Skip to main content
SpringerLink
Log in

In vitro and ex vivo models indicate that the molecular clock in fast skeletal muscle of Atlantic cod is not autonomous

  • Published:
Molecular Biology Reports Aims and scope Submit manuscript

Abstract

The notion that the circadian rhythm is exclusively regulated by a central clock has been challenged by the discovery of peripheral oscillators. These peripheral clocks are known to have a direct influence on the biological processes in a tissue or cell. In fish, several peripheral clocks respond directly to light, thus raising the hypothesis of autonomous regulation. Several clock genes are expressed with daily rhythmicity in Atlantic cod (Gadus morhua) fast skeletal muscle. In the present study, myosatellite cell culture and short-term cultured fast skeletal muscle explant models were developed and characterized, in order to investigate the autonomy of the clock system in skeletal muscle of Atlantic cod. Myosatellite cells proliferated and differentiated in vitro, as shown by the changes in cellular and myogenic gene markers. The high expression of myogenic differentiation 1 during the early days post-isolation implied the commitment to myogenic lineage and the increasing mRNA levels of proliferating cell nuclear antigen (pcna) indicated the proliferation of the cells in vitro. Transcript levels of myogenic marker genes such as pcna and myogenin increased during 5 days in culture of skeletal muscle explants, indicating that the muscle cells were proliferating and differentiating under ex vivo conditions. Transcript levels of the clock gene aryl hydrocarbon receptor nuclear translocator-like 2 (arntl2) in myosatellite cells showed no daily oscillation regardless of photoperiod manipulation. On the other hand, mRNA levels of the clock gene circadian locomotor output cycles kaput (clock) showed circadian rhythmicity in 5-day-old skeletal muscle explant under different photoperiod regimes. The expression of arntl2, cryptochrome2 (cry2), period 2a (per2a) and nuclear receptor subfamily 1, group D, member 1 was not rhythmic in muscle explants but photoperiod manipulation had a significant effect on mRNA levels of cry2 and per2a. Taken together, the lack of rhythmicity of molecular clocks in vitro and ex vivo indicate that the putative peripheral clock in Atlantic cod fast skeletal muscle is not likely to be autonomous.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Kulczykowska E, Popek W, KBG (2010) Biological clock in fish. Science Publishers, Enfield

    Book  Google Scholar 

  2. Dardente H, Cermakian N (2007) Molecular circadian rhythms in central and peripheral clocks in mammals. Chronobiol Int 24:195–213

    Article  CAS  PubMed  Google Scholar 

  3. Vatine G, Vallone D, Gothilf Y, Foulkes NS (2011) It’s time to swim! Zebrafish and the circadian clock. FEBS Lett 585:1485–1494

    Article  CAS  PubMed  Google Scholar 

  4. Sánchez JA, Madrid JA, Sánchez-Vázquez FJ (2010) Molecular cloning, tissue distribution, and daily rhythms of expression of per1 gene in European sea bass (Dicentrarchus labrax). Chronobiol Int 27:19–33

    Article  PubMed  Google Scholar 

  5. Amaral IPG, Johnston IA (2012) Circadian expression of clock and putative clock controlled genes in skeletal muscle of the zebrafish. Am J Physiol Regul Integr Comp Physiol 302:R193–R206

    Article  CAS  PubMed  Google Scholar 

  6. Whitmore D, Foulkes NS, Strähle U, Sassone-Corsi P (1998) Zebrafish Clock rhythmic expression reveals independent peripheral circadian oscillators. Nat Neurosci 1(701):707

    Google Scholar 

  7. Velarde E, Haque R, Iuvone PM, Azpeleta C, Alonso-Gomez AL, Delgado MJ (2009) Circadian clock genes of goldfish, Carassius auratus: CDNA cloning and rhythmic expression of period and cryptochrome transcripts in retina, liver, and gut. J Biol Rhythms 24:104–113

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  8. Patiño MAL, Rodríguez-Illamola A, Conde-Sieira M, Soengas JL, Míguez JM (2011) Daily rhythmic expression patterns of clock1a, bmal1, and per1 genes in retina and hypothalamus of the rainbow trout, Oncorhynchus mykiss. Chronobiol Int 28(381):389

    Google Scholar 

  9. Kowalska E, Brown SA (2007) Peripheral clocks: keeping up with the master clock. Cold Spring Harb Symp Quant Biol 72:301–305

    Article  CAS  PubMed  Google Scholar 

  10. Carr AJF, Whitmore D (2005) Imaging of single light-responsive clock cells reveals fluctuating free-running periods. Nat Cell Biol 7:319–321

