Skeletal muscle biochemical and molecular changes

review article

Skeletal muscle biochemical and molecular changes due to aging Alterações bioquímicas e moleculares do músculo esquelético devidas ao envelhecimento

Caroline Pieta Dias1, Bruno Manfredini Baroni1, Marco Aurélio Vaz1

Recebido em 15/10/2010 Aceito em 1/2/2011

RESUMO O envelhecimento é um processo naturalmente acompanhado por decréscimos significativos da quantidade e da qualidade do tecido muscular esquelético. Essa perda de massa muscular, também conhecida por sarcopenia, é uma das principais responsáveis pela redução da capacidade musculoesquelética dos idosos, ocasionando perda funcional e consequente redução da qualidade de vida. Embora as causas da sarcopenia ainda não estejam completamente elucidadas pela literatura, a redução da sobrecarga mecânica sofrida pela musculatura devida à redução de uso observada com o envelhecimento pode ser citada como um dos principais agentes causadores. Além disso, evidências sugerem que o envelhecimento é acompanhado por uma série de alterações em fatores fisiológicos relacionados ao ganho ou perda de tecido muscular. Assim, o presente estudo de revisão objetiva sintetizar o comportamento dos principais fatores bioquímicos e moleculares atuantes na degeneração muscular associada ao envelhecimento: (1) Enzimas musculares; (2) fatores regulatórios de crescimento e (3) moléculas de sinalização e adesão celular. Palavras-chave: Envelhecimento, músculo esquelético, sarcopenia.

ABSTRACT Aging is a naturally accompanied process by significant decreases of quantity and quality of skeletal muscle tissue. This muscle mass loss, also called sarcopenia, is one of the major responsible for musculoskeletal capacity reduction in elderly, causing a functional loss and consequent reduction of life quality. Although the causes of sarcopenia are not fully understood in the literature, the reduction of mechanic overload suffered by the musculature due to use reduced observed with aging can be cited as one of the main causative agents. Moreover, evidences suggest that aging is accompanied by a lot of changes in physiologic factors related to gain or loss of muscle tissue. Therefore, the present review study aims to synthesize the behavior of the main biochemical and molecular factors acting on the muscle degeneration associated with aging: (1) Muscle enzymes; (2) regulatory growth factors; and (3) signaling and adhesion cellular molecules. Keywords: Aging, skeletal muscle, sarcopenia.

1 Exercise Research Laboratory, School of Physical Education, Federal University of Rio Grande do Sul Sul (UFRGS), Porto Alegre, RS, Brazil.

Correspondence address to: Caroline Pieta Dias • Federal University of Rio Grande do Sul, School of Physical Education, Exercise Research Laboratory • Street Felizardo, 750 – 90690-200 – Porto Alegre, RS, Brazil • Phone: (+55 51) 3308-5859 • FAX: (+55 51) 3308-5858 • E-mail: [email protected]

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Geriatria & Gerontologia. 2010;4(4):229-237

INTRODUCTION

gration of stimulators, regulation of growth factors, and protein transcription and translation11,12.

It is well known that the muscle mass decrease (or sarcopenia) is a normal phenomena associated with aging1,2. Sarcopenia affects more than 50% of population above 80 years of age3, it seems to be more pronounced in men than women and occurs predominantly in lower limb muscle groups4-6. The muscle mass reduction with aging is related to both a loss of sarcomeres organized in parallel (i.e. a reduction of fiber diameter due to loss in the number of myofibrils) and in series (i.e. a reduction in fiber length). These changes have been indirectly documented in humans as a reduction in muscle cross-sectional area, muscle thickness, fascicle length and pennation angle fibers in muscle architecture studies7. Added to the predominant atrophy of type II fibers (fast-twitch)1, these morphologicaly adaptations affect muscle function, changing the force-length and force-velocity relationships. Since these adaptations lead to a decrease in muscle force and velocity, sarcopenia is also responsible for the reduction in mobility and for the increased functional incapacity and dependence in the elderly1,2. Therefore, great attention has been given to the socio-economic impact of elderly health care in developed countries8.

