Acta Oto-Laryngologica. 2015; 135: 713–717
ORIGINAL ARTICLE
Preventive effects of bortezomib on denervation-induced atrophy of the intrinsic laryngeal muscles: an experimental study in the rat
HIROFUMI SEI1, AKI TAGUCHI2, NAOYA NISHIDA1, NAOHITO HATO1 & KIYOFUMI GYO3 1
Department of Otolaryngology, Ehime University, School of Medicine, Toon-city Shitsukawa, Ehime, Department of Otolaryngology, Matsuyama Red Cross Hospital, Matsuyama-city, Ehime and 3 Department of Otolaryngology, Takanoko Hospital, Matsuyama-city, Ehime, Japan 2
Abstract Conclusion: Bortezomib was effective in attenuating atrophy of the posterior cricoarytenoid (PCA) muscle, but not the thyroarytenoid (TA) muscle. This was probably due to differences in the fiber composition of the two muscles. The PCA muscle is composed of a combination of fast- and slow-twitch fibers, and therefore is more resistant to atrophy than the TA muscle, which is composed solely of fast-twitch fibers. Objectives: To investigate the preventive effects of bortezomib on denervation-induced atrophy of the TA and PCA muscles in the rat. Methods: Following transection of the left recurrent laryngeal nerve, bortezomib (100 mg/kg) was administered subcutaneously on post-denervation days 1 and 4, followed by a 10-day rest period every 14 days; each 2-week period constituted a single treatment cycle. In controls, saline was administered instead. Animals were killed for histological examination at 4 (n = 6), 8 (n = 7), and 12 (n = 7) weeks post-denervation. Muscle atrophy was assessed using three indices: wet muscle weight, muscle fiber cross-sectional area, and the number of muscle fibers/mm2. The effects of bortezomib were evaluated by comparing the left (L) and right (R) muscles, with sequential changes in the L/R ratio assessed. Results: In saline-administered animals, atrophy of the left-sided TA and PCA muscles progressed rapidly during the first 4 weeks post-denervation, following which progression slowed. Atrophy was greater in the TA compared with the PCA muscle, although this difference was not statistically significant. In bortezomib-administered animals, atrophy of the PCA muscle was attenuated significantly at post-denervation weeks 8 and 12; no such reduction in atrophy was observed for the TA muscle.
Keywords: Recurrent laryngeal nerve palsy, intrinsic laryngeal muscle atrophy, prevention of proteolysis, ubiquitin-proteasome pathway, muscle fiber composition
Introduction The recurrent laryngeal nerve innervates all intrinsic laryngeal muscles (except the cricothyroid muscle), controls the production of voice sounds, and regulates respiration and deglutition. When the recurrent laryngeal nerve is severed, atrophy of the intrinsic laryngeal muscles occurs, resulting in breathiness or aphonic hoarseness. Various procedures have been used to treat this condition, such as injection of collagen or fat into the vocal cords [1], laryngeal implantation of
cartilage or Gore-Tex (Gore & Associates, Livingston, UK) [2], adduction of the arytenoid cartilage, and nerve-muscle pedicle grafting [3]. However, all of these treatments require surgical intervention; in patients refusing surgery hoarseness will remain, thereby reducing quality of life. No atrophypreventing medication has been developed for the intrinsic laryngeal muscles. Recently, bortezomib (Velcade or PS-341; Millenium Pharmaceuticals, Cambridge, MA, USA), a dipeptidyl boronic acid, proved effective
Correspondence: Hirofumi Sei, Department of Otolaryngology, Ehime University, School of Medicine, Toon-city Shitsukawa, Ehime 91-0295, Japan. Tel: +81 89 960 5366. Fax: +81 89 960 5368. E-mail: [email protected]
(Received 19 November 2014; accepted 22 December 2014) ISSN 0001-6489 print/ISSN 1651-2251 online 2015 Informa Healthcare DOI: 10.3109/00016489.2015.1006793
714
H. Sei et al.
for attenuating lower-limb muscle atrophy in an experimental animal model [4]. Bortezomib is a selective proteasome inhibitor in the ubiquitin-proteasome pathway, which plays an important role in regulating muscle protein breakdown, including that of the intrinsic laryngeal muscle [4,5]. According to Adams [6], more than 80% of all cellular proteins are processed by the proteasome pathway; the remaining proteins, including cathepsins, calpains, and caspases, are destroyed by the lysosomal system [7]. We investigated whether bortezomib attenuated denervation-induced atrophy of the thyroarytenoid (TA) and posterior cricoarytenoid (PCA) muscles in the rat. The TA is one of several larynx adductor muscles; the PCA is the sole abductor muscle.
