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Increased skeletal muscle expression of the endoplasmic reticulum chaperone GRP78 in patients with myasthenia gravis
JNI-475910; No of Pages 5 Journal of Neuroimmunology xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Journal of Neuroimmunology journal homepage: www.elsevier.com/locate/jneuroim
Increased skeletal muscle expression of the endoplasmic reticulum chaperone GRP78 in patients with myasthenia gravis Kazuo Iwasa a,⁎, Yoshinori Nambu a, Yuko Motozaki b, Yutaka Furukawa c, Hiroaki Yoshikawa d, Masahito Yamada a a
Department of Neurology and Neurobiology of Aging, Kanazawa University Graduate School of Medical Science, 13-1 Takara-Machi, Kanazawa 920-8640, Japan Department of Neurology, Iou National Hospital, Ni 73-1 Iwade-Machi, Kanazawa 920-0192, Japan Department of Neurology, Takaoka Koseiren Hospital, 5-10 Eiraku-Cho, Takaoka 933-8555, Japan d Health Service Center, Kanazawa University, Kakuma-Machi, Kanazawa 920-1192, Japan b c
a r t i c l e
i n f o
Article history: Received 7 March 2014 Received in revised form 11 May 2014 Accepted 13 May 2014 Available online xxxx Keywords: Myasthenia gravis Endoplasmic reticulum (ER) stress response Glucose-regulated protein 78 (GRP78) Skeletal muscle
a b s t r a c t In myasthenia gravis (MG), damage to neuromuscular junctions may induce endoplasmic reticulum (ER) stress in skeletal muscles. In the current study, skeletal muscles obtained from patients with MG exhibited upregulation of glucose-regulated protein 78 (GRP78) mRNA that was activated by ER stress. Furthermore, GRP78 mRNA expression was higher in patients with MG and myositis than in patients with non-myopathy. We also observed a significant positive correlation between GRP78 mRNA expression and GRP78 protein levels and between GRP78 mRNA expression and age of MG onset. Our findings suggest that muscle weakness in MG might be caused by both neuromuscular junction disruption and ER stress. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Acquired myasthenia gravis (MG) is an organ-specific, autoimmune disorder that stems from antibodies directed against skeletal muscle receptors and proteins at the neuromuscular junction. In most cases, autoantibodies against the acetylcholine receptor (AChR) impair the postsynaptic membrane by inducing complement-mediated damage, blocking acetylcholine interactions with the AChR, and inducing internalization and degradation of the AChR (Meriggioli and Sanders, 2009). In other cases, non-AChR components of the postsynaptic muscle end-plate, such as muscle-specific receptor tyrosine kinase (MuSK) and low-density lipoprotein receptor-related protein 4 (Lrp-4), serve as targets for the autoimmune attack (Hoch et al., 2001; Higuchi et al., 2011). Impaired receptor function results in diminished neuromuscular transmission, leading to symptoms of weakness and fatigability in patients with MG. Interestingly, early studies have demonstrated a poor correlation between the levels of autoantibodies and the severity of disease symptoms (Somnier, 1993; Meriggioli and Sanders, 2012).
⁎ Corresponding author at: Department of Neurology and Neurobiology of Aging, Kanazawa University Graduate School of Medical Science, 13-1 Takara-Machi, Kanazawa 920-8640, Japan. Tel.: +81 76 265 2292; fax: +81 76 234 4253. E-mail address: [email protected] (K. Iwasa).
An endoplasmic reticulum (ER) stress response has been observed in some muscular diseases, including myotonic dystrophy and myositis (Vattemi et al., 2004; Ikezoe et al., 2007). ER stress in muscle cells might influence muscle weakness and degeneration or apoptosis (Rayavarapu et al., 2012). Glucose-regulated protein 78 (GRP78), also known as immunoglobulin heavy chain binding protein (BiP), belongs to the heat shock protein 70 kDa (Hsp70) family and is a major ER chaperone protein, which can aid in the repair of unfolded proteins, as well as control the activation of ER transmembrane signaling molecules (Wang et al., 2009; Pfaffenbach and Lee, 2011). Furthermore, GRP78 induction has been widely used as a marker of ER stress (Wang et al., 2009). There have been reports of GRP94 overexpression in skeletal muscles (Suzuki et al., 2011) as well as an upregulation of major histocompatibility complex (MHC) class I expression in patients with MG (Iwasa et al., 2010). Levels of GRP94, an ER stress protein of the heat shock protein 90 kDa (Hsp90) family, has also been shown to be greatly enhanced in damaged muscle cells (Rayavarapu et al., 2012). The upregulation of MHC class I in muscles associated with myopathy has been attributed to the ER stress response (Nagaraju et al., 2005; Needham et al., 2007). It is plausible that in MG, disruption of neuromuscular junctions may induce ER stress in skeletal muscles. However, current information about ER stress in myasthenic muscle is lacking, and no study has reported on GRP78 overexpression in MG skeletal muscle. Therefore, the aim of the current study was to evaluate the upregulation of the ER stress chaperone protein, GRP78, in the skeletal muscles of patients with MG.
http://dx.doi.org/10.1016/j.jneuroim.2014.05.006 0165-5728/© 2014 Elsevier B.V. All rights reserved.
