Endocrine DOI 10.1007/s12020-014-0271-5
ORIGINAL ARTICLE
Thyroid hormones regulate skeletal muscle regeneration after acute injury Anna Lu´cia R. C. Leal • Joa˜o Paulo C. Albuquerque • Marina S. Matos • Rodrigo S. Fortunato • Denise P. Carvalho Doris Rosenthal • Vaˆnia Maria Correˆa da Costa
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Received: 4 February 2014 / Accepted: 8 April 2014 Ó Springer Science+Business Media New York 2014
Abstract We evaluated the effects of hypo- and hyperthyroid statuses during the initial phase of skeletal muscle regeneration in rats. To induce hypo- or hyperthyroidism, adult male Wistar rats were treated with methimazole (0.03 %) or T4 (10 lg/100 g), respectively, for 10 days. Three days before sacrifice, a crush injury was produced in the solear muscles of one half of the animals, while the other half remained intact. T3, T4, TSH, and leptin serum levels were not affected by the injury. Serum T3 and T4 levels were significantly increased in hyperthyroid and hyper-injury animals. Hypothyroidism was confirmed by the significant increase in serum TSH levels in hypothyroid and hypo-injury animals. Injury increased cell infiltration and macrophage accumulation especially in hyperthyroid animals. Both type 2 and type 3 deiodinases were induced by lesion, and the opposite occurred with the type 1 isoform, at least in the control and hyperthyroid groups. Injury increased both MyoD and myogenin expression in all the studied groups, but only MyoD expression was increased by thyroidal status only at the protein level. We conclude that thyroid hormones Electronic supplementary material The online version of this article (doi:10.1007/s12020-014-0271-5) contains supplementary material, which is available to authorized users. A. L. R. C. Leal J. P. C. Albuquerque M. S. Matos R. S. Fortunato D. P. Carvalho D. Rosenthal V. M. C. da Costa Laborato´rio de Fisiologia Endo´crina Doris Rosenthal, Instituto de Biofı´sica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, CCS, bloco G, Cidade Universita´ria, Ilha do Funda˜o, Rio de Janeiro 21949-900, Brazil V. M. C. da Costa (&) VM Correˆa da Costa, Instituto de Biofı´sica Carlos Chagas Filho, CCS Bloco G, Cidade Universita´ria, Ilha do Funda˜o, Rio de Janeiro 21949-900, Brazil e-mail: [email protected]; [email protected]
modulate skeletal muscle regeneration possibly by regulating the inflammatory process, as well as MyoD and myogenin expression in the injured tissue. Keywords Skeletal muscle Thyroid hormones Regeneration Injury
Introduction The regeneration of skeletal muscle is a complex process involving different cell types, such as inflammatory cells (neutrophils and macrophages) and muscle precursor cells (satellite cells), which are responsible for the restoration and maintenance of skeletal muscles in postnatal life. Muscular damage leads to death of muscle fibers. In an attempt to restore the functionality of the damaged region, extracellular matrix components, growth factors, cytokines, and chemokines are secreted, and activate the muscle precursor cells, which begin to express the typical muscle transcription factors: MyoD (initial differentiation), and Myogenin (terminal differentiation). These transcription factors induce the synthesis of muscle-specific proteins, such as desmin and myosin [1, 2]. In the second stage of the muscle fiber regeneration process, macrophages acquire a phenotype associated with tissue remodeling, acting together with myogenic cells in muscle repair [3, 4]. The presence of macrophages is a crucial factor for the regeneration of skeletal muscle cells, probably by increasing the replication and differentiation of muscle precursor cells [5, 6]. It is possible that at all stages of muscle damage degeneration, regeneration, and fibrosis, the satellite cells’ activity is modulated by mediators such as inflammatory cytokines, chemokines, and growth factors released by the activated macrophages [3, 7]. Summan et al. [8] reported
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that the process of muscle repair was impaired in the absence of local and systemic monocyte/macrophages. The skeletal muscle contains, in addition to macrophages, other cell types such as endothelial cells and fibroblasts, besides myocytes and myoblasts. Myocytes and myoblasts are able to produce and release cytokines and chemokines that act as chemoattractant for neutrophils and macrophages. IL-1, IL-6, IL-8, and TNF-alpha are the examples of molecules that directly or indirectly influence the chemotaxis of neutrophils and macrophages. Peterson and Pizza and coworkers [9] clearly demonstrated that skeletal muscle cellderived IL-8 and GM-CSF, in synergy, promote neutrophil chemotaxis after injurious mechanical strain. Thyroid hormone (TH) is essential for skeletal muscle differentiation, in vivo myogenesis, and regeneration of skeletal muscle tissue, participating in the activation of myoblasts through maturation in differentiated muscle fibers. In adult skeletal muscles, hypothyroidism increases the expression of the slow MHC isoforms in both skeletal and cardiac muscles, whereas hyperthyroidism inhibits the slow MHC isoform expression in soleus and diaphragm of rats [10]. In general, the action of thyroid hormones on gene transcription is mediated by their nuclear receptors, but THs are also able to modulate cytoplasmatic signal transduction pathways. D’Arezzo et al. [11] showed that thyroid hormones’ signal transduction in rat L6 myoblasts is a rapid nongenomic effect that requires a tyrosine kinasedependent protein phosphorylation of phospholipase C, protein kinase C, and MAPK (mitogen-activated protein kinase). Activation of the Ras protein is able to control myogenesis through the kinases MEK and MAPK, by mechanisms still poorly understood [12]. Perry et al. [13] investigated the mechanism by which MAPK signaling pathway regulates the transcription factor MyoD and the role of intermediate MEK1 in myogenesis. Their data demonstrate that MEK1 regulates the activity of MyoD negatively through MEK1-binding mechanism involving the nuclear complex of MyoD transcription. Here, we aim to evaluate the influences of hypo- and hyperthyroidisms on the early stages of the skeletal muscle regeneration process in rats submitted to acute muscle injury.
