Calcineurin-NFATc Regulates Type 2 Inositol 1,4,5-Trisphosphate Receptor (InsP3R2) Expression during Cardiac Remodeling*

Background: InsP3R2 is overexpressed during cardiac remodeling. The transcriptional regulation of InsP3R2 is not known. Results: InsP3R2 gene expression is regulated by calcineurin-NFATc signaling pathway. Conclusion: Calcineurin-NFATc signaling is a downstream target of the InsP3R2 that regulates InsP3R2 expression in a positive feedback loop. Significance: InsP3R2 Ca2+ channel-calcineurin-NFATc signaling network regulates ITPR2 gene and fetal gene expression during cardiac remodeling. In heart, the type 2 inositol 1,4,5-triphosphate receptor (InsP3R2) is the predominant isoform expressed and is localized in the nuclear membrane of ventricular myocytes. InsP3R2-mediated Ca2+ release regulates hypertrophy specific gene expression by modulating CaMKIIδ, histone deacetylase, and calcineurin-NFATc signaling pathways. InsP3R2 protein is a hypertrophy specific marker and is overexpressed in heart failure animal models and in humans. However, the regulation of InsP3R2 mRNA and protein expression during cardiac hypertrophy and heart failure is not known. Here we show the transcriptional regulation of the Itpr2 gene in adult cardiomyocytes. Our data demonstrates that, InsP3R2 mRNA and protein expression is activated by hypertrophic agonists and attenuated by InsP3R inhibitors 2-aminoethoxyldiphenyl borate and xestospongin-C. The Itpr2 promoter is regulated by the calcineurin-NFATc signaling pathway. NFATc1 regulates Itpr2 gene expression by directly binding to the Itpr2 promoter. The calcineurin-NFATc mediated up-regulation of the Itpr2 promoter was attenuated by cyclosporine-A. InsP3R2 mRNA and protein expression was up-regulated in calcineurin-A transgenic mice and in human heart failure. Collectively, our data suggests that ITPR2 and hypertrophy specific gene expression is regulated, in part, by a positive feedback regulation between InsP3R2 and calcineurin-NFATc signaling pathways.

tional network (2)(3)(4). At the cellular level, this leads to an increase in cell size by the activation of protein synthesis and re-activation of the fetal gene program (5). The differential gene expression includes the Ca 2ϩ handling protein genes that are modulated in response to the diverse hypertrophic stimuli for maintaining Ca 2ϩ homeostasis (6).
Inositol 1,4,5-triphosphate receptors (InsP 3 Rs) 3 are a family of Ca 2ϩ channels modulated by InsP 3 released in response to neurohumoral factor-mediated ␣/␤-adrenergic receptor activation (7). Three types of InsP 3 receptors (types 1, 2, and 3) are expressed in human cells and the expression of these receptors are cell-type specific (7,8). The expression of these receptors are modulated by physiological and pathological stimuli during development and differentiation (7). In heart, the type 2 InsP 3 receptor (InsP 3 R2) is the major isoform expressed and localized predominantly in the nuclear envelope (3,9). The nuclear membrane-enriched InsP 3 R2 regulates Ca 2ϩ release into the cytosol and nucleus (10) and regulates Ca 2ϩ dependent events in both the nucleoplasm and cytoplasm (11)(12)(13)(14)(15)(16). The InsP 3 R2mediated nuclear Ca 2ϩ transient regulate cardiomyocyte functions by activating Ca 2ϩ -dependent signaling cascades independently of the global Ca 2ϩ changes during every heart beat by excitation-contraction coupling (11,12,17). InsP 3 R2-mediated Ca 2ϩ mobilization modulates the excitation-contraction coupling in myocytes and increased expression of InsP 3 R2 in atria has been shown to induce arrhythmias (11,(17)(18)(19). Additionally, InsP 3 R2-mediated Ca 2ϩ release is instrumental in excitation-transcription coupling to activate hypertrophic gene expression by modulating CaMKII␦, histone deacetylase, and calcineurin-NFATc signaling pathways (12,13,16). 4 Recent studies have shown that there was an elevated expression of InsP 3 R2 in human and animal heart failure models (20,21). Nevertheless, the molecular mechanism that regulates the expression of InsP 3 R2 in cardiac myocytes is not well understood. In this study we have delineated the mechanism of transcriptional regulation of InsP 3 R2 expression.

