Requirement of Nuclear Factor of Activated T-cells in Calcineurin-mediated Cardiomyocyte Hypertrophy*

The calcium-activated phosphatase calcineurin has been implicated as a critical intracellular signal transducer of cardiomyocyte hypertrophy. Although previous data suggested the nuclear factor of activated T-cells (NFAT) as its sole transcriptional effector, the absolute requirement of NFAT as a mediator of calcineurin signaling has not been examined in the heart. We therefore investigated the expression and activation profile of NFAT genes in the heart. Four members (NFATc1–c4) are expressed in cardiomyocytes, elicit nuclear translocation upon calcineurin activation, and are able to drive transactivation of cardiac promoter luciferase constructs. To define the necessary function of NFAT factors as hypertrophic transducers, a dominant negative NFAT construct was created, encompassing part of the N-terminal region of NFATc4 containing a conserved calcineurin-binding motif. Cotransfection of this construct dose-dependently abrogated promoter activation, irrespective of the NFAT isoform used, whereas a control construct with the calcineurin-binding motif mutated displayed no such effects. Adenoviral gene transfer of dominant negative NFAT rendered cardiomyocytes resistant toward all aspects of calcineurin or agonist-induced cardiomyocyte hypertrophy, whereas adenoviral gene transfer of the control construct had no discernable effect on these parameters. These results indicate that multiple NFAT isoforms are expressed in cardiomyocytes where they function as necessary transducers of calcineurin in facilitating cardiomyocyte hypertrophy.

The calcium-activated phosphatase calcineurin has been implicated as a critical intracellular signal transducer of cardiomyocyte hypertrophy. Although previous data suggested the nuclear factor of activated T-cells (NFAT) as its sole transcriptional effector, the absolute requirement of NFAT as a mediator of calcineurin signaling has not been examined in the heart. We therefore investigated the expression and activation profile of NFAT genes in the heart. Four members (NFATc1-c4) are expressed in cardiomyocytes, elicit nuclear translocation upon calcineurin activation, and are able to drive transactivation of cardiac promoter luciferase constructs. To define the necessary function of NFAT factors as hypertrophic transducers, a dominant negative NFAT construct was created, encompassing part of the N-terminal region of NFATc4 containing a conserved calcineurin-binding motif. Cotransfection of this construct dose-dependently abrogated promoter activation, irrespective of the NFAT isoform used, whereas a control construct with the calcineurin-binding motif mutated displayed no such effects. Adenoviral gene transfer of dominant negative NFAT rendered cardiomyocytes resistant toward all aspects of calcineurin or agonist-induced cardiomyocyte hypertrophy, whereas adenoviral gene transfer of the control construct had no discernable effect on these parameters. These results indicate that multiple NFAT isoforms are expressed in cardiomyocytes where they function as necessary transducers of calcineurin in facilitating cardiomyocyte hypertrophy.
Heart failure is a leading cause of morbidity and mortality in industrial countries, affecting over 10 million Americans and Western Europeans, with a 5-year mortality approaching 50% despite current medical therapy (1). 1 These mortality figures reflect the lack of biologically efficacious therapies directed against the underlying disease processes that lead to maladaptive left ventricular remodeling and, ultimately, failure itself. In response to a plethora of intra-and extracardiac stimuli, cardiomyocytes exhibit cellular enlargement or hypertrophy as a compensatory adaptation to increased ventricular wall stress (3). However, sustained cardiac hypertrophy is the single most important risk factor for the development of heart failure (4,5). Because intracellular signaling pathways are thought to both initiate and perpetuate the cardiac hypertrophic response and its transition to dilated failure, recent investigation has attempted the identification of key regulatory factors with the goal of defining novel therapeutic targets (3).
One recently characterized intracellular signaling pathway that links extracellular stimuli to a hypertrophic transcriptional response employs the phosphatase calcineurin and its downstream transcriptional effector nuclear factor of activated T-cells (NFAT). 2 Four of the five NFAT proteins (NFATc1, NFAT2, or NFATc; NFATc2, NFAT1, or NFATp; NFATc3, NFAT4, or NFATx; and NFATc4 or NFAT3) reside in the cytoplasm in unstimulated cells but quickly translocate to the nucleus in response to stimulation that promote Ca 2ϩ mobilization (6). The Ca 2ϩ -calmodulin-activated phosphatase calcineurin physically interacts with NFAT members within the cytoplasm, where it directly dephosphorylates multiple serine residues within the N-terminal regulatory domain of NFAT, resulting in the unmasking of two nuclear localization sequences required for nuclear import (7)(8)(9).