Materials and methods This study was approved by the Institutional Committee for Use of Animals in Research (IBCCF 080), and the procedures used were compliant with the International Guiding Principles for Biomedical Research Involving Animals, the Council for International Organizations of Medical Sciences (Geneva, Switzerland), and the guiding principles for care and use of the American Physiological Society. Male
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adult Wistar rats weighing 220–270 g were kept from birth in a temperature-controlled (22–25 °C) animal room, with a 12-h light:12-h darkness cycle. Pelleted commercial chow (Purina, Sa˜o Paulo, Brazil, iodine content: 2 mg/kg) and water were available ad libitum. To induce hypothyroidism (Hypo), the Wistar rats received 2-mercapto-1-methylimidazole (MMI, Sigma, USA), 0.03 % in the drinking water, for 10 days. During the same period, another group (Hyper) was given a supraphysiological amount of T4 in saline (10 lg/100 g bw, sc) for the same 10 days, a dose of T4 that was previously shown to induce hyperthyroidism in these animals [14, 15]. Control rats received only NaCl 0.9 % solution, sc, during the same period. The animals were euthanized by decapitation, the soleus muscles were excised and quickly frozen in liquid N2, and blood samples were collected. The blood was centrifuged at 1,2009g and 4 °C for 15 min, and the serum was stored at -20 °C for T3, T4, TSH, and leptin measurements. Serum T3 and T4 levels were determined by specific coated-tube radioimmunoassay (RIA) kits (Diagnostic Systems Laboratories. Inc, TX, USA). Leptin serum levels were determined using a specific RIA for rat leptin (Linco Research, St Charles, MO). Serum TSH RIA measurements were done, as previously described [16], using a kit supplied by the National Hormone and Peptide Program, NIDDK (Bethesda, MD, USA) and expressed in terms of the preparation (RP-3) provided. After treatment, each group was subdivided into two: half were kept intact until the end of treatment, and the other half suffered an acute crushing injury of the soleus muscle. Thus, the following groups were formed: control, control ? injury, hypothyroid, hypo ? injury, hyperthyroid, and hyper ? injury. After 7 days of treatment, control, hypo- and hyperthyroid rats were sedated and anesthetized using a combination of ketamine (50 mg/kg) and xylazine (5 mg/kg). After locating the soleus, a Gaspin forceps was placed in parallel over the muscle, and the injury was induced by 20 clip holds in its maximum amplitude. After surgery, the incision was sutured and cleansed quickly using 70 % alcohol solution. The animals were kept in a cage with water and food ad libitum. On the third post-injury day (total 10 days of treatment), animals were euthanized by decapitation, and the soleus muscles of all groups were removed and frozen in liquid nitrogen. The soleus muscles of each experimental group were homogenized using an Ultra-Turrax (JankeÒ& Kunk/IkaLabortechnik) in Tris–HCl buffer 62.5 mM, pH 6.8, containing 10 % glycerol, 3 % sodium dodecyl sulfate (SDS), and 1 mM phenylmethylsulfonyl fluoride (PMSF). Protein concentration was determined by the method of Bradford [17], before the addition of 5 % of mercaptoethanol and 0.01 % bromophenol blue. 50–100 lg of the homogenate protein of the homogenate was separated by polyacrilamide
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gel electrophoresis (SDS-PAGE): 7.5 % gel for pERK, pMEK, MyoD, and Myogenin; and 12 % gel for Ras. After transfer to a PVDF membrane (Millipore) and hybridization with specific anti-H-Ras, anti-pERK, anti-ERK 1, antipMEK, anti-Myo D, or anti-Myogenin antibodies (Santa Cruz Biothechnogy, Inc), or anti-GAPDH (Millipore), the immunoblots were resolved using an ECL kit (Amersham International). Real-time PCR was done as described previously [18]. Total RNA was extracted from soleus muscle using the RNeasyÒ Plus Mini Kit (Qiagen), following the fabricant recommendations. After DNAse treatment, reverse transcription was followed by real-time PCR. Specific oligonucleotides, described in supplementary data, were purchased from Applied Biosystems (Foster City, CA, USA). The threshold cycle (CT) was measured in duplicate for each sample. Each reaction was normalized versus GAPDH gene expression. The relative abundance of mRNAs was calculated using the 2-DDCT method [18]. For histological analysis, the muscle tissue slices were fixed in 10 % buffered formalin solution, dehydrated in absolute ethanol, and embedded in paraffin. Solear samples were sectioned (5 lm), rested in laminated slides with poly-L-lysine (Sigma), and stained with picrosirius red and hematoxylin–eosin. The images were observed in an optical microscope (Zeiss, Oberkochen, Germany) using 2009 (normal) or 10009 (high) magnification, and scanned with the attached Color View XS Soft Imaging System. Quantification was estimated by the percentage of stained area in comparison to the total area of the fields examined, using Image-Pro Plus 5.0 (Media Cybernetics, Bethesda, MD, United States) image analysis software. For the immunohistochemistry evaluation, paraffin sections were washed in xylene and rehydrated in decreasing solutions of ethanol. Tissues were incubated at 5 % sodium tetraborate for 15 min, and antigen retrieval was done using citrate buffer 0.01 M, pH 6.0 for 30 min, and then were blocked with TBS-BSA 1 % Triton 0.1 %, for 30 min at room temperature and incubated with rat monoclonal anti-ED1 antibody (Serotec) at 4 °C, overnight. Then, the sections were incubated with the biotinylated secondary antibody from the Applied Kit (Enzinger-LSAB) for 20 min at room temperature. After three times of TBS washes, streptavidin (LSAB-Kit Enzigas) was added to react for 20 min at room temperature. The colorimetric detection was carried out using a diaminobenzamide tetrahydrochloride (DAB) peroxidase system (Dako, K069) and counterstained for 3 min with Harris hematoxilin. Tissue sections were observed and photographed using a Zeiss microscope. For quantification of Macrophage (ED1) and cellular infiltration (HE), seven fields per histological section of soleus muscle were quantified at normal magnification
(n = 11 per group). Using Image ProÒ software, we observed staining detected in each section and the nuclei stained by HE, generating an average area of staining tissue from the total area of the cut muscle. The data are expressed as mean ± standard error. The differences between groups were compared by two-way analysis of variance, followed by Tukey’s or Holm–Sidak’s multiple comparisons tests when appropriate. Densitometric data were expressed as arbitrary units using GAPDH or total ERK as loading controls. Graph Pad Prism software (Graphpad Prism 6.0 Software Inc., USA) was used for all analyses.
