Eur Arch Otorhinolaryngol (2015) 272:137–141 DOI 10.1007/s00405-014-3226-9
LARYNGOLOGY
Expression of atrophy-related transcription factors in the process of intrinsic laryngeal muscle atrophy after denervation Hirofumi Sei • Aki Taguchi • Naoya Nishida Naohito Hato • Kiyofumi Gyo
•
Received: 10 June 2014 / Accepted: 29 July 2014 / Published online: 7 August 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract We examined changes in the expressions of three atrophy-related transcription factors (FOXO3a, P-FOXO3a, and PGC-1a) in the process of intrinsic laryngeal muscle atrophy after denervation. In total, 51 Wistar rats were used. After transection of the unilateral recurrent laryngeal nerve, the thyroarytenoid (TA) muscle and the posterior cricoarytenoid (PCA) muscle were excised and subjected to histological and Western blot studies. Relationships between the expressions of transcription factors during atrophy of the intrinsic laryngeal muscles were investigated by comparing the results of the treated side (T) with those of the untreated side (U), and sequential changes in the T/U ratio after denervation were assessed. Loss of wet muscle weight, together with a decrease in muscle fiber cross-sectional area and increase in the number of muscle fibers/mm2, occurred more quickly in TA muscle than in PCA muscle. Muscle atrophy progressed rapidly between 7 and 28 days after denervation, while expression of FOXO3a was maximal on day 7, in both TA and PCA muscles. By contrast, P-FOXO3a expression decreased gradually after denervation. Expression of PGC1a increased slowly until day 7, and then it declined. Denervation-induced atrophy of the intrinsic laryngeal muscles was closely linked with the expression of FOXO3a and PGC-1a, suggesting that atrophy of these muscles may involve the actions of these transcription factors. In
H. Sei (&) A. Taguchi N. Nishida N. Hato Department of Otolaryngology, Ehime University School of Medicine, Shitsukawa, Toon, Ehime 91-0295, Japan e-mail: [email protected] K. Gyo Department of Otolaryngology, Takanoko Hospital, Takanoko Town 525-1, Matsuyama, Ehime 790-0925, Japan
addition, muscle atrophy progressed faster in TA muscle than in PCA muscle, due mainly to differences in muscle fiber composition. Keywords Laryngeal muscle atrophy Intrinsic laryngeal muscle FOXO3a Phosphorylated FOXO3a PGC-1a
Introduction The intrinsic laryngeal muscles consist of five muscles, including the thyroarytenoid (TA) and posterior cricoarytenoid (PCA) muscles, and are innervated by the recurrent laryngeal nerve, except for the cricothyroid muscle. If this nerve is resected, the muscles undergo atrophy, resulting in breathy or aphonic hoarseness. Muscle atrophy due to denervation differs by fiber type, because they have different contractile and metabolic properties. Oxidative muscles are mainly composed of slow-twitch fibers and generally more resistant to atrophy than glycolytic muscles, which are mainly composed of fast-twitch fibers [1]. Given that the TA muscle is solely composed of fast-twitch fibers and the PCA muscle is composed of a combination of fastand slow-twitch fibers, denervation-induced atrophy may differ somewhat between the muscles. Proteolysis of skeletal muscle after denervation is controlled by the transcription factors that regulate atrogenes [1–3]. Recently, atrophy-related transcription factors such as Forkhead box O3a (FOXO3a), phosphorylated FOXO3a (P-FOXO3a), and peroxisome coactivator-1a (PGC-1a) have been shown to play important roles in the atrophy program necessary for the expression of the rate-limiting enzymes of the ubiquitin–proteasome and autophagy– lysosome systems [4]. Presently, the roles of these transcription factors in the atrophy process of the intrinsic
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laryngeal muscles remain unclear. In the present study, we assessed the relationships between the expression of atrophy-related transcription factors and the progression of intrinsic laryngeal muscle atrophy after transection of the unilateral recurrent laryngeal nerve.
