The cardiac calsequestrin gene transcription is modulated at the promoter by NFAT and MEF-2 transcription factors

Figures

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  Fig 1. Inhibition of NFAT activity with CsA and INCA-6 decreases CASQ2 mRNA synthesis in neonatal cardiomyocytes. A) Neonatal rat cardiomyocytes were treated with DMSO as the vehicle (Control) or CsA for 12 h. The cells were then harvested and CASQ2 mRNA was determined by real-time PCR. GAPDH mRNA was used as normalizing gene B) Neonatal rat cardiomyocytes were treated with vehicle (Control) or INCA-6 for 30 h. The cells were then harvested, and CASQ2 mRNA was determined by real-time PCR. GAPDH mRNA was used as normalizing gene. Results of RT-qPCR data are expressed as the mean of three independent experiments +/- SEM. *p values < 0.05 vs control. C) Neonatal rat cardiomyocytes were treated with INCA-6 for 60 h. The cells were then harvested and CASQ2 protein levels were analyzed by Western blot. β-actin protein levels were used for normalization. A picture of a representative the gel is shown. D) Densitometry analysis of representative experiments (n = 2) was made using the Image-Studio Lite 5.2.5 software, LI-COR, NE, USA. (The data showed in the graphs can be found as supporting information in the S1 Dataset file. The complete image of the blot can be found in S1 Fig file).

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  Fig 2. Inhibition of NFAT activity with CsA and INCA-6 reduced the transcriptional activity of the hCASQ2 promoter constructs. The two hCASQ2 gene chimeric constructs were transfected into neonatal rat cardiomyocytes cultures. A) Diagram of the hCASQ2 gene promoter constructs. The boundaries of the two hCASQ2 gene chimeric constructs (-3102/+176 bp and -288/+176 bp) are indicated. As well, the relative locations of potential binding sites for transcription factors found by sequence analysis are shown. B) Luciferase activity of neonatal cardiomyocytes transfected with the pGL3-hCASQ2prom/Luc -3102/+176 bp construct (black bars) or the pGL3-hCASQ2prom/Luc -288/+176 bp construct (gray bars) and treated with vehicle (Control) or with CsA for 12 h. C) Luciferase activity of neonatal cardiomyocytes transfected with the pGL3-hCASQ2prom/Luc -3102/+176 bp (black bars) or the pGL3-hCASQ2prom/Luc -288/+176 bp construct (gray bars) and treated with vehicle (Control) or INCA-6 for 16 h. D) Luciferase activity of neonatal cardiomyocytes transfected with the pGL3-promoter construct, containing the SV-40 promoter (Promega) and treated with vehicle or INCA-6 for 16 h. Results are expressed as the mean…

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  Fig 3. Overexpression of NFAT increased transcriptional activity of hCASQ2 gene constructs. A) To verify the NFAT overexpression, the NFATc1 or NFATc3 expression vectors were transfected into neonatal rat cardiomyocytes. After 48 h, the cells were harvested and NFATc1 and NFATc3 protein levels were evaluated by Western blot. A picture of a representative gel is shown. Densitometry analysis of the blot was made using the Image-Studio Lite 5.2.5 software, LI-COR, NE, USA. B) Luciferase activity of neonatal cardiomyocytes co-transfected with pGL3-hCASQ2prom/Luc -3102/+176 bp (black bars) or pGL3-hCASQ2prom/Luc -288/+176 bp (gray bars) and the NFATc1 or NFATc3 expression vectors. After 24 h, the cells were harvested and luciferase activity was measured. C) Luciferase activity of neonatal cardiomyocytes co-transfected with pGL3-hCASQ2prom/Luc -3102/+176 bp construct and NFATc1 or NFATc3 expression vectors as above. Then, cells were treated with vehicle (black bars) or CsA (gray bars). After 24 h, the cells were harvested, and luciferase activity was measured. Results for functional assays (Fig 3B and 3C) are expressed as the mean of three independent experiments +/- SEM. The luciferase…

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  Fig 4. Sequence analysis of hCASQ2 gene promoter. The sequence analysis was made using MacVector 6.5.3 and BLAST tool from NCBI. The 5´-regulatory region is highly conserved among species. The analysis revealed a potential binding site for NFAT located between -230 to -222 bp, which is conserved among species. Within this region, there are also the binding sites for MEF-2 and SRF transcription factors between -130 to -120 bp and -103-93 bp, respectively (Modified from Reyes-Juarez JL. et al. 2007).

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  Fig 5. The NFAT-induced transactivation of hCASQ2 gene promoter is mediated by the -133 bp MEF-2 binding site. A) Luciferase activity of neonatal cardiomyocytes co-transfected with the pGL3-hCASQ2prom/Luc -288/+176 bp wild-type construct (black bars) or the -230 bp NFAT site mutated construct (gray bars) and the NFATc3 expression vector. B) Luciferase activity of neonatal cardiomyocytes co-transfected with pGL3-hCASQ2prom/Luc -288/+176 bp wild-type construct (black bars), the -103 bp SRF site mutated construct (gray bars) or the -133 bp MEF-2 site mutated construct (white bars) and the NFATc3 expression vector. After 24 h, the cells were harvested, and luciferase activity was determined. Results are expressed as the mean of three independent experiments +/- SEM. The -288 bp construct luciferase activity is given the arbitrary value of 1. *p values <0.05 were considered statistically significant. (The data showed in the graphs can be found as supporting information in the S1 Dataset file).

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  Fig 6. MEF-2 and NFAT transcription factors bind to the CASQ2 gene promoter and interact with each other. A) Chromatin Immunoprecipitation (ChIP) assay of the rat CASQ2 gene promoter; fragmented chromatin was incubated with antibodies against MEF-2c, NFATc3, Sp1 and IgG. PCR was performed to amplify the region between -259 to -21 bp of CASQ2 gene promoter. A picture of a representative gel is shown. B) Densitometry analysis of representative experiments (n = 2) was made with the Image-Studio Lite 5.2.5 software, (LI-COR, NE, USA). C) Total protein extracts were immunoprecipitated (IP) with anti-NFAT or anti-MEF-2 antibodies as indicated. Western blot analysis of immunoprecipitated proteins was performed using antibody against MEF-2 or NFAT as indicated. The identity of IP proteins is as follows: Lane 1, Mock (Protein A Sepharose antibody-binding beads without antibody); Lane 2, NFAT IP; Lane 3 MEF-2 IP. (The data showed in the graphs can be found as supporting information in the S1 Dataset file. The complete image of the blot can be found in S3 Fig file).

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  Fig 7. Proposed transcriptional regulation of hCASQ2 gene in cardiomyocytes. CASQ2 is the main SR Ca2+-binding protein. In association with Triadin (T) and Junctin (J) proteins, CASQ2 is capable to regulate the activity of the SR Ca2+-release channel RyR2. NFAT transcription factor interacts with MEF-2 bound to its -133 bp binding site at the hCASQ2 gene promoter. In this way, NFAT cooperates with MEF-2 in the transcriptional activation of the hCASQ2 gene. Inhibition of NFAT activation with cyclosporine A (CsA) or INCA-6 blocks NFAT activation and down-regulates hCASQ2 gene transcription. Abbreviations: CaM, calmodulin; CN, calcineurin; L, L-type Ca2+ channels; MEF-2, myocyte enhancer factor 2; NCX, Na+/Ca2+ exchanger; NFAT, nuclear factor of activated T cells; PLN, phospholamban; SRF, serum response factor; SR, sarcoplasmic reticulum.