreative environment, it is also possible to find an excessive PE practice as a result of the exacerbated concern that subjects have with body image (Vieira et al., 2010). This PE practice is often performed without the accompaniment of qualified professionals, which increases the occurrence of mistakes in the training schedule as well as in the activities’ performance. These faults mostly happen due to the incorrect technical execution, and by the demanding workloads use concomitantly with inappropriate recovery periods (Vieira et al., 2010).

2.5.5.1. Explosive sports versus endurance sports

The ability to perform a maximal effort in repeated exercise sessions is influenced by the imposed work and recovery periods (Tomlin & Wenger, 2001). Greater homeostasis disturbances require longer recovery periods in order to avoid physiological problems and, consequently, future performance disturbances. Thus, strenuous sports such as weightlifting or the 100-meter race will require longer and full recovery periods in order to maintain performance levels in the following work sessions, without potentiate the OTS development (Tomlin & Wenger, 2001). Sports with mostly explosive actions are responsible for inducing greater disturbances in the organic homeostasis compared to sports with prolonged actions (Brehm & Gutin, 1986). According to literature, the training programs prescribed for these sports are generally characterized by low training volumes, with huge intensities, small number of repetitions per set, and a low to moderate number of sets per exercise (Azevedo et al., 2007; Stone & Coulter, 1994). Further, the repetitions can differ between 10 seconds (anaerobic power) and 120 seconds (anaerobic capacity) (Powers & Howley, 2005a). On the other hand, immediate recovery periods between 3 and 5 minutes are used in the exercise series recovery (Powers & Howley, 2005a). These sports present short

performance times and high intensities, needing large amounts of energy available during the effort. Thus, the organism uses metabolic pathways with high energetic power, namely the anaerobic metabolic pathways, as glycolysis, to suppress this need (Powers & Howley, 2005b). The anaerobic glycolysis increase during the sport performance causes a H+ and lactate concentration increment, and consequently, a pH decrease, which promote disturbances in the muscular contractile processes, affecting the sport performance (Tomlin & Wenger, 2001). According to Simão et al. (2008), after a high intensity exercise are required recovery periods between 3 and 5 minutes and between 4 and 10 minutes in order to observe an adenosine triphosphate (ATP) levels recovery and a lactate concentration decrease, respectively. Thus, the adoption of complete recovery periods, between 3 and 5 minutes, is essential to not compromise the performance (Powers & Howley, 2005a; Tomlin & Wenger, 2001). Contrary, the prolonged sports, between the 3 and the 12 minutes (aerobic power) and from the 12 minutes (aerobic capacity) present training methodologies usually related with the continuous load application, without recovery times, such as the duration method (Gueths & Flor, 2004; Powers & Howley, 2005a). However, the interval training, competitive and control methods are used too. The interval training method shows an incomplete recovery, usually active, while the competitive and control methods are based on the competitive activities’ performance (Gueths & Flor, 2004). As seen, the increased exercise time induces a muscle glycogen reserves decrease. The longer exercises cause a higher glycogen decrease, and consequently, a faster occurrence of asthenia (Ament & Verkerke, 2009). The explanation is since the use of the available glucose promotes a glycolysis speed decrease, which consequently causes a muscular pyruvate concentration reduction (Powers & Howley, 2005b). The pyruvate is a precursor of several intermediates of the Krebs cycle, such as oxaloacetate and malate, which in large quantity promote the speed increase of this cycle activity. On the other hand, the pyruvate production decrease causes a decline in the Krebs cycle intermediates production, which promotes a reduce in this cycle activity, resulting in a decrease of ATP aerobic production. The decrease of ATP production influences the

amount of ATP available to the exercise, causing asthenia and performance problems (Powers & Howley, 2005b). In summary, the prolonged sports are characterized by incomplete recovery periods, where it is hard to restore the needed substrates for normal performance (Gueths & Flor, 2004; Powers & Howley, 2005b). On the other hand, explosive sports exhibit complete recovery periods, evidencing an ability to maintain the energy levels during the training sessions (Powers & Howley, 2005a; Tomlin & Wenger, 2001). Thus, the prolonged sports are more susceptible to the OTS conditions development among athletes (Myrick, 2015).

