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Calcineurin regulates slow myosin, but not fast myosin or metabolic enzymes, during fast‐to‐slow transformation in rabbit skeletal muscle cell culture
Author(s) -
Meißner Joachim D.,
Gros Gerolf,
Scheibe Renate J.,
Scholz Michael,
Kubis HansPeter
Publication year - 2001
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1111/j.1469-7793.2001.0215b.x
Subject(s) - downregulation and upregulation , calcineurin , myosin , messenger rna , biology , skeletal muscle , ionophore , glyceraldehyde 3 phosphate dehydrogenase , endocrinology , medicine , microbiology and biotechnology , biochemistry , transplantation , membrane , gene
The addition of cyclosporin A (500 ng ml −1 ) ‐ an inhibitor of the Ca 2+ ‐calmodulin‐regulated serine/threonine phosphatase calcineurin ‐ to primary cultures of rabbit skeletal muscle cells had no influence on the expression of fast myosin heavy chain (MHC) isoforms MHCIIa and MHCIId at the level of protein and mRNA, but reduced the expression of slow MHCI mRNA. In addition, no influence of cyclosporin A on the expression of citrate synthase (CS) and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) mRNA was found. The level of enzyme activity of CS was also not affected. When the Ca 2+ ionophore A23187 (4 × 10 −7 m ) was added to the medium, a partial fast‐to‐slow transformation occurred. The level of MHCI mRNA increased, and the level of MHCIId mRNA decreased. Cotreatment with cyclosporin A was able to prevent the upregulation of MHCI at the level of mRNA as well as protein, but did not reverse the decrease in MHCIId expression. The expression of MHCIIa was also not influenced by cyclosporin A. Cyclosporin A was not able to prevent the upregulation of CS mRNA under Ca 2+ ionophore treatment and failed to reduce the increased enzyme activity of CS. The expression of GAPDH mRNA was reduced under Ca 2+ ionophore treatment and was not altered under cotreatment with cyclosporin A. When the myotubes in the primary muscle culture were electrostimulated at 1 Hz for 15 min periods followed by pauses of 30 min, a partial fast‐to‐slow transformation was induced. Again, cotreatment with cyclosporin A prevented the upregulation of MHCI at the level of mRNA and protein without affecting MHCIId expression. The nuclear translocation of the calcineurin‐regulated transcription factor nuclear factor of activated thymocytes (NFATc1) during treatment with Ca 2+ ionophore, and the prevention of the translocation in the presence of cyclosporin A, were demonstrated immunocytochemically in the myotubes of the primary culture. The effects of cyclosporin A demonstrate the involvement of calcineurin‐dependent signalling pathways in controlling the expression of MHCI, but not of MHCIIa, MHCIId, CS and GAPDH, during Ca 2+ ionophore‐ and electrostimulation‐induced fast‐to‐slow transformations. The data indicate a differential regulation of MHCI, of MHCII and of metabolism. Calcineurin alone is not sufficient to mediate the complete transformation.