Please cite this article as: Iwasa, K., et al., Increased skeletal muscle expression of the endoplasmic reticulum chaperone GRP78 in patients with myasthenia gravis, J. Neuroimmunol. (2014), http://dx.doi.org/10.1016/j.jneuroim.2014.05.006
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K. Iwasa et al. / Journal of Neuroimmunology xxx (2014) xxx–xxx
2. Materials and methods 2.1. Patients Patients with MG included six individuals with thymoma and seven without thymoma (Table 1). All patients tested positive for anti-AChR antibodies, the edrophonium test, and/or exhibited decremented responses to repetitive nerve stimulation. In addition, serum creatine kinase levels of these patients were normal, and all patients provided written informed consent prior to muscle biopsy. In addition, five patients with inflammatory myopathy (four with polymyositis [PM] and one with dermatomyositis [DM]) and five patients with nonmyopathy provided biopsy specimens from their upper or lower limb muscles as control samples. During thymectomy, tissue samples were resected from the musculus pectoralis major and then immediately frozen in liquid nitrogen and stored at −80 °C until immunohistochemical staining and mRNA extraction were carried out. Standard protocols and patient consents for this study have been approved by the medical ethics committee of Kanazawa University School of Medicine. 2.2. Immunohistochemistry Routine hematoxylin–eosin (H & E) staining was performed, and immunohistochemistry was completed according to standard methodology. Briefly, 6-μm sections of frozen tissue from patients and controls were prepared and fixed in cold acetone. Non-specific protein staining was blocked using Protein Block, Serum-Free (Dako, Denmark), and sections were then incubated with rabbit polyclonal GRP78 antibody (1:400, Abcam, Cambridge, UK) at 4 °C overnight. After washing, sections were incubated in goat anti-rabbit IgG antibody conjugated with Cromeo546 (1:750, Abcam) for 2 h at room temperature. Images of sections were then acquired with an Olympus BX51 microscope equipped with a DP72 system (Olympus, Japan). Samples for negative controls were obtained from patients with peripheral neuropathy. Dermatomyositis muscle samples with consistent GRP78 expression were used as a positive control. Two experimenters, who were blind to patient backgrounds and the RT-PCR results, empirically assessed an immunohistochemical grading scale for GRP78 expression. The percentage of immunoreactivity was calculated as follows: number of GRP78-positive muscle cells / total muscle cells × 100.
Quantitative reverse transcriptase polymerase chain reaction (qRTPCR) was performed using a TaqMan® gene expression master mix (Applied Biosystems) with specific primers and TaqMan® probes for target genes and for an endogenous control gene (GRP78; Hs00946084_g1 and GAPDH; Hs03929097_g1). Amplifications were carried out in 20 μl volumes, in duplicate, for at least three replicates. The cycling parameters were: incubation at 50 °C for 2 min, incubation at 95 °C for 10 min, and 50 cycles of PCR at 95 °C for 15 s and at 60 °C for 1 min on an ABI Prism 7300 (Applied Biosystems). Expression of target genes was normalized to that of the GAPDH endogenous control gene, and results were shown as relative mRNA expression. Fold changes, which were calculated using the 2−ΔΔCt method, were considered biologically significant when mRNA levels varied more than 1.8 fold. 2.4. Statistical analysis The Statistical Package for the Social Sciences (SPSS) statistical software (version 19.0, IBM, USA) was used for statistical analyses. Owing to the small sample size in this study, exact non-parametric tests were used to test for significance. Comparisons between data regarding GRP78 expression were performed using the Kruskal–Wallis method of multiple range testing. Correlations were assessed using Pearson's correlation analysis. All reported p values are based on two-tailed statistical tests with a significance level of 0.05. 3. Results 3.1. Immunohistochemistry Immunostaining was performed to determine if the skeletal muscle fibers associated with ER stress exhibited increased GRP78 expression. Twelve out of the 13 patients with MG and all five patients with myositis demonstrated positive staining with the anti-GRP78 antibody on the cell surface and within cytoplasmic regions of the muscle (Fig. 1). In patients with MG, not all muscle fibers were positively labeled for GRP78; the muscles showed partly heterogeneous expression patterns, with expression in approximately 28–86% of the fibers. In contrast, muscle fibers from patients affected by myositis exhibited diffuse expression of GRP78 in the sarcolemmal and/or sarcoplasmic regions. Moreover, we observed a correlation between GRP78 protein expression and age of MG onset (r = 0.59, p = 0.034; data not shown). 3.2. qRT-PCR of GRP78 mRNA
2.3. Quantitative real-time PCR of GRP78 mRNA Total RNA was extracted from frozen specimens using the Ambion® PARIS™ kit (Ambion, Austin, TX, USA), and subjected to cDNA synthesis using a high capacity cDNA RT kit (Applied Biosystems, Foster City, CA, USA), according to the manufacturer's instructions.
qRT-PCR analysis revealed that muscle fibers obtained from nine out of the 13 patients with MG and all the patients with myositis exhibited higher GRP78 mRNA expression than in fibers obtained from patients with non-myopathy (Fig. 2). In addition, GRP78 mRNA expression showed a significant positive correlation with GRP78 protein
Table 1 Demographic data of patients with MG. Case
Age at onset (year)
Sex
Thymoma type
MGFA
QMG score
Anti-AChR antibody (nM)
1 2 3 4 5 6 7 8 9 10 11 12 13
24 13 22 29 21 30 26 39 53 48 67 39 36
F F F M M M M F F F F F M
No thymoma No thymoma No thymoma No thymoma No thymoma No thymoma No thymoma B1 B2 B3 B1 B2 B3
II a II a II b II a II b II b III a I II b II b III b III b II b
7 10 21 23 9 20 25 9 nd 10 13 22 nd
71.6 7.5 39.3 162 14.7 135 10.9 33.4 43.1 30.7 64.4 123 25.7
F = female; M = male; MGFA = Myasthenia Gravis Foundation of America classification; QMG = quantitative MG score for disease severity; and AChR = acetylcholine receptor.
