Involvement of natriuretic peptide system in C2C12 myocytes

  • Kiyoshi IshikawaEmail author
  • Taiki Hara
  • Kana Kato
  • Takeshi Shimomura
  • Kenji Omori


The natriuretic peptide system, a key regulator of cGMP signaling, comprises three types of natriuretic peptides, osteocrin/musclin (OSTN), and their natriuretic peptide receptors. Although this system plays important roles in many organs, its physiological roles in skeletal muscle have not been clearly described. In the present study, we investigated the role of the natriuretic peptide system in C2C12 myocytes. All three natriuretic peptide receptors were expressed by cells differentiating from myoblasts to myotubes, and natriuretic peptide receptor B (NPR-B) transcripts were detected at the highest levels. Further, higher levels of cGMP were generated in response to stimulation with C-type natriuretic peptide (CNP) versus atrial natriuretic peptide (ANP), which reflected receptor expression levels. A cGMP analog downregulated the expression of a few ER stress-related genes. Furthermore, OSTN gene expression was strongly upregulated after 20 days of differentiation. Augmented gene expression was found to correlate closely with endoplasmic reticulum (ER) stress, and C/EBP [CCAAT-enhancer-binding protein] homologous protein (CHOP), which is known to be activated by ER stress, affected the expression of OSTN. Together, these results suggest a role for natriuretic peptide signaling in the ER stress response of myocytes.


Natriuretic peptide Cyclic GMP ER stress Skeletal muscle Cell signaling 



Atrial natriuretic peptide


Activating transcription factor


Brain natriuretic peptide


C/EBP homologous protein


C-type natriuretic peptide


Endoplasmic reticulum


Enzyme immunoassay


Growth hormone


Myogenic differentiation


Myosin heavy chain


Myogenic factor 5


Natriuretic peptide receptor A


Natriuretic peptide receptor B


Natriuretic peptide receptor C




PKR-like ER kinase


Unfolded protein response


X-box binding protein 1



We thank Shinji Kojima of the Mitsubishi Tanabe Pharma Corporation, Japan, for his technical assistance. This work was partly supported by Mitsubishi Tanabe Pharma Corporation, Japan. The authors would like to thank Enago ( for the English language review.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest with the contents of this article.

Ethical approval

All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.