Eur Arch Otorhinolaryngol (2015) 272:137–141
measuring the MFCSA and for counting the numbers of muscle fibers/mm2. Muscle atrophy was assessed by comparing the results of the treated (T) and untreated (U) sides using NIH ImageJ software. Sequential changes in the T/U ratio were investigated. Western blot analysis
Materials and methods Animal model This study was conducted with the approval of the Ethics Committee of Ehime University Graduate School of Medicine, following the guidelines for animal experimentation at our institute. All efforts were made to minimize the number of animals and suffering following the experimental protocol. Fifty-one male Wistar rats (weighing 210–250 g, 9- to 10-week old) were used. They were housed in an animal room at a temperature of 21–23 °C and a 12/12-h light/dark cycle (lights on 7:00 a.m. to 7:00 p.m.). The animals were allowed free access to food and water until the end of the experiment. Each animal was anesthetized with an intramuscular injection of ketamine 150 mg/kg, and fixed on an experimental table in a supine position. After skin incision at the neck, one side of the recurrent laryngeal nerve was transected at the level of the seventh tracheal ring. The distal and proximal ends of the nerve were ligatured, and the latter was embedded in the sternocleidomastoid muscle to prevent any future neural contact between the cut ends. Following these procedures, the animals were returned to the animal room. Animals were killed using an overdose of ketamine administered intraperitoneally before and 1, 4, 7, 14, 28, 56, and 84 days after denervation. The TA and PCA muscles were excised bilaterally for histological and Western blot studies. The wet weight of each muscle was measured immediately after excision.
Each pair of muscles was homogenized on ice in buffer solution [Tris–HCl, NaCl, 1 % Triton X-100, Na2EDTAH2O and protease inhibitor cocktail (Sigma-Aldrich Japan, Tokyo, Japan)]. Following homogenization, the concentrations of protein in the samples were measured using a BCA protein assay kit (Pierce, Pittsburgh, PA, USA). Then, 20 lg/lane of protein was loaded onto 7.5 % SDS–polyacrylamide gels for electrophoresis. Separated proteins were transferred to PVDF membranes. The primary antibodies used were anti-FOXO3a (#ab47285, dilution 1:500; Abcam, Tokyo, Japan), anti-P-FOXO3a (#9466, dilution 1:500; Cell Signaling Technology Japan, Tokyo, Japan), and anti-PGC-1a (#AF1817a, dilution 1:500; Abgent, San Diego, CA, USA). All membranes were blocked with 5 % non-fat dry milk in Tris-buffered saline with 0.1 % Tween (TBST). The membranes were incubated overnight with the respective antibody. After a third serial wash with TBST, they were incubated with a horseradish peroxidase-conjugated secondary antibody (anti-rabbit immunoglobulin; IgG; dilution 1:5,000; Vector, Burlingame, CA, USA) in blocking buffer for 1 h, followed by another wash in TBST. Immunocomplexes were visualized using enhanced chemiluminescence reagents (ECL plus western blotting detection reagents; GE Healthcare, Tokyo, Japan). Finally, enhanced signals were detected using an ImageQuant LAS4000 (GE Healthcare) and analyzed with ImageQuant TL software (GE Healthcare). As in assessing muscle atrophy, the expression of atrophy-related transcription factors was evaluated by comparing the results of the T and U sides. The sequential changes in the T/U ratio were also investigated.
Histological study Statistical analyses The severity of muscle atrophy was evaluated not only by measuring wet muscle weight, but also by assessing muscle fiber cross-sectional area (MFCSA) and by counting the number of muscle fibers/mm2. Muscle specimens were embedded in OCT compound (#4583; Sakura Finetek; USA), snap-frozen in liquid nitrogen, and stored at -80 °C. They were cut into 10-lm sections in the coronal plane using a cryostat (CM1900; Leica; Wetzlar, Germany). Then they were stained with hematoxylin and eosin, and observed under a microscope (BZ9000 Biorevo; Keyence Japan). Muscle samples from three different sections were examined and the mean values were used for
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All data are presented as mean ± SD. An unpaired Student’s t test was used for the statistical analyses. Differences were considered to be statistically significant at P \ 0.05.