Please cite this article as: Iwasa, K., et al., Increased skeletal muscle expression of the endoplasmic reticulum chaperone GRP78 in patients with myasthenia gravis, J. Neuroimmunol. (2014), http://dx.doi.org/10.1016/j.jneuroim.2014.05.006
K. Iwasa et al. / Journal of Neuroimmunology xxx (2014) xxx–xxx
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Fig 1. Immunostaining for GRP78 in muscle sections. GRP78 was upregulated in the sarcolemmal and sarcoplasmic in polymyositis (A) and exhibited a heterogeneous pattern in myasthenia gravis (B). There was no overexpression in muscle tissue obtained from patients with peripheral neuropathy (C). Scale bar = 100 μm.
expression, as evaluated by immunohistochemistry (Fig. 3A). Furthermore, GRP78 mRNA was found at higher levels in aged patients with MG (Fig. 3B). Lastly, we found no association between GRP78 mRNA gene expression levels and titers of anti-AChR antibodies, the Myasthenia Gravis of Foundation of America criteria scores, or Quantitative MG Scores for Disease Severity (Fig. 3C, D, E). 4. Discussion In this study, we obtained skeletal muscle samples from patients with MG and found a positive correlation between mRNA and protein expression levels of the ER chaperone protein, GRP78. It should be noted that the muscles from which the biopsy samples were obtained differed between the MG and control groups, and each muscle has a different amount of type I and type II fibers. While this difference might influence GRP78 mRNA expression, the ER stress response in the muscles should not be activated during the non-stressed condition. GRP78 belongs to the Hsp70 family and is a major chaperone protein (Wang et al., 2009; Pfaffenbach and Lee, 2011) found constitutively within the ER. Under normal conditions, this chaperone protein functions under moderate levels of basal expression and assists in protein folding and assembly (Pfaffenbach and Lee, 2011). GRP78 has been
Fig 2. qRT-PCR analysis of GRP78 mRNA in muscle tissue. Relative GRP78 mRNA expression levels were significantly increased in patients with MG and inflammatory myositis when compared to patients with peripheral neuropathy (MG vs. PN p = 0.042, PM/DM vs. PN p = 0.006).
shown to be upregulated under conditions of ER stress and, for this reason, has been widely used as a marker for this type of defensive cellular response (Wang et al., 2009). In MG, neuromuscular junction damage may induce ER stress in the skeletal muscles and may be implicated in GRP78 expression. Following muscle inflammation, ER stress has been observed in the skeletal muscles in response to cytokine release from inflammatory cells (Nagaraju et al., 2005). A number of patients with MG have been reported to show lymphocytic infiltration in their muscles (Nakano and Engel, 1993; Iwasa et al., 2010). Lymphocytic infiltration in MG muscles may lead to secretion of cytokines that could then stimulate the ER and induce GRP78 expression. Other causes of muscle damage, including viral infection, mitochondrial dysfunction, and chemical depletion of ubiquinone function, may also lead to induction of the ER stress response. As previously discussed, neuromuscular dysfunction in MG results in diminished neuromuscular transmission leading to symptoms of weakness and fatigability (Meriggioli and Sanders, 2009). The ER stress response is known to be activated in the muscle tissue of patients with myositis. In fact, ER stress may play a role in the muscle fiber damage and dysfunction characteristic of myositis. Our results showed one outlier in the PM/DM patient group in terms of GRP78 mRNA expression. However, the cause for this outlier is unknown; possibly, the severe muscle damage in this patient influenced the GRP78 mRNA levels. It is possible that in patients with both myositis and MG, ER stress is responsible for the reduction in muscle strength (Vattemi et al., 2004; Nagaraju et al., 2005). Overexpression of GRP78 might prevent muscle cell damage and apoptosis. For example, under ischemic cell conditions, GRP78 has been shown to protect cells through a reduction of Ca2+ flux from the ER to the mitochondria and to play a role in preserving mitochondrial function during cellular stress (Ouyang et al., 2011; Belaidi et al., 2013). In addition, ER stress in muscles is important for muscle repair after injury (Wang et al., 2009). Thus, upregulation of GRP78 in muscle cells may play an important role in muscle repair and improvement of MG. On the other hand, GRP78 has been shown to have immunomodulatory properties; GRP78-specific T-cells can produce IL-4 and IL-5 (Brownlie et al., 2006; Corrigall et al., 2009; Franco et al., 2010). GRP78 also induces IL-10 secretion from human monocytes. Therefore, an increase of GRP78 expression in the muscles may play immunoregulatory roles in MG, serving as a defense mechanism against immunological attack and modulating the immune system itself. Another interesting finding of our analysis was that GRP78 gene and protein expression were correlated with age of MG onset. Therefore, the muscle tissue of older patients affected by MG might comprise of different pathological conditions (Aarli, 2008). The increased GRP78 levels observed in aged patients may represent reactions of aged muscle cells, which involve different stress factors like mitochondria dysfunction, membrane dysplasia of the muscular reticulum, and hyper loading of Ca2+ ions (Puzianowska-Kuznicka and Kuznicki, 2009; Brown and Naidoo, 2012). One animal model of aging has supported this, showing
Please cite this article as: Iwasa, K., et al., Increased skeletal muscle expression of the endoplasmic reticulum chaperone GRP78 in patients with myasthenia gravis, J. Neuroimmunol. (2014), http://dx.doi.org/10.1016/j.jneuroim.2014.05.006
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K. Iwasa et al. / Journal of Neuroimmunology xxx (2014) xxx–xxx
Fig 3. Relative GRP78 mRNA expression in MG muscle tissue was significantly correlated with % of GRP78 expression in muscle cells (A) (as assessed through immunohistochemistry) (r = 0.73, p = 0.004) and onset age (B) (r = 0.61, p = 0.028). A comparison of relative GRP78 mRNA expression with anti-AChR antibody titer (C), MGFA stage (D), and QMG score (E) showed no significant correlation for patients with MG, as assessed by Pearson's correlation coefficient.