  1. 1.
    Walsh MC, Hunter GR, Livingstone MB (2006) Sarcopenia in premenopausal and postmenopausal women with osteopenia, osteoporosis and normal bone mineral density. Osteoporos Int 17:61–67. CrossRefPubMedGoogle Scholar
  2. 2.
    Spencer EM, Liu CC, Si EC, Howard GA (1991) In vivo actions of insulin-like growth factor-I (IGF-I) on bone formation and resorption in rats. Bone 12:21–26CrossRefPubMedGoogle Scholar
  3. 3.
    Kassem M, Blum W, Ristelli J, Mosekilde L, Eriksen EF (1993) Growth hormone stimulates proliferation and differentiation of normal human osteoblast-like cells in vitro. Calcif Tissue Int 52:222–226CrossRefPubMedGoogle Scholar
  4. 4.
    Weber MM (2002) Effects of growth hormone on skeletal muscle. Horm Res 58(Suppl 3):43–48. CrossRefPubMedGoogle Scholar
  5. 5.
    Elkasrawy MN, Hamrick MW (2010) Myostatin (GDF-8) as a key factor linking muscle mass and bone structure. J Musculoskelet Neuronal Interact 10:56–63PubMedCentralPubMedGoogle Scholar
  6. 6.
    Matsuzaki S, Hiratsuka T, Taniguchi M, Shingaki K, Kubo T, Kiya K, Fujiwara T, Kanazawa S, Kanematsu R, Maeda T, Takamura H, Yamada K, Miyoshi K, Hosokawa K, Tohyama M, Katayama T (2015) Physiological ER stress mediates the differentiation of fibroblasts. PLoS ONE 10:e0123578. CrossRefPubMedCentralPubMedGoogle Scholar
  7. 7.
    Schroder M, Kaufman RJ (2005) ER stress and the unfolded protein response. Mutat Res 569:29–63. CrossRefPubMedGoogle Scholar
  8. 8.
    Zhong J, Rao X, Xu JF, Yang P, Wang CY (2012) The role of endoplasmic reticulum stress in autoimmune-mediated beta-cell destruction in type 1 diabetes. Exp Diabetes Res 2012:238980. CrossRefPubMedCentralPubMedGoogle Scholar
  9. 9.
    Fox RM, Andrew DJ (2015) Transcriptional regulation of secretory capacity by bZip transcription factors. Front Biol (Beijing) 10:28–51. CrossRefPubMedCentralGoogle Scholar
  10. 10.
    Yamamoto K, Sato T, Matsui T, Sato M, Okada T, Yoshida H, Harada A, Mori K (2007) Transcriptional induction of mammalian ER quality control proteins is mediated by single or combined action of ATF6alpha and XBP1. Dev Cell 13:365–376. CrossRefPubMedGoogle Scholar
  11. 11.
    Weintraub H, Davis R, Tapscott S, Thayer M, Krause M, Benezra R, Blackwell TK, Turner D, Rupp R, Hollenberg S et al (1991) The myoD gene family: nodal point during specification of the muscle cell lineage. Science 251:761–766CrossRefPubMedGoogle Scholar
  12. 12.
    Rudnicki MA, Schnegelsberg PN, Stead RH, Braun T, Arnold HH, Jaenisch R (1993) MyoD or Myf-5 is required for the formation of skeletal muscle. Cell 75:1351–1359CrossRefPubMedGoogle Scholar
  13. 13.
    Buono R, Vantaggiato C, Pisa V, Azzoni E, Bassi MT, Brunelli S, Sciorati C, Clementi E (2012) Nitric oxide sustains long-term skeletal muscle regeneration by regulating fate of satellite cells via signaling pathways requiring Vangl2 and cyclic GMP. Stem Cells 30:197–209. CrossRefPubMedGoogle Scholar
  14. 14.
    Cordani N, Pisa V, Pozzi L, Sciorati C, Clementi E (2014) Nitric oxide controls fat deposition in dystrophic skeletal muscle by regulating fibro-adipogenic precursor differentiation. Stem Cells 32:874–885. CrossRefPubMedGoogle Scholar
  15. 15.
    Mitsuishi M, Miyashita K, Itoh H (2008) cGMP rescues mitochondrial dysfunction induced by glucose and insulin in myocytes. Biochem Biophys Res Commun 367:840–845. CrossRefPubMedGoogle Scholar
  16. 16.
    Miyashita K, Itoh H, Tsujimoto H, Tamura N, Fukunaga Y, Sone M, Yamahara K, Taura D, Inuzuka M, Sonoyama T, Nakao K (2009) Natriuretic peptides/cGMP/cGMP-dependent protein kinase cascades promote muscle mitochondrial biogenesis and prevent obesity. Diabetes 58:2880–2892. CrossRefPubMedCentralPubMedGoogle Scholar
  17. 17.
    Potter LR, Abbey-Hosch S, Dickey DM (2006) Natriuretic peptides, their receptors, and cyclic guanosine monophosphate-dependent signaling functions. Endocr Rev 27:47–72. CrossRefPubMedGoogle Scholar
  18. 18.