Results As expected, serum T3 and T4 levels were significantly increased in hyperthyroid and hyper-injury animals, and decreased in hypothyroid and hypo-injury rats, although these decreases did not reach statistical significance compared to control and control-injury animals (Fig. 1a, b). Hypothyroidism was confirmed by the significant increase in serum TSH levels in hypothyroid and hypo-injury animals (Fig. 1c). Hyperthyroid animals, submitted to injury or not, had a significant decrease in serum leptin levels (Fig. 1d). The total cellular infiltrate was significantly increased in control-injury, hypo-injury, and hyper-injury animals in comparison to the intact controls; this increase was significantly greater in the hyper-injury group. Both hypo- and hyperthyroid intact animals also had greater cellular infiltration in solear muscles than the control animals (Fig. 2a). Interestingly, the increase in cellular infiltration caused by thyroid dysfunction could not be attributed to macrophage infiltration, since the ED1 staining revealed that macrophage accumulation only occurred in the acutely crushed soleus muscle (Fig. 2b). Solear Ras expression was not affected by thyroidal status per se, but injury in hypothyroid or hyperthyroid rats was able to significantly increase Ras expression, especially in hyperthyroid animals (Fig. 3). Injury increased ERK phosphorylation in the muscle of control and hypothyroid animals, but this effect was not detected in hyperthyroid animals, suggesting that the high serum T3 levels of hyperthyroid animals increase ERK phosphorylation (Fig. 4). Injury increased MyoD expression, a typical muscle transcription factor, activated in the early stages of skeletal muscle differentiation at mRNA level (Fig. 5a) as well as at protein level in all groups studied (Fig. 5b). The inductions of hypo- and hyperthyroidism per se increased MyoD expression only at protein level. Injury also increased myogenin expression both in hypo- and
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Fig. 1 Total serum T3 (a), total serum TSH (b), total serum T4 (c), and total serum leptin (d) concentrations in control, control ? injury, hypothyroid, hypothyroid ? injury, hyperthyroid, and hyperthyroid ? injury
rats. #p \ 0.05 versus control and hypothyroid animals injured or not. *p \ 0.05 versus control and hyperthyroid animals injured or not
hyperthyroid animals at both mRNA (Fig. 6a) and protein (Fig. 6b) levels. Thyroidal status did not alter myogenin solear response to injury neither at mRNA nor protein levels (Figs. 6a, b). Concerning mRNA expression, solear thyroid hormone metabolism seems to be modulated by lesion. Type 2 and type 3 iodothyronines deiodinases mRNAs were positively regulated by injury, while type 1 iodothyronine deiodinase mRNA seems to be negatively regulated, at least in control and hyperthyroid animals after injury (Fig. 7). As expected, increased T3 serum present in hyperthyroid animals positively regulates type 1 activity (Fig. 7).
