Activation and Cellular Localization of the Cyclosporine A-sensitive Transcription Factor NF-AT in Skeletal Muscle Cells

Abstract

The widely used immunosuppressant cyclosporine A (CSA) blocks nuclear translocation of the transcription factor, NF-AT (nuclear factor of activated T cells), preventing its activity. mRNA for several NF-AT isoforms has been shown to exist in cells outside of the immune system, suggesting a possible mechanism for side effects associated with CSA treatment. In this study, we demonstrate that CSA inhibits biochemical and morphological differentiation of skeletal muscle cells while having a minimal effect on proliferation. Furthermore, in vivo treatment with CSA inhibits muscle regeneration after induced trauma in mice. These results suggest a role for NF-AT-mediated transcription outside of the immune system. In subsequent experiments, we examined the activation and cellular localization of NF-AT in skeletal muscle cells in vitro. Known pharmacological inducers of NF-AT in lymphoid cells also stimulate transcription from an NF-AT-responsive reporter gene in muscle cells. Three isoforms of NF-AT (NF-ATp, c, and 4/x/c3) are present in the cytoplasm of muscle cells at all stages of myogenesis tested. However, each isoform undergoes calcium-induced nuclear translocation from the cytoplasm at specific stages of muscle differentiation, suggesting specificity among NF-AT isoforms in gene regulation. Strikingly, one isoform (NF-ATc) can preferentially translocate to a subset of nuclei within a single multinucleated myotube. These results demonstrate that skeletal muscle cells express functionally active NF-AT proteins and that the nuclear translocation of individual NF-AT isoforms, which is essential for the ability to coordinate gene expression, is influenced markedly by the differentiation state of the muscle cell.

Calcineurin signaling in avian cardiovascular development

“…Together, these studies demonstrate that the early differentiation and diversification of cardiac myocytes is not dependent on calcineurin signal transduction. This finding is in contrast to the effects of CsA treatment or loss of CnA isoforms on the differentiation of cultured skeletal muscle myocytes and on the diversification of slow vs. fast skeletal muscle lineages (Abbott et al, 1998;Chin et al, 1998;Delling et al, 2000;Parsons et al, 2003). In general, calcineurin inhibition leads to loss of slow muscle fibers in cultured skeletal myocytes or genetically altered mice.…”

“…In addition, cultured mouse embryos treated with CsA beginning at E8.5 exhibited a variety of malformations and embryonic growth retardation, supporting the requirement for calcineurin signaling at these later stages (Uhing et al, 1993). In cell culture experiments, CsA treatment inhibits the differentiation of skeletal and vascular smooth muscle cells and also inhibits contractile activity and calcium handling in cardiomyocytes (Abbott et al, 1998;Janssen et al, 2000;Robida et al, 2000;Ohkawa et al, 2003). Further studies are necessary to identify specific populations of cells in the embryo affected by calcineurin inhibition in vivo and to determine the timing of sensitivity of different cell lineages during development.…”

“…In skeletal muscle cells, calcineurin inhibition prevents myogenic differentiation and promotes fast vs. slow skeletal muscle fiber types (Abbott et al, 1998;Delling et al, 2000;Friday et al, 2000). The possibility that calcineurin has a similar function in the developing heart was investigated by treatment of avian embryos in ovo with cyclosporin A (CsA).…”

Recent research developments in regeneration of skeletal muscle

“…In addition, it was shown that the inhibition of calcineurin by CsA induced extensive inflammation, marked fiber atrophy, and the appearance of immature myotubes in regenerating muscle compared with placebo-treated mice 60) . Several other studies indicated such defects in skeletal muscle regeneration when calcineurin was inhibited 62,63) , whereas transgenic activation of calcineurin is known to markedly promote the remodeling of muscle fibers after damage 50,64) .…”

Effect of calcineurin inhibitors on myotoxic activity of crotoxin and Bothrops asper phospholipase A2 myotoxins in vivo and in vitro

“…This phosphatase has been involved in muscle differentiation (Abbott et al, 1998), fiber type determination (Chin et al, 1998) and hypertrophy (Musaro et al, 1999). Calcineurin binds to and activates nuclear factor of activated T cells (NFAT) (Rao et al, 1997;Crabtree, 1999), which targets the nucleus, dimerizes with other nuclear factors and modulates transcription of target genes (Rao et al, 1997).…”