In vivo studies suggest that muscle atrophy results from both reduction in protein synthesis and increased muscle degradation. Studies have demonstrated that the atrophy is caused by changes in the levels of RNAm of proteolysis specific regulators, protein synthesis and energy metabolism13-15. Mechanotransduction and mechanoquimiotransduction are two processes directly related to this phenomenon that have been recently described. The first relates to the energy conversion of mechanical energy into biochemical reactions, involving the stimulation of mechanoreceptors located in the cellular membrane and other structures of the extracellular matrix (ECM), such as the integrins and the cytoskeleton complex16. The second characterizes the transmission of mechanical signals of the EMC to the cytoskeleton through sarcoplasmatic structures (integrins, dystroglicans and calcium channels) followed by the induction of biochemical signals, which regulate protein synthesis14.

The loss of muscle mass quantification has been made mainly through the evaluation of imaging analysis methods (such as ultrasound, computed tomography, magnetic resonance imaging and dual-energy X-ray absorptiometry), measurements of total body potassium or creatinine excretion, and/or histochemical analysis of muscle fibres in either biopsy or autopsy specimens2. Despite of that it is well established that humans suffer significant reduction in the amount of muscle mass, and in the quality of muscle tissue (i.e. capacity to generate force per unit of muscle area)1. Although the factors leading to sarcopenia are not fully understood, evidences haves suggested that creatine kinase, protein kinase 5 AMP activated, serine/ threonine kinase AKT, mammalian target of rapamycin, insulin growth factor, mechanical growth factor, myogenic regulatory factors, inflammatory cytocins and signaling and adhesion cellular molecules are factors directly related to the loss of muscle mass with aging1,9,10. Changes in muscle mass are usually attributed to changes in the relation between protein synthesis and degradation. Therefore, the loss of muscle mass can be directly attributed to a decrease in protein regulation. It has been postulated that several factors are involved in this process, such as the inte-

Both routes directly or indirectly lead to the signaling of specific enzymes: fosfaditil-inositol-3 kinase (P13K), protein kinase B, the mammalian target of rapamicyn (mTOR) and and p70 S6 Kinase (P70S6k). These enzymes, in turn, lead to a reduction in the activation of targets required for protein synthesis. Knowledge about these mechanisms becomes important in order to explain the involvement of specific proteins and regulatory factors in the biochemical routes of muscle atrophy. Since the literature presents few studies regarding the molecular mechanisms of muscle atrophy with aging10,14,15,17-20 and the necessity of a better understanding of skeletal muscle biochemical and molecular changes with aging, the aim of the present study was to review the main biochemistry markers that are used to indicate skeletal muscle degeneration and the main proteins associated with mechanotransduction. This review was organized in the following topics: (1) Muscle enzymes; (2) Regulatory growth factors; and (3) Signaling and adhesion cellular molecules.

MUSCLE ENZYMES Creatine kinase (CK) The serum level of skeletal muscle enzymes is a marker of the muscle tissue functional status and varies widely in both pathological and physiological conditions. Creatine kinase (CK) is a dimeric globular protein consisting of two subunits (B and M) with a molecu-

Skeletal muscle biochemical and molecular

lar mass of 43 kDa. At least five isoforms of CK exist: three isoenzymes in cytoplasm (CK-BB, CK-MB and CK-MM) and two isoenzymes (non-sarcomeric and sarcomeric) in mitochondria21. CK-BB or CK-1 is predominantly in the brain, while CK-MB or CK-2 (a hybrid form) is predominantly in the myocardium and CK-MM is specifically bound to the myofibrillar M-Line structure sarcomere21-24. The function of CK in the skeletal muscle tissue is to help in adenosine tryphosphate (ATP) re-synthesis, which is the first energetic route and also the simplest way for ATP phosphorylation25. The presence of MM-CK in skeletal muscle myofibrils suggests that the sarcomere M-line structure has a functional role for regenerating ATP, thus offering myosin with sufficient ATP to work under strenuous conditions21. In normal serum, total CK is provided mainly by the skeletal muscle and is almost entirely of the MM type. Total CK levels depend on age, gender, race, muscle mass, physical activity and climatic condition. There are marked sex differences in CK serum levels at rest and after exercise, with lower values in females than in males. In addition, resting levels are higher in athletes compared to sedentary subjects, and black people appear to have higher concentrations compared to caucasians21,26. Young adults have high serum levels of CK, which decline slightly with age during the geriatric period. Besides being a marker of aging muscle, the monitoring of CK and characterization of its isoenzymes are widely used in the diagnosis of myopathies, cardiomyopathies and encephalopathies. Elevated CK has also been described in various neuromuscular conditions as a result of muscle damage and in many muscular dystrophies. In normal conditions, CK-MM is a cytoplasmatic molecule and does not cross the sarcolemma. However, when this membrane is ruptured, the serum levels of CK increase, and it is frequently described as one of the best indirect markers of muscle tissue damage27-29. Protein kinase 5´-AMP-activated (AMPK) The protein kinase 5´-activated AMP (AMPK) is a heterotrimetric molecule that contains a catalytic subunit (α), with two isoforms (α1 and α2) and two regulatory subunits (β and γ)30. AMPK and its functional kinase activity were initially identified in liver. After mRNA and protein expression analysis, it was found that AMPK had a role in skeletal muscle, because its mRNA expression was seven-fold higher than that in liver31.