EXPERIMENTAL PROCEDURES
Heart Samples-LV tissue samples were obtained from four failing human hearts after explanations in patients undergoing cardiac transplantation for end-stage dilated cardiomyopathy (DCM) performed at the Loyola University Chicago Hospital. Four non-failing (control) human heart samples were also from Loyola University Chicago Hospital. The study was approved by the Human Studies Committee of Loyola University Chicago, Maywood, IL. Hearts from calcineurin-A overexpressing transgenic (CnA-TG) mice (22) and WT littermates were a kind gift from Dr. Jeffery D. Molkentin, Cincinnati Children's Hospital, Cincinnati, OH.
Cardiomyocytes and Cells-Adult rat ventricular myocytes (ARVMs) were isolated by standard methods and the procedures complied with guidelines established in the Guide for the Care and Use of Laboratory Animals (NIH Publication 65-23), core facility, Department of Cell and Molecular Physiology, Loyola University. ARVMs were cultured in 35-or 60-mm culture dishes using rat cardiomyocyte media (Cell Applications Inc.) and treated with endothelin-1 (ET-1, 100 nM), angiotensin II (AngII, 200 nM), phenylephrine (PE, 1 M), CaMKII inhibitor KN-93 (5 M), and InsP 3 R inhibitor 2-ABP (2 M) and xestospongin-C (Xes-C, 3 M) for different time points as indicated in the figures and figure legends. For immunofluorescence experiments the cells were cultured in 4-well chambered microscopic slides. For promoter assay (live cell imaging) the cells were cultured in glass bottom 4-well chamber slides. H9C2 (rat cardiomyotube cell line) was cultured in DMEM (Cellgro) with 10% FBS, 1% penicillin/streptomycin, and 2 mM glutamine in a humidified CO 2 (5%) incubator at 37°C.
Western Blotting and Immunofluorescence-Total protein extract was prepared from human heart samples as described previously (3) and cardiomyocytes by using Hunter's buffer. Protein concentration was quantified using microBCA assay (Thermo Fisher). Approximately 20 -30 g of total proteins were resolved on 4 -15% gradient gels (Bio-Rad Laboratories) and transferred to nitrocellulose membrane by wet transfer. Membranes were probed with anti-InsP 3 R2 (T2NH and GMC2T; 1:1000 (3)) and GAPDH-HRP (1:50,000; Sigma) overnight at 4°C. Membranes were then incubated with secondary antibodies (anti-mouse and anti-rabbit-HRP, 1:10,000; Santa Cruz) for 1 h at room temperature and the bands were visualized using chemiluminescence reagent (Millipore), the images were acquired using Chemidoc instrument (Bio-Rad). Quantification by densitometry was carried out using the Image Lab software (Bio-Rad).
Real-Time qPCR-Total RNA from human samples, mouse hearts, and ARVMs were isolated using TRIzol reagent (Invitrogen) as per the manufacturer's instruction. The RNA was treated with RNase-free DNase (Ambion) and precipitated with ethanol. About 1 g of total RNA was reverse transcribed using Moloney murine leukemia virus reverse transcriptase and random primers as per the manufacturer's instruction (Invitrogen). The samples were diluted and approximately 12.5 ng of cDNA were used for quantification using Fast Start SYBR Green master mix (Roche Diagnostics GmbH) in the CFX96 Real-time PCR detection system (Bio-Rad) in a 96-well format and the results analyzed using CFX manager software (Bio-Rad). The specificity of each primer set was analyzed by the melt curve and all the primers used gave a single peak. The primers used in this study are provided in Table 1.
Construction of Rat Itpr2 Gene Promoter-To construct the rat InsP 3 R2 promoter, genomic DNA was isolated from frozen rat heart (Pel-Freeze Biologicals) by standard procedures. The mouse promoter sequence (23) was aligned with the rat genome and the conserved region was amplified using AccuPrime Pfx DNA polymerase (Invitrogen) with the following primer pairs: forward, 5Ј-AGGATCCTAGGTATCGTGT-CCCAATTTTCCTCT-3Ј, reverse, 5Ј-AAGCTGGTACCGCT-TCACGCTCGTGAGGC-3Ј. The primers were designed with 5Ј SnaBI and 3Ј KpnI sites. Initially, the RFP cassette from pTagRFP-C (Evrogen) was digested with SalI and NotI and inserted in pShuttle vector after digesting with the same enzymes and this was used to construct the Ad-RFP control virus. The amplified rat InsP 3 R2 promoter sequence (SnaBI/KpnI fragment) was inserted in pShuttle-RFP vector after excising the CMV promoter with the same enzymes. The recombinant plasmids were verified by PCR and DNA sequencing and transcription factor binding sites were identified using Genomatix Software. The deletion constructs were also made with similar strategy. To construct the adenoviruses expressing the promoter constructs, the FL and del-1 promoter cassette in pShuttle vector were used and the adenovirus was prepared using AdEasy adenoviral vector systems as described in the manufacturer's instructions (Agilent Technologies).