Material and methods Animal preparation Following approval from the Ethics Committee of Ehime University Graduate School of Medicine, this study was conducted according to the animal experimentation guidelines of our institution. Forty 9–10-week-old male Wistar rats, weighing between 210 and 250 g, were used. Each animal was anesthetized with an intramuscular injection of ketamine 150 mg/kg, and fixed on an experimental table in the prone position. Following incision of the skin of the neck, the left recurrent laryngeal nerve was transected at the level of the seventh tracheal ring. The proximal end of the nerve was ligatured and embedded in the sternocleidomastoid muscle to prevent future contact between the cut ends. The nerve of the right side remained intact. Animals were divided into bortezomib-treated and non-treated (vehicle control) groups (bortezomib group, n = 20). Following transection of the left recurrent laryngeal nerve, bortezomib (100 mg/kg) was administered subcutaneously on postdenervation days 1 and 4, followed by a 10-day rest period every 14 days; this 2-week period constituted a single treatment cycle. Thereafter, the treatment cycle was repeated until the end of the experiment as per the procedure of Orlowski [8]. The vehicle control group (n = 20) was administered saline at identical intervals. Animals were then returned to the animal room, which was maintained at 21–23 C under a 12/12 h light/dark cycle (lights on between 7:00 a.m. and 7:00 p.m.). Animals were granted access to food and water ad libitum until the experiment ended. During the experiment, every effort was made to minimize the suffering and the number of animals that were used.
Animals were killed at 4 (n = 6), 8 (n = 7), and 12 weeks (n = 7) post-denervation by intraperitoneal injection of an excessive dose of ketamine. The TA and PCA muscles were excised bilaterally, with their wet muscle weights separately measured immediately following excision. The muscle specimens were then embedded in OCT compound (#4583; Sakura Finetek, Torrance, CA, USA), snap-frozen in liquid nitrogen, and stored at –80 C. Histological study For the histological study, the larynx was symmetrically placed on a plate and cut perpendicularly along its longitudinal axis (10 mm thickness) with a cryostat (CM1900; Leica, Wetzlar, Germany). The slices were then stained with hematoxylin and eosin (HE) and observed under a microscope (BZ9000 Biorevo; Keyence, Japan). We measured the muscle fiber cross-sectional area (MFCSA) and the number of muscle fibers/mm2, as described by Kumai et al. [3]. Cross-sectional area was measured by calculating the sum of the muscle fiber areas in a cross-section using the ImageJ software package (NIH, Bethesda, MD, USA). The number of muscle fibers was determined by counting the number of fibers in a 1 1 mm area of a muscle cross-section. Muscle samples from at least three different sections were examined, with the mean values then calculated. The progress of muscle atrophy was evaluated by comparing the muscles of the left (L) and right (R) sides. Sequential changes in the L/R ratio with and without bortezomib treatment were investigated. Statistical analyses All data are presented as means ± SD. An unpaired Student’s t test was used for the statistical analyses. A value of p < 0.05 was taken to indicate statistical significance. Results Figures 1 and 2 depict representative cross-sections of the TA and PCA muscles, respectively, with or without bortezomib treatment, at post-denervation week 12. Bortezomib treatment appeared to attenuate PCA muscle atrophy on the left (denervated) side; TA muscle atrophy continued to progress. Bortezomib did not appear to cause any histological side effects in the muscles of the right (undenervated) side. Sequential changes in denervation-induced muscle atrophy with or without bortezomib treatment are summarized in Figures 3,4,5. In controls, atrophy of the TA and PCA muscles on the left side progressed rapidly during the first 4 weeks after
Bortezomib and intrinsic laryngeal muscle atrophy Left denervated side
Left denervated side
Right side
Bortezomib
Bortezomib
Saline
Saline
Bar =100 µm
715
Right side
Bar =100 µm
Figure 1. Cross-section of the thyroarytenoid (TA) muscle with or without bortezomib administration at post-denervation week 12. HE staining; original magnification 400. Scale bar = 100 mm.