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When AMPK is phosphorylated, it activates paths responsible for ATP increase, which has an important metabolic role in muscle metabolism, controlling both glucose consumption and fatty acids32. Different experiments point out that both in situ and in vitro electrically stimulated contractions in rats and voluntary contractions during exercise in humans increase significantly the activity of the AMPK33-35. It has been suggested that the activation of AMPK can be involved in the mediation of skeletal muscle biochemical adaptations36. It is well known that ageing is accompanied by a loss of muscle mass mainly in fast-twitch fibres, which causes muscle strength and power decrease1. Thomson and Gordon19 examined the effect of compensatory overload-induced hypertrophy in plantaris and soleus muscles of young adult and old rats, and showed that AMPK phosphorylation is increased with age and is correlated with overload-induced hypertrophy in the fast-twitch plantaris muscle but not in the slow-twitch soleus muscles. This supports the idea that elevated AMPK phosphorylation may contribute to the decreased hypertrophy observed in aged fast-twitch skeletal muscle. The AMPK hyper phosphorylation may be a strong mechanism by which translational signaling and overload-induced growth is decreased in aged fast-twitch muscles19,20. Furthermore, there was a strong negative correlation between the quantity of AMPK phosphorylation and the quantity of hypertrophy in the overloaded muscles, implicating AMPK as an important negative regulator of overload-induced skeletal muscle hypertrophy20.

REGULATORY GROWTH FACTORS Serine/threonine kinase AKT or protein kinase B (PKB) The serine/threonine kinase AKT (also known as protein kinase B, PKB) is activated by innumerable growth factors and immune receptor through phosphatidylinositol (PI) 3-kinase lipidic products. The AKT can connect pathways that regulate the glucose metabolism or cellular survival. AKT is activated by the PI 3-kinase37,38, and has an important role in gene expression regulation and in cellular growth in response to several types of stimulation39. AKT can be activated as a result of IGF-I stimulation causing activation of phosphatidyl-inositol-3 kinase (PI3K) to phosphorylate AKT. When AKT is

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phosphorylated it acts in different ways that influence protein synthesis, cell survival and cell proliferation9. AKT activation is also know to occur by other stimulation types, such as growth factors, citocins and hormones, which are also dependent of phosphatidyl-inositol-3 kinase (PI3K) and act on muscle cells. Evidences suggest that a reduction of AKT expression in an animal model decrease muscle growth39, whereas mice with AKT over-expression presented muscle hypertrophy40. Mammalian target of tapamycin (AKT/ mTOR) AKT/mTOR (mammalian target of rapamycin) has been investigated in several in vivo models of skeletal muscle hypertrophy and atrophy. The main activator of the protein kinase B/AKT and mTOR is IGF1 (Insulin-like growth factor), which marks the mechanical growth factor (MGF). When IGF-I is binding to a specific receptor, it activates the phosphatidyl-inositol-3 kinase (PI3K) to phosphorylate AKT, which acts on protein synthesis. Since phosphorylation of mTOR by AKT occurs, the p70S6k (70 kDa ribosomal protein S6 kinase) activation is possible. This event is important for the promotion of muscle growth because p70S6k stimulates protein synthesis9. The AKT/mTOR pathway is high-expressed during hypertrophy and low-expressed during muscle atrophy41. The genetic activation of AKT/mTOR regulation was enough to cause hypertrophy and to prevent atrophy in vivo, but the genetic blockade hinders hypertrophy in vivo. However, the activation of AKT/mTOR pathway, with its low concentration targets (e.g. protein p70S6K) is involved in the regulation of the skeletal muscle fibres size, which may induce muscular atrophy due to disuse observed with aging42. The importance of the AKT/mTOR/p70S6k pathway is the fostering of muscle growth. In addition, evidences showed that AKT phosphorylation and expression are increased on muscle hypertrophy and decreased during atrophy9. Insulin growth factors (IGFs) The IGFs are factors of growth promotion with homologous molecular structure to the insulin, found in the form of IGF-1 and IGF-243. These factors are synthesized by the liver and by the majority of the cells in response to the activation promoted by growth hormone (GH) or GH-independent form. The IGFs can influence growth, differentiation and cellu-