Promoter Assay in H9C2 Cells and ARVMs-For the promoter assay in H9C2 cells, the cells were seeded in 12-well plates, the next day the promoter constructs (ϳ1 g) were transfected using Lipofectamine 2000 (Invitrogen) reagent as per the manufacturer's instruction. After 16 h post-transfection, the cells were made quiescent by growing in serum-free media for an additional 48 h. For normalizing the transfection, the promoter constructs were co-transfected with GFP plasmid (10:1 ratio). Then the cells were treated with serum (10%), ET-1 (100 nM), AngII (200 nM), and PE (1 M) and harvested at different time points, lysed in lysis buffer (Promega), and cleared by centrifugation. The lysate (50 l) was used to measure the fluorescence intensity using a plate reader in a 96-well format (BioTek). The red fluorescence was normalized with the green fluorescence and the promoter activity was plotted. To assay the promoter activity in ARVMs the cells were seeded in cover glass bottom chamber dishes and the cells were infected with 50 -100 multiplicity of infection of adenoviruses. After 24 h post-infection the cells were treated with ET-1 (100 nM) and CsA (10 M) for 6 -12 h and live cell imaging was carried out using confocal microscopy (Leica SP5 confocal microscope equipped with a 1.3 N.A. ϫ63 water immersion objective). Fluorescence intensity from 50 to 60 cells was measured and plotted.
Luciferase Assay-Cultured ARVMs were infected with adenovirus expressing the NFATc-Luc-GFP construct. The cells were treated with ET-1 or with or without 2-APB and xestospongin-C for 12 h. The cells were lysed with passive lysis buffer (Promega) and luciferase activity was measured using the luciferase assay system (Promega). The luciferase activity was normalized with GFP fluorescence.
Electrophoretic Mobility Shift Assay (EMSA)-EMSA was carried out essentially as described (24). H9C2 cells were infected with adenoviruses expressing GFP-NFATc1 and NFATc3 (25) for 24 h and the cells were stimulated with ET-1 to induce nuclear translocation of NFATc proteins. Nuclear extracts prepared from these cells were incubated with the annealed end labeled oligonucleotides corresponding to the NFATc binding site of the Itpr2 promoter. The binding reaction was carried out with 2 g of nuclear extract and resolved on a 5% native polyacrylamide gel in 0.5ϫ TBE buffer (Tris borate-EDTA), dried, and autoradiographed. The oligonucleotides used for EMSA were NFATc-WT forward, 5Ј-TCGTGT-CGGCAATTTTCCTCCAAACC-3Ј; reverse, 5Ј-GGTTTGGA-GGAAAATTGCCGACACGA-3Ј; and NFATc-Mut forward, 5Ј-TCGTGTCGGCAATTTTttcCCAAACC-3Ј, reverse, 5Ј-GGT-TTGGgaaAAAATTGCCGACACGA-3Ј. The core NFATc binding sequences are underlined and the mutated nucleotides are shown in lowercase letters.
Statistical Analysis-All data are represented as average Ϯ S.E. Statistical tests were either Student's paired or unpaired t tests and p Ͻ 0.05 was considered as statistically significant.

