Abstract
Resting intracellular Ca(2+) can be raised, in neonatal rat cardiac myocytes, by exposure to very low concentration of thapsigargin (TG). Such a Ca(2+) rise yields calcineurin (CN) activation demonstrated by increased expression of transfected luciferase cDNA under control of nuclear factor of activated T-cells (NFAT) promoter and increased translocation of NFAT to nuclei. We found that exposure of cardiac myocytes to TG is followed by increase of sarcroplasmic reticulum Ca(2+) transport ATPase (SERCA2) expression, which is further increased when CN inactivation by CAMKII (calmodulin-dependent kinase) is prevented with KN93 (CAMKII inhibitor). On the other hand, SERCA2 expression is reduced by CN inhibition with cyclosporine. We have now induced calcineurin A (CNA) α- or β-subunit gene silencing with small interfering RNA (siRNA) and observed strong interference with expression of SERCA2, both in control myocytes and following exposure to TG. Such interference is also obtained following NFAT displacement from CN with 9,10-dihydro-9,10[1',2']-benzenoanthracene-1,4-dione (INCA-6). We have also observed analogous effects on expression of phospholamban (PLB) and Na(+)/Ca(2+) exchanger (NCX). Pertinent to these findings, we have identified, by in-silico analysis, NFAT binding sites in SERCA2, PLB, and NCX1 promoters. Our experiments indicate that activation of the calcineurin-NFAT pathway by rise of resting cytosolic Ca(2+) elevates transcription/expression of SERCA2, PLB, and NCX1, providing a homeostatic mechanism for long-term control of cytosolic Ca(2+).