lar metabolism and they are connected to carrying proteins called IGFBPs (IGFBPs 1,2,3,4,5 and 6)44,45. The GH-insulin-like growth factor (IGF-I) path is a system that integrates mediators, receivers and linking proteins that modulate the development of many tissues and are also involved with adaptation to exercise46. Evidence suggests that the IGF-I has an essential role in the formation and maintenance of skeletal muscle. Animal experimental models demonstrate that large amounts of IGF-I are related skeletal muscle hypertrophy11,47. Muscle overload seems to be associated with IGF-I increase, whereas this improved expression is observed in skeletal muscle fibers after resistance training exercises in rats46. Furthermore, in vivo experiments have shown that IGF-I is an important regulator of muscle growth and demonstrated that the manipulation of IGF-I levels in muscle can cause an increase in muscle mass9. The affinity of IGF-I and MGF for the linking receivers is regulated by the stretching transduction sensitive signals, overload and muscle contraction. These growth factors stimulate the proliferation of satellites cells and the differentiation of these cells, promoting muscle growth18. However, the changes in IGFBPs proteins expression can be decreased in old animals, inducing muscle atrophy with aging18. Mechanical growth factor (MGF) One of the IGF-I isoforms has been identified as being produced exclusively in skeletal muscles and called MGF (mechanical growth factor). MGF RNAm levels were observed to increased in muscles submitted to continuous stretching12,47. It has been postulated that factors IGF-I and MGF, are mechanotransductors activated by mechanical stimuli, such as stretching. Mechanical stimulation produces a chemical signal through IGF-I and MGF interaction, which are signaling molecules that interact with the extracellular matrix and cytoskeleton, increasing gene expression related with muscle hypertrophy12. Haddad and Adams48 showed that after exercise MGF is expressed earlier than IGF-I. Another study also found that the IGF-I gene is expressed before MGF expression in response to mechanical strain and muscle damage in rodents49. When damage occurs in a normal muscle, regeneration is initiated by MGF, which is not the case of dystrophic muscle, indicating incapacity to renew the satellite cells and resulting in muscle fibers injury50. In young rats submitted to muscle overload, an increased MGF expression was observed, middle-


-aged rats presented just a moderate raise and old rats showed a very low response50. Similar results were observed on a human study where an increased MGF expression was observed in muscles of young but not in elderly subjects50. However, in some cases, the inability to express MGF is linked to muscle loss15,44. Myogenic regulatory factors (MRFs) The myogenic regulatory factor (MRF) family is made up of a set of transcriptional factors that determine the myogenic weirdly of cells51. With disuse and aging, satellites cells proliferation and differentiation are reduced by myostatin expression. Similarly, cyclin-dependent kinase inhibitor p21 is responsible for the interruption of the cell cycle and cell differentiation51. Myostatin expression increase leads to a kinase cyclin dependent up regulation inhibiting p21 and hindering the expression of determinative myoblast factors. Myogenic regulatory factors (MRFs) have been suggested to be involved in this process52. Skeletal muscles are made of muscle-specific proteins, known as MRFs, which bind to the genes regulatory regions and activate its transcription53. MRF subfamily encompasses the MyoD (or Myf-3), Myf-5, myogenin (or Myf-1) and MRF-4 (or Myf-6/ herculin) proteins54. In accordance with the literature, evidence shows that the MRFs Myo-D, MRF-4, myogenin and myf-5 had increased expression and/or had been detected after continuous stretching stimulation, indicating that these factors are probably part of the skeletal muscle hypertrophy mechanism55,56. Possibly MRFs involvement in the hypertrophy or atrophy mechanism can be attributed to the role of myostatin. It has been suggested that myostatin is a negative skeletal muscle growth regulator, inhibiting the myoblasts proliferation51,57. Since myostatin is a myoblasts proliferation regulator, its absence leads a disordered proliferation of these cells, leading to muscle fibers hypertrophy and hyperplasia. In adult skeletal muscle, myostatin expression can increase in response to glycocorticoids increase, inhibiting satellites cells proliferation and inducing muscle proteolysis, hindering muscle fibers hypertrophy58. However, it has been described that, during disuse periods, such as observed in aging, myostatin increase can occur59. The MyoD family activates the expression of muscle specific genes, where each family member regulates different stages of muscle development. Myf-5 and Myo-D act in the early stage of myoblasts differentiation and myogenin and Myf-4 in later stages10. MyoD has a role during embryogenesis and regulates