InsP 3 R2 Is Overexpressed in Human Heart
Failure-The type 2 InsP 3 R is the isoform predominantly expressed in cardiomyocytes and is localized in the nuclear membrane (3,9). Recent studies have shown increased expression of this protein in spontaneously hypertensive rat animal models and ischemic dilated cardiomyopathy patient samples in the perinuclear region (20,21). To gain insights on the increased expression of InsP 3 R2 we have used human DCM samples with different etiologies 4 and studied expression of the ITPR2 gene. Western blot analysis revealed that there was an increase in expression of the InsP 3 R2 protein in human DCM heart failure samples compared with control non-failing hearts (Fig. 1A). Quantification data shows a severalfold increase of InsP 3 R2 protein (2.7 Ϯ 0.59; p Ͻ 0.05, n ϭ 4) in DCM samples compared with non-failing control hearts (Fig. 1B) and is consistent with previous studies (20,21). ITPR2 (InsP 3 R2) mRNA expression was also quantified by real-time qPCR and the results show that there was a significant increase in ITPR2 gene expression (1.5 Ϯ 0.2, p Ͻ 0.05) in DCM samples compared with the control (Fig. 1C). Expression of the ANF transcript, the well documented hypertrophic gene marker, was also increased (ϳ30 Ϯ 0.46; p Ͻ 0.01) in DCM samples compared with the controls (Fig. 1D). We then analyzed the mRNA expression levels of ITPR1 and ITPR3 (type 1 and type 3 InsP 3 R genes, respectively) in these samples. Of the four HF samples, one sample (#2) showed a 1.8-fold increase in ITPR1 mRNA and another sample (#3) showed a 2.5-fold increase in ITPR3 mRNA expression compared with the controls. However, there was no significant difference in either ITPR1 or ITPR3 mRNA expression between normal and HF samples (Fig. 1, E and F).

Name
Direction Sequence

Mouse RT-qPCR primers
Our data suggests that increased expression of ITPR2 but not ITPR1 or ITPR3 is common in heart failure with different etiologies and is regulated at the transcriptional level. The heterogeneity observed in expression levels for ITPR1 and ITPR3 may be a function of different etiologies between patients with DCM and the functional significance is unknown.
GPCR Activation Regulates InsP 3 R Expression-The molecular mechanism(s) that activates the expression of InsP 3 R2 in the heart during the development of hypertrophy and heart failure has not been addressed. To examine this, we carried out in vitro studies using ARVMs that were stimulated with hypertrophy inducing agonists and expression of the InsP 3 R2 protein was evaluated. GPCR agonists have been linked to activation of hypertrophy specific fetal genes during cardiac remodeling and heart failure (15,18,19,26). To test whether InsP 3 R2 protein expression is activated by ET-1, AngII, and PE, ARVMs were stimulated with ET-1 (100 nM), AngII (200 nM), and PE (1 M) for 16 -18 h and analyzed for InsP 3 R2 protein expression. Immunoblot analysis followed by quantification showed a significant increase in InsP 3 R2 protein levels with ET-1 (3.2 Ϯ 0.08; p Ͻ 0.05), AngII (2.5 Ϯ 0.15; p Ͻ 0.05), and PE (3.7 Ϯ 0.19; p Ͻ 0.05) stimulation for 16 h compared with untreated control cells ( Fig. 2A).
Next, we analyzed the mRNA expression for Itpr2 and Anf in ET-1-stimulated ARVMs. Total RNA isolated from ET-1-stimulated cells was reverse transcribed and real-time PCR was used to quantify the expression of Anf and Itpr2. Consistent with InsP 3 R2 protein expression, ET-1 activated expression of the Itpr2 gene transcript ϳ3-fold at 6 and 12 h and declined to 1.6-fold at 24 h (Fig. 2B). ET-1 stimulation activated Anf transcript expression ϳ7-fold at 6 h and these were sustained to 24 h (Fig. 2C). A similar increase in Itpr2 mRNA expression was observed in AngII-and PE-treated cells (data not shown). Immunofluorescence using a InsP 3 R2 specific antibody (T2NH) (3) followed by confocal microscopy showed that ET-1 increased InsP 3 R2 immunoreactivity significantly at 6 h compared with untreated cells (data not shown). The InsP 3 R2 positive cells were scored from ϳ200 cells from three different experiments and the results show that ϳ70% (67.8 Ϯ 1.4; p Ͻ 0.01) of the cells were positive for InsP 3 R2 at 6 h and there was an incremental increase in staining at 12 and 24 h (71.8 Ϯ 2.12; p Ͻ 0.01 and 74.3 Ϯ 1.93; p Ͻ 0.01), respectively (Fig. 2D). Similarly, ANF positive cells were also increased in a time-dependent manner (Fig. 2F).
These results demonstrate that GPCR activation by ET-1, AngII, and PE activates expression of the Itpr2 gene transcript and InsP 3 R2 protein expression in parallel to Anf transcript and protein. The difference in the mRNA expression pattern of these two genes may be due to their regulation by differential transcriptional programs. Whereas the mRNA expression of the Itpr2 gene was down-regulated at 24 h, the protein level remained elevated suggesting that apart from transcriptional regulation, the post-transcriptional mechanism(s) also stabilizes the InsP 3 R2 protein. Overall, these results reveal that GPCR activation up-regulates the InsP 3 R2 mRNA and protein by transcriptional and post-transcriptional mechanisms suggesting that InsP 3 R2 is a hypertrophy specific gene marker as described earlier (20,21).