Figure 2. Cross-section of the cricoarytenoid (PCA) muscle with or without bortezomib administration at post-denervation week 12. HE staining; original magnification 400. Scale bar = 100 mm.
denervation; subsequent progress was markedly less rapid. Atrophy was greater in the TA compared with the PCA muscle, although this difference was not significant. Bortezomib administration attenuated atrophy of the PCA muscle but not the TA muscle. Figure 3 describes sequential changes in wet muscle weight. In controls, the L/R ratio of the TA and PCA muscles was 36 ± 5.9% and 39.9 ± 5.6%, respectively, at post-denervation week 12 (n = 7). When treated with bortezomib, L/R ratios were 34.3 ± 3. 6% and 64.9 ± 6.4%, respectively (n = 7). Bortezomib administration significantly attenuated PCA (p < 0.01), but not TA, muscle atrophy (p = 0.12). Similar results were also noted for the MFCSA and number of muscle fibers/mm2.
ubiquitin-proteasome system is a major pathway involved in the break-down of intracellular proteins, including myofibrillar proteins such as actin and the myosin heavy chain [9]. This system targets proteins by attaching at least four copies of ubiquitin, a small polypeptide. Ubiquitination is mediated by a series of enzymes: E1, or ubiquitin-activating enzyme; E2, or ubiquitin-conjugating enzyme; and E3, or ubiquitin ligase. When a substrate is polyubiquitinated, it is targeted for destruction by the proteasome, a large multi-subunit protease complex, which breaks them into short oligopeptides that are degraded rapidly to amino acids by cytosolic peptidases [10]. Therefore, inhibition of the ubiquitin-proteasome system can prevent atrophy of the intrinsic laryngeal muscles following denervation. As demonstrated in the present study, bortezomib was effective in attenuating atrophy of the PCA muscle but not the TA muscle. This latter result was unexpected, and might be explained by the different
Discussion Various mechanisms are involved in the process of muscle protein destruction; the ATP-dependent TA muscle
PCA muscle
(L/R)
(L/R)
1
1
0.8
0.8
0.6
0.6
Control
0.4
0.4
Bortezomib
0.2
0.2
*:p < 0.01
0
0 4w 8w (n = 6) (n = 7)
12 w (n = 7)
4w 8w (n = 6) (n = 7)
12 w (n = 7)
Figure 3. Effects of bortezomib treatment on the left/right (L/R) ratio of wet muscle weight at post-denervation weeks 4, 8, and 12. Bortezomib attenuated denervation-induced atrophy of the cricoarytenoid (PCA) but not the thyroarytenoid (TA) muscle.
716
H. Sei et al. TA muscle
PCA muscle
(L/R)
(L/R)
1
1
0.8
0.8
0.6
0.6
Control
0.4
0.4
Bortezomib
0.2
0.2
*:p < 0.01
0
4w 8w (n = 6) (n = 7)
0
12 w (n = 7)
4w 8w (n = 6) (n = 7)
12 w (n = 7)
Figure 4. Effects of bortezomib treatment on the left/right (L/R) ratio of the muscle fiber cross-sectional area (MFCSA). Bortezomib attenuated the decrease in the MFCSA of the cricoarytenoid (PCA) muscle but not the thyroarytenoid (TA) muscle.