Calcineurin-NFAT signaling has been implicated as a critical regulator of the cardiac hypertrophic growth response. Molkentin et al. (10,11) generated several lines of transgenic mice expressing activated mutants of either calcineurin or NFATc4 in a cardiac-selective manner, which developed robust hypertrophy that quickly transitioned to ventricular dilation and overt heart failure. The identification of calcineurin as a signaling factor has attracted considerable interest, in part due to the demonstration that the calcineurin inhibitory drugs cy-closporin A and FK506 were shown to abrogate the cardiomyopathic response in several, but not all, rodent models of congenital and acquired forms of hypertrophic heart disease (reviewed in Refs. 12 and 13). A central role for calcineurin in the cardiac hypertrophic response was substantiated by the observation that hearts from transgenic mice expressing either MCIP1, a dominant negative calcineurin mutant, or the calcineurin inhibitory domains of Cain or AKAP79, were largely resistant to pleiotropic, hypertrophic stimuli (14 -16). More recently, calcineurin A␤ gene-targeted mice were generated and shown to be defective in mounting a cardiac hypertrophic response due to pressure overload or agonist infusion (17). Although a large number of studies have convincingly demonstrated the importance of calcineurin as a hypertrophic mediator, the importance of the downstream NFAT factors has not been evaluated in cardiomyocytes.
In the present study we demonstrate the presence of all four calcineurin-sensitive members of the NFAT family (NFATc1, -c2, -c3, and -c4) in the ventricular cardiomyocyte cell lineage. All four isoforms displayed calcineurin-dependent nuclear translocation and the ability to transactivate cardiac promoters. To simultaneously inhibit all myocardial NFAT factors in an effort to effectively examine their necessary function as hypertrophic transducers, a dominant negative NFAT strategy was developed. Dominant negative NFAT dose-dependently abrogated calcineurin-NFAT-dependent transactivation of MCIP1 and BNP promoter luciferase constructs. Adenoviralmediated gene transfer of dominant negative NFAT in cultured cardiomyocytes efficiently inhibited calcineurin-and agonistinduced cardiomyocyte hypertrophy. Taken together, these data demonstrate a previously unexpected level of redundancy of the downstream targets of calcineurin and establish their requirement in pathophysiological signaling in the cardiomyocyte.
Tissue Culture and Transient Transfection Assays-Adult mouse ventricular myocytes were isolated as described previously (19), pelleted by centrifugation, and stored at Ϫ80°C. Isolation and culture of neonatal rat ventricular cardiomyocytes was performed as described before in detail (20). Low passage COS-7 and HEK 293 cells were grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum. COS-7 cells were grown in 12-well plates and transfected using 5 l of FuGENE 6 reagent (Roche Molecular Biochemicals, Indianapolis) and a total of 2 g of DNA, consisting of the above luciferase reporter constructs, in the presence or absence of expression vectors for ⌬CnA; NFATc1, -c2, -c3, or -c4; pECE-NFATc4(PXIXIT); pCDNA3-NFATc4(Ala mut); pECE-(⌬315)NFATc3; or pCG-GATA4 as indicated. In addition, 20 ng of pRL-CMV (Promega), an expression vector containing the Renilla luciferase gene under control of a CMV promoter, was included in each experiment to correct for transfection efficiency (see below). Empty expression vector was used to normalize the DNA amount. The cultures were harvested for luciferase activities 48 h after transfection. Fifty microliters of cell extract (100 l) was assayed for luciferase activity for 3 s in a Biocounter M1500 luminometer (Lumac, Netherlands) using the Dual Luciferase assay system (Promega), where firefly luciferase activity is normalized for Renilla luciferase activity to control for variations in transfection efficiency according to the manufacturer's procedures.
RT-PCR and Northern Blot Analysis-Total RNA was isolated from the indicated murine tissues or cell types using TRIzol reagent (Invitrogen). The presence