    Koller KJ, Goeddel DV (1992) Molecular biology of the natriuretic peptides and their receptors. Circulation 86:1081–1088CrossRefPubMedGoogle Scholar
  19. 19.
    Thomas G, Moffatt P, Salois P, Gaumond MH, Gingras R, Godin E, Miao D, Goltzman D, Lanctot C (2003) Osteocrin, a novel bone-specific secreted protein that modulates the osteoblast phenotype. J Biol Chem 278:50563–50571. CrossRefPubMedGoogle Scholar
  20. 20.
    Nishizawa H, Matsuda M, Yamada Y, Kawai K, Suzuki E, Makishima M, Kitamura T, Shimomura I (2004) Musclin, a novel skeletal muscle-derived secretory factor. J Biol Chem 279:19391–19395. CrossRefPubMedGoogle Scholar
  21. 21.
    Matsukawa N, Grzesik WJ, Takahashi N, Pandey KN, Pang S, Yamauchi M, Smithies O (1999) The natriuretic peptide clearance receptor locally modulates the physiological effects of the natriuretic peptide system. Proc Natl Acad Sci USA 96:7403–7408CrossRefPubMedGoogle Scholar
  22. 22.
    Moffatt P, Thomas G, Sellin K, Bessette MC, Lafreniere F, Akhouayri O, St-Arnaud R, Lanctot C (2007) Osteocrin is a specific ligand of the natriuretic Peptide clearance receptor that modulates bone growth. J Biol Chem 282:36454–36462. CrossRefPubMedGoogle Scholar
  23. 23.
    Rollin R, Mediero A, Roldan-Pallares M, Fernandez-Cruz A, Fernandez-Durango R (2004) Natriuretic peptide system in the human retina. Mol Vis 10:15–22PubMedGoogle Scholar
  24. 24.
    Bostrom P, Wu J, Jedrychowski MP, Korde A, Ye L, Lo JC, Rasbach KA, Bostrom EA, Choi JH, Long JZ, Kajimura S, Zingaretti MC, Vind BF, Tu H, Cinti S, Hojlund K, Gygi SP, Spiegelman BM (2012) A PGC1-alpha-dependent myokine that drives brown-fat-like development of white fat and thermogenesis. Nature 481:463–468. CrossRefPubMedCentralPubMedGoogle Scholar
  25. 25.
    Davidson B, Abeler VM, Forsund M, Holth A, Yang Y, Kobayashi Y, Chen L, Kristensen GB, Shih Ie M, Wang TL (2014) Gene expression signatures of primary and metastatic uterine leiomyosarcoma. Hum Pathol 45:691–700. CrossRefPubMedGoogle Scholar
  26. 26.
    Yasui A, Nishizawa H, Okuno Y, Morita K, Kobayashi H, Kawai K, Matsuda M, Kishida K, Kihara S, Kamei Y, Ogawa Y, Funahashi T, Shimomura I (2007) Foxo1 represses expression of musclin, a skeletal muscle-derived secretory factor. Biochem Biophys Res Commun 364:358–365. CrossRefPubMedGoogle Scholar
  27. 27.
    Steinhelper ME (1993) Structure, expression, and genomic mapping of the mouse natriuretic peptide type-B gene. Circ Res 72:984–992CrossRefPubMedGoogle Scholar
  28. 28.
    Huang H, Acuff CG, Steinhelper ME (1996) Isolation, mapping, and regulated expression of the gene encoding mouse C-type natriuretic peptide. Am J Physiol 271:H1565–H1575. CrossRefPubMedGoogle Scholar
  29. 29.
    Gogos JA, Thompson R, Lowry W, Sloane BF, Weintraub H, Horwitz M (1996) Gene trapping in differentiating cell lines: regulation of the lysosomal protease cathepsin B in skeletal myoblast growth and fusion. J Cell Biol 134:837–847CrossRefPubMedGoogle Scholar
  30. 30.
    Zammit PS, Partridge TA, Yablonka-Reuveni Z (2006) The skeletal muscle satellite cell: the stem cell that came in from the cold. J Histochem Cytochem 54:1177–1191. CrossRefPubMedGoogle Scholar
  31. 31.
    Averous J, Bruhat A, Jousse C, Carraro V, Thiel G, Fafournoux P (2004) Induction of CHOP expression by amino acid limitation requires both ATF4 expression and ATF2 phosphorylation. J Biol Chem 279:5288–5297. CrossRefPubMedGoogle Scholar
  32. 32.
    Nakanishi K, Sudo T, Morishima N (2005) Endoplasmic reticulum stress signaling transmitted by ATF6 mediates apoptosis during muscle development. J Cell Biol 169:555–560. CrossRefPubMedCentralPubMedGoogle Scholar
  33. 33.
    Yamazaki T, Ohmi A, Kurumaya H, Kato K, Abe T, Yamamoto H, Nakanishi N, Okuyama R, Umemura M, Kaise T, Watanabe R, Okawa Y, Takahashi S, Takahashi Y (2010) Regulation of the human CHOP gene promoter by the stress response transcription factor ATF5 via the AARE1 site in human hepatoma HepG2 cells. Life Sci 87:294–301. CrossRefPubMedGoogle Scholar
