No abstract

SummaryThe cytosolic calcium concentration ([Ca 21 ] c ) is key for the regulation of many cellular processes, such cell signaling and proliferation, metabolism, and muscle contraction. In cardiomyocytes, Ca 21 is an important regulator in many cellular functions such electrophysiological processes, excitation-contraction coupling, regulation of contractile proteins activity, energy metabolism, cell death, and transcriptional regulation by the activation of Ca 21 -dependent transcriptional pathways.

The precise control of Ca2+levels during the contraction–relaxation cycle in cardiac myocytes is extremely important for normal beat-to-beat contractile activity. The sarcoplasmic reticulum (SR) plays a key role controlling calcium concentration in the cytosol. The SR Ca2+-ATPase (SERCA2) transports Ca2+inside the SR lumen during relaxation of the cardiac myocyte. Calsequestrin (Casq2) is the main protein in the SR lumen, functioning as a Ca2+buffer and participating in Ca2+release by interacting with the ryanodine receptor 2 (RyR2) Ca2+-release channel. Alterations in normal Ca2+handling significantly contribute to the contractile dysfunction observed in cardiac hypertrophy and in heart failure. Transcriptional regulation of the SERCA2 gene has been extensively studied and some of the mechanisms regulating its expression have been elucidated. Overexpression of Sp1 factor in cardiac hypertrophy downregulates SERCA2 gene expression and increased levels of thyroid hormone up-regulates its transcription. Other hormones such norepinephrine, angiotensin II, endothelin-1, parathyroid hormone, prostaglandin-F2α, as well the cytokines tumor necrosis factor-α and interleukin-6 also downregulate SERCA2 expression. Calcium acting through the calcineurin–NFAT (nuclear factor of activated T cells) pathway has been suggested to regulate SERCA2 and CASQ2 gene expression. This review focuses on the current knowledge regarding transcriptional regulation of SERCA2 and CASQ2 genes in the normal and pathologic heart.

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.