fiber types that make up the two muscles. The TA muscle is composed solely of fast-twitch fibers, whereas the PCA muscle is composed of a combination of fast- and slow-twitch fibers [11]. Because fasttwitch fibers are more fragile, TA muscle proteins atrophied more rapidly compared with the PCA muscle proteins [12] such that bortezomib administration could not prevent TA muscle atrophy. It is also possible that the protein degradation systems of the TA and PCA muscles differ. Muscle protein is destroyed not only by the ubiquitin-proteasome system, but also by the lysosomal system, which involves cathepsins, calpains, and caspases. The relative contributions of the respective proteolysis systems vary within different muscles and animals [13]. In an experimental study of hindlimb-denervated rats, Beehler et al. [4] reported that bortezomib attenuated atrophy of the soleus muscle (type I, slow-twitch fibers) but exerted less effect on the extensor
digitorum longus (EDL) muscle (type II, fast-twitch fibers). These authors speculated that the reduced effect of bortezomib on the EDL muscle was due to the dominant role of a non-ubiquitin-proteasome system in its atrophic process. At present, the protein degradation system of the intrinsic laryngeal muscles remains poorly understood. The bortezomib dose (100 mg/kg) suggested by Orlowski [8] might be insufficient to prevent atrophy of the TA muscle. However, excessive doses of bortezomib are toxic. In our preliminary study, bortezomib at 200 mg/kg killed 40% of the animals within 1 week; at 250 mg/kg, 80% of the animals died. In this study, bortezomib prevented TA muscle atrophy. We plan to investigate whether other medicines, or combinations of medicines, including ubiquitin-proteasome and lysosomal system inhibitors such as MG132 [10], torbafylline [14], N-acetyll-leucinyl-l-leucinal-l-norleucinal (LLnL) [14],
TA muscle
PCA muscle
(L/R)
(L/R)
2
2
1.5
1.5
1
1
Control
0.5
*
0.5
0 4w (n = 6)
8w 12 w (n = 7) (n = 7)
Bortezomib
*
*:p < 0.01
0 4w (n = 6)
8w 12 w (n = 7) (n = 7)
Figure 5. Effects of bortezomib treatment on the left/right (L/R) ratio of the number of muscle fibers/mm2. Bortezomib decreased the number of muscle fibers/mm2 in the cricoarytenoid (PCA) muscle but not the thyroarytenoid (TA) muscle. The decreased L/R ratio in the PCA muscle was statistically significant at post-denervation weeks 8 and 12 (p < 0.01), but not at week 4.
Bortezomib and intrinsic laryngeal muscle atrophy lactacystin, and b-lactone [15] can also prevent intrinsic laryngeal muscle atrophy, including that of the TA muscle.
Conclusion Bortezomib reduced denervation-induced atrophy of the PCA muscle, but not the TA muscle. Declaration of interest: The authors have no financial or other conflicts of interest to declare, and are solely responsible for the content of this manuscript.
References [1] Remacle M, Lawson G. Results with collagen injection into the vocal folds for medialization. Curr Opin Otolaryngol Head Neck Surg 2007;15:148–52. [2] Isshiki N. Progress in laryngeal framework surgery. Acta Otolaryngol 2000;120:120–7. [3] Kumai Y, Ito T, Udaka N, Yumoto E. Effects of a nervemuscle pedicle on the denervated rat thyroarytenoid muscle. Laryngoscope 2006;116:1027–32. [4] Beehler BC, Sleph PG, Benmassaoud L, Grover GJ. Reduction of skeletal muscle atrophy by a proteasome inhibitor in a rat model of denervation. Exp Biol Med 2006;231:335–41. [5] Jackman RW, Kandarian SC. The molecular basis of skeletal muscle atrophy. Am J Physiol Cell Physiol 2004;287:C834– 43. [6] Adams J. Development of the proteasome inhibitor PS-341. Oncologist 2002;7:9–16.