increases in both cell and macrophage infiltration after injury, independent of thyroidal status. Hyperthyroidism seems to potentiate both cellular and macrophage infiltration: the maximal cellular and macrophage infiltration were detected in the solear muscles of hyperthyroid-injury animals. Furthermore, the number of resident macrophages in the uninjured muscle did not change in either hypo- or hyperthyroidism. There are few data demonstrating the involvement of cytokines in muscle regeneration. Pedersen and Febbraio [20] demonstrated that skeletal muscle contraction leads to secretion of cytokines that can influence the metabolism of other tissues or organs. These authors have shown that IL-6 increases in muscle during exercise, and it is noteworthy that this response is directly linked to muscle damage and regeneration. The active thyroid hormone, T3, derives either from the thyroid gland directly or from the 50 -monodeiodination of T4. Two iodothyronines deiodinases can catalyze this reaction (D1 and D2). In skeletal muscle, D2 expression can be modulated and seems to be required for normal myogenesis and during muscle regeneration [21]. After skeletal muscle injury in vivo, there is a marked increase in D2 activity [22];
Discussion Monocyte and macrophage depletion induced by clodronate liposome treatment is associated with an impaired repair process after skeletal muscle injury [8]. In fact, the regenerative process and the recovery of muscle function are associated with macrophage infiltration and chemokine response in injured skeletal muscles [19]. Our data in solear skeletal muscles support these findings, since we detected
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Fig. 4 Densitometric analysis: pERK expression, 3 days after crush injury, in soleus muscles of control, hypothyroid, and hyperthyroid rats. n = 3 animals/group. *p \ 0.05 versus paired controls. # p \ 0.05 versus intact control and hypothyroid animals
Fig. 2 Histomorphometric analysis of infiltrating cells (a) and percentage of area occupied by macrophages (ED1) (b), 3 days after crush injury, in soleus muscles of control, hypothyroid, and hyperthyroid rats. n C 7 animals/group. *p \ 0.05 versus paired controls. # p \ 0.05 versus intact control animals
Fig. 3 Densitometric analysis of Ras expression, 3 days after crush injury, in soleus muscles of control, hypothyroid, and hyperthyroid rats. n = 3 animals/group. *p \ 0.05 versus paired controls
this D2 upregulation suggests an important role of T3 during this process. D3 repression was also reported in C2C12 cells [23]. Recently, Salvatore et al. [24] proposed a sequential
Fig. 5 Densitometric analysis: expression of mRNA (a) and protein (b) of MyoD, 3 days after crush injury, in soleus muscles of control, hypothyroid, and hyperthyroid rats. n = 4 animals/group. *p \ 0.05 versus paired controls. #p \ 0.05 versus intact control animals
expression of D3 followed by D2 in order to tightly control intracellular T3 levels during myogenesis. D2 highly expressed in myoblast precursor cells is important to
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Fig. 6 Densitometric analysis: expression of mRNA (a) and protein (b) of myogenin, 3 days after crush injury, in soleus muscles of control, hypothyroid, and hyperthyroid rats. n = 3 animals/group. *p \ 0.05 versus paired controls
increase intracellular T3 and induce Myo D expression. Here, we detected an important increase in both type 2 and type 3 iodothyronine deiodinase expression after injury, in agreement with the importance of local thyroid hormone availability during solear regenerating process (Fig. 7), and we do attribute this to the presence of myoblasts in different stages of differentiation and so requiring different amounts of T3, in solear samples studied. In fact, Rozing et al. [25], associated high free iodothyronines levels to a higher production capacity of proinflammatory cytokines; our results are in agreement with this idea, since the higher levels of both cellular and macrophage infiltration were detected in the hyper-injury animals. Ras–MAPK pathway modulation by skeletal muscle injury has already been proposed both in vitro [26] and in vivo [27]. Induction of the Ras–MAPK pathway by nerve activity in the regenerating soleus muscle was previously demonstrated [27], indicating that Ras–MAPK signaling is involved in the promotion of nerve-activity-dependent differentiation of slow muscle fibers in vivo. Our data showed increased Ras–pERK expressions in control and hypothyroid animals submitted to injury, but in hyperthyroid animals, ERK phosphorylation is already higher, and no further
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Fig. 7 Densitometric analysis: mRNA expression of type 1 (a), type 2 (b), and type 3 (c) iodothyronines deiodinases, 3 days after crush injury, in soleus muscles of control, hypothyroid, and hyperthyroid rats. n = 3 animals/group. *p \ 0.05 versus paired controls. # p \ 0.05 versus intact control animals
increase could be detected in hyperthyroid-injury animals, suggesting that in solear muscles from the hyper-injury group, Ras protein maybe signaling downstream by another pathway instead of pERK. Activation of the Ras–MAPK pathway is able to control myogenesis by still poorly understood mechanisms [12]. Perry et al. [13] studying the mechanisms by which MAPK signaling pathway regulates the transcription factor MyoD showed that MEK1 negatively regulates the activity of MyoD by binding of activated MEK1 to the nuclear MyoD transcription complex. In this study, we detected an increase in MyoD expression after injury in all the studied
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groups, but without any clear relation with pMEK expression (data not shown). Increase in MyoD expression after injury indicates that this myogenic regulatory factor plays an important role after acute injury. Myogenin expression is frequently associated with terminal muscle differentiation; nevertheless, we detected an important increase in this other myogenic regulatory factor as early as 3 days after injury, but without any relation to the thyroid status. Skeletal muscle has a remarkable ability to adapt to physiological demands such as growth and exercise. Regular endurance exercise enhances skeletal muscle interleukin-15-mediating muscle adaptation during training [28]. In fact, exercise and nutrition interventions, such as alkali and vitamin D supplementation, were suggested to promote skeletal mass muscle preservation, frequently reduced during aging process [29–31]. Regeneration is a sequential process involving degeneration, proliferation of satellite cells, differentiation and fusion of myoblasts, and growth and maturation of myofibers. The involvement of different cell types, such as inflammatory cells and muscle precursor cells, is well established, but recently, Cianferotti and Brandi [32] pointed out that also bone tissue is able to modulate muscle metabolism, and possibly may be involved in regeneration process after muscle injury. Herein, we demonstrate that thyroid hormones are involved in skeletal muscle regeneration, possibly by regulating inflammatory process, Ras– MAPK pathway, MyoD, and myogenin expression in the injured tissue. Acknowledgments The authors gratefully acknowledge the technical assistance of Advaldo Nunes Bezerra, Jose´ Humberto Tavares de Abreu, Norma Lima de Arau´jo Faria, and Wagner Nunes Bezerra. This work was supported by the grants from Fundac¸a˜o Carlos Chagas Filho de Amparo ‘a Pesquisa do Estado do Rio de Janeiro (FAPERJ), and Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq). Conflict of interest
Authors declare no conflict of interest.