CHAPTER 2

EXPERIMENTAL STUDY

1. EXPERIMENTAL STUDY

Chronic skeletal muscle damage induced by a demanding physical training is not a major physiopathological factor for overtraining development in Wistar rats

Francisco Leite a*, Antonio Bovolini a, Catarina C. Costa a, José Alberto Duarte a

a CIAFEL - Laboratory of Biochemistry and Experimental Morphology, Sports Faculty, University of Porto; Porto, Portugal.

*Corresponding author: Francisco LEITE, CIAFEL, Sports Faculty, University of Porto, R. Dr. Plácido da Costa 91, 4200-450, Porto, Portugal, [email protected]

Submitted to The Journal of Sports Medicine and Physical Fitness

Abstract

BACKGROUND: This study aimed to test the assumption that successive muscle damage accumulation due to the training overload is in the origin of the overtraining syndrome (OTS), using the soleus (SOL) and tibialis anterior (TA) muscles of Wistar rats exposed to a demanding exercise training protocol. METHODS: Animals were randomized distributed to a control group (CG, n=5) or to an exercised training group (EE, n=10), which performed a treadmill running training (-20º; from 25m/min, with progressive increase of 1.25m/min per day; 60 min) for 6 times/week being animals sacrificed 1 (EE1, n=5) and 3 weeks (EE3, n=5) after the beginning of the training program. Body weight, food intake, hair appearance, ability to perform work, and animals’ behavior were measured during the protocol. After sacrifice, SOL and TA muscles were collected for histological analysis. RESULTS: Both showed muscle damage signs in EE1 and EE3 through the increase of cell degeneration, tissue necrosis, and inflammatory activity. However, the exercise training protocol was not able to induce OTS. In parallel to the occurrence of muscle injury, adaptative signs in the exercised muscles were found, such as enhanced collagen content and cross-sectional area were also observed. CONCLUSIONS: the great amount of chronic muscular damage in SOL and TA is not associated with the OTS in the short and medium-term. Further, muscle damage demonstrates different behaviors according to the type of work that each muscle performs, questioning the use of systemic markers of muscle damage as reliable methods to study the relationship between skeletal muscle damage and OTS.

Key words: TRAINING OVERLOAD; MUSCLE INJURY; ECCENTRIC EXERCISE; SOLEUS MUSCLE; TIBIALIS ANTERIOR MUSCLE;

Introduction

Strenuous and unusual physical exercise (PE) performances are the origin of an anatomopathological condition named exercise myopathy (EM) (Duarte, 1993; Duarte et al., 2001). The EM is characterized by an amount of acute changes in skeletal muscle tissue with local and systemic repercussions (de Almeida et al., 2017). Specifically, strenuous and unusual PE, particularly when composed by eccentric contractions, induces short-term muscle damage such as striated muscle pattern disorganization or necrosis areas as well as increased inflammatory response (Duarte et al., 2001). However, between two to three weeks after exercise, it’s possible to observe the return to normal muscle tissue structure (Duarte, 1993). Continuous PE practice confers to skeletal muscle a greater adaptative capacity which causes a smaller muscle damage amplitude after PE practice sessions (Lira et al., 2013; Zanchi et al., 2010). Indeed, the training seems to provide adaptations in the myofibrillar structure and in other cellular compartments which, consequently, reduce the muscle damage and the consequent tissue inflammatory response (Friden et al., 1983). Competitive athletes are confronted daily with the limits of their physical abilities (Carfagno & Hendrix, 2014). Currently, these limitations appear in several contexts and are not exclusive of elite athletes (Vieira et al., 2010; Walters et al., 2018), once they are also commonly observed in young categories and in recreative environments (Vieira et al., 2010; Walters et al., 2018) specially when the training programs are characterized by high intensity loads and short rest periods (Vieira et al., 2010; Walters et al., 2018). Indeed, training programs that combine elevated demands, great amount of eccentric contractions, and reduced rest periods provide to athletes a greater risk of exceeding the limits of their adaptive abilities and, subsequently, evidencing progressively a lesser muscle adaptative ability to training stimuli (Kentta & Hassmen, 1998).