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[7] Lecker SH, Solomon V, Mitch WE, Goldberg AL. Muscle protein breakdown and the critical role of the ubiquitinproteasome pathway in normal and disease states. J Nutr 1999;129:227S–37S. [8] Orlowski RZ. Proteasome inhibitors in cancer therapy. Methods Mol Biol 1999;301:339–50. [9] Ikemoto M, Nikawa T, Takeda S, Watanabe C, Kitano T, Baldwin KM, et al. Space shuttle flight (STS-90) enhances degradation of rat myosin heavy chain in association with activation of ubiquitin-proteasome pathway. FASEB J 2001; 15:1279–81. [10] Jamart C, Raymackers JM, Li An G, Deldicque L, Francaux M. Preservation of muscle disuse atrophy by MG132 proteasome inhibitor. Muscle Nerve 2011;43: 708–16. [11] Okamoto T, Torii S, Machida S. 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 2011;61: 537–46. [12] Sei H, Taguchi A, Nishida N, Hato N, Gyo K. Expression of atrophy-related transcription factors in the process of intrinsic laryngeal muscle atrophy after denervation. Eur Arch Otorhinolaryngol 2015;272:137–41. [13] Tisdale MJ. The ubiquitin-proteasome pathway as a therapeutic target for muscle wasting. J Support Oncol 2005;3: 209–17. [14] Combaret L, Tilignac T, Claustre A, Voisin L, Taillandier D, Obled C, et al. Torbafylline (HWA 448) inhibits enhanced skeletal muscle ubiquitin–proteasomedependent proteolysis in cancer and septic rats. Biochem J 2002;361:185–92. [15] Fang CH, Wang JJ, Hobler S, Guo B, Fischer JE, Hasselgren PO. Proteasome blockers inhibit protein breakdown in skeletal muscle after burn injury in rats. Clin Sci 1998;95:225–33.
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ORIGINAL ARTICLE
Preventive effects of bortezomib on denervation-induced atrophy of the intrinsic laryngeal muscles: an experimental study in the rat
HIROFUMI SEI1, AKI TAGUCHI2, NAOYA NISHIDA1, NAOHITO HATO1 & KIYOFUMI GYO3 1
Department of Otolaryngology, Ehime University, School of Medicine, Toon-city Shitsukawa, Ehime, Department of Otolaryngology, Matsuyama Red Cross Hospital, Matsuyama-city, Ehime and 3 Department of Otolaryngology, Takanoko Hospital, Matsuyama-city, Ehime, Japan 2
Abstract Conclusion: Bortezomib was effective in attenuating atrophy of the posterior cricoarytenoid (PCA) muscle, but not the thyroarytenoid (TA) muscle. This was probably due to differences in the fiber composition of the two muscles. The PCA muscle is composed of a combination of fast- and slow-twitch fibers, and therefore is more resistant to atrophy than the TA muscle, which is composed solely of fast-twitch fibers. Objectives: To investigate the preventive effects of bortezomib on denervation-induced atrophy of the TA and PCA muscles in the rat. Methods: Following transection of the left recurrent laryngeal nerve, bortezomib (100 mg/kg) was administered subcutaneously on post-denervation days 1 and 4, followed by a 10-day rest period every 14 days; each 2-week period constituted a single treatment cycle. In controls, saline was administered instead. Animals were killed for histological examination at 4 (n = 6), 8 (n = 7), and 12 (n = 7) weeks post-denervation. Muscle atrophy was assessed using three indices: wet muscle weight, muscle fiber cross-sectional area, and the number of muscle fibers/mm2. The effects of bortezomib were evaluated by comparing the left (L) and right (R) muscles, with sequential changes in the L/R ratio assessed. Results: In saline-administered animals, atrophy of the left-sided TA and PCA muscles progressed rapidly during the first 4 weeks post-denervation, following which progression slowed. Atrophy was greater in the TA compared with the PCA muscle, although this difference was not statistically significant. In bortezomib-administered animals, atrophy of the PCA muscle was attenuated significantly at post-denervation weeks 8 and 12; no such reduction in atrophy was observed for the TA muscle.
