Calcineurin a Subunit Silencing Reduces Serca2 Expression in Cardiac Myocytes

Abstract

Resting intracellular Ca2+ can be raised, in neonatal rat cardiac myocytes, by exposure to very low concentration of thapsigargin (TG). Such a Ca2+ rise yields calcineurin (CN) activation, demonstrated by increased expression of transfected luciferase cDNA under control of nuclear factor of activated T-cells (NFAT) promoter. We found that exposure of cardiac myocytes to TG is followed by increase of 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) alpha or beta subunit gene silencing with 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 calcineruin with INCA-6. We have also observed analogous effects on expression of phospholamban (PLB) and Na+/Ca2+ exchanger (NCX). Pertinent to these findings, we have identified, by in-silico analysis, NFAT binding sites in SERCA2, PLB and NCX promoters. Our experiments indicate that activation of the calcineurin/NFAT pathway by rise of resting cytosolic Ca2+ elevates transcription/expression of SERCA2, PLB and NCX, providing a homeostatic mechanism for long term control of cytosolic Ca2+ (Supported by 5 R01 HL069830-08).