When the adaptive ability limit is exceeded, skeletal muscles becomes intolerant to exercise due to the chronic damage caused by repeated PE stress, originating a long-term muscle injury accumulation (Tiidus, 1998). It is believed that this successive muscle damage accumulation due to the training overload is the origin of a chronic condition designated by overtraining syndrome (OTS) (Ruzicic et al., 2016; Tiidus, 1998). The OTS is described by a decreased functional performance capacity and intolerance to exercise, accompanied by modifications in the nervous, endocrine, and immune systems functionality (Cardoos, 2015; Kreher, 2016). Of note that to support the association between muscle injury and the occurrence of OTS the literature just present indirect markers of muscle damage, such as the seric levels of creatine kinase (CK). However, seric CK response during muscular damage shows a huge interindividual variability, which reducing the sensitivity and specificity of this parameter as indicator of damage, makes the association of OTS and chronic muscle injury less reliable (Magal et al., 2010). On the other hand, there is no scientific direct evidences associating the chronic structural muscle damage induced by training overload with the occurrence of OTS. Therefore, the aim of this study was to verify in soleus (SOL) and tibialis anterior (TA) muscles of Wistar rats, the relationship between chronic muscle damage induced by a demanding exercise training and the occurrence of the overtraining syndrome. The exercise protocol was designed to promote a high level of acute and chronic muscle damage since it has combined a high running velocity performed with a negative slope and short rest periods between exercise bouts. Based on the widely belief, we hypothesize that the great amount of chronic muscle damage in the studied muscles is closely associated with OTS signs, in the short and medium term after the beginning of the training protocol.

Materials and methods

Animals and experimental design

Fifteen male Wistar rats from Charles River Laboratories (Barcelona, Spain) with 4-weeks old were used in this study. The animals were housed in collective cages (2 per cage) with a temperature and humidity-controlled environment (21ºC-22ºC and 50%-60%, respectively) and 12h light/dark inverted cycle. The animals had food and water access ad libitum (standard laboratory diet 4RF21®, Mucedola, Italy). After 1-week of acclimatization, the animals were randomly distributed in 2 groups: control group (CG, n=5, body weight 161.8±7.05) and exercise training group (EE group, n=10, body weight 164.7±16.82). The animals of this group were sacrificed in different subgroups, at sort-term, in the end of first week (EE1 subgroup, n=5, body weight 158.8±16.32) and at medium-term, in the end of third week (EE3 subgroup, n=5, body weight 170.6±16.83). The exercised rats had one-week adaptation to the treadmill (10 minute/day without slope and a low intensity), and then, both experimental groups were submitted to a treadmill training protocol (Treadmill Control LE 8710, Harvard Apparatus, USA) with a 20º negative slope. The experimental protocol consisted in a progressive increase of intensity running ranging from 25 to 48 meters/minute for 1 hour/day, 6 days per week throughout 1-weeks and 3-weeks, respectively. The running protocol starts with an intensity of 25 meters/minute which intensity increased 1.25 meters/minute in each training session, reaching at the end of first and third weeks, a maximal intensity of 32.5 meters/minute and 48 meters/minute, respectively. The animals of CG remained limited to cage’s space during all the study. Animals weight and amount of food consumption were daily assessed before each training session.

Criteria to assess overtraining

In humans it is possible to identify signs of overtraining not only by the decrease of the sport performance but also by behavioral changes, such as the increase of the irritability, the aggressiveness and the anxiety or the lack of appetite (Johnson & Thiese, 1992). However, in animals there is no set of validated parameters that allow us to evaluate. Thus, in this study, in order to evaluate and quantify the occurrence of OTS in the exercised groups were adapted and used some parameters: i) changes in the body weight (BW), ii) evolution of food intake (FI), iii) the animals´ behavior, iv) their hair appearance, v) signs of nose bleed, and vi) the animals’ ability to perform and tolerate the imposed running demand. Namely, it was observed whether the experimental group showed decreases in weight and food intake as well as abnormal behavior, characterized by a passive attitude in the periphery of an open field, without exploratory appetence (Lezak et al., 2017). Moreover, it was noted if the animals had bristly hairs and signs of nose bleed (Council, 2008; Rivera, 2006). As a functional criterion, we observed the rats’ ability to perform the physical effort imposed in each training session (Hohl et al., 2009).