Keywords: Recurrent laryngeal nerve palsy, intrinsic laryngeal muscle atrophy, prevention of proteolysis, ubiquitin-proteasome pathway, muscle fiber composition
Introduction The recurrent laryngeal nerve innervates all intrinsic laryngeal muscles (except the cricothyroid muscle), controls the production of voice sounds, and regulates respiration and deglutition. When the recurrent laryngeal nerve is severed, atrophy of the intrinsic laryngeal muscles occurs, resulting in breathiness or aphonic hoarseness. Various procedures have been used to treat this condition, such as injection of collagen or fat into the vocal cords [1], laryngeal implantation of
cartilage or Gore-Tex (Gore & Associates, Livingston, UK) [2], adduction of the arytenoid cartilage, and nerve-muscle pedicle grafting [3]. However, all of these treatments require surgical intervention; in patients refusing surgery hoarseness will remain, thereby reducing quality of life. No atrophypreventing medication has been developed for the intrinsic laryngeal muscles. Recently, bortezomib (Velcade or PS-341; Millenium Pharmaceuticals, Cambridge, MA, USA), a dipeptidyl boronic acid, proved effective
Correspondence: Hirofumi Sei, Department of Otolaryngology, Ehime University, School of Medicine, Toon-city Shitsukawa, Ehime 91-0295, Japan. Tel: +81 89 960 5366. Fax: +81 89 960 5368. E-mail: [email protected]
(Received 19 November 2014; accepted 22 December 2014) ISSN 0001-6489 print/ISSN 1651-2251 online 2015 Informa Healthcare DOI: 10.3109/00016489.2015.1006793
714
H. Sei et al.
for attenuating lower-limb muscle atrophy in an experimental animal model [4]. Bortezomib is a selective proteasome inhibitor in the ubiquitin-proteasome pathway, which plays an important role in regulating muscle protein breakdown, including that of the intrinsic laryngeal muscle [4,5]. According to Adams [6], more than 80% of all cellular proteins are processed by the proteasome pathway; the remaining proteins, including cathepsins, calpains, and caspases, are destroyed by the lysosomal system [7]. We investigated whether bortezomib attenuated denervation-induced atrophy of the thyroarytenoid (TA) and posterior cricoarytenoid (PCA) muscles in the rat. The TA is one of several larynx adductor muscles; the PCA is the sole abductor muscle.
Material and methods Animal preparation Following approval from the Ethics Committee of Ehime University Graduate School of Medicine, this study was conducted according to the animal experimentation guidelines of our institution. Forty 9–10-week-old male Wistar rats, weighing between 210 and 250 g, were used. Each animal was anesthetized with an intramuscular injection of ketamine 150 mg/kg, and fixed on an experimental table in the prone position. Following incision of the skin of the neck, the left recurrent laryngeal nerve was transected at the level of the seventh tracheal ring. The proximal end of the nerve was ligatured and embedded in the sternocleidomastoid muscle to prevent future contact between the cut ends. The nerve of the right side remained intact. Animals were divided into bortezomib-treated and non-treated (vehicle control) groups (bortezomib group, n = 20). Following transection of the left recurrent laryngeal nerve, bortezomib (100 mg/kg) was administered subcutaneously on postdenervation days 1 and 4, followed by a 10-day rest period every 14 days; this 2-week period constituted a single treatment cycle. Thereafter, the treatment cycle was repeated until the end of the experiment as per the procedure of Orlowski [8]. The vehicle control group (n = 20) was administered saline at identical intervals. Animals were then returned to the animal room, which was maintained at 21–23 C under a 12/12 h light/dark cycle (lights on between 7:00 a.m. and 7:00 p.m.). Animals were granted access to food and water ad libitum until the experiment ended. During the experiment, every effort was made to minimize the suffering and the number of animals that were used.