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the myogenic program in satellite cells. MyoD is associated with cell determination, is highly expressed in actively proliferating myoblasts and may be critical for proliferation in response to skeletal muscle injury57. After birth and during adult life the MRF4 levels remain elevated, whereas the greatest amount of MyoD, myf5 and myogenin all decline within one week of birth. With ageing these factors are enabled again to restart the myogenic programme and repair the damage54, leading to atrophy. However, evidences suggested that old people submitted to strength training showed decreased myostatin expression, reaching muscle mass gains60. Atrogin has been characterized as an atrophy gene, as its expression has been shown to increase during processes that induce muscle atrophy, as sepsis, caquexy due to cancer, diabetes mellitus, uremi, food deprivation, immobilization and denervation61,62. Atrogin has been suggested to be a regulatory nuclear protein that plays an important role in the mechanisms of protein synthesis and degradation, as is increased during skeletal muscle atrophy. This inhibitory effect in the satellites cells proliferation and the myostatin differentiation results in myonucleus number reduction, leading to the muscle atrophy process. However, the mechanisms that regulate the MGF expression are still unknown. What is known until now is that the IGF-I expression increases with increased hormone growth and testosterone levels and decreases in response to increased glucocorticoids and inflammatory citocins levels (factor of tumoral necrosis (TNF) α and interleucins (IL) 1β)53,57. Inflammatory cytocins Evidences have suggested that the TNF-α and the IL-1β have an important role in muscle mass decline with age. Although the accurate suppression mechanism of GH-IGF-I is unknown, Scheett et al.63 consider the pro-inflammatory cytocins IL-1β and TNF-α (tumor necrosis factor α) involved with exercise, whose concentrations are raised by the intense exercise in the blood serum. These cytocins can induce the reduction of IGF-I circulating concentration, indicating a catabolic activity63. TNF-α was originally identified by its powerful cytotoxic effect against tumor cells. It is a trimetric polypeptides mainly produced by monocits and macrophages activated, as well as by other cells such as lynfocits, fibroblasts, neutrofils, smooth muscle and mastocits64,65. This cytocin can act in almost all nucleated cells type through an interaction with mem-

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brane receptors or soluble molecules. Moreover, the adult mammalian cardiac myocite is capable of producing TNF-α after extracellular stimulations, such as endotoxins, hypoxia or mechanical stress increase66. TNF-α acts at the cellular level through two receptor types, type I (TNFI) and type II (TNFII). The extracellular domain fragments of both TNF-α receptors can be set free of the cellular membrane and detected in its soluble form (sTNFRI and sTNFRII) in urine and plasma67. The catabolic stimulations increase with aging has been pointed as a probable cause for the muscle mass reduction. There is evidence that treatments with function-blocking anti-TNF-α can promote slow muscle growth and regeneration9. Roubenoff and Hughes17 described that the production of pro-inflammatory citocins (IL-6, TNF-α and IL-1) increase in elderly subjects. Thus, the aging process presents high levels of muscle injury biochemical marker at rest, such as IL-6, TNF-α and sTNF-R1, having higher neutrofils concentration. These mononucleus cells in the elderly have an increased capacity to produce pro-inflammatory cytocins68. Intense compared to moderate exercise induces increased levels of cytocins and interleukins as TNF-α concentration, IL-6, IL-1 and IL-1 increased in response to vigorous exercise69.