Animals sacrifice and samples collection

One day after the end of the experimental protocol as described before, the animals were weighed and anesthetized with Ketamine (90 mg/kg, Merial, France), Xylazine (10mg/kg, Bayer, German) and euthanized by exsanguination. After animals’ weight assessment, the soleus and tibialis anterior were harvested, washed in PBS (pH 7.2), weighed in a precision balance (resolution 0.01mg; Kern 870, Balingen, Germany), and processed immediately for light microscopy. After SOL and TA weights mensuration (SW and TAW, respectively), relative Soleus and tibialis anterior weights (rSW and rTAW, respectively) were calculated by the respective ratio for BW and expressed as percentage (%).

Histological analysis

After soleus and tibialis anterior excision, the muscles were 24h fixed in 4% paraformaldehyde solution at 4ºC, dehydrated through graded ethanol solutions, cleared in xylene and mounted in paraffin. 5µm thick sections from both muscles were cut by microtome (Leica, RM 2125) and used for assessing fibrous tissue, cross-sectional area, muscle fibers’ nucleus position and rates of muscular damage. The sections were analyzed with a light microscope (Axior Imager A1, Carl Zeiss; Germany) and images recorded with a coupled digital camera (Leica DM4000B, Nussloch, Germany).

Total collagen content analysis

To fibrous tissue accumulation assessment, SOL and TA sections were stained with Picrosirius Red according to the method of Sweat et al. (1964) by incubation on 0.1% Sirius red picric acid for 90 minutes. Them, sections were rinsed in 0.5% acetic acid, dehydrated in ethanol and cleared in xylene. The collagen and the remained muscular tissue stains in red light and yellow, respectively. The software Image-Pro Plus 6.0 (Media Cybernetics, Inc) was used to quantify the percentage of area (µm2) covered by the colors corresponding to the respective tissues. For each muscle, 6 photos per animal were analyzed.

Cross-sectional area and muscle fibers nucleus position analysis

SOL and TA sections were stained with hematoxylin-eosin (H&E). The H&E protocol consisted of immersing the sections in Mayer's hematoxylin solution for 5 min, followed by its immersion in 1% eosin solution for 10 minutes, alcoholic dehydration and immersion in xylene for 5 min succeeded by posterior laminar assembly with DPX (dibutyl xylene phthalate; Shandon EZ-Mount, Thermo Electron Corporation, USA). Then the sections were photographed by an optical microscope (Axio Imager A1; Carl Zeiss, Oberkochen, Germany) coupled

to a digital camera (×400, LEICA DM4000B; Nussloch, Germany). The NIH ImageJ software (Image Processing and Analysis in Java, USA) was utilized to analyze muscles fibers cross sectional area (CSA) and nuclei position (NP). In both analyses 6 photos per animal were used. To analyze nuclei position, all the fibers contained in the picture were analyzed. The number of central nuclei per analyzed area was expressed as a percentage of total fibers analyzed. A total of 5920 muscle fibers from SOL were analyzed, specifically 2488 fibers from CG, 2217 fibers from EE1 subgroup and 1215 fibers from EE3 subgroup. Meanwhile, a total of 8324 muscle fibers from TA analyzed were 8324, namely 3154 fibers from CG, 3108 fibers from EE1 subgroup and 2062 fibers from EE3 subgroup.

Muscle damage evaluation

Muscular damage evidences were measured according to a semi- quantitative procedure (da Silva Teixeira, 2017; Dinis-Oliveira et al., 2007), adapted to the skeletal muscle tissue. Randomly photos stained with H&E were evaluated in accordance with 4 parameters: 1) the inflammatory activity (IA); 2) the necrosis extent (N); 3) the tissue organization (TO) and 4) the cellular degeneration (CD). The inflammatory activity (i.e., interstitial and inner leucocytes) was scored as follows: grade 0 = no cellular infiltration; grade 1 = mild leukocyte infiltration (1 to 3 cells per image); grade 2 = moderate infiltration (4 to 6 leukocytes per visual field); grade 3 = heavy infiltration by leucocytes. The severity of necrosis extent (i.e., necrotic and eosinophilic muscular fibers) was scored as: grade 0 = no necrosis; grade 1 = dispersed necrotic foci; grade 2 = confluent necrotic areas; grade 3 = massive necrosis. Tissue disorganization was scored as: score 0 = normal structure; score 1 = less than one-third of the image; score 2 = greater than one-third and less than two-thirds of the image; score 3 = greater than two-thirds of the image. The cellular degeneration (i.e., fibers dilatation, sarcoplasm vacuolization and density) was scored as per percentage of the affected tissue: score 0 = no change from normal; score 1 = a limited number of isolated cells (up to 5% of each total cell number); score 2 = groups of

cells (5 to 30% of total cell number); score 3 = diffuse cell damage (more than 30% of total cell number). After, all the previous parameters were summed to produce a total muscle damage (TD) score. For each muscle, at least 10 microscopic visual field were analyzed using an objective of 40x.