Animals were killed at 4 (n = 6), 8 (n = 7), and 12 weeks (n = 7) post-denervation by intraperitoneal injection of an excessive dose of ketamine. The TA and PCA muscles were excised bilaterally, with their wet muscle weights separately measured immediately following excision. The muscle specimens were then embedded in OCT compound (#4583; Sakura Finetek, Torrance, CA, USA), snap-frozen in liquid nitrogen, and stored at –80 C. Histological study For the histological study, the larynx was symmetrically placed on a plate and cut perpendicularly along its longitudinal axis (10 mm thickness) with a cryostat (CM1900; Leica, Wetzlar, Germany). The slices were then stained with hematoxylin and eosin (HE) and observed under a microscope (BZ9000 Biorevo; Keyence, Japan). We measured the muscle fiber cross-sectional area (MFCSA) and the number of muscle fibers/mm2, as described by Kumai et al. [3]. Cross-sectional area was measured by calculating the sum of the muscle fiber areas in a cross-section using the ImageJ software package (NIH, Bethesda, MD, USA). The number of muscle fibers was determined by counting the number of fibers in a 1 1 mm area of a muscle cross-section. Muscle samples from at least three different sections were examined, with the mean values then calculated. The progress of muscle atrophy was evaluated by comparing the muscles of the left (L) and right (R) sides. Sequential changes in the L/R ratio with and without bortezomib treatment were investigated. Statistical analyses All data are presented as means ± SD. An unpaired Student’s t test was used for the statistical analyses. A value of p < 0.05 was taken to indicate statistical significance. Results Figures 1 and 2 depict representative cross-sections of the TA and PCA muscles, respectively, with or without bortezomib treatment, at post-denervation week 12. Bortezomib treatment appeared to attenuate PCA muscle atrophy on the left (denervated) side; TA muscle atrophy continued to progress. Bortezomib did not appear to cause any histological side effects in the muscles of the right (undenervated) side. Sequential changes in denervation-induced muscle atrophy with or without bortezomib treatment are summarized in Figures 3,4,5. In controls, atrophy of the TA and PCA muscles on the left side progressed rapidly during the first 4 weeks after
Bortezomib and intrinsic laryngeal muscle atrophy Left denervated side
Left denervated side
Right side
Bortezomib
Bortezomib
Saline
Saline
Bar =100 µm
715
Right side
Bar =100 µm
Figure 1. Cross-section of the thyroarytenoid (TA) muscle with or without bortezomib administration at post-denervation week 12. HE staining; original magnification 400. Scale bar = 100 mm.
Figure 2. Cross-section of the cricoarytenoid (PCA) muscle with or without bortezomib administration at post-denervation week 12. HE staining; original magnification 400. Scale bar = 100 mm.
denervation; subsequent progress was markedly less rapid. Atrophy was greater in the TA compared with the PCA muscle, although this difference was not significant. Bortezomib administration attenuated atrophy of the PCA muscle but not the TA muscle. Figure 3 describes sequential changes in wet muscle weight. In controls, the L/R ratio of the TA and PCA muscles was 36 ± 5.9% and 39.9 ± 5.6%, respectively, at post-denervation week 12 (n = 7). When treated with bortezomib, L/R ratios were 34.3 ± 3. 6% and 64.9 ± 6.4%, respectively (n = 7). Bortezomib administration significantly attenuated PCA (p < 0.01), but not TA, muscle atrophy (p = 0.12). Similar results were also noted for the MFCSA and number of muscle fibers/mm2.
ubiquitin-proteasome system is a major pathway involved in the break-down of intracellular proteins, including myofibrillar proteins such as actin and the myosin heavy chain [9]. This system targets proteins by attaching at least four copies of ubiquitin, a small polypeptide. Ubiquitination is mediated by a series of enzymes: E1, or ubiquitin-activating enzyme; E2, or ubiquitin-conjugating enzyme; and E3, or ubiquitin ligase. When a substrate is polyubiquitinated, it is targeted for destruction by the proteasome, a large multi-subunit protease complex, which breaks them into short oligopeptides that are degraded rapidly to amino acids by cytosolic peptidases [10]. Therefore, inhibition of the ubiquitin-proteasome system can prevent atrophy of the intrinsic laryngeal muscles following denervation. As demonstrated in the present study, bortezomib was effective in attenuating atrophy of the PCA muscle but not the TA muscle. This latter result was unexpected, and might be explained by the different
Discussion Various mechanisms are involved in the process of muscle protein destruction; the ATP-dependent TA muscle
PCA muscle
(L/R)
(L/R)
1
1
0.8
0.8
0.6
0.6
Control
0.4
0.4
Bortezomib
0.2
0.2
*:p < 0.01
0
0 4w 8w (n = 6) (n = 7)
12 w (n = 7)
4w 8w (n = 6) (n = 7)
12 w (n = 7)
Figure 3. Effects of bortezomib treatment on the left/right (L/R) ratio of wet muscle weight at post-denervation weeks 4, 8, and 12. Bortezomib attenuated denervation-induced atrophy of the cricoarytenoid (PCA) but not the thyroarytenoid (TA) muscle.
