Histochemistry and Cell Biology

, Volume 135, Issue 1, pp 11–20 | Cite as

Silencing SERCA1b in a few fibers stimulates growth in the entire regenerating soleus muscle

  • Ernő ZádorEmail author
  • Grzegorz Owsianik
  • Frank Wuytack
Original Paper


The neonatal isoform of the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 1 (SERCA1b) is a dominant Ca2+ pump in the young fibers of regenerating muscle. In vivo transfection of about 1% of the fibers with SERCA1b RNAi plasmid resulted in no apparent change in the transfected fibers, but enhanced the increase of fresh weight and fiber size in the whole regenerating rat soleus muscle, until the normal size was reached. Co-transfection of calcineurin inhibitor cain/cabin-1 with SERCA1b RNAi was sufficient to cut down the widespread growth stimulation, but the subsequent transfection of cain into the SERCA1b RNAi transfected muscle did not inhibit muscle growth. The SERCA1b RNAi preferably upregulated the expression of the NFAT reporter lacZ compared to controls when co-transfected into the fibers. Notably, perimuscular injection of interleukin-4 (IL-4) antibody but not that of an unrelevant antibody completely abolished the growth-promoting effect of SERCA1b RNAi. This indicates that silencing SERCA1b in a few fibers stimulates the calcineurin-NFAT-IL-4 pathway and fiber growth in the whole regenerating soleus. These results suggest the presence of an autocrine–paracrine coordination of growing muscle fibers, and put forward a new method to stimulate skeletal muscle regeneration.


SERCA1b Muscle regeneration Calcineurin Interleukin-4 Fiber growth 



Eukaryotic green fluorescent protein




Slow type myosin heavy chain


Nuclear factor of activated T-cells




Reverse transcriptase polymerase chain reaction


Adult fast type sarcoplasmic/endoplasmic reticulum Ca2+ ATPase


Neonatal sarcoplasmic/endoplasmic reticulum Ca2+ ATPase


Sarcoplasmic reticulum



Thank you to Dr. Paul Rosenberg for the NRElacZ plasmid, to Dr. Peter Vangheluwe and Dr. Magdolna Kósa for technical help. This work was supported by the TéT B-3/99, B-9/02, B-20/04 and the ETT 168/2003 grants in Hungary and the BIL 99/19 and BIL 02/18 from the Ministerie Vlaamse Gemeenschap, Belgium.

Supplementary material

418_2010_766_MOESM1_ESM.ppt (4.1 mb)
Supplementary material 1 (PPT 4213 kb)