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ORIGINAL ARTICLE
Thyroid hormones regulate skeletal muscle regeneration after acute injury Anna Lu´cia R. C. Leal • Joa˜o Paulo C. Albuquerque • Marina S. Matos • Rodrigo S. Fortunato • Denise P. Carvalho Doris Rosenthal • Vaˆnia Maria Correˆa da Costa
•
Received: 4 February 2014 / Accepted: 8 April 2014 Ó Springer Science+Business Media New York 2014
Abstract We evaluated the effects of hypo- and hyperthyroid statuses during the initial phase of skeletal muscle regeneration in rats. To induce hypo- or hyperthyroidism, adult male Wistar rats were treated with methimazole (0.03 %) or T4 (10 lg/100 g), respectively, for 10 days. Three days before sacrifice, a crush injury was produced in the solear muscles of one half of the animals, while the other half remained intact. T3, T4, TSH, and leptin serum levels were not affected by the injury. Serum T3 and T4 levels were significantly increased in hyperthyroid and hyper-injury animals. Hypothyroidism was confirmed by the significant increase in serum TSH levels in hypothyroid and hypo-injury animals. Injury increased cell infiltration and macrophage accumulation especially in hyperthyroid animals. Both type 2 and type 3 deiodinases were induced by lesion, and the opposite occurred with the type 1 isoform, at least in the control and hyperthyroid groups. Injury increased both MyoD and myogenin expression in all the studied groups, but only MyoD expression was increased by thyroidal status only at the protein level. We conclude that thyroid hormones Electronic supplementary material The online version of this article (doi:10.1007/s12020-014-0271-5) contains supplementary material, which is available to authorized users. A. L. R. C. Leal J. P. C. Albuquerque M. S. Matos R. S. Fortunato D. P. Carvalho D. Rosenthal V. M. C. da Costa Laborato´rio de Fisiologia Endo´crina Doris Rosenthal, Instituto de Biofı´sica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, CCS, bloco G, Cidade Universita´ria, Ilha do Funda˜o, Rio de Janeiro 21949-900, Brazil V. M. C. da Costa (&) VM Correˆa da Costa, Instituto de Biofı´sica Carlos Chagas Filho, CCS Bloco G, Cidade Universita´ria, Ilha do Funda˜o, Rio de Janeiro 21949-900, Brazil e-mail: [email protected]; [email protected]
modulate skeletal muscle regeneration possibly by regulating the inflammatory process, as well as MyoD and myogenin expression in the injured tissue. Keywords Skeletal muscle Thyroid hormones Regeneration Injury
Introduction The regeneration of skeletal muscle is a complex process involving different cell types, such as inflammatory cells (neutrophils and macrophages) and muscle precursor cells (satellite cells), which are responsible for the restoration and maintenance of skeletal muscles in postnatal life. Muscular damage leads to death of muscle fibers. In an attempt to restore the functionality of the damaged region, extracellular matrix components, growth factors, cytokines, and chemokines are secreted, and activate the muscle precursor cells, which begin to express the typical muscle transcription factors: MyoD (initial differentiation), and Myogenin (terminal differentiation). These transcription factors induce the synthesis of muscle-specific proteins, such as desmin and myosin [1, 2]. In the second stage of the muscle fiber regeneration process, macrophages acquire a phenotype associated with tissue remodeling, acting together with myogenic cells in muscle repair [3, 4]. The presence of macrophages is a crucial factor for the regeneration of skeletal muscle cells, probably by increasing the replication and differentiation of muscle precursor cells [5, 6]. It is possible that at all stages of muscle damage degeneration, regeneration, and fibrosis, the satellite cells’ activity is modulated by mediators such as inflammatory cytokines, chemokines, and growth factors released by the activated macrophages [3, 7]. Summan et al. [8] reported
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that the process of muscle repair was impaired in the absence of local and systemic monocyte/macrophages. The skeletal muscle contains, in addition to macrophages, other cell types such as endothelial cells and fibroblasts, besides myocytes and myoblasts. Myocytes and myoblasts are able to produce and release cytokines and chemokines that act as chemoattractant for neutrophils and macrophages. IL-1, IL-6, IL-8, and TNF-alpha are the examples of molecules that directly or indirectly influence the chemotaxis of neutrophils and macrophages. Peterson and Pizza and coworkers [9] clearly demonstrated that skeletal muscle cellderived IL-8 and GM-CSF, in synergy, promote neutrophil chemotaxis after injurious mechanical strain. Thyroid hormone (TH) is essential for skeletal muscle differentiation, in vivo myogenesis, and regeneration of skeletal muscle tissue, participating in the activation of myoblasts through maturation in differentiated muscle fibers. In adult skeletal muscles, hypothyroidism increases the expression of the slow MHC isoforms in both skeletal and cardiac muscles, whereas hyperthyroidism inhibits the slow MHC isoform expression in soleus and diaphragm of rats [10]. In general, the action of thyroid hormones on gene transcription is mediated by their nuclear receptors, but THs are also able to modulate cytoplasmatic signal transduction pathways. D’Arezzo et al. [11] showed that thyroid hormones’ signal transduction in rat L6 myoblasts is a rapid nongenomic effect that requires a tyrosine kinasedependent protein phosphorylation of phospholipase C, protein kinase C, and MAPK (mitogen-activated protein kinase). Activation of the Ras protein is able to control myogenesis through the kinases MEK and MAPK, by mechanisms still poorly understood [12]. Perry et al. [13] investigated the mechanism by which MAPK signaling pathway regulates the transcription factor MyoD and the role of intermediate MEK1 in myogenesis. Their data demonstrate that MEK1 regulates the activity of MyoD negatively through MEK1-binding mechanism involving the nuclear complex of MyoD transcription. Here, we aim to evaluate the influences of hypo- and hyperthyroidisms on the early stages of the skeletal muscle regeneration process in rats submitted to acute muscle injury.
