Competitive NFAT Bottleneck For Transcriptional Activation of Endogenous Ca2+ ATPASE (SERCA2) in Adrenergic Hypertrophy

Abstract

A prominent feature of cardiac hypertrophy and failure is reduced SERCA2 expression and deficient Ca2+ signaling. To uncover a causal relationship between hypertrophy and downregulation of SERCA2, we use neonatal rat cardiac myocytes where SERCA2 transcription can be increased by exposure to 10 nM thapsigargin (TG), which allows cytosolic Ca2+ rise and calmodulin activation of calcineurin (CN). This SERCA2 rise is markedly reduced by cyclosporine (CsA), which inhibits CN phosphatase, and increased by KN-93, a calmodulin activated kinase (CAMKII) inhibitor which relieves CAMKII dependent phosphorylation and inhibition of CN. These CsA and KN-93 effects are also produced on TG enhanced luciferase expression, under the control of an NFAT (Nuclear factor of activated T Cells) dependent promoter. We conclude that NFAT dephosphorylation is a limiting factor for SERCA2 transcription. Exposure of myocytes to PE yields adrenergic hypertrophy, with rise of Atrial Natriuretic Factor (ANF) transcript, protein incorporation of 14C-phenylalanine and fluorescent staining of actin, while SERCA2 is downregulated. The adrenergic response can be reproduced by direct stimulation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate, indicating that inhibition of glycogen synthase kinase (GSK3β) by PKC and consequent reduced NFAT phosphorylation, as well as histone deacetylase (HDAC) phosphorylation by PKC activated MAPK, are involved in the mechanism of adrenergic hypertrophy. The hypertrophy response is markedly reduced by CN inhibition by CsA, indicating that CN dephosphorylation and nuclear import of NFAT play an important role in the development of hypertrophy, and SERCA2 downregulation is produced by competitive utilization of NFAT by the hypertrophy program. Interestingly, CAMKII inhibition limits the development of hypertrophy, emphasizing a rate limiting role of CAMKII dependent phosphorylation and nuclear export of HDAC in the extensive transcriptional hypertrophic program (Supported by 5 R01 HL069830-08).