Abstract
Calsequestrin-2 (CASQ2) is the main Ca2+-binding protein inside the sarcoplasmic reticulum of cardiomyocytes. Previously, we demonstrated that MEF-2 and SRF binding sites within the human CASQ2 gene (hCASQ2) promoter region are functional in neonatal cardiomyocytes. In this work, we investigated if the calcineurin/NFAT pathway regulates hCASQ2 expression in neonatal cardiomyocytes. The inhibition of NFAT dephosphorylation with CsA or INCA-6, reduced both the luciferase activity of hCASQ2 promoter constructs (-3102/+176 bp and -288/+176 bp) and the CASQ2 mRNA levels in neonatal rat cardiomyocytes. Additionally, NFATc1 and NFATc3 over-expressing neonatal cardiomyocytes showed a 2-3-fold increase in luciferase activity of both hCASQ2 promoter constructs, which was prevented by CsA treatment. Site-directed mutagenesis of the -133 bp MEF-2 binding site prevented trans-activation of hCASQ2 promoter constructs induced by NFAT overexpression. Chromatin Immunoprecipitation (ChIP) assays revealed NFAT and MEF-2 enrichment within the -288 bp to +76 bp of the hCASQ2 gene promoter. Besides, a direct interaction between NFAT and MEF-2 proteins was demonstrated by protein co-immunoprecipitation experiments. Taken together, these data demonstrate that NFAT interacts with MEF-2 bound to the -133 bp binding site at the hCASQ2 gene promoter. In conclusion, in this work, we demonstrate that the Ca2+-calcineurin/NFAT pathway modulates the transcription of the hCASQ2 gene in neonatal cardiomyocytes.
Highlights
In cardiomyocytes, the sarcoplasmic reticulum (SR) is the main intracellular Ca2+ reservoir
Our experiments showed that both MEF-2 and NFAT transcription factors are present in the CASQ2 gene proximal promoter and they physically interact with each other in neonatal cardiomyocytes
In order to verify if the inhibition of calcineurin/NFAT pathway reduces the CASQ2 expression in our experimental conditions, we treated neonatal rat cardiomyocytes in culture with cyclosporine A (CsA) during 12 h
Summary
The sarcoplasmic reticulum (SR) is the main intracellular Ca2+ reservoir. The SR has a main role in the Ca2+ homeostasis control of cardiomyocytes [1]. The SR is a complex network of membranous structures constituted by longitudinal tubules interconnected by wide cisterns. Inside the SR there are Ca2+ binding proteins that play fundamental roles in the Ca2+ homeostasis [2, 3]. The concerted action of the Ca2+-binding protein calsequestrin (CASQ), the Ca2+-release channel (RyR) and the Ca2+-ATPase pump (SERCA) control storage, release, and re-uptake of Ca2+, respectively, regulating contraction and Ca2+ homeostasis in skeletal and cardiac muscles [1, 4, 5].
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