  34. 34.
    Haze K, Yoshida H, Yanagi H, Yura T, Mori K (1999) Mammalian transcription factor ATF6 is synthesized as a transmembrane protein and activated by proteolysis in response to endoplasmic reticulum stress. Mol Biol Cell 10:3787–3799CrossRefPubMedCentralPubMedGoogle Scholar
  35. 35.
    Yoshida H, Okada T, Haze K, Yanagi H, Yura T, Negishi M, Mori K (2000) ATF6 activated by proteolysis binds in the presence of NF-Y (CBF) directly to the cis-acting element responsible for the mammalian unfolded protein response. Mol Cell Biol 20:6755–6767CrossRefPubMedCentralPubMedGoogle Scholar
  36. 36.
    Cai WQ, Terenghi G, Bodin P, Burnstock G, Polak JM (1993) In situ hybridization of atrial natriuretic peptide mRNA in the endothelial cells of human umbilical vessels. Histochemistry 100:277–283CrossRefPubMedGoogle Scholar
  37. 37.
    Ishikawa K, Yoshida K, Kanie K, Omori K, Kato R (2018) Morphology-Based Analysis of Myoblasts for Prediction of Myotube Formation. SLAS Discov. CrossRefPubMedGoogle Scholar
  38. 38.
    Garbers DL (1999) The guanylyl cyclase receptors. Methods 19:477–484. CrossRefPubMedGoogle Scholar
  39. 39.
    Kelsall CJ, Chester AH, Sarathchandra P, Singer DR (2006) Expression and localization of C-type natriuretic peptide in human vascular smooth muscle cells. Vascul Pharmacol 45:368–373. CrossRefPubMedGoogle Scholar
  40. 40.
    Suga S, Itoh H, Komatsu Y, Ogawa Y, Hama N, Yoshimasa T, Nakao K (1993) Cytokine-induced C-type natriuretic peptide (CNP) secretion from vascular endothelial cells-evidence for CNP as a novel autocrine/paracrine regulator from endothelial cells. Endocrinology 133:3038–3041. CrossRefPubMedGoogle Scholar
  41. 41.
    Peake NJ, Hobbs AJ, Pingguan-Murphy B, Salter DM, Berenbaum F, Chowdhury TT (2014) Role of C-type natriuretic peptide signalling in maintaining cartilage and bone function. Osteoarthritis Cartilage 22:1800–1807. CrossRefPubMedGoogle Scholar
  42. 42.
    Nakao K, Osawa K, Yasoda A, Yamanaka S, Fujii T, Kondo E, Koyama N, Kanamoto N, Miura M, Kuwahara K, Akiyama H, Bessho K, Nakao K (2015) The Local CNP/GC-B system in growth plate is responsible for physiological endochondral bone growth. Sci Rep 5:10554. CrossRefPubMedCentralPubMedGoogle Scholar
  43. 43.
    Salminen A, Kaarniranta K (2010) ER stress and hormetic regulation of the aging process. Ageing Res Rev 9:211–217. CrossRefPubMedGoogle Scholar
  44. 44.
    Gong W, Duan Q, Cai Z, Chen C, Ni L, Yan M, Wang X, Cianflone K, Wang DW (2013) Chronic inhibition of cGMP-specific phosphodiesterase 5 suppresses endoplasmic reticulum stress in heart failure. Br J Pharmacol 170:1396–1409. CrossRefPubMedCentralPubMedGoogle Scholar
  45. 45.
    Ma H, Butler MR, Thapa A, Belcher J, Yang F, Baehr W, Biel M, Michalakis S, Ding XQ (2015) cGMP/Protein Kinase G signaling suppresses inositol 1,4,5-trisphosphate receptor phosphorylation and promotes endoplasmic reticulum stress in photoreceptors of cyclic nucleotide-gated channel-deficient mice. J Biol Chem 290:20880–20892. CrossRefPubMedCentralPubMedGoogle Scholar
  46. 46.
    Ramji DP, Foka P (2002) CCAAT/enhancer-binding proteins: structure, function and regulation. Biochem J 365:561–575. CrossRefPubMedCentralPubMedGoogle Scholar
  47. 47.
    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. CrossRefPubMedGoogle Scholar
  48. 48.
    Iwasa K, Nambu Y, Motozaki Y, Furukawa Y, Yoshikawa H, Yamada M (2014) Increased skeletal muscle expression of the endoplasmic reticulum chaperone GRP78 in patients with myasthenia gravis. J Neuroimmunol 273:72–76. CrossRefPubMedGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Sohyaku Innovative Research DivisionMitsubishi Tanabe Pharma CorporationTodaJapan
  2. 2.Department of Basic Medicinal Sciences, Graduate School of Pharmaceutical SciencesNagoya UniversityNagoyaJapan
  3. 3.Department of Pathology, Faculty of MedicineUniversity of MiyazakiMiyazakiJapan

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