Endothelin-1 Activates IPTR2 Promoter-The increased expression of InsP 3 R2 in ARVMs treated with hypertrophic agonists prompted us to look at regulation of the Itpr2 pro-moter. The mouse Itpr2 proximal promoter region has been cloned and reported (23). To study regulation of the Itpr2 gene promoter we constructed the conserved proximal promoter sequence of ITPR2 from the rat heart upstream of the red fluorescent protein (RFP) as a reporter. Additionally, we made a series of 5Ј deletion constructs (Fig. 4, A and B). Transcription factor binding sites were predicted using Genomatix software and compared with the previously reported mouse sequence. The rat Itpr2 promoter showed putative binding sites for several known cardiac-specific transcription factors MyoD, CEBP, E-box, and CARE including NFATc (NFB/c-Rel).
Because calcineurin-NFATc signaling is downstream of InsP 3 R2 Ca 2ϩ signaling and regulates hypertrophy specific gene expression (12,13,22), we examined regulation of the Itpr2 promoter by this signaling pathway. Initially we studied regulation of the Itpr2 promoter in H9C2 cells (immortalized rat cardiomyotubes). The promoter constructs were transfected together with GFP in H9C2 cells and serum starved for 48 h. The cells were then stimulated with ET-1 (100 nM, 12 h) and the results showed that ET-1 activated the ITPR2 promoter in quiescent (serum-starved) H9C2 cells. Deletion of the NFATc site completely abolished InsP 3 R2 promoter activity and further deletion did not show any additional activity (Fig.  4B). Similarly, addition of serum and other hypertrophic agonists, AngII and PE, also activated the Itpr2 promoter in quiescent H9C2 cells (data not shown) suggesting that in the immortalized rat cardiomyotube cell line, GPCR activation could increase expression of the Itpr2 gene.

Calcineurin-NFATc Regulates Itpr2
Promoter-In adult cardiomyocytes, the activation of hypertrophic gene expression is modulated by specific and distinct pathways elicited by different agonists. To gain insights on regulation of the InsP 3 R2 promoter in adult cardiomyocytes, we constructed adenoviruses expressing Itpr2-RFP reporter constructs with (Ad-Itpr2-RFP-FL) and without the NFATc site (Ad-Itpr2-RFP-del-1) (Fig.  5A).
ARVMs were infected with both of the reporter constructs and then induced with ET-1 for 12 h. Live cell imaging was carried out to measure Itpr2 promoter activity as judged by RFP fluorescence. ET-1 induced Itpr2 promoter activity in cells infected with the full-length promoter (Ad-Itpr2-RFP-FL) compared with un-stimulated control cells (Fig. 5B). In contrast, del-1 (Ad-Itpr2-RFP-del-1)-infected cells showed no increase in RFP fluorescence in either ET-1-stimulated or non-stimulated cells (Fig. 5C). Quantification of RFP fluorescence intensity from 40 to 60 cells showed that there was a significant increase (ϳ3-fold) in Itpr2 promoter activity in full-length promoter-infected ET-1-stimulated cells compared with the control (un-stimulated) and del-1 construct expressing ET-1stimulated and non-stimulated cells (Fig. 5D). For control experiments, we infected adenovirus expressing RFP protein alone and there was no significant difference in RFP protein expression in both ET-1-stimulated and non-stimulated cells (data not shown). These results suggest that the Itpr2 promoter is regulated by the calcineurin-NFATc signaling pathway and NFATc directly binds to the Itpr2 promoter to regulate its expression.
To further evaluate NFATc-mediated regulation of the Itpr2 promoter we used CsA, the potent inhibitor of calcineurin-NFAT signaling (22). ARVMs were infected with the full-length Itpr2 promoter construct (Ad-Itpr2-RFP-FL) for 24 h and then stimulated with ET-1 (100 nM) with or without CsA. ITPR2 promoter activity was measured after 12 h. As shown in Fig. 6A in cells expressing the full-length Itpr2 promoter, CsA alone had no effect and showed only a basal level of RFP fluorescence. However, when CsA-treated cells were stimulated with ET-1 (100 nM), the ET-1-mediated increase in RFP fluorescence was attenuated (Fig. 6, A and B). These data corroborate previous results and suggest that the Itpr2 promoter is regulated by direct binding of NFATc and addition of CsA attenuates ITPR2 expression by inhibiting calcineurin-NFATc signaling.