    Article  CAS  PubMed  Google Scholar 

  11. Tamai TK, Young LC, Whitmore D (2007) Light signaling to the zebrafish circadian clock by Cryptochrome 1a. Proc Natl Acad Sci USA 104:14712–14717

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  12. Whitmore D, Foulkes NS, Sassone-Corsi P (2000) Light acts directly on organs and cells in culture to set the vertebrate circadian clock. Nature 404:87–91

    Article  CAS  PubMed  Google Scholar 

  13. Plautz JD, Kaneko M, Hall JC, Kay SA (1997) Independent photoreceptive circadian clocks throughout Drosophila. Science 278:1632–1635

    Article  CAS  PubMed  Google Scholar 

  14. Giebultowicz JM (2001) Peripheral clocks and their role in circadian timing: insights from insects. Philos Trans R Soc Lond B Biol Sci 356:1791–1799

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  15. Lazado CC, Nagasawa K, Babiak I, Kumaratunga HPS, Fernandes JMO (2014) Circadian rhythmicity and photic plasticity of myosin gene transcription in fast skeletal muscle of Atlantic cod (Gadus morhua). Mar Genomics. doi:10.1016/j.margen.2014.04.011

    PubMed  Google Scholar 

  16. Lazado CC, Kumaratunga HPS, Nagasawa K, Babiak I, Giannetto A, Fernandes JMO (2014) Daily rhythmicity of clock gene transcripts in Atlantic cod fast skeletal muscle. PLoS ONE 9(6):e99172

  17. Nagasawa K, Giannetto A, Fernandes JMO (2012) Photoperiod influences growth and MLL (mixed-lineage leukaemia) expression in Atlantic cod. PLoS ONE 7(5):e36908

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  18. Almeida FFL, Taranger GL, Norberg B, Karlsen Ø, Bogerd J, Schulz RW (2009) Photoperiod-modulated testis maturation in Atlantic cod (Gadus morhua, L.). Biol Reprod 80:631–640

    Article  CAS  PubMed  Google Scholar 

  19. Imsland AK, Foss A, Koedijk R, Folkvord A, Stefansson SO, Jonassen TM (2007) Persistent growth effects of temperature and photoperiod in Atlantic cod Gadus morhua. J Fish Biol 71:1371–1382

    Article  Google Scholar 

  20. Giannetto A, Nagasawa K, Fasulo S, Fernandes JMO (2013) Influence of photoperiod on expression of DNA (cytosine-5) methyltransferases in Atlantic cod. Gene 519(222):230

    Google Scholar 

  21. Koumans JTM, Akster HA, Dulos GJ, Osse JWM (1990) Myosatellite cells of Cyprinus carpio (Teleostei) in vitro: isolation, recognition and differentiation. Cell Tissue Res 261:173–181

    Article  Google Scholar 

  22. Bower NI, Johnston IA (2009) Selection of reference genes for expression studies with fish myogenic cell cultures. BMC Mol Biol 10:80

    Article  PubMed Central  PubMed  Google Scholar 

  23. Stern-Straeter J, Bran G, Riedel F, Sauter A, Hörmann K, Goessler UR (2008) Characterization of human myoblast cultures for tissue engineering. Int J Mol Med 21:49–56

    CAS  PubMed  Google Scholar 

  24. Funkenstein B, Balas V, Skopal T, Radaelli G, Rowlerson A (2006) Long-term culture of muscle explants from Sparus aurata. Tissue Cell 38:399–415

    Article  CAS  PubMed  Google Scholar 

  25. Fernandes JMO, Mommens M, Hagen Ø, Babiak I, Solberg C (2008) Selection of suitable reference genes for real-time PCR studies of Atlantic halibut development. Comp Biochem Physiol B: Biochem Mol Biol 150:23–32

    Article  Google Scholar 

  26. Giannetto A, Fernandes JMO, Nagasawa K, Mauceri A, Maisano M et al (2014) Influence of continuous light treatment on expression of stress biomarkers in Atlantic cod. Dev Comp Immunol 44:30–34

    Article  CAS  PubMed  Google Scholar 

  27. Koumans JTM, Akster HA (1995) Myogenic cells in development and growth of fish. Comp Biochem Physiol A Physiol 110:3–20

    Article  Google Scholar 

  28. Fauconneau B, Paboeuf G (2000) Effect of fasting and refeeding on in vitro muscle cell proliferation in rainbow trout (Oncorhynchus mykiss). Cell Tissue Res 301:459–463

    Article  CAS  PubMed  Google Scholar 

  29. Gabillard JC, Sabin N, Paboeuf G (2010) In vitro characterization of proliferation and differentiation of trout satellite cells. Cell Tissue Res 342:471–477