GRP78 upregulation in the calf muscles of rats (Ogata et al., 2009). Additionally, autoantibodies directed against non-AChR skeletal muscle proteins are more frequent in older patients with MG (Aarli, 2008; Meriggioli and Sanders, 2012) and in those with more severe disease states. It is possible that a heterogeneous immune attack against muscle antigens could induce ER stress in muscle cells. In summary, the muscles of patients with MG might be damaged not only in the neuromuscular junction membrane, but also in intracellular areas. Our findings may help to understand muscle responses to stress conditions in MG. However, more studies are needed to elucidate the mechanisms underlying GRP78 increases following ER stress. Acknowledgments This work was supported by a grant from the Japanese Ministry of Education, Science, Sports and Culture (No. 25461273) and a Neuroimmunological Disease Research Committee grant from the Japanese Ministry of Health, Labor, and Welfare. We would like to thank Ms. Yumiko Kakuda and Ms. Yukari Yamaguchi for their excellent technical assistance in performing the immunohistochemistry studies. References Aarli, J.A., 2008. Myasthenia gravis in the elderly: is it different? Ann. N. Y. Acad. Sci. 1132, 238–243. Belaidi, E., Decorps, J., Augeul, L., Durand, A., Ovize, M., 2013. Endoplasmic reticulum stress contributes to heart protection induced by cyclophilin D inhibition. Basic Res. Cardiol. 108, 363. Brown, M.K., Naidoo, N., 2012. The endoplasmic reticulum stress response in aging and age-related diseases. Front. Physiol. 3, 263.
Brownlie, R.J., Myers, L.K., Wooley, P.H., Corrigall, V.M., Bodman-Smith, M.D., Panayi, G.S., Thompson, S.J., 2006. Treatment of murine collagen-induced arthritis by the stress protein BiP via interleukin-4-producing regulatory T cells: a novel function for an ancient protein. Arthritis Rheum. 54, 854–863. Corrigall, V.M., Vittecoq, O., Panayi, G.S., 2009. Binding immunoglobulin protein-treated peripheral blood monocyte-derived dendritic cells are refractory to maturation and induce regulatory T-cell development. Immunology 128, 218–226. Franco, A., Almanza, G., Burns, J.C., Wheeler, M., Zanetti, M., 2010. Endoplasmic reticulum stress drives a regulatory phenotype in human T-cell clones. Cell. Immunol. 266, 1–6. Higuchi, O., Hamuro, J., Motomura, M., Yamanashi, Y., 2011. Autoantibodies to low-density lipoprotein receptor-related protein 4 in myasthenia gravis. Ann. Neurol. 69, 418–422. Hoch, W., McConville, J., Helms, S., Newsom-Davis, J., Melms, A., Vincent, A., 2001. Autoantibodies to the receptor tyrosine kinase MuSK in patients with myasthenia gravis without acetylcholine receptor antibodies. Nat. Med. 7, 365–368. Ikezoe, K., Nakamori, M., Furuya, H., Arahata, H., Kanemoto, S., Kimura, T., Imaizumi, K., Takahashi, M.P., Sakoda, S., Fujii, N., Kira, J., 2007. Endoplasmic reticulum stress in myotonic dystrophy type 1 muscle. Acta Neuropathol. 114, 527–535. Iwasa, K., Kato-Motozaki, Y., Furukawa, Y., Maruta, T., Ishida, C., Yoshikawa, H., Yamada, M., 2010. Up-regulation of MHC class I and class II in the skeletal muscles of myasthenia gravis. J. Neuroimmunol. 225, 171–174. Meriggioli, M.N., Sanders, D.B., 2009. Autoimmune myasthenia gravis: emerging clinical and biological heterogeneity. Lancet Neurol. 8, 475–490. Meriggioli, M.N., Sanders, D.B., 2012. Muscle autoantibodies in myasthenia gravis: beyond diagnosis? Expert. Rev. Clin. Immunol. 8, 427–438. Nagaraju, K., Casciola-Rosen, L., Lundberg, I., Rawat, R., Cutting, S., Thapliyal, R., Chang, J., Dwivedi, S., Mitsak, M., Chen, Y.W., Plotz, P., Rosen, A., Hoffman, E., Raben, N., 2005. Activation of the endoplasmic reticulum stress response in autoimmune myositis: potential role in muscle fiber damage and dysfunction. Arthritis Rheum. 52, 1824–1835. Nakano, S., Engel, A.G., 1993. Myasthenia gravis: quantitative immunocytochemical analysis of inflammatory cells and detection of complement membrane attack complex at the end-plate in 30 patients. Neurology 43, 1167–1172. Needham, M., Fabian, V., Knezevic, W., Panegyres, P., Zilko, P., Mastaglia, F.L., 2007. Progressive myopathy with up-regulation of MHC-I associated with statin therapy. Neuromuscul. Disord. 17, 194–200. Ogata, T., Machida, S., Oishi, Y., Higuchi, M., Muraoka, I., 2009. Differential cell death regulation between adult-unloaded and aged rat soleus muscle. Mech. Ageing Dev. 130, 328–336.