Calcineurin-NFATc Regulates Itpr2 Transcription-To confirm the previous results we examined the regulation of Itpr2 expression by the calcineurin-NFATc signaling pathway. To study activation of the NFATc transcriptional activity, ARVMs were infected with the adenovirus expressing NFATc-Luc-GFP construct for 24 h. The cells were then stimulated with ET-1(100 nM) with and without either 2-APB or Xes-C for 6 h. The cells were lysed and the luciferase activity was measured and normalized to the GFP fluorescence.
The results presented in Fig. 7A show that there was approximately a 4-fold increase in NFATc luciferase activity in ET-1stimulated cells compared with control (p Ͻ 0.05). However, both of the InsP 3 R antagonists 2-APB and Xes-C significantly inhibited ET-1-induced NFATc luciferase activity. These results suggest that ET-1 stimulation activates NFATc activity through InsP 3 R2-mediated Ca 2ϩ release and that it was attenuated by the InsP 3 R antagonists consistent with the previous studies (13).
In heart both NFATc1 and NFATc3 have been implicated in he regulation of hypertrophic gene expression (13,25). To study the transcriptional regulation of InsP 3 R2 by NFATc isoforms, we infected ARVMs with adenoviruses expressing GFP-NFATc1 and GFP-NFATc3 for 24 h followed by treatment with ET-1 (100 nM) for 12 h. Total RNA extracted from these cells was reverse transcribed and the ITPR2 mRNA level was analyzed by RT-qPCR. The results show that NFATc1-infected ET-1-stimulated cells activated Itpr2 mRNA expression (7-fold; p Ͻ 0.05) compared with NFATc1-infected non-stimulated cells. There was no significant difference in Itpr2 mRNA expression between ET-1-stimulated and non-stimulated cells infected with NFATc3 (Fig. 7B). To confirm these results, ARVMs were treated with the calcineurin-NFATc antagonist CsA and stimulated with ET-1. The results show that there was significant inhibition of Itpr2 gene expression in ET-1-stimulated cells treated with CsA compared with CsA alone control cells (Fig. 7C). These results confirm that ET-1-stimulated expression of Itpr2 was inhibited by CsA. Together these results demonstrate that InsP 3 R2-mediated Ca 2ϩ release regulates the calcineurin-NFATc signaling pathway and inhibition of either InsP 3 -mediated Ca 2ϩ release or calcineurin-NFATc signaling inhibits the activation of Itpr2 gene expression.
NFATc1 Binds to the Itpr2 Gene Promoter-To further evaluate the NFATc isoform-specific regulation of Itpr2 gene expression, we carried out EMSA as described under "Experimental Procedures." H9C2 cells were infected with adenoviruses expressing GFP-NFATc1 and GFP-NFATc3. The cells were stimulated with ET-1 for 6 h for nuclear translocation of NFATc proteins and the nuclear extract prepared from these cells was incubated with oligonucleotide corresponding to the NFAT binding site of the Itpr2 gene promoter. GFP-NFATc1 protein formed a complex with the DNA (lane 2) and this was supershifted by the GFP antibody (lane 3) confirming the presence of the NFATc1 complex (Fig. 7D). Competition assays carried out with ϫ20 WT oligo reduced the intensity of the complex (lane 4), whereas the NFATc-mut oligo retained the complex (lane 5) suggesting that NFATc1 forms a complex.
When GFP-NFATc3 nuclear extract was used in the binding reaction, it formed a less intense band (Fig. 7, lane 6) and GFP antibody did not show any supershift (lane 7). These results suggest that NFATc1 strongly binds to the Itpr2 gene promoter compared with NFATc3. These results are consistent with our RT-qPCR results, which showed elevated InsP 3 R2 mRNA expression in NFATc1-expressed ARVMs compared with NFATc3-expressed cells.