Statistical analysis

The statistical analysis was performed using GraphPad Prism® (version 8.00, GraphPad Software, San Diego, California, USA). The Kolmogorov- Smirnov test was performed to investigate the data normality in variables with n>50 from SOL and TA cross-sectional area (SOLCSA and TACSA, respectively) and SOL muscle damage (SOLMD), while the Shapiro-Wilk test was performed to investigate the data normality when n<50 from BW, SW, TAW, rSW, rTAW, SOL and TA collagen content (SOLCC and TACC, respectively), SOL and TA nucleus position (SOLNP and TANP, respectively), TA muscle damage (TAMD) and the FI. The one-way ANOVA followed by the Tukey post hoc comparison test was used to analyze data with normal distribution (BW, SW, SOLCC, TACC and FI). The data with not normal distribution were then analyzed with Kruskal-Wallis followed by the Dunn´s post hoc comparison test (rSW, TAW, rATW, SOLCSA, TACSA, SOLANP, TANP, SOLMD and TAMD). Differences were considered significant at p˂0.001, and the obtained data were expressed as a mean ± standard deviation for normal distributed data or as median with percentiles 25 and 75 (P25-P75) for abnormal distributed data.

Results

Overtraining outcomes

The results allusive to body weight and food intake evolution are expressed in Figure 1. In both weeks were demonstrated significative differences in BW. The EE1 subgroup presented a significant BW decrease when compared to CG. Further, the EE3 subgroup exhibited a significant BW increase in comparison to EE1 subgroup. Regarding to the FI, although EE3 subgroup (24.4±3.37) presented a higher mean comparatively to CG (23.9±2.91) and EE1 subgroup (21.2±2.51), no significant differences were detected from the comparison among them. Moreover, no significant differences were observed in the animal behavior and the state of the hair as well as in the ability to perform the imposed demand. Although the animals showed a BW decrease in the first week, this was recovered throughout the experiment. Thus, from the evaluation criteria analysis, we can observe that the exercised subgroups didn’t show overtraining signs.

Figure 1. The mean value with standard deviation of body weight and the food intake for the CG and exercised group at short and medium-term are depicted in A and B, respectively.

* p<0.001 vs. CG; # p<0.001 vs. EE1.

Muscles outcomes

As we can observe in Table I, no significant differences were presented by the absolute and relative weight relative weight from SOL and TA muscles.

Table I. Muscles weights

SW rSW TAW rTAW

CG 0.118±0.019 0.040 (0.035-0.050) 0.470 (0.450-0.510) 0.180 (0.160-0.185)

EE1 0.094±0.036 0.040 (0.035-0.060) 0.340 (0.140-0.420) 0.170 (0.070-0.195)

EE3 0.118±0.025 0.040 (0.040-0.045) 0.520 (0.520-0.640) 0.190 (0.190-0.200) The results are presented as mean ± standard deviation or median (P25-P75) of control group (CG), 1-week eccentric exercise (EE1) and 3-weeks eccentric exercise (EE3) subgroups. Soleus weight (SW); Relative soleus weight (rSW); Tibialis anterior weight (TAW); Relative tibialis anterior weight (rTAW). * p<0.001 vs. CG; # p<0.001 vs. EE1.

Cross-sectional area outcomes

Soleus CSA results are labelled in Figure 2. The EE3 subgroup presented the largest CSA among all groups. Specifically, the EE3 subgroup presented a pronounced and significant increase in CSA compared to CG and EE1 subgroup. Further, the EE1 subgroup also demonstrated a significant increase in CSA comparatively to CG.

stained area for the CG and each exercised subgroup, indicative of tissue cross-sectional area, are shown in B

* p<0.001 vs. CG; # p<0.001 vs. EE1.

The results regarding TA CSA are shown in Figure 3. EE3 subgroup exhibited a significantly increase of TA CSA compared to CG and EE1 subgroup. In contrast, no statistical differences were found in TA CSA between EE1 subgroup and CG.