    Article  PubMed  Google Scholar 

  30. Rudnicki MA, Schnegelsberg PNJ, Stead RH, Braun T, Arnold HH, Jaenisch R (1993) MyoD or Myf-5 is required for the formation of skeletal muscle. Cell 75:1351–1359

    Article  CAS  PubMed  Google Scholar 

  31. Richardson BE, Nowak SJ, Baylies MK (2008) Myoblast fusion in fly and vertebrates: new genes, new processes and new perspectives. Traffic 9:1050–1059

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  32. Tamai TK, Young LC, Cox CA, Whitmore D (2012) Light acts on the zebrafish circadian clock to suppress rhythmic mitosis and cell proliferation. J Biol Rhythms 27:226–236

    Article  CAS  PubMed  Google Scholar 

  33. Escobar C, Cailotto C, Angeles-Castellanos M, Delgado RS, Buijs RM (2009) Peripheral oscillators: the driving force for food-anticipatory activity. Eur J Neurosci 30(1665):1675

    Google Scholar 

  34. Dibner C, Schibler U, Albrecht U (2010) The mammalian circadian timing system: organization and coordination of central and peripheral clocks. Annu Rev Physiol 72:517–549

    Article  CAS  PubMed  Google Scholar 

  35. Cavallari N, Frigato E, Vallone D, Fröhlich N, Lopez-Olmeda JF et al (2011) A blind circadian clock in cavefish reveals that opsins mediate peripheral clock photoreception. PLoS Biol 9:e1001142

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  36. Cassone VM (1998) Melatonin’s role in vertebrate orcadian rhythms. Chronobiol Int 15:457–473

    Article  CAS  PubMed  Google Scholar 

  37. Jung-Hynes B, Huang W, Reiter RJ, Ahmad N (2010) Melatonin resynchronizes dysregulated circadian rhythm circuitry in human prostate cancer cells. J Pineal Res 49:60–68

    PubMed Central  CAS  PubMed  Google Scholar 

  38. McDonald MJ, Rosbash M (2001) Microarray analysis and organization of circadian gene expression in Drosophila. Cell 107:567–578

    Article  CAS  PubMed  Google Scholar 

  39. Balsalobre A, Damiola F, Schibler U (1998) A serum shock induces circadian gene expression in mammalian tissue culture cells. Cell 93:929–937

    Article  CAS  PubMed  Google Scholar 

  40. Moore HA, Whitmore D (2014) Circadian rhythmicity and light sensitivity of the zebrafish brain. PLoS ONE 9:e86176

    Article  PubMed Central  PubMed  Google Scholar 

  41. Dixit AS, Sougrakpam R (2013) Circadian rhythmicity in photoperiodic regulation of reproductive responses in the Yellow-breasted bunting. Biol Rhythm Res 44:589–600

    Article  CAS  Google Scholar 

  42. Glickman G, Webb IC, Elliott JA, Baltazar RM, Reale ME, Lehman MN, Gorman MR (2012) Photic sensitivity for circadian response to light varies with photoperiod. J Biol Rhythms 27:308–318

    Article  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by a grant from the Research Council of Norway (ref. 190350/S40) to J.M.O. Fernandes. The technical assistance of Hilde Ribe and Katrine Klippenberg (University of Nordland) is also gratefully acknowledged. The two anonymous reviewers are also acknowledged for their constructive criticisms, which greatly improved this paper.

Author information

Authors and Affiliations

  1. Faculty of Biosciences and Aquaculture, University of Nordland, 8049, Bodø, Norway

    Carlo C. Lazado, Hiruni P. S. Kumaratunga, Kazue Nagasawa, Igor Babiak, Christopher Marlowe A. Caipang & Jorge M. O. Fernandes

  2. Department of Zoology, Faculty of Applied Sciences, University of Sri Jayewardenepura, Nugegoda, 10250, Sri Lanka

    Hiruni P. S. Kumaratunga

  3. Laboratory of Aquacultural Biology, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Sendai, 981-8555, Japan

    Kazue Nagasawa

  4. School of Applied Science, Temasek Polytechnic, Singapore, 529757, Singapore

    Christopher Marlowe A. Caipang

Authors
  1. Carlo C. Lazado
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hiruni P. S. Kumaratunga
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kazue Nagasawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Igor Babiak
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Christopher Marlowe A. Caipang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jorge M. O. Fernandes
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jorge M. O. Fernandes.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Lazado, C.C., Kumaratunga, H.P.S., Nagasawa, K. et al. In vitro and ex vivo models indicate that the molecular clock in fast skeletal muscle of Atlantic cod is not autonomous. Mol Biol Rep 41, 6679–6689 (2014). https://doi.org/10.1007/s11033-014-3551-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11033-014-3551-5

Keywords

Search

Navigation