Results The T/U ratios of weight loss in TA and PCA muscles decreased gradually after denervation. As shown in Fig. 1a, loss of muscle weight was faster in TA muscle than in PCA
Eur Arch Otorhinolaryngol (2015) 272:137–141
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Fig. 2 Sequential changes in expression of atrophy-related transcription factors after denervation. a FOXO3a, b P-FOXO3a, and c PGC1a Fig. 1 Sequential changes in intrinsic laryngeal muscle atrophy after denervation. a Wet muscle weight, b muscle fiber cross-sectional area (MFCA), and c number of muscle fibers/mm2
muscle. Until day 4, no substantial difference in the progression of muscle atrophy could be seen between the muscles; however, on days 7 and 14, loss of muscle weight was more severe in TA muscle than in PCA muscle (P \ 0.05 and P \ 0.01, respectively). Thereafter, the severity of atrophy did not differ between the two muscles. Sequential changes in muscle fiber cross-sectional area (MFCA) were the same as those of wet muscle weight: atrophy of the TA muscle was faster than that of the PCA muscle on days 14 and 28 (Fig. 1b). Figure 1c shows the sequential changes in T/U ratios concerning the number of muscle fibers/mm2. The value increased gradually after denervation, in accordance with the progression of muscle fiber atrophy. As with the other indices, the progression of
muscle atrophy was faster in TA muscle than in PCA muscle. Figure 2a shows the sequential changes in the T/U ratios of FOXO3a expression in both the TA and PCA muscles. They increased gradually until day 7, declined on day 14, and then became stable. Note that the increase in FOXO3a expression was remarkable on day 7 in both muscles; the increase in the T/U ratio was greater in the TA muscle than in the PCA muscle (P \ 0.01). By contrast, P-FOXO3a expression decreased gradually with time after denervation: the difference was significant on day 7 and after (Fig. 2b). The decrease in P-FOXO3a expression did not differ between the two muscles, except on day 84. PGC-1a expression also increased with time until day 7; then it decreased in both TA and PCA muscles (Fig. 2c). No statistically significant difference in
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Eur Arch Otorhinolaryngol (2015) 272:137–141
the T/U ratio of expression was seen between the muscle types.
Discussion As shown in this study, muscle atrophy progressed more rapidly in TA muscle than in PCA muscle, as assessed using three indices: loss of wet muscle weight; decrease in MFCSA; and increase in the number of muscle fibers/mm2. This was likely because TA muscle is composed solely of fast-twitch fibers (type II fibers), which are more susceptible to denervation than PCA muscle. According to Shiotani et al. [5], the myosin heavy chain composition in TA muscle is 67 % type II B fibers and 33 % type II L fibers, whereas that of PCA muscle is 6.5 % type I fibers (slow-twitch fibers), 12 % type II A, 26.8 % type II X, 46.3 % type II B, and 8.3 % type II L in adult rats. Nishida et al. [6] reported similar myosin heavy chain compositions in TA and PCA muscles. In Files et al. [7], PCA muscle was spared from muscle atrophy in an animal model of acute respiratory distress syndrome, because the muscle lacked E3 ubiquitin ligases such as muscle ring finger-1 (MuRF1) and atrogin-1, both of which were identified following transcript profiling in fasting and immobilization models of muscle atrophy. Other characteristics of PCA muscle, including its neuromuscular junction structure or satellite cell regenerative potential, may also contribute to its protection from atrophy [8, 9]. In this study, atrophy of the intrinsic laryngeal muscle progressed rapidly between 7 and 28 days after denervation. Then it gradually subsided. Similar results have been reported in other skeletal muscles after denervation. Sacheck et al. [10] reported that atrophy of the gastrocnemius muscle occurred maximally at around 14 days after denervation in an experiment in rats. Goldspink et al. [11] investigated the denervation-induced atrophy of the soleus and the extensor digitorum longus muscles in the rat, and reported that it began 5–7 days after resection of the sciatic nerve and peaked on day 14. These findings suggest that the progression of denervation-induced atrophy in these muscles does not greatly differ from that in the intrinsic laryngeal muscles. The underlying mechanisms of skeletal muscle atrophy are complex. FOXO3a has been implicated as a major mediator of muscle atrophy, but how its subcellular location and activity are controlled during the progression of muscle atrophy remains unclear. Wei et al. [12] reported that the process of denervation-induced atrophy was maximal on day 7. In our study, the FOXO3a expression was maximal on day 7, in both the TA and PCA muscles, when the muscle atrophy began to progress rapidly. This might indicate that FOXO3a expression preceded the progression