  1. Abbott KL, Friday BB, Thaloor D, Murphy TJ, Pavlath GK (1998) Activation and cellular localization of the cyclosporine A-sensitive transcription factor NF-AT in skeletal muscle cells. Mol Biol Cell 9:2905–2916PubMedGoogle Scholar
  2. Bassel-Duby R, Olson EN (2006) Signaling pathways in skeletal muscle remodeling. Ann Rev Biochem 75:19–37CrossRefPubMedGoogle Scholar
  3. Brandl CJ, Green NM, Korczak B, MacLennan DH (1986) Two Ca2+ ATPase genes: homologies and mechanistic implications of deduced amino acid sequences. Cell 44:597–607CrossRefPubMedGoogle Scholar
  4. Brandl CJ, DeLeon S, Martin DR, MacLennan DH (1987) Adult forms of the Ca2+ ATPase of sarcoplasmic reticulum. Expression in developing skeletal muscle. J Biol Chem 262:3768–3774PubMedGoogle Scholar
  5. Brody IA (1969) Muscle contracture induced by exercise. A syndrome attributable to decreased relaxing factor. N Engl J Med 281:187–192CrossRefPubMedGoogle Scholar
  6. Brummelkamp TR, Bernards R, Agami R (2002) A system for stable expression of short interfering RNAs in mammalian cells. Science 296:550–553CrossRefPubMedGoogle Scholar
  7. Carlson BM (2005) Some principles of regeneration in mammalian systems. Anat Rec B New Anat 287:4–13PubMedGoogle Scholar
  8. Fenyvesi R, Rácz G, Wuytack F, Zádor E (2004) The calcineurin activity and MCIP1.4 mRNA levels are increased by innervation in regenerating soleus muscle. Biochem Biophys Res Commun 320:599–605CrossRefPubMedGoogle Scholar
  9. Harris JB (2003) Myotoxic phospholipases A2 and the regeneration of skeletal muscles. Toxicon 42:933–945CrossRefPubMedGoogle Scholar
  10. Harris JB, Johnson MA, Karlsson E (1975) Pathological responses of rat skeletal muscle to a single subcutaneous injection of a toxin isolated from the venom of the Australian tiger snake, Notechis scutatus scutatus. Clin Exp Pharmacol Physiol 2:383–404CrossRefGoogle Scholar
  11. Horsley V, Pavlath GK (2004) Forming a multinucleated cell: molecules that regulate myoblast fusion. Cells Tissues Organs 176:67–78CrossRefPubMedGoogle Scholar
  12. Horsley V, Friday BB, Matteson S, Kegley KM, Gephart J, Pavlath GK (2001) Regulation of the growth of multinucleated muscle cells by an NFATC2-dependent pathway. J Cell Biol 153:329–338CrossRefPubMedGoogle Scholar
  13. Horsley V, Jansen KM, Mills ST, Pavlath GK (2003) Il-4 acts as a myoblast recruitment factor during mammalian muscle growth. Cell 113:483–494CrossRefPubMedGoogle Scholar
  14. Korczak B, Zarain-Herzberg A, Brandl CJ, Ingles CJ, Green MN, MacLennan DH (1988) Structure of the rabbit fast-twitch skeletal muscle Ca2+ ATPase gene. J Biol Chem 263:4813–4819PubMedGoogle Scholar
  15. Lai MM, Burnett PE, Wolosker H, Blackshaw H, Snyder SH (1998) Cain, a novel physiologic inhibitor of calcineurin. J Biol Chem 273:18325–18331CrossRefPubMedGoogle Scholar
  16. Maruyama K, MacLennan DH (1988) Mutation of aspartic acid-351, lysine-352, and lysine-515 alters the Ca2+ transport activity of the Ca2+-ATPase expressed in COS-1 cells. Proc Natl Acad Sci USA 85:3314–3318CrossRefPubMedGoogle Scholar
  17. Mendler L, Zádor E, Dux L, Wuytack F (1998) mRNA levels of myogenic regulatory factors in rat slow and fast muscles regenerating from notexin-induced necrosis. Neuromuscul Disord 8:533–541CrossRefPubMedGoogle Scholar
  18. Moss FP, Leblond CP (1971) Satellite cells as the source of nuclei in muscles of growing rats. Anat Rec 170:421–435CrossRefPubMedGoogle Scholar
  19. Murgia M, Serrano AL, Calabria E, Pallafacchina G, Lomo T, Schiaffino S et al (2000) Ras is involved in nerve-activity-dependent regulation of muscle genes. Nat Cell Biol 2:142–147CrossRefPubMedGoogle Scholar
  20. Nakanishi K, Dohmae N, Morishima N (2007) Endoplasmic reticulum stress increases myofiber formation in vitro. FASEB J 21:2994–3003CrossRefPubMedGoogle Scholar
  21. Pan Y, Zvaritch E, Tupling RW, Rice J, de Leon S, Rudnicki M et al (2003) Targeted disruption of the ATP2A1 gene encoding the sarco(endo)plasmic reticulum Ca2+ ATPase isoform 1 (SERCA1) impairs diaphragm function and is lethal in neonatal mice. J Biol Chem 278:13367–13375CrossRefPubMedGoogle Scholar