Please cite this article as: Iwasa, K., et al., Increased skeletal muscle expression of the endoplasmic reticulum chaperone GRP78 in patients with myasthenia gravis, J. Neuroimmunol. (2014), http://dx.doi.org/10.1016/j.jneuroim.2014.05.006
K. Iwasa et al. / Journal of Neuroimmunology xxx (2014) xxx–xxx Ouyang, Y.B., Xu, L.J., Emery, J.F., Lee, A.S., Giffard, R.G., 2011. Overexpressing GRP78 influences Ca2+ handling and function of mitochondria in astrocytes after ischemia-like stress. Mitochondrion 11, 279–286. Pfaffenbach, K.T., Lee, A.S., 2011. The critical role of GRP78 in physiologic and pathologic stress. Curr. Opin. Cell Biol. 23, 150–156. Puzianowska-Kuznicka, M., Kuznicki, J., 2009. The ER and ageing II: calcium homeostasis. Ageing Res. Rev. 8, 160–172. Rayavarapu, S., Coley, W., Nagaraju, K., 2012. Endoplasmic reticulum stress in skeletal muscle homeostasis and disease. Curr. Rheumatol. Rep. 14, 238–243. Somnier, F.E., 1993. Clinical implementation of anti-acetylcholine receptor antibodies. J. Neurol. Neurosurg. Psychiatry 56, 496–504.
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Suzuki, S., Utsugisawa, K., Iwasa, K., Satoh, T., Nagane, Y., Yoshikawa, H., Kuwana, M., Suzuki, N., 2011. Autoimmunity to endoplasmic reticulum chaperone GRP94 in myasthenia gravis. J. Neuroimmunol. 237, 87–92. Vattemi, G., Engel, W.K., McFerrin, J., Askanas, V., 2004. Endoplasmic reticulum stress and unfolded protein response in inclusion body myositis muscle. Am. J. Pathol. 164, 1–7. Wang, M., Wey, S., Zhang, Y., Ye, R., Lee, A.S., 2009. Role of the unfolded protein response regulator GRP78/BiP in development, cancer, and neurological disorders. Antioxid. Redox Signal. 11, 2307–2316.
Please cite this article as: Iwasa, K., et al., Increased skeletal muscle expression of the endoplasmic reticulum chaperone GRP78 in patients with myasthenia gravis, J. Neuroimmunol. (2014), http://dx.doi.org/10.1016/j.jneuroim.2014.05.006
Contents lists available at ScienceDirect
Journal of Neuroimmunology journal homepage: www.elsevier.com/locate/jneuroim
Increased skeletal muscle expression of the endoplasmic reticulum chaperone GRP78 in patients with myasthenia gravis Kazuo Iwasa a,⁎, Yoshinori Nambu a, Yuko Motozaki b, Yutaka Furukawa c, Hiroaki Yoshikawa d, Masahito Yamada a a
Department of Neurology and Neurobiology of Aging, Kanazawa University Graduate School of Medical Science, 13-1 Takara-Machi, Kanazawa 920-8640, Japan Department of Neurology, Iou National Hospital, Ni 73-1 Iwade-Machi, Kanazawa 920-0192, Japan Department of Neurology, Takaoka Koseiren Hospital, 5-10 Eiraku-Cho, Takaoka 933-8555, Japan d Health Service Center, Kanazawa University, Kakuma-Machi, Kanazawa 920-1192, Japan b c
a r t i c l e
i n f o
Article history: Received 7 March 2014 Received in revised form 11 May 2014 Accepted 13 May 2014 Available online xxxx Keywords: Myasthenia gravis Endoplasmic reticulum (ER) stress response Glucose-regulated protein 78 (GRP78) Skeletal muscle
a b s t r a c t In myasthenia gravis (MG), damage to neuromuscular junctions may induce endoplasmic reticulum (ER) stress in skeletal muscles. In the current study, skeletal muscles obtained from patients with MG exhibited upregulation of glucose-regulated protein 78 (GRP78) mRNA that was activated by ER stress. Furthermore, GRP78 mRNA expression was higher in patients with MG and myositis than in patients with non-myopathy. We also observed a significant positive correlation between GRP78 mRNA expression and GRP78 protein levels and between GRP78 mRNA expression and age of MG onset. Our findings suggest that muscle weakness in MG might be caused by both neuromuscular junction disruption and ER stress. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Acquired myasthenia gravis (MG) is an organ-specific, autoimmune disorder that stems from antibodies directed against skeletal muscle receptors and proteins at the neuromuscular junction. In most cases, autoantibodies against the acetylcholine receptor (AChR) impair the postsynaptic membrane by inducing complement-mediated damage, blocking acetylcholine interactions with the AChR, and inducing internalization and degradation of the AChR (Meriggioli and Sanders, 2009). In other cases, non-AChR components of the postsynaptic muscle end-plate, such as muscle-specific receptor tyrosine kinase (MuSK) and low-density lipoprotein receptor-related protein 4 (Lrp-4), serve as targets for the autoimmune attack (Hoch et al., 2001; Higuchi et al., 2011). Impaired receptor function results in diminished neuromuscular transmission, leading to symptoms of weakness and fatigability in patients with MG. Interestingly, early studies have demonstrated a poor correlation between the levels of autoantibodies and the severity of disease symptoms (Somnier, 1993; Meriggioli and Sanders, 2012).