InsP 3 R2 Expression in Calcineurin-A Overexpressing Trans-
genic Animals-Our data demonstrates that the calcineurin-NFATc signaling pathway regulates expression of the Itpr2 gene by regulating a component of its promoter activity. To confirm our in vitro data we studied the expression of InsP 3 R2 mRNA in CnA-TG mouse hearts (22) and compared this with the WT littermates. We found increased expression of the InsP 3 R2 protein in CnA-TG animal hearts compared with WT. 4 Here we studied Itpr2 mRNA expression. RT-qPCR analysis showed that there was an approximate 2-fold activation of Itpr2 mRNA expression in CnA-TG mouse hearts compared with WT hearts. Similarly, in other hypertrophic gene markers, Anf, Acta1, and ␤-Mhc, gene expression was also increased in CnA-TG mouse hearts (Fig. 8). These results are consistent with previous studies (22) and confirm that expression of the Itpr2 gene is regulated by the calcineurin-NFATc signaling pathway during cardiac hypertrophy and heart failure that results in the increased expression of InsP 3 R2 protein.

DISCUSSION
In the heart, InsP 3 R2 is the predominant isoform expressed among the three types of InsP 3 Rs and largely localized to the nuclear membrane (3,9). The InsP 3 R2 channel released nuclear Ca 2ϩ regulates excitation-transcription coupling and regulates different Ca 2ϩ -sensitive signaling pathways that activate hypertrophic gene expression (4,11,12,14,15,26,28).
Recent studies have shown that the InsP 3 R2 protein is a hypertrophy specific marker and it is overexpressed during cardiac hypertrophy and heart failure in animal models and humans (20,21). However, the molecular mechanism(s) regulating the increased expression of InsP 3 R2 and its functional consequences during cardiac hypertrophy and heart failure are not known. In this study we demonstrate that InsP 3 -mediated Ca 2ϩ release activates transcriptional regulation of the Itpr2 gene through the calcineurin-NFATc signaling pathway (13). Hypertrophy agonists ET-1, AngII, and PE activate InsP 3 R2 mRNA and protein expression in cardiomyocytes and this activation is attenuated by InsP 3 R and calcineurin-NFATc signaling inhibitors 2-ABP, Xes-C, and CsA, respectively. We show that NFATc1, but not NFATc3, directly binds to the Itpr2 gene promoter to regulate its expression. Deletion of the NFATc binding region or inhibition of calcineurin-NFATc signaling attenuated activation of Itpr2 gene promoter. Consistent with these in vitro studies, InsP 3 R2 protein and mRNA expression was activated in calcineurin-A transgenic mouse hearts and human heart failure samples.
The hypertrophy agonists that activate ␣/␤-adrenergic receptors have been shown to regulate hypertrophic gene expression in cultured cardiomyocytes, animal models, and humans (3,12,15). Consistent with these studies, our data dem-onstrates that hypertrophy specific genes are activated along with the Itpr2 gene in agonist-stimulated ARVMs.
Our data show that the Itpr2 gene is one of the early hypertrophic genes expressed after agonist treatment and can be detected as early as 6 h post-stimulation. These data demonstrate that Itpr2 gene expression is regulated at early time points by distinct hypertrophic transcriptional program.
Accumulating evidence shows that G-protein coupled and growth hormone receptors present in the nuclear membrane and perinuclear region are involved in the activation of hypertrophic gene expression (29 -32). However, all these signaling mechanisms rely on InsP 3 R2 for increased nuclear Ca 2ϩ to activate hypertrophic gene transcription. InsP 3 R2-mediated Ca 2ϩ release in the activation of hypertrophic gene expression has been shown by many studies. In all these studies blocking InsP 3 R2-mediated Ca 2ϩ release using InsP 3 R2 specific antagonists 2-APB, xestospongin-C, and InsP 3 -Sponge attenuates the expression of hypertrophic gene expression (3,12,13,28). In close agreement with these studies Itpr2 gene expression was inhibited by InsP 3 R inhibitors 2-APB and Xes-C and suggests that Itpr2 and other hypertrophic genes are regulated by the InsP 3 R2-mediated nuclear Ca 2ϩ release.