Figure 3. Representative light micrographs of Tibialis Anterior muscles from control group (CG), 1- week eccentric exercise subgroup (EE1), and 3-weeks eccentric exercise subgroup (EE3), stained with Hematoxylin & Eosin, are depicted in A. The boxplot graphic presents the median value with 25 and 75 percentiles of red stained area for the CG and each exercised subgroup, indicative of tissue cross-sectional area, are shown in B

* p<0.001 vs. CG; # p<0.001 vs. EE1.

Muscle fibers nucleus position outcomes

As can be seen in Figure 4, the EE3 subgroup shown a significant increase of muscle fibers with central nuclei position in comparison to EE1 subgroup. However, EE3 and EE1 subgroups didn’t exhibit statistical differences comparatively to CG.

# p<0.001 vs. EE1.

Although EE3 subgroup presented a median of 0.0% (0.00%-2.07%) comparatively to 0.7% (0.00%-1.33%) from CG and 0.7% (0.00%-1.22%) from EE1 subgroup, no significant differences were detected from the comparison among groups in the TA nuclei position.

Muscular damage outcomes

Through Table II, it’s possible to note that there were significant differences in some muscular damage parameters of the SOL muscle. Namely, the EE1 and EE3 subgroups presented a significant increase in IA, N, CD and TD comparatively to CG. Contrary, EE3 subgroup exhibited a significant IA, N and TD decrease when compared to EE1 subgroup. No statistical differences were found among groups in TO parameter.

Table II. Muscular damage in soleus muscle

Inflammatory Tissue Cellular Total Necrosis activity organization degeneration damage

CG 0 (0-0) 0 (0-0) 0 (0-0) 0 (0-0.5) 0 (0-0.5)

EE1 2 (1-2) * 1 (1-2) * 0 (0-0) 1 (1-2) * 4 (3-6) *

EE3 1 (1-1) * # 0 (0-1) * # 0 (0-0) 1 (1-2) * 3 (2-4) * #

The results are presented as median (P25-P75) of control group (CG), 1-week eccentric exercise (EE1) and 3-weeks eccentric exercise (EE3) subgroups. * p<0.001 vs. CG; # p<0.001 vs. EE1.

As we noted in Table III, EE3 subgroup demonstrated significant IA, N and TD increases comparatively to CG and EE1 subgroup. Further, EE1 subgroup shown bigger CD and TD scores than CG. Finally, EE3 subgroup also exhibited a significant CD increase in comparison to CG. No statistical differences were found among groups in TO parameter.

Table III. Muscular damage in tibialis anterior muscle

Inflammatory Tissue Cellular Total Necrosis activity organization degeneration damage

CG 0 (0-0) 0 (0-0) 0 (0-0) 0 (0-1) 0 (0-1)

EE1 0 (0-0) 0 (0-0) 0 (0-0) 1 (0-1) * 1 (1-2) *

EE3 1 (0-1) * # 1 (1-1) * # 0 (0-0) 1 (1-1) * 3 (2-4) * #

The results are presented as median and percentiles (P25-P75) of control group (CG), 1-week eccentric exercise (EE1) and 3-weeks eccentric exercise (EE3) subgroups. * p<0.001 vs. CG; # p<0.001 vs. EE1.

Collagen outcomes

According Figure 5, there are significant collagen content differences in exercised groups. The EE1 subgroup and EE3 subgroup shown a significant increase in SOL collagen content comparatively to CG. Also, no significant difference was observed between groups EE1 and EE3 subgroups, it’s important to note a trend towards a collagen content decrease in animals with 3-weeks of eccentric exercise.

Figure 5. Representative light micrographs of Soleus muscles from control group (CG), 1-week eccentric exercise subgroup (EE1), and 3-weeks eccentric exercise subgroup (EE3), stained with Sirius red, are depicted in A. The mean value with standard deviation of red stained area for the CG and each exercised subgroup, indicative of tissue collagen content, are shown in B

* p<0.001 vs. CG; # p<0.001 vs. EE1.

Based on analysis of Figure 6, it’s possible to observe no statistical differences in both exercise groups related to collagen area. Although EE1 subgroup presented an increase in TA collagen content in comparison to CG, this increase is not significant. In turn, despite EE3 subgroup demonstrated a collagen deposition decrease when compared to the EE1 subgroup, no significant differences were noted.