Materials and methods This study was approved by the Institutional Committee for Use of Animals in Research (IBCCF 080), and the procedures used were compliant with the International Guiding Principles for Biomedical Research Involving Animals, the Council for International Organizations of Medical Sciences (Geneva, Switzerland), and the guiding principles for care and use of the American Physiological Society. Male
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adult Wistar rats weighing 220–270 g were kept from birth in a temperature-controlled (22–25 °C) animal room, with a 12-h light:12-h darkness cycle. Pelleted commercial chow (Purina, Sa˜o Paulo, Brazil, iodine content: 2 mg/kg) and water were available ad libitum. To induce hypothyroidism (Hypo), the Wistar rats received 2-mercapto-1-methylimidazole (MMI, Sigma, USA), 0.03 % in the drinking water, for 10 days. During the same period, another group (Hyper) was given a supraphysiological amount of T4 in saline (10 lg/100 g bw, sc) for the same 10 days, a dose of T4 that was previously shown to induce hyperthyroidism in these animals [14, 15]. Control rats received only NaCl 0.9 % solution, sc, during the same period. The animals were euthanized by decapitation, the soleus muscles were excised and quickly frozen in liquid N2, and blood samples were collected. The blood was centrifuged at 1,2009g and 4 °C for 15 min, and the serum was stored at -20 °C for T3, T4, TSH, and leptin measurements. Serum T3 and T4 levels were determined by specific coated-tube radioimmunoassay (RIA) kits (Diagnostic Systems Laboratories. Inc, TX, USA). Leptin serum levels were determined using a specific RIA for rat leptin (Linco Research, St Charles, MO). Serum TSH RIA measurements were done, as previously described [16], using a kit supplied by the National Hormone and Peptide Program, NIDDK (Bethesda, MD, USA) and expressed in terms of the preparation (RP-3) provided. After treatment, each group was subdivided into two: half were kept intact until the end of treatment, and the other half suffered an acute crushing injury of the soleus muscle. Thus, the following groups were formed: control, control ? injury, hypothyroid, hypo ? injury, hyperthyroid, and hyper ? injury. After 7 days of treatment, control, hypo- and hyperthyroid rats were sedated and anesthetized using a combination of ketamine (50 mg/kg) and xylazine (5 mg/kg). After locating the soleus, a Gaspin forceps was placed in parallel over the muscle, and the injury was induced by 20 clip holds in its maximum amplitude. After surgery, the incision was sutured and cleansed quickly using 70 % alcohol solution. The animals were kept in a cage with water and food ad libitum. On the third post-injury day (total 10 days of treatment), animals were euthanized by decapitation, and the soleus muscles of all groups were removed and frozen in liquid nitrogen. The soleus muscles of each experimental group were homogenized using an Ultra-Turrax (JankeÒ& Kunk/IkaLabortechnik) in Tris–HCl buffer 62.5 mM, pH 6.8, containing 10 % glycerol, 3 % sodium dodecyl sulfate (SDS), and 1 mM phenylmethylsulfonyl fluoride (PMSF). Protein concentration was determined by the method of Bradford [17], before the addition of 5 % of mercaptoethanol and 0.01 % bromophenol blue. 50–100 lg of the homogenate protein of the homogenate was separated by polyacrilamide
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gel electrophoresis (SDS-PAGE): 7.5 % gel for pERK, pMEK, MyoD, and Myogenin; and 12 % gel for Ras. After transfer to a PVDF membrane (Millipore) and hybridization with specific anti-H-Ras, anti-pERK, anti-ERK 1, antipMEK, anti-Myo D, or anti-Myogenin antibodies (Santa Cruz Biothechnogy, Inc), or anti-GAPDH (Millipore), the immunoblots were resolved using an ECL kit (Amersham International). Real-time PCR was done as described previously [18]. Total RNA was extracted from soleus muscle using the RNeasyÒ Plus Mini Kit (Qiagen), following the fabricant recommendations. After DNAse treatment, reverse transcription was followed by real-time PCR. Specific oligonucleotides, described in supplementary data, were purchased from Applied Biosystems (Foster City, CA, USA). The threshold cycle (CT) was measured in duplicate for each sample. Each reaction was normalized versus GAPDH gene expression. The relative abundance of mRNAs was calculated using the 2-DDCT method [18]. For histological analysis, the muscle tissue slices were fixed in 10 % buffered formalin solution, dehydrated in absolute ethanol, and embedded in paraffin. Solear samples were sectioned (5 lm), rested in laminated slides with poly-L-lysine (Sigma), and stained with picrosirius red and hematoxylin–eosin. The images were observed in an optical microscope (Zeiss, Oberkochen, Germany) using 2009 (normal) or 10009 (high) magnification, and scanned with the attached Color View XS Soft Imaging System. Quantification was estimated by the percentage of stained area in comparison to the total area of the fields examined, using Image-Pro Plus 5.0 (Media Cybernetics, Bethesda, MD, United States) image analysis software. For the immunohistochemistry evaluation, paraffin sections were washed in xylene and rehydrated in decreasing solutions of ethanol. Tissues were incubated at 5 % sodium tetraborate for 15 min, and antigen retrieval was done using citrate buffer 0.01 M, pH 6.0 for 30 min, and then were blocked with TBS-BSA 1 % Triton 0.1 %, for 30 min at room temperature and incubated with rat monoclonal anti-ED1 antibody (Serotec) at 4 °C, overnight. Then, the sections were incubated with the biotinylated secondary antibody from the Applied Kit (Enzinger-LSAB) for 20 min at room temperature. After three times of TBS washes, streptavidin (LSAB-Kit Enzigas) was added to react for 20 min at room temperature. The colorimetric detection was carried out using a diaminobenzamide tetrahydrochloride (DAB) peroxidase system (Dako, K069) and counterstained for 3 min with Harris hematoxilin. Tissue sections were observed and photographed using a Zeiss microscope. For quantification of Macrophage (ED1) and cellular infiltration (HE), seven fields per histological section of soleus muscle were quantified at normal magnification
(n = 11 per group). Using Image ProÒ software, we observed staining detected in each section and the nuclei stained by HE, generating an average area of staining tissue from the total area of the cut muscle. The data are expressed as mean ± standard error. The differences between groups were compared by two-way analysis of variance, followed by Tukey’s or Holm–Sidak’s multiple comparisons tests when appropriate. Densitometric data were expressed as arbitrary units using GAPDH or total ERK as loading controls. Graph Pad Prism software (Graphpad Prism 6.0 Software Inc., USA) was used for all analyses.