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Fig. 3 Molecular mechanisms of muscle atrophy after denervation (modified from Sandri et al. [3])
of muscle atrophy. Figure 3 shows the mechanisms of atrophy-related transcription factors in the process of the skeletal muscle atrophy [3]. When the denervation signal is received by the cell membrane of the atrophying muscle, P-FOXO3a in the cytoplasm is converted into FOXO3a and phosphate by activation of the AKT/PKB pathway. Then FOXO3a enters the nucleus and binds to DNA, which then facilitates degradation of intracellular proteins, including myofibrillar proteins, by transcription of ubiquitin ligaserelated genes such as muscle atrophy F-box (MAFbx)/ Atrogin-1 and muscle ring finger-1 (MuRF1), resulting in a subsequent dramatic loss of muscle mass [13]. Another transcription factor that plays an essential role in muscle atrophy is NF-jB [8, 9]. Although activation of the NF-jB pathway is apparently sufficient to induce muscle atrophy, its precise role and the factors that control its activity in muscle are still poorly understood. By contrast, PGC-1a protects against muscle atrophy by downregulating FOXO3a transcription via inhibiting the AKT pathway [2, 13], although the effect is limited in denervation-induced atrophy. As shown in the present study, PGC-1a expression increased gradually after denervation, peaked on day 7, and then decreased. According to Sandri et al. [13], PGC-1a protects against skeletal muscle atrophy by suppressing the actions of FOXO3, which stimulates transcription of atrophy-specific genes. We also found that elevated PGC-1a prevented proteolysis via induction of autophagy and atrophy-specific ubiquitin ligases. As summarized by Brault et al. [14], PGC-1a overexpression inhibits denervation atrophy of the skeletal muscles by suppressing ubiquitin ligase induction and NF-jB transcription. By analyzing the molecular background of muscle atrophy, it is possible to address clinical issues such as prevention or treatment of the atrophy of intrinsic laryngeal
Eur Arch Otorhinolaryngol (2015) 272:137–141
muscles. Since blocking FOXO3a or stimulating PGC-1a might prevent the progression of muscle atrophy, we are now investigating the effects of bortezomib, a selective inhibitor of proteasome in the ubiquitin–proteasome pathway, for the treatment of muscle atrophy.
Conclusions Denervation-induced intrinsic laryngeal muscle atrophy is closely related to the expression of FOXO3a and PGC-1a. Loss of wet muscle weight, together with other muscle atrophy indices, was preceded by expression of FOXO3a, suggesting that FOXO3a may control the process of atrophy in the TA and PCA muscles. The present study also showed that atrophy of the TA muscle was faster than that of the PCA muscle, likely because the former is composed solely of fast-twitch fibers. Conflict of interest The authors disclose no financial or other conflict of interest. The authors are solely responsible for the content and writing of the paper.
References 1. Okamoto T, Torii S, Machida S (2011) Differential gene expression of muscle-specific ubiquitin ligase MAFbx/Atrogin-1 and MuRF1 in response to immobilization-induced atrophy of slow-twitch and fast-twitch muscles. J Physiol Sci 61:537–546. doi:10.1007/s12576-011-0175-6 2. Stitt TN, Drujan D, Clarke BA, Panaro F, Timofeyva Y, Kline WO, Gonzalez M, Yancopoulos GD, Glass DJ (2004) The IGF-1/ PI3K/Akt pathway prevents expression of muscle atrophyinduced ubiquitin ligases by inhibiting FOXO transcription factors. Mol Cell 14:395–403 3. Sandri M, Sandri C, Gilbert A, Skurk C, Calabria E, Picard A, Walsh K, Schiaffino S, Lecker SH, Goldberg AL (2004) Foxo transcription factors induce the atrophy-related ubiquitin ligase atrogin-1 and cause skeletal muscle atrophy. Cell 117:399–412