Figure 6. Representative light micrographs of Tibialis Anterior muscles from control group (CG), 1- week eccentric exercise subgroup (EE1), and 3-weeks eccentric exercise subgroup (EE3), stained with Sirius red, are depicted in A. The mean value with standard deviation of red stained area for the CG and each exercised subgroup, indicative of tissue collagen content, are shown in B

Discussion

The obtained results do not support our working hypothesis since any signal of overtraining was found although the effectiveness of PE protocol to induce early and mid-term structural alterations either in SOL and TA muscles. Even though the training protocol presented all the characteristics to induce an overtraining syndrome, such as a large percentage of eccentric contractions, a high running speed and short periods of rest between exercises, the exercised group showed no signs of OTS. Of note that the adoption of a protocol with negative slope in our study is justified by the greater eccentric work performed during the running, causing a greater mechanical effort due to the distribution of the mechanical stress by a smaller number of muscular fibers and, consequently, having a greater injurious role (Douglas et al., 2017; Hyldahl & Hubal, 2014). Indeed, at the end of the first week, a significant decrease in BW was observed, suggesting a training protocol negative impact, however this decrease was not accompanied by any alteration in the other OTS criteria evaluated. This initial weight loss in the exercised group could be explained by increased energy expenditure and proteolysis under persistent workloads (Pereira et al., 2016) since no diet uptake changes were registered. Furthermore, at the end of the third week, a total BW recovery was reported to similar levels of the control group, along with maintenance of the remaining parameters of OTS at normal levels. Indeed, despite the high mechanical and metabolic demands imposed, the animals were able to maintain motor performance throughout the experiment, showing no sign of nasal bleeding or anomaly in their normal behavior, nor manifesting significant changes in the food intake evolution or in their hair appearance. Although the existence of a negative impact on body weight at the first week, these results support that the imposed training protocol did not induce signs of OTS in these three weeks. Although the training program implemented did not provoke signs of OTS, it was able to induce severe muscle damage in both muscles at the end of the 1st and 3rd week, confirming evidence already presented in literature using similar

methodologies (Pereira et al., 2015; Silva et al., 2013). However, the studied muscles behavior to exercise-induced muscle damage was different. It must be highlighted that the selection of the evaluated muscles in our experimental design was associated with the type of action they perform in the running protocol. The results showed that SOL muscle presented a greater damage than TA muscle throughout the study, visible by the amount of cell degeneration and tissue necrosis, accompanied by the elevation of inflammatory response. Nevertheless, at the end of the third week, though the muscle damage rates remained high in SOL, there were signs of injury reduction when compared to the first week. On the other hand, despite a lower lesion score than the SOL muscle, the TA muscle showed worsening signs of muscle damage at the end of the experiment, observable through the levels of cell degeneration and necrosis associated with an intensification of inflammatory process. While the TA muscle showed a continuous increase of muscle damage throughout the study, the SOL muscle expressed at the end of the 3rd week a slight decrease of this damage compared to the 1st week. These differences observed in the muscle damage responses to the training protocol in the studied muscles can be explained by the different types of muscle contraction performed during exercise as well as by the different muscle fibers type composition (Gonzalez‐Izal et al., 2014; Qaisar et al., 2016). Effectively, during the running realized in negative slope there is a greater eccentric work in the active muscles comparatively to the horizontal slope (Alkahtani et al., 2019). Nevertheless, comparing the two muscles in the negative slope, the SOL performed a higher eccentric work than the TA, which justifies its greater damage (Giandolini et al., 2017). Besides that, the SOL muscle is more trained and prepared for this type of eccentric work than the TA muscle as a result of his daily action, which may explain the differences in the responses of both muscles over time (Hessel & Nishikawa, 2017). Additionally, the fact that TA muscle has a predominance of fast-twitch fibers compared to the SOL muscle may explain the progressive worsening of muscle damage signs obtained in this muscle, because these type of fibers appear to be more susceptible to problems