  22. Roberts-Wilson TK, Reddy RN, Bailey JL, Zheng B, Ordas R, Gooch JL, Price SR (2010) Calcineurin signaling and PGC-1 alpha expression are suppressed during muscle atrophy due to diabetes. Biochim Biophys Acta 1803:960–967CrossRefPubMedGoogle Scholar
  23. Rosenberg P, Hawkins A, Stiber J, Shelton JM, Hutcheson K, Bassel-Duby R et al (2004) TRPC3 channels confer cellular memory of recent neuromuscular activity. Proc Natl Acad Sci USA 101:9387–9392CrossRefPubMedGoogle Scholar
  24. Schiaffino S (2010) Fibre types in skeletal muscle: a personal account. Acta Physiol 199:451–463Google Scholar
  25. Seigneurin-Venin S, Parrish E, Marty I, Rieger F, Romey G, Villaz M et al (1996) Involvement of the dihydropyridine receptor and internal Ca2+ stores in myoblast fusion. Exp Cell Res 223:301–307CrossRefPubMedGoogle Scholar
  26. Serrano AL, Murgia M, Pallafacchina G, Calabria E, Coniglio P, Lømo T et al (2001) Calcineurin controls nerve activity-dependent specification of slow skeletal muscle fibers but not muscle growth. Proc Natl Acad Sci USA 98:13108–13113CrossRefPubMedGoogle Scholar
  27. Tothova J, Blaauw B, Pallafacchina G, Rudolf R, Argentini C, Reggiani C, Schiaffino S (2006) NFATc1 nucleocytoplasmic shuttling is controlled by nerve activity in skeletal muscle. J Cell Sci 119:1604–1611CrossRefPubMedGoogle Scholar
  28. Utvik JK, Njå A, Gundersen K (1999) DNA injection into single cells of intact mice. Hum Gene Ther 10:291–300CrossRefPubMedGoogle Scholar
  29. Whalen RG, Harris JB, Butler-Browne GS, Sesodia S (1990) Expression of myosin isoforms during notexin-induced regeneration of rat soleus muscles. Dev Biol 141:24–40CrossRefPubMedGoogle Scholar
  30. Yang J, Rothermel B, Vega RB, Frey N, McKinsey TA, Olson EN, Bassel-Duby R, Williams RS (2000) Independent signals control expression of the calcineurin inhibitory proteins MCIP1 and MCIP2 in striated muscles. Circ Res 87:E61–E68PubMedGoogle Scholar
  31. Zádor E (2008) dnRas stimulates autocrine-paracrine growth of regenerating muscle via calcineurin-NFAT-IL-4 pathway. Biochem Biophys Res Commun 375:265–270CrossRefPubMedGoogle Scholar
  32. Zádor E, Wuytack F (2003) Expression of SERCA2a is independent of innervation in regenerating soleus muscle. Am J Physiol Cell Physiol 285:C853–C861PubMedGoogle Scholar
  33. Zádor E, Mendler L, Ver Heyen M, Dux L, Wuytack F (1996) Changes in mRNA levels of the sarcoplasmic-reticulum Ca2+ ATPase isoforms in the rat soleus muscle regenerating from notexin-induced necrosis. Biochem J 320:461–464Google Scholar
  34. Zádor E, Szakonyi G, Rácz G, Mendler L, Ver Heyen M, Lebacq J et al (1998) Expression of sarcoplasmic/endoplasmic reticulum Ca2+ ATPases in the rat extensor digitorum longus (EDL) muscle regenerating from notexin-induced necrosis. Acta Histochem 100:355–369PubMedGoogle Scholar
  35. Zádor E, Mendler L, Takács V, De Bleecker J, Wuytack F (2001) Regenerating soleus and extensor digitorum longus muscles of the rat show elevated levels of TNF-alpha and its receptors, TNFR-60 and TNFR-80. Muscle Nerve 24:1058–1067CrossRefPubMedGoogle Scholar
  36. Zádor E, Bottka S, Wuytack F (2002) Antisense inhibition of myoD expression in regenerating rat soleus muscle is followed by an increase in the mRNA levels of myoD, myf-5 and myogenin and by a retarded regeneration. BBA Mol Cell Res 1590:52–63Google Scholar
  37. Zádor E, Fenyvesi R, Wuytack F (2005) Expression of SERCA2a is not regulated by calcineurin or upon mechanical unloading in skeletal muscle regeneration. FEBS Lett 579:749–752CrossRefPubMedGoogle Scholar
  38. Zádor E, Vangheluwe P, Wuytack F (2007) The expression of the neonatal sarcoplasmic reticulum Ca(2+) pump (SERCA1b) hints to a role in muscle growth and development. Cell Calcium 41:379–388CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Ernő Zádor
    • 1
    • 4
    Email author
  • Grzegorz Owsianik
    • 2
  • Frank Wuytack
    • 3
  1. 1.Institute of Biochemistry, Faculty of MedicineUniversity of SzegedSzegedHungary
  2. 2.Department of Molecular Cell Biology, Laboratory of Ion Channel ResearchCatholic University of LeuvenLeuvenBelgium
  3. 3.Department of Molecular Cell Biology, Laboratory for Ca2+ transport ATPasesCatholic University of LeuvenLeuvenBelgium
  4. 4.Institute of Biochemistry, Faculty of MedicineUniversity of SzegedSzegedHungary

Personalised recommendations