⁎ Corresponding author at: Department of Neurology and Neurobiology of Aging, Kanazawa University Graduate School of Medical Science, 13-1 Takara-Machi, Kanazawa 920-8640, Japan. Tel.: +81 76 265 2292; fax: +81 76 234 4253. E-mail address: [email protected] (K. Iwasa).
An endoplasmic reticulum (ER) stress response has been observed in some muscular diseases, including myotonic dystrophy and myositis (Vattemi et al., 2004; Ikezoe et al., 2007). ER stress in muscle cells might influence muscle weakness and degeneration or apoptosis (Rayavarapu et al., 2012). Glucose-regulated protein 78 (GRP78), also known as immunoglobulin heavy chain binding protein (BiP), belongs to the heat shock protein 70 kDa (Hsp70) family and is a major ER chaperone protein, which can aid in the repair of unfolded proteins, as well as control the activation of ER transmembrane signaling molecules (Wang et al., 2009; Pfaffenbach and Lee, 2011). Furthermore, GRP78 induction has been widely used as a marker of ER stress (Wang et al., 2009). There have been reports of GRP94 overexpression in skeletal muscles (Suzuki et al., 2011) as well as an upregulation of major histocompatibility complex (MHC) class I expression in patients with MG (Iwasa et al., 2010). Levels of GRP94, an ER stress protein of the heat shock protein 90 kDa (Hsp90) family, has also been shown to be greatly enhanced in damaged muscle cells (Rayavarapu et al., 2012). The upregulation of MHC class I in muscles associated with myopathy has been attributed to the ER stress response (Nagaraju et al., 2005; Needham et al., 2007). It is plausible that in MG, disruption of neuromuscular junctions may induce ER stress in skeletal muscles. However, current information about ER stress in myasthenic muscle is lacking, and no study has reported on GRP78 overexpression in MG skeletal muscle. Therefore, the aim of the current study was to evaluate the upregulation of the ER stress chaperone protein, GRP78, in the skeletal muscles of patients with MG.
http://dx.doi.org/10.1016/j.jneuroim.2014.05.006 0165-5728/© 2014 Elsevier B.V. All rights reserved.
Please cite this article as: Iwasa, K., et al., Increased skeletal muscle expression of the endoplasmic reticulum chaperone GRP78 in patients with myasthenia gravis, J. Neuroimmunol. (2014), http://dx.doi.org/10.1016/j.jneuroim.2014.05.006
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K. Iwasa et al. / Journal of Neuroimmunology xxx (2014) xxx–xxx
2. Materials and methods 2.1. Patients Patients with MG included six individuals with thymoma and seven without thymoma (Table 1). All patients tested positive for anti-AChR antibodies, the edrophonium test, and/or exhibited decremented responses to repetitive nerve stimulation. In addition, serum creatine kinase levels of these patients were normal, and all patients provided written informed consent prior to muscle biopsy. In addition, five patients with inflammatory myopathy (four with polymyositis [PM] and one with dermatomyositis [DM]) and five patients with nonmyopathy provided biopsy specimens from their upper or lower limb muscles as control samples. During thymectomy, tissue samples were resected from the musculus pectoralis major and then immediately frozen in liquid nitrogen and stored at −80 °C until immunohistochemical staining and mRNA extraction were carried out. Standard protocols and patient consents for this study have been approved by the medical ethics committee of Kanazawa University School of Medicine. 2.2. Immunohistochemistry Routine hematoxylin–eosin (H & E) staining was performed, and immunohistochemistry was completed according to standard methodology. Briefly, 6-μm sections of frozen tissue from patients and controls were prepared and fixed in cold acetone. Non-specific protein staining was blocked using Protein Block, Serum-Free (Dako, Denmark), and sections were then incubated with rabbit polyclonal GRP78 antibody (1:400, Abcam, Cambridge, UK) at 4 °C overnight. After washing, sections were incubated in goat anti-rabbit IgG antibody conjugated with Cromeo546 (1:750, Abcam) for 2 h at room temperature. Images of sections were then acquired with an Olympus BX51 microscope equipped with a DP72 system (Olympus, Japan). Samples for negative controls were obtained from patients with peripheral neuropathy. Dermatomyositis muscle samples with consistent GRP78 expression were used as a positive control. Two experimenters, who were blind to patient backgrounds and the RT-PCR results, empirically assessed an immunohistochemical grading scale for GRP78 expression. The percentage of immunoreactivity was calculated as follows: number of GRP78-positive muscle cells / total muscle cells × 100.