The initial Ca 2ϩ released through InsP 3 R binds to calmodulin and regulates multiple Ca 2ϩ -sensitive signaling pathways to activate hypertrophic response (33). At present the major signaling pathways that are regulated by InsP 3 R2-mediated Ca 2ϩ release are CaMKII␦, histone deacetylase, and calcineurin-NFATc (12,13,16). These pathways have been shown to regulate hypertrophic development by deregulating hypertrophy specific fetal gene expression (22,34,35). The InsP 3 R inhibitors attenuated the activation of these signaling pathways followed by hypertrophic gene expression. Among these signaling pathways, calcineurin-NFATc signaling pathway-mediated fetal gene expression has been well established in the development of hypertrophy (22). Calcineurin-A is a Ca 2ϩ -calmodulin-dependent phosphatase that dephosphorylates cytosolic NFATc. The dephosphorylated NFATc translocates into the nucleus and activates hypertrophic gene expression either by binding directly to its target sequence or in concert with other transcription factors such as AP-1, GATA4, NF-B, MEF2, cAMPresponse element-binding protein, c-Myc, and IRF-1 and also the chromatin modifying proteins hypoxanthine/aminopterin/ thymidines and histone deacetylases interact with NFATc (22). Activation of calcineurin has been shown to be regulated by InsP 3 R2-mediated Ca 2ϩ release and InsP 3 R2-mediated NFATc nuclear translocation into the nucleus have been reported in rabbit cardiomyocytes (13) and during early heart development (36). In these studies, either inhibition of InsP 3 R Ca 2ϩ release or the calcineurin-NFATc pathway attenuated NFATc nuclear translocation. Moreover in ventricular cardiomyocytes, NFATc1 but not NFATc3 nuclear translocation was induced by ET-1 stimulation (37). Our results are consistent with these studies and show that Itpr2 gene expression is activated in vivo in human heart failure and CnA-TG mouse hearts. Our in vitro studies using ARVMs stimulated with hypertrophy agonists  clearly demonstrate InsP 3 R2 transcriptional and translational activation. The activation of InsP 3 R2 mRNA expression was regulated by NFATc1 compared with NFATc3. This NFATc1mediated activation of InsP 3 R2 was attenuated by InsP 3 R2 inhibitors 2-APB and Xes-C, and calcineurin-NFATc inhibitor CsA. Quantification of InsP 3 R2 transcripts in animal hearts is controversial as shown by recent studies. Using different mouse models Cooley et al. (38) showed InsP 3 R2 mRNA expression by qPCR from mouse hearts yet concluded that the InsP 3 R2 did not contribute to progression of DCM or hypertrophy. However, Drawnel et al. (39) showed that there was no increase in InsP 3 R2 mRNA expression in animal hearts, and microRNA, mi133a, regulates the increased InsP 3 R2 protein expression during cardiac hypertrophy and heart failure. This difference in Itpr2 gene expression in animal hearts may be due to the temporally constrained regulation of Itpr2 gene expression. Our in vitro data provide evidence that the Itpr2 gene is regulated during early time points and declines after 24 h suggesting that Itpr2 mRNA expression and stability are tightly regulated during early time points after ␣/␤-adrenergic receptor activation.
Several studies have shown NFATc in the regulation of the InsP 3 R1 (type 1) expression in neuronal cells and those predominantly express InsP 3 R1 (40,41). A recent study has shown that during hypertension InsP 3 R1 expression is activated in vascular smooth muscle cells in a NFATc-dependent manner (42). This increase in InsP 3 R1 sensitizes IP 3 -mediated Ca 2ϩ signaling. All these studies have shown activation of InsP 3 R1 by calcineurin-NFATc-mediated signaling. However, in the heart the InsP 3 R2 is the major isoform and this is the first report describing the regulation of InsP 3 R2 by the calcineurin-NFATc signaling pathway at the transcriptional level. Together, the positive feedback loop between the InsP 3 R2 Ca 2ϩ channel and calcineurin-NFATc pathway regulates hypertrophic gene expression including the Itpr2 gene during cardiac remodeling.
In conclusion, our data provide evidence that InsP 3 R2-mediated activation of the calcineurin-NFATc signaling pathway regulates Itpr2 and hypertrophy specific gene expression in a positive feedback loop (Fig. 9). The increased expression of InsP 3 R2 during cardiac hypertrophy and heart failure is regulated by both transcriptional and post-transcriptional mechanisms that may be important to maintain Ca 2ϩ homeostasis during cardiac remodeling. FIGURE 9. A working model showing the regulation if InsP 3 R2 expression during cardiac remodeling. ␣/␤-Adrenergic stimulation followed by phospholipase-C activation releases the second messenger InsP 3 that stimulates the InsP 3 R2 channel to release Ca 2ϩ . This Ca 2ϩ associates with calmodulin and Ca 2ϩ -CaM regulates the activity of calcineurin-A. The activated CnA dephosphorylates the phospho-NFATc transcription factor in the cytosol. The dephosphorylated NFATc translocates into the nucleus and binds to the Itpr2 gene promoter to regulate its expression in a positive feedback loop. Similarly, NFATc transcription factors activate the responsive hypertrophy specific fetal genes during cardiac remodeling process.