SIGNALLING AND ADHESION CELLULAR MOLECULES Among the many sensing structures during cell mechanical loading (stretching, overload, etc.) the transarcolemmal proteins have important functions in muscle mass regulation. They are called integrins, dystroglycans and stretching sensible calcium channels. The integrins and the dystroglycans are the main adhesion proteins complexes, called costamers, which bind the extracellular matrix to the sarcomere Z-disc through cytoskeleton70,71. Due to the linking of these proteins to sarcomere through filaments γ-actin and other cytoskeletal proteins (talin, vinculin and α-actin), the costamers are also related to the lateral transmission of the force generated by myofibrils to the extracellular matrix72. Studies suggest that the integrins, transmembrane glycoproteins, that keep the cytoplasm interactions with the extracellular matrix and other cells, play an important role in mechanical signal sensing and transmission to the cytoplasm, and activation of cascade reactions73. The events generated by the integrins activation after mechanical sti-

mulation include alterations of gene expression and growth factors, as well as proliferation, differentiation and apoptosis70,71. Signal transmission through integrin interaction with extracellular matrix initially occurs for the formation of a focal adhesion complex, that involves proteins such as vinculin, talin and actin, which are necessary for the linking between the integrins to the cytoskeletal74. Evidence indicates that exercise increase the focal adhesion complex and the focal adhesion kinase (FAK) fosforilation, one of the initial signals for the mechanical signal transduction. Thus, the focal adhesion complex can be considered as the “key” for signal sensing during mechanical stimulations that induce muscle hypertrophy75. After the focal adhesion kinase (FAK) activation, and also of proteins P pertaining to the RAS of small GTPases family (guanosin-triphosphatase), they interact with some signaling paths, such as that of the protein kinase and PI3k-mTOR, regulating protein synthesis. Evidence suggested that chronic loads reaching skeletal muscles cause hypertrophy and this is followed by an up regulation of the FAK activity and increased content75. In vivo and in vitro animal studies suggest that costamers FAK congregate to construct a regulating canal for the sarcomere number in response to mechanical loading changes74-76. Therefore, the integrins complex and dystroglicans act in the mechanoreceptors transmitting information between the external and internal muscle fiber sides. These proteins are also involved in the conversion of the mechanical stimulus into biochemical signals, thus regulating protein synthesis. It is probable that these proteins complex also have a role in disuse atrophy as its presence is decreased in rat muscles subject to tenotomy76. Since aging is accompanied by disuse, the reduction of these molecules may be related with muscle mass decrease in elderly. In addition to the integrins and dystroglicans, it is also suggested that the cytoskeletal protein called desmin is involved in sarcomere remodeling in response to load or to its lack. This protein forms intermediate filaments in sarcomere Z-discs. The desmin filaments connect the sarcomeres in lateral direction and form connections for the nucleus and costamers in the sarcolema. Due to its sarcomere structural localization, desmin appears to be involved in mechanical signal transduction regulating muscle mass7, but does not appear to be essential for sarcomere remodeling in response to disuse or aging.


CONCLUSION There are different transduction mechanisms involving the extracellular matrix and cytoskeleton, which lead to gene expression or to the induction of biochemical signals that regulate biochemical protein synthesis. Therefore, vectors generated for protein synthesis may indicate the muscle mass decline and sarcomere remodeling in response to aging. In summary, this review showed the relation between several markers and their respective role in the mechanisms of skeletal muscle adaptation due to aging. We may conclude that the changes which modulate atrophy or hypertrophy with aging are related with several intracellular mechanisms mediated by mechanical loading such as increased use, disuse or training. However, there is a lack in the literature regarding the exercise effects on the biochemichal repair of skeletal muscle during aging. The difficulty of performing in vivo studies in this area lead to the necessity of relying on the evidences of animal studies that show the involvement of biochemichal muscle markers in order to explain their relation during physical activity with aging in humans.

ACKNOWLEDGMENTS The authors would like to thank National Council for Scientific and Technological Development (CNPq), Brazil and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes), Brazil for financial support.

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