Quantitative reverse transcriptase polymerase chain reaction (qRTPCR) was performed using a TaqMan® gene expression master mix (Applied Biosystems) with specific primers and TaqMan® probes for target genes and for an endogenous control gene (GRP78; Hs00946084_g1 and GAPDH; Hs03929097_g1). Amplifications were carried out in 20 μl volumes, in duplicate, for at least three replicates. The cycling parameters were: incubation at 50 °C for 2 min, incubation at 95 °C for 10 min, and 50 cycles of PCR at 95 °C for 15 s and at 60 °C for 1 min on an ABI Prism 7300 (Applied Biosystems). Expression of target genes was normalized to that of the GAPDH endogenous control gene, and results were shown as relative mRNA expression. Fold changes, which were calculated using the 2−ΔΔCt method, were considered biologically significant when mRNA levels varied more than 1.8 fold. 2.4. Statistical analysis The Statistical Package for the Social Sciences (SPSS) statistical software (version 19.0, IBM, USA) was used for statistical analyses. Owing to the small sample size in this study, exact non-parametric tests were used to test for significance. Comparisons between data regarding GRP78 expression were performed using the Kruskal–Wallis method of multiple range testing. Correlations were assessed using Pearson's correlation analysis. All reported p values are based on two-tailed statistical tests with a significance level of 0.05. 3. Results 3.1. Immunohistochemistry Immunostaining was performed to determine if the skeletal muscle fibers associated with ER stress exhibited increased GRP78 expression. Twelve out of the 13 patients with MG and all five patients with myositis demonstrated positive staining with the anti-GRP78 antibody on the cell surface and within cytoplasmic regions of the muscle (Fig. 1). In patients with MG, not all muscle fibers were positively labeled for GRP78; the muscles showed partly heterogeneous expression patterns, with expression in approximately 28–86% of the fibers. In contrast, muscle fibers from patients affected by myositis exhibited diffuse expression of GRP78 in the sarcolemmal and/or sarcoplasmic regions. Moreover, we observed a correlation between GRP78 protein expression and age of MG onset (r = 0.59, p = 0.034; data not shown). 3.2. qRT-PCR of GRP78 mRNA
2.3. Quantitative real-time PCR of GRP78 mRNA Total RNA was extracted from frozen specimens using the Ambion® PARIS™ kit (Ambion, Austin, TX, USA), and subjected to cDNA synthesis using a high capacity cDNA RT kit (Applied Biosystems, Foster City, CA, USA), according to the manufacturer's instructions.
qRT-PCR analysis revealed that muscle fibers obtained from nine out of the 13 patients with MG and all the patients with myositis exhibited higher GRP78 mRNA expression than in fibers obtained from patients with non-myopathy (Fig. 2). In addition, GRP78 mRNA expression showed a significant positive correlation with GRP78 protein
Table 1 Demographic data of patients with MG. Case
Age at onset (year)
Sex
Thymoma type
MGFA
QMG score
Anti-AChR antibody (nM)
1 2 3 4 5 6 7 8 9 10 11 12 13
24 13 22 29 21 30 26 39 53 48 67 39 36
F F F M M M M F F F F F M
No thymoma No thymoma No thymoma No thymoma No thymoma No thymoma No thymoma B1 B2 B3 B1 B2 B3
II a II a II b II a II b II b III a I II b II b III b III b II b
7 10 21 23 9 20 25 9 nd 10 13 22 nd
71.6 7.5 39.3 162 14.7 135 10.9 33.4 43.1 30.7 64.4 123 25.7
F = female; M = male; MGFA = Myasthenia Gravis Foundation of America classification; QMG = quantitative MG score for disease severity; and AChR = acetylcholine receptor.
Please cite this article as: Iwasa, K., et al., Increased skeletal muscle expression of the endoplasmic reticulum chaperone GRP78 in patients with myasthenia gravis, J. Neuroimmunol. (2014), http://dx.doi.org/10.1016/j.jneuroim.2014.05.006
K. Iwasa et al. / Journal of Neuroimmunology xxx (2014) xxx–xxx
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Fig 1. Immunostaining for GRP78 in muscle sections. GRP78 was upregulated in the sarcolemmal and sarcoplasmic in polymyositis (A) and exhibited a heterogeneous pattern in myasthenia gravis (B). There was no overexpression in muscle tissue obtained from patients with peripheral neuropathy (C). Scale bar = 100 μm.
expression, as evaluated by immunohistochemistry (Fig. 3A). Furthermore, GRP78 mRNA was found at higher levels in aged patients with MG (Fig. 3B). Lastly, we found no association between GRP78 mRNA gene expression levels and titers of anti-AChR antibodies, the Myasthenia Gravis of Foundation of America criteria scores, or Quantitative MG Scores for Disease Severity (Fig. 3C, D, E). 4. Discussion In this study, we obtained skeletal muscle samples from patients with MG and found a positive correlation between mRNA and protein expression levels of the ER chaperone protein, GRP78. It should be noted that the muscles from which the biopsy samples were obtained differed between the MG and control groups, and each muscle has a different amount of type I and type II fibers. While this difference might influence GRP78 mRNA expression, the ER stress response in the muscles should not be activated during the non-stressed condition. GRP78 belongs to the Hsp70 family and is a major chaperone protein (Wang et al., 2009; Pfaffenbach and Lee, 2011) found constitutively within the ER. Under normal conditions, this chaperone protein functions under moderate levels of basal expression and assists in protein folding and assembly (Pfaffenbach and Lee, 2011). GRP78 has been
Fig 2. qRT-PCR analysis of GRP78 mRNA in muscle tissue. Relative GRP78 mRNA expression levels were significantly increased in patients with MG and inflammatory myositis when compared to patients with peripheral neuropathy (MG vs. PN p = 0.042, PM/DM vs. PN p = 0.006).