Results As expected, serum T3 and T4 levels were significantly increased in hyperthyroid and hyper-injury animals, and decreased in hypothyroid and hypo-injury rats, although these decreases did not reach statistical significance compared to control and control-injury animals (Fig. 1a, b). Hypothyroidism was confirmed by the significant increase in serum TSH levels in hypothyroid and hypo-injury animals (Fig. 1c). Hyperthyroid animals, submitted to injury or not, had a significant decrease in serum leptin levels (Fig. 1d). The total cellular infiltrate was significantly increased in control-injury, hypo-injury, and hyper-injury animals in comparison to the intact controls; this increase was significantly greater in the hyper-injury group. Both hypo- and hyperthyroid intact animals also had greater cellular infiltration in solear muscles than the control animals (Fig. 2a). Interestingly, the increase in cellular infiltration caused by thyroid dysfunction could not be attributed to macrophage infiltration, since the ED1 staining revealed that macrophage accumulation only occurred in the acutely crushed soleus muscle (Fig. 2b). Solear Ras expression was not affected by thyroidal status per se, but injury in hypothyroid or hyperthyroid rats was able to significantly increase Ras expression, especially in hyperthyroid animals (Fig. 3). Injury increased ERK phosphorylation in the muscle of control and hypothyroid animals, but this effect was not detected in hyperthyroid animals, suggesting that the high serum T3 levels of hyperthyroid animals increase ERK phosphorylation (Fig. 4). Injury increased MyoD expression, a typical muscle transcription factor, activated in the early stages of skeletal muscle differentiation at mRNA level (Fig. 5a) as well as at protein level in all groups studied (Fig. 5b). The inductions of hypo- and hyperthyroidism per se increased MyoD expression only at protein level. Injury also increased myogenin expression both in hypo- and
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Fig. 1 Total serum T3 (a), total serum TSH (b), total serum T4 (c), and total serum leptin (d) concentrations in control, control ? injury, hypothyroid, hypothyroid ? injury, hyperthyroid, and hyperthyroid ? injury
rats. #p \ 0.05 versus control and hypothyroid animals injured or not. *p \ 0.05 versus control and hyperthyroid animals injured or not
hyperthyroid animals at both mRNA (Fig. 6a) and protein (Fig. 6b) levels. Thyroidal status did not alter myogenin solear response to injury neither at mRNA nor protein levels (Figs. 6a, b). Concerning mRNA expression, solear thyroid hormone metabolism seems to be modulated by lesion. Type 2 and type 3 iodothyronines deiodinases mRNAs were positively regulated by injury, while type 1 iodothyronine deiodinase mRNA seems to be negatively regulated, at least in control and hyperthyroid animals after injury (Fig. 7). As expected, increased T3 serum present in hyperthyroid animals positively regulates type 1 activity (Fig. 7).