in excitation-contraction coupling than slow-twitch fibers, promoting a greater muscle damage degree (Qaisar et al., 2016). On the other hand, the results demonstrate that, despite the high demand of the training protocol, both muscles presented phenotypic signs of favorable adaptations to training load, which may contribute to explain the absence of OTS signs. Indeed, the SOL muscle showed signs of tissue repair, namely not only by the healing process, observed by the increase in collagen content, but also trough the regenerative process characterized by an increased number of centralized nuclei in the muscle fibers. The increased collagen content is important to change the mechanical properties of the skeletal muscle, improving the mechanical forces distribution and decreasing the susceptibility to mechanical stress and damage (Takagi et al., 2016). Further, during the muscle regenerative process following injury, the satellite cells activation with consequent fusion to the existing fibers promotes an increased number of centralized nuclei, which subsequently explains the significant increase in the cross-sectional area that the SOL muscle exhibited throughout the study (Liu et al., 2013; Song et al., 2019). The increased cross-sectional area facilitates the dissipation and distribution of the mechanical stress exerted, improving the tolerance of muscular fibers to mechanic stress overload (Hedayatpour & Falla, 2015). In contrast, despite the continuous increase of muscle damage in the TA muscle, it is also possible to observe signs of adaptation, through the increase of the cross-sectional area at the end of the 3rd week compared to the 1st week. Although our study aims to verify the relationship between chronic muscle damage induced by demanding exercise training and overtraining syndrome in SOL and TA of Wistar rats, the duration of the protocol as well as the lack of biochemical and functional evaluations may be considered limitations of our study. Indeed, the duration of the protocol was determined in order to observe the evolution of muscle damage and the OTS signs in the short-term (1st week) and medium-term (3rd weeks) after the beginning of the training protocol. Nevertheless, although the absence of OTS signs during this period of time, the experimental protocol does not guarantee the absence of OTS with a prolonged period of physical training.

Conclusions

It was expected that both muscles did not show tolerance to the continuous increase of the training demand, however despite the occurrence of muscle damage, no OTS signs were observed in the first three weeks. Contrary to what is believed, the results found in this study do not support the concept that the great amount of chronic muscular damage is associated with the overtraining syndrome signs in the short and medium-term. Furthermore, the results support the idea that in the absence of OTS signals, the muscle damage seems to occur simultaneously with the increase of adaptive signals. Finally, our results showed different muscle damage behaviors according to the type of work that each muscle performs, questioning the use of systemic markers of muscle damage as reliable methods to study the relationship between skeletal muscle damage and OTS.

References

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NOTES

Conflicts of interest. - The authors declare that they have no conflict of interest. Funding. - This study was financed by the Fundação para a Ciência e Tecnologia (UID/DTP/00617/2019). Authors’ contributions. – Francisco LEITE: work conception, laboratory work, data collection and processing, and article writing. Antonio BOVOLINI: laboratory work and article writing. Catarina C. COSTA: laboratory work article writing. José A. DUARTE: work conception and article writing. Acknowledgements. - The authors would like to thank Mrs. Celeste Resende for technical assistance in sample preparation for histological analysis.

CHAPTER 3

CONCLUSIONS

1. CONCLUSIONS

Based on the proposed objectives as well as on the results obtained it is possible to conclude the following points regarding muscle damage and overtraining syndrome:

• The overtraining syndrome affects subjects in diverse contexts, such as recreational or in young ages, and is not limited to high-level athletes. • Overtraining syndrome is a condition that does not exclusively affect the muscular system, but also other systems such as nervous, endocrine and immune, presenting a diversity of symptoms. • Correct training planning is essential. The training programs should consider the balance in the periodization of training loads and the rest and recovery periods, in order to prevent overtraining. • The continuity of physical exercise practice, disregarding the recovery periods, compromises the capacity for homeostatic recovery and the acquisition of a beneficial adaptive phenotype, promoting the overtraining syndrome development. • Despite the continuous demand increase, a large amount of chronic muscle damage is not associated with overtraining syndrome in the short and medium term. • In the absence of OTS signals, muscle damage appears to occur simultaneously with the increase of adaptive signals. • Muscle damage demonstrates different behaviors depending on the type of work that the muscle performs. • The measurement of systemic signs of muscle damage is not a reliable method for relating muscle damage and OTS.

The study does not support the hypothesis initially presented that muscle damage induced by a demanding exercise program with negative slope promotes the development of short- and medium-term overtraining syndrome. In future studies, it is suggested to increase the duration of the protocol to induce overtraining syndrome, as well as the inclusion of biochemical and

functional evaluations in order to confer greater robustness to the evaluated parameters.

CHAPTER 4

REFERENCES

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