shown to be upregulated under conditions of ER stress and, for this reason, has been widely used as a marker for this type of defensive cellular response (Wang et al., 2009). In MG, neuromuscular junction damage may induce ER stress in the skeletal muscles and may be implicated in GRP78 expression. Following muscle inflammation, ER stress has been observed in the skeletal muscles in response to cytokine release from inflammatory cells (Nagaraju et al., 2005). A number of patients with MG have been reported to show lymphocytic infiltration in their muscles (Nakano and Engel, 1993; Iwasa et al., 2010). Lymphocytic infiltration in MG muscles may lead to secretion of cytokines that could then stimulate the ER and induce GRP78 expression. Other causes of muscle damage, including viral infection, mitochondrial dysfunction, and chemical depletion of ubiquinone function, may also lead to induction of the ER stress response. As previously discussed, neuromuscular dysfunction in MG results in diminished neuromuscular transmission leading to symptoms of weakness and fatigability (Meriggioli and Sanders, 2009). The ER stress response is known to be activated in the muscle tissue of patients with myositis. In fact, ER stress may play a role in the muscle fiber damage and dysfunction characteristic of myositis. Our results showed one outlier in the PM/DM patient group in terms of GRP78 mRNA expression. However, the cause for this outlier is unknown; possibly, the severe muscle damage in this patient influenced the GRP78 mRNA levels. It is possible that in patients with both myositis and MG, ER stress is responsible for the reduction in muscle strength (Vattemi et al., 2004; Nagaraju et al., 2005). Overexpression of GRP78 might prevent muscle cell damage and apoptosis. For example, under ischemic cell conditions, GRP78 has been shown to protect cells through a reduction of Ca2+ flux from the ER to the mitochondria and to play a role in preserving mitochondrial function during cellular stress (Ouyang et al., 2011; Belaidi et al., 2013). In addition, ER stress in muscles is important for muscle repair after injury (Wang et al., 2009). Thus, upregulation of GRP78 in muscle cells may play an important role in muscle repair and improvement of MG. On the other hand, GRP78 has been shown to have immunomodulatory properties; GRP78-specific T-cells can produce IL-4 and IL-5 (Brownlie et al., 2006; Corrigall et al., 2009; Franco et al., 2010). GRP78 also induces IL-10 secretion from human monocytes. Therefore, an increase of GRP78 expression in the muscles may play immunoregulatory roles in MG, serving as a defense mechanism against immunological attack and modulating the immune system itself. Another interesting finding of our analysis was that GRP78 gene and protein expression were correlated with age of MG onset. Therefore, the muscle tissue of older patients affected by MG might comprise of different pathological conditions (Aarli, 2008). The increased GRP78 levels observed in aged patients may represent reactions of aged muscle cells, which involve different stress factors like mitochondria dysfunction, membrane dysplasia of the muscular reticulum, and hyper loading of Ca2+ ions (Puzianowska-Kuznicka and Kuznicki, 2009; Brown and Naidoo, 2012). One animal model of aging has supported this, showing
Please cite this article as: Iwasa, K., et al., Increased skeletal muscle expression of the endoplasmic reticulum chaperone GRP78 in patients with myasthenia gravis, J. Neuroimmunol. (2014), http://dx.doi.org/10.1016/j.jneuroim.2014.05.006
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K. Iwasa et al. / Journal of Neuroimmunology xxx (2014) xxx–xxx
Fig 3. Relative GRP78 mRNA expression in MG muscle tissue was significantly correlated with % of GRP78 expression in muscle cells (A) (as assessed through immunohistochemistry) (r = 0.73, p = 0.004) and onset age (B) (r = 0.61, p = 0.028). A comparison of relative GRP78 mRNA expression with anti-AChR antibody titer (C), MGFA stage (D), and QMG score (E) showed no significant correlation for patients with MG, as assessed by Pearson's correlation coefficient.
GRP78 upregulation in the calf muscles of rats (Ogata et al., 2009). Additionally, autoantibodies directed against non-AChR skeletal muscle proteins are more frequent in older patients with MG (Aarli, 2008; Meriggioli and Sanders, 2012) and in those with more severe disease states. It is possible that a heterogeneous immune attack against muscle antigens could induce ER stress in muscle cells. In summary, the muscles of patients with MG might be damaged not only in the neuromuscular junction membrane, but also in intracellular areas. Our findings may help to understand muscle responses to stress conditions in MG. However, more studies are needed to elucidate the mechanisms underlying GRP78 increases following ER stress. Acknowledgments This work was supported by a grant from the Japanese Ministry of Education, Science, Sports and Culture (No. 25461273) and a Neuroimmunological Disease Research Committee grant from the Japanese Ministry of Health, Labor, and Welfare. We would like to thank Ms. Yumiko Kakuda and Ms. Yukari Yamaguchi for their excellent technical assistance in performing the immunohistochemistry studies. References Aarli, J.A., 2008. Myasthenia gravis in the elderly: is it different? Ann. N. Y. Acad. Sci. 1132, 238–243. Belaidi, E., Decorps, J., Augeul, L., Durand, A., Ovize, M., 2013. Endoplasmic reticulum stress contributes to heart protection induced by cyclophilin D inhibition. Basic Res. Cardiol. 108, 363. Brown, M.K., Naidoo, N., 2012. The endoplasmic reticulum stress response in aging and age-related diseases. Front. Physiol. 3, 263.
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Please cite this article as: Iwasa, K., et al., Increased skeletal muscle expression of the endoplasmic reticulum chaperone GRP78 in patients with myasthenia gravis, J. Neuroimmunol. (2014), http://dx.doi.org/10.1016/j.jneuroim.2014.05.006