increases in both cell and macrophage infiltration after injury, independent of thyroidal status. Hyperthyroidism seems to potentiate both cellular and macrophage infiltration: the maximal cellular and macrophage infiltration were detected in the solear muscles of hyperthyroid-injury animals. Furthermore, the number of resident macrophages in the uninjured muscle did not change in either hypo- or hyperthyroidism. There are few data demonstrating the involvement of cytokines in muscle regeneration. Pedersen and Febbraio [20] demonstrated that skeletal muscle contraction leads to secretion of cytokines that can influence the metabolism of other tissues or organs. These authors have shown that IL-6 increases in muscle during exercise, and it is noteworthy that this response is directly linked to muscle damage and regeneration. The active thyroid hormone, T3, derives either from the thyroid gland directly or from the 50 -monodeiodination of T4. Two iodothyronines deiodinases can catalyze this reaction (D1 and D2). In skeletal muscle, D2 expression can be modulated and seems to be required for normal myogenesis and during muscle regeneration [21]. After skeletal muscle injury in vivo, there is a marked increase in D2 activity [22];
Discussion Monocyte and macrophage depletion induced by clodronate liposome treatment is associated with an impaired repair process after skeletal muscle injury [8]. In fact, the regenerative process and the recovery of muscle function are associated with macrophage infiltration and chemokine response in injured skeletal muscles [19]. Our data in solear skeletal muscles support these findings, since we detected
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Fig. 4 Densitometric analysis: pERK expression, 3 days after crush injury, in soleus muscles of control, hypothyroid, and hyperthyroid rats. n = 3 animals/group. *p \ 0.05 versus paired controls. # p \ 0.05 versus intact control and hypothyroid animals
Fig. 2 Histomorphometric analysis of infiltrating cells (a) and percentage of area occupied by macrophages (ED1) (b), 3 days after crush injury, in soleus muscles of control, hypothyroid, and hyperthyroid rats. n C 7 animals/group. *p \ 0.05 versus paired controls. # p \ 0.05 versus intact control animals
Fig. 3 Densitometric analysis of Ras expression, 3 days after crush injury, in soleus muscles of control, hypothyroid, and hyperthyroid rats. n = 3 animals/group. *p \ 0.05 versus paired controls
this D2 upregulation suggests an important role of T3 during this process. D3 repression was also reported in C2C12 cells [23]. Recently, Salvatore et al. [24] proposed a sequential
Fig. 5 Densitometric analysis: expression of mRNA (a) and protein (b) of MyoD, 3 days after crush injury, in soleus muscles of control, hypothyroid, and hyperthyroid rats. n = 4 animals/group. *p \ 0.05 versus paired controls. #p \ 0.05 versus intact control animals
expression of D3 followed by D2 in order to tightly control intracellular T3 levels during myogenesis. D2 highly expressed in myoblast precursor cells is important to
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Fig. 6 Densitometric analysis: expression of mRNA (a) and protein (b) of myogenin, 3 days after crush injury, in soleus muscles of control, hypothyroid, and hyperthyroid rats. n = 3 animals/group. *p \ 0.05 versus paired controls
increase intracellular T3 and induce Myo D expression. Here, we detected an important increase in both type 2 and type 3 iodothyronine deiodinase expression after injury, in agreement with the importance of local thyroid hormone availability during solear regenerating process (Fig. 7), and we do attribute this to the presence of myoblasts in different stages of differentiation and so requiring different amounts of T3, in solear samples studied. In fact, Rozing et al. [25], associated high free iodothyronines levels to a higher production capacity of proinflammatory cytokines; our results are in agreement with this idea, since the higher levels of both cellular and macrophage infiltration were detected in the hyper-injury animals. Ras–MAPK pathway modulation by skeletal muscle injury has already been proposed both in vitro [26] and in vivo [27]. Induction of the Ras–MAPK pathway by nerve activity in the regenerating soleus muscle was previously demonstrated [27], indicating that Ras–MAPK signaling is involved in the promotion of nerve-activity-dependent differentiation of slow muscle fibers in vivo. Our data showed increased Ras–pERK expressions in control and hypothyroid animals submitted to injury, but in hyperthyroid animals, ERK phosphorylation is already higher, and no further
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Fig. 7 Densitometric analysis: mRNA expression of type 1 (a), type 2 (b), and type 3 (c) iodothyronines deiodinases, 3 days after crush injury, in soleus muscles of control, hypothyroid, and hyperthyroid rats. n = 3 animals/group. *p \ 0.05 versus paired controls. # p \ 0.05 versus intact control animals
increase could be detected in hyperthyroid-injury animals, suggesting that in solear muscles from the hyper-injury group, Ras protein maybe signaling downstream by another pathway instead of pERK. Activation of the Ras–MAPK pathway is able to control myogenesis by still poorly understood mechanisms [12]. Perry et al. [13] studying the mechanisms by which MAPK signaling pathway regulates the transcription factor MyoD showed that MEK1 negatively regulates the activity of MyoD by binding of activated MEK1 to the nuclear MyoD transcription complex. In this study, we detected an increase in MyoD expression after injury in all the studied
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groups, but without any clear relation with pMEK expression (data not shown). Increase in MyoD expression after injury indicates that this myogenic regulatory factor plays an important role after acute injury. Myogenin expression is frequently associated with terminal muscle differentiation; nevertheless, we detected an important increase in this other myogenic regulatory factor as early as 3 days after injury, but without any relation to the thyroid status. Skeletal muscle has a remarkable ability to adapt to physiological demands such as growth and exercise. Regular endurance exercise enhances skeletal muscle interleukin-15-mediating muscle adaptation during training [28]. In fact, exercise and nutrition interventions, such as alkali and vitamin D supplementation, were suggested to promote skeletal mass muscle preservation, frequently reduced during aging process [29–31]. Regeneration is a sequential process involving degeneration, proliferation of satellite cells, differentiation and fusion of myoblasts, and growth and maturation of myofibers. The involvement of different cell types, such as inflammatory cells and muscle precursor cells, is well established, but recently, Cianferotti and Brandi [32] pointed out that also bone tissue is able to modulate muscle metabolism, and possibly may be involved in regeneration process after muscle injury. Herein, we demonstrate that thyroid hormones are involved in skeletal muscle regeneration, possibly by regulating inflammatory process, Ras– MAPK pathway, MyoD, and myogenin expression in the injured tissue. Acknowledgments The authors gratefully acknowledge the technical assistance of Advaldo Nunes Bezerra, Jose´ Humberto Tavares de Abreu, Norma Lima de Arau´jo Faria, and Wagner Nunes Bezerra. This work was supported by the grants from Fundac¸a˜o Carlos Chagas Filho de Amparo ‘a Pesquisa do Estado do Rio de Janeiro (FAPERJ), and Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq). Conflict of interest
Authors declare no conflict of interest.
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