141 4. Bertaggia E, Coletto L, Sandri M (2012) Posttranslational modifications control FoxO3 activity during denervation. Am J Physiol Cell Physiol 302:C587–C596. doi:10.1152/ajpcell.00142. 2011 5. Shiotani A, Flint W (1998) Myosin heavy chain composition in rat laryngeal muscles after denervation. Laryngoscope 108:1225– 1229 6. Nishida N, Taguchi A, Motoyoshi K, Hyodo M, Gyo K, Desaki J (2013) Age-related changes in rat intrinsic laryngeal muscles: analysis of muscle fibers, muscle fiber proteins, and subneural apparatuses. Eur Arch Otorhinolaryngol 270:975–984. doi:10. 1007/s00405-012-2231-0 7. Files DC, Xiao K, Zhang T, Liu C, Qian J, Zhao W, Morris PE, Delbono O, Feng X (2014) The posterior cricoarytenoid muscle is spared from MuRF1-mediated muscle atrophy in mice with acute lung injury. PLoS ONE 31(9):e87587. doi:10.1371/journal.pone. 0087587 8. Feng X, Zhang T, Ralston E, Ludlow CL (2012) Differences in neuromuscular junctions of laryngeal and limb muscles in rats. Laryngoscope 122:1093–1098. doi:10.1002/lary.23218 9. Donghui C, Shicai C, Wei W, Fei L, Jianjun J, Gang C, Hongliang Z (2010) Functional modulation of satellite cells in longterm denervated human laryngeal muscle. Laryngoscope 120:353–358. doi:10.1002/lary.20796 10. Sacheck JM, Hyatt JK, Raffaello A, Jagoe RT, Roy RR, Edgerton VR, Lecker SH, Goldberg AL (2007) Rapid disuse and denervation atrophy involve transcriptional changes similar to those of muscle wasting during systemic diseases. FASEB J 21:140–155 11. Goldspink DF (1976) The effects of denervation on protein turnover of rat skeletal muscle. Biochem J 15(156):71–80 12. Wei B, Dui W, Liu D, Xing Y, Yuan Z, Ji G (2013) MST1, a key player, in enhancing fast skeletal muscle atrophy. BMC Biol 1(11):12. doi:10.1186/1741-7007-11-12 13. Sandri M, Lin J, Handschin C, Yang W, Arany ZP, Lecker SH, Goldberg AL, Spiegelman BM (2006) PGC-1alpha protects skeletal muscle from atrophy by suppressing FoxO3 action and atrophy-specific gene transcription. Proc Natl Acad Sci USA 103:16260–16265 14. Brault JJ, Jespersen JG, Goldberg AL (2010) Peroxisome proliferator-activated receptor c coactivator 1a or 1b overexpression inhibits muscle protein degradation, induction of ubiquitin ligases, and disuse atrophy. J Biol Chem 285(25):19460–19471. doi:10.1074/jbc.M110.113092
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LARYNGOLOGY
Expression of atrophy-related transcription factors in the process of intrinsic laryngeal muscle atrophy after denervation Hirofumi Sei • Aki Taguchi • Naoya Nishida Naohito Hato • Kiyofumi Gyo
•
Received: 10 June 2014 / Accepted: 29 July 2014 / Published online: 7 August 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract We examined changes in the expressions of three atrophy-related transcription factors (FOXO3a, P-FOXO3a, and PGC-1a) in the process of intrinsic laryngeal muscle atrophy after denervation. In total, 51 Wistar rats were used. After transection of the unilateral recurrent laryngeal nerve, the thyroarytenoid (TA) muscle and the posterior cricoarytenoid (PCA) muscle were excised and subjected to histological and Western blot studies. Relationships between the expressions of transcription factors during atrophy of the intrinsic laryngeal muscles were investigated by comparing the results of the treated side (T) with those of the untreated side (U), and sequential changes in the T/U ratio after denervation were assessed. Loss of wet muscle weight, together with a decrease in muscle fiber cross-sectional area and increase in the number of muscle fibers/mm2, occurred more quickly in TA muscle than in PCA muscle. Muscle atrophy progressed rapidly between 7 and 28 days after denervation, while expression of FOXO3a was maximal on day 7, in both TA and PCA muscles. By contrast, P-FOXO3a expression decreased gradually after denervation. Expression of PGC1a increased slowly until day 7, and then it declined. Denervation-induced atrophy of the intrinsic laryngeal muscles was closely linked with the expression of FOXO3a and PGC-1a, suggesting that atrophy of these muscles may involve the actions of these transcription factors. In
H. Sei (&) A. Taguchi N. Nishida N. Hato Department of Otolaryngology, Ehime University School of Medicine, Shitsukawa, Toon, Ehime 91-0295, Japan e-mail: [email protected] K. Gyo Department of Otolaryngology, Takanoko Hospital, Takanoko Town 525-1, Matsuyama, Ehime 790-0925, Japan
addition, muscle atrophy progressed faster in TA muscle than in PCA muscle, due mainly to differences in muscle fiber composition. Keywords Laryngeal muscle atrophy Intrinsic laryngeal muscle FOXO3a Phosphorylated FOXO3a PGC-1a