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H. Sei et al. TA muscle
PCA muscle
(L/R)
(L/R)
1
1
0.8
0.8
0.6
0.6
Control
0.4
0.4
Bortezomib
0.2
0.2
*:p < 0.01
0
4w 8w (n = 6) (n = 7)
0
12 w (n = 7)
4w 8w (n = 6) (n = 7)
12 w (n = 7)
Figure 4. Effects of bortezomib treatment on the left/right (L/R) ratio of the muscle fiber cross-sectional area (MFCSA). Bortezomib attenuated the decrease in the MFCSA of the cricoarytenoid (PCA) muscle but not the thyroarytenoid (TA) muscle.
fiber types that make up the two muscles. The TA muscle is composed solely of fast-twitch fibers, whereas the PCA muscle is composed of a combination of fast- and slow-twitch fibers [11]. Because fasttwitch fibers are more fragile, TA muscle proteins atrophied more rapidly compared with the PCA muscle proteins [12] such that bortezomib administration could not prevent TA muscle atrophy. It is also possible that the protein degradation systems of the TA and PCA muscles differ. Muscle protein is destroyed not only by the ubiquitin-proteasome system, but also by the lysosomal system, which involves cathepsins, calpains, and caspases. The relative contributions of the respective proteolysis systems vary within different muscles and animals [13]. In an experimental study of hindlimb-denervated rats, Beehler et al. [4] reported that bortezomib attenuated atrophy of the soleus muscle (type I, slow-twitch fibers) but exerted less effect on the extensor
digitorum longus (EDL) muscle (type II, fast-twitch fibers). These authors speculated that the reduced effect of bortezomib on the EDL muscle was due to the dominant role of a non-ubiquitin-proteasome system in its atrophic process. At present, the protein degradation system of the intrinsic laryngeal muscles remains poorly understood. The bortezomib dose (100 mg/kg) suggested by Orlowski [8] might be insufficient to prevent atrophy of the TA muscle. However, excessive doses of bortezomib are toxic. In our preliminary study, bortezomib at 200 mg/kg killed 40% of the animals within 1 week; at 250 mg/kg, 80% of the animals died. In this study, bortezomib prevented TA muscle atrophy. We plan to investigate whether other medicines, or combinations of medicines, including ubiquitin-proteasome and lysosomal system inhibitors such as MG132 [10], torbafylline [14], N-acetyll-leucinyl-l-leucinal-l-norleucinal (LLnL) [14],
TA muscle
PCA muscle
(L/R)
(L/R)
2
2
1.5
1.5
1
1
Control
0.5
*
0.5
0 4w (n = 6)
8w 12 w (n = 7) (n = 7)
Bortezomib
*
*:p < 0.01
0 4w (n = 6)
8w 12 w (n = 7) (n = 7)
Figure 5. Effects of bortezomib treatment on the left/right (L/R) ratio of the number of muscle fibers/mm2. Bortezomib decreased the number of muscle fibers/mm2 in the cricoarytenoid (PCA) muscle but not the thyroarytenoid (TA) muscle. The decreased L/R ratio in the PCA muscle was statistically significant at post-denervation weeks 8 and 12 (p < 0.01), but not at week 4.
Bortezomib and intrinsic laryngeal muscle atrophy lactacystin, and b-lactone [15] can also prevent intrinsic laryngeal muscle atrophy, including that of the TA muscle.
Conclusion Bortezomib reduced denervation-induced atrophy of the PCA muscle, but not the TA muscle. Declaration of interest: The authors have no financial or other conflicts of interest to declare, and are solely responsible for the content of this manuscript.
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