Figure 7. Calsarcin-2 inhibits calcineurin activity in vitro and in vivo.
C2C12 cells containing an NFAT reporter plasmid (NFATluc) were transfected with (A) full-length calcineurin A (CnA), an NFAT expression plasmid, and an increasing amount of calsarcin-2 or (B) constitutively active calcineurin (ΔC) and NFAT expression plasmid, resulting in induction of the NFAT-luciferase reporter (P < 0.01). (A) After transfection, cells underwent treatment with the calcineurin stimulators PMA and ionomycin (PMA+Iono). PMA and ionomycin strongly induced NFAT reporter activity, while increasing amounts of calsarcin-2 completely abrogated the increase in reporter gene activity. (B) The presence of calsarcin-2 inhibited the calcineurin activity in a dose-dependent and significant manner. (C) Western blot analysis (left) and densitometric quantification (right) showing induction of the endogenous calcineurin-responsive Rcan1-4 gene in Myoz1 KO mice and (D) an increase of nuclear NFATc4 in the gastrocnemius of Myoz1 KO mice compared with that of WT littermates. *P < 0.05, †P < 0.01, ‡P < 0.001.