Introduction The intrinsic laryngeal muscles consist of five muscles, including the thyroarytenoid (TA) and posterior cricoarytenoid (PCA) muscles, and are innervated by the recurrent laryngeal nerve, except for the cricothyroid muscle. If this nerve is resected, the muscles undergo atrophy, resulting in breathy or aphonic hoarseness. Muscle atrophy due to denervation differs by fiber type, because they have different contractile and metabolic properties. Oxidative muscles are mainly composed of slow-twitch fibers and generally more resistant to atrophy than glycolytic muscles, which are mainly composed of fast-twitch fibers [1]. Given that the TA muscle is solely composed of fast-twitch fibers and the PCA muscle is composed of a combination of fastand slow-twitch fibers, denervation-induced atrophy may differ somewhat between the muscles. Proteolysis of skeletal muscle after denervation is controlled by the transcription factors that regulate atrogenes [1–3]. Recently, atrophy-related transcription factors such as Forkhead box O3a (FOXO3a), phosphorylated FOXO3a (P-FOXO3a), and peroxisome coactivator-1a (PGC-1a) have been shown to play important roles in the atrophy program necessary for the expression of the rate-limiting enzymes of the ubiquitin–proteasome and autophagy– lysosome systems [4]. Presently, the roles of these transcription factors in the atrophy process of the intrinsic
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laryngeal muscles remain unclear. In the present study, we assessed the relationships between the expression of atrophy-related transcription factors and the progression of intrinsic laryngeal muscle atrophy after transection of the unilateral recurrent laryngeal nerve.
Eur Arch Otorhinolaryngol (2015) 272:137–141
measuring the MFCSA and for counting the numbers of muscle fibers/mm2. Muscle atrophy was assessed by comparing the results of the treated (T) and untreated (U) sides using NIH ImageJ software. Sequential changes in the T/U ratio were investigated. Western blot analysis
Materials and methods Animal model This study was conducted with the approval of the Ethics Committee of Ehime University Graduate School of Medicine, following the guidelines for animal experimentation at our institute. All efforts were made to minimize the number of animals and suffering following the experimental protocol. Fifty-one male Wistar rats (weighing 210–250 g, 9- to 10-week old) were used. They were housed in an animal room at a temperature of 21–23 °C and a 12/12-h light/dark cycle (lights on 7:00 a.m. to 7:00 p.m.). The animals were allowed free access to food and water until the end of the experiment. Each animal was anesthetized with an intramuscular injection of ketamine 150 mg/kg, and fixed on an experimental table in a supine position. After skin incision at the neck, one side of the recurrent laryngeal nerve was transected at the level of the seventh tracheal ring. The distal and proximal ends of the nerve were ligatured, and the latter was embedded in the sternocleidomastoid muscle to prevent any future neural contact between the cut ends. Following these procedures, the animals were returned to the animal room. Animals were killed using an overdose of ketamine administered intraperitoneally before and 1, 4, 7, 14, 28, 56, and 84 days after denervation. The TA and PCA muscles were excised bilaterally for histological and Western blot studies. The wet weight of each muscle was measured immediately after excision.
Each pair of muscles was homogenized on ice in buffer solution [Tris–HCl, NaCl, 1 % Triton X-100, Na2EDTAH2O and protease inhibitor cocktail (Sigma-Aldrich Japan, Tokyo, Japan)]. Following homogenization, the concentrations of protein in the samples were measured using a BCA protein assay kit (Pierce, Pittsburgh, PA, USA). Then, 20 lg/lane of protein was loaded onto 7.5 % SDS–polyacrylamide gels for electrophoresis. Separated proteins were transferred to PVDF membranes. The primary antibodies used were anti-FOXO3a (#ab47285, dilution 1:500; Abcam, Tokyo, Japan), anti-P-FOXO3a (#9466, dilution 1:500; Cell Signaling Technology Japan, Tokyo, Japan), and anti-PGC-1a (#AF1817a, dilution 1:500; Abgent, San Diego, CA, USA). All membranes were blocked with 5 % non-fat dry milk in Tris-buffered saline with 0.1 % Tween (TBST). The membranes were incubated overnight with the respective antibody. After a third serial wash with TBST, they were incubated with a horseradish peroxidase-conjugated secondary antibody (anti-rabbit immunoglobulin; IgG; dilution 1:5,000; Vector, Burlingame, CA, USA) in blocking buffer for 1 h, followed by another wash in TBST. Immunocomplexes were visualized using enhanced chemiluminescence reagents (ECL plus western blotting detection reagents; GE Healthcare, Tokyo, Japan). Finally, enhanced signals were detected using an ImageQuant LAS4000 (GE Healthcare) and analyzed with ImageQuant TL software (GE Healthcare). As in assessing muscle atrophy, the expression of atrophy-related transcription factors was evaluated by comparing the results of the T and U sides. The sequential changes in the T/U ratio were also investigated.
Histological study Statistical analyses The severity of muscle atrophy was evaluated not only by measuring wet muscle weight, but also by assessing muscle fiber cross-sectional area (MFCSA) and by counting the number of muscle fibers/mm2. Muscle specimens were embedded in OCT compound (#4583; Sakura Finetek; USA), snap-frozen in liquid nitrogen, and stored at -80 °C. They were cut into 10-lm sections in the coronal plane using a cryostat (CM1900; Leica; Wetzlar, Germany). Then they were stained with hematoxylin and eosin, and observed under a microscope (BZ9000 Biorevo; Keyence Japan). Muscle samples from three different sections were examined and the mean values were used for
123
All data are presented as mean ± SD. An unpaired Student’s t test was used for the statistical analyses. Differences were considered to be statistically significant at P \ 0.05.
Results The T/U ratios of weight loss in TA and PCA muscles decreased gradually after denervation. As shown in Fig. 1a, loss of muscle weight was faster in TA muscle than in PCA
Eur Arch Otorhinolaryngol (2015) 272:137–141
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Fig. 2 Sequential changes in expression of atrophy-related transcription factors after denervation. a FOXO3a, b P-FOXO3a, and c PGC1a Fig. 1 Sequential changes in intrinsic laryngeal muscle atrophy after denervation. a Wet muscle weight, b muscle fiber cross-sectional area (MFCA), and c number of muscle fibers/mm2
muscle. Until day 4, no substantial difference in the progression of muscle atrophy could be seen between the muscles; however, on days 7 and 14, loss of muscle weight was more severe in TA muscle than in PCA muscle (P \ 0.05 and P \ 0.01, respectively). Thereafter, the severity of atrophy did not differ between the two muscles. Sequential changes in muscle fiber cross-sectional area (MFCA) were the same as those of wet muscle weight: atrophy of the TA muscle was faster than that of the PCA muscle on days 14 and 28 (Fig. 1b). Figure 1c shows the sequential changes in T/U ratios concerning the number of muscle fibers/mm2. The value increased gradually after denervation, in accordance with the progression of muscle fiber atrophy. As with the other indices, the progression of
muscle atrophy was faster in TA muscle than in PCA muscle. Figure 2a shows the sequential changes in the T/U ratios of FOXO3a expression in both the TA and PCA muscles. They increased gradually until day 7, declined on day 14, and then became stable. Note that the increase in FOXO3a expression was remarkable on day 7 in both muscles; the increase in the T/U ratio was greater in the TA muscle than in the PCA muscle (P \ 0.01). By contrast, P-FOXO3a expression decreased gradually with time after denervation: the difference was significant on day 7 and after (Fig. 2b). The decrease in P-FOXO3a expression did not differ between the two muscles, except on day 84. PGC-1a expression also increased with time until day 7; then it decreased in both TA and PCA muscles (Fig. 2c). No statistically significant difference in
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Eur Arch Otorhinolaryngol (2015) 272:137–141
the T/U ratio of expression was seen between the muscle types.
Discussion As shown in this study, muscle atrophy progressed more rapidly in TA muscle than in PCA muscle, as assessed using three indices: loss of wet muscle weight; decrease in MFCSA; and increase in the number of muscle fibers/mm2. This was likely because TA muscle is composed solely of fast-twitch fibers (type II fibers), which are more susceptible to denervation than PCA muscle. According to Shiotani et al. [5], the myosin heavy chain composition in TA muscle is 67 % type II B fibers and 33 % type II L fibers, whereas that of PCA muscle is 6.5 % type I fibers (slow-twitch fibers), 12 % type II A, 26.8 % type II X, 46.3 % type II B, and 8.3 % type II L in adult rats. Nishida et al. [6] reported similar myosin heavy chain compositions in TA and PCA muscles. In Files et al. [7], PCA muscle was spared from muscle atrophy in an animal model of acute respiratory distress syndrome, because the muscle lacked E3 ubiquitin ligases such as muscle ring finger-1 (MuRF1) and atrogin-1, both of which were identified following transcript profiling in fasting and immobilization models of muscle atrophy. Other characteristics of PCA muscle, including its neuromuscular junction structure or satellite cell regenerative potential, may also contribute to its protection from atrophy [8, 9]. In this study, atrophy of the intrinsic laryngeal muscle progressed rapidly between 7 and 28 days after denervation. Then it gradually subsided. Similar results have been reported in other skeletal muscles after denervation. Sacheck et al. [10] reported that atrophy of the gastrocnemius muscle occurred maximally at around 14 days after denervation in an experiment in rats. Goldspink et al. [11] investigated the denervation-induced atrophy of the soleus and the extensor digitorum longus muscles in the rat, and reported that it began 5–7 days after resection of the sciatic nerve and peaked on day 14. These findings suggest that the progression of denervation-induced atrophy in these muscles does not greatly differ from that in the intrinsic laryngeal muscles. The underlying mechanisms of skeletal muscle atrophy are complex. FOXO3a has been implicated as a major mediator of muscle atrophy, but how its subcellular location and activity are controlled during the progression of muscle atrophy remains unclear. Wei et al. [12] reported that the process of denervation-induced atrophy was maximal on day 7. In our study, the FOXO3a expression was maximal on day 7, in both the TA and PCA muscles, when the muscle atrophy began to progress rapidly. This might indicate that FOXO3a expression preceded the progression
123
Fig. 3 Molecular mechanisms of muscle atrophy after denervation (modified from Sandri et al. [3])
of muscle atrophy. Figure 3 shows the mechanisms of atrophy-related transcription factors in the process of the skeletal muscle atrophy [3]. When the denervation signal is received by the cell membrane of the atrophying muscle, P-FOXO3a in the cytoplasm is converted into FOXO3a and phosphate by activation of the AKT/PKB pathway. Then FOXO3a enters the nucleus and binds to DNA, which then facilitates degradation of intracellular proteins, including myofibrillar proteins, by transcription of ubiquitin ligaserelated genes such as muscle atrophy F-box (MAFbx)/ Atrogin-1 and muscle ring finger-1 (MuRF1), resulting in a subsequent dramatic loss of muscle mass [13]. Another transcription factor that plays an essential role in muscle atrophy is NF-jB [8, 9]. Although activation of the NF-jB pathway is apparently sufficient to induce muscle atrophy, its precise role and the factors that control its activity in muscle are still poorly understood. By contrast, PGC-1a protects against muscle atrophy by downregulating FOXO3a transcription via inhibiting the AKT pathway [2, 13], although the effect is limited in denervation-induced atrophy. As shown in the present study, PGC-1a expression increased gradually after denervation, peaked on day 7, and then decreased. According to Sandri et al. [13], PGC-1a protects against skeletal muscle atrophy by suppressing the actions of FOXO3, which stimulates transcription of atrophy-specific genes. We also found that elevated PGC-1a prevented proteolysis via induction of autophagy and atrophy-specific ubiquitin ligases. As summarized by Brault et al. [14], PGC-1a overexpression inhibits denervation atrophy of the skeletal muscles by suppressing ubiquitin ligase induction and NF-jB transcription. By analyzing the molecular background of muscle atrophy, it is possible to address clinical issues such as prevention or treatment of the atrophy of intrinsic laryngeal
Eur Arch Otorhinolaryngol (2015) 272:137–141
muscles. Since blocking FOXO3a or stimulating PGC-1a might prevent the progression of muscle atrophy, we are now investigating the effects of bortezomib, a selective inhibitor of proteasome in the ubiquitin–proteasome pathway, for the treatment of muscle atrophy.
Conclusions Denervation-induced intrinsic laryngeal muscle atrophy is closely related to the expression of FOXO3a and PGC-1a. Loss of wet muscle weight, together with other muscle atrophy indices, was preceded by expression of FOXO3a, suggesting that FOXO3a may control the process of atrophy in the TA and PCA muscles. The present study also showed that atrophy of the TA muscle was faster than that of the PCA muscle, likely because the former is composed solely of fast-twitch fibers. Conflict of interest The authors disclose no financial or other conflict of interest. The authors are solely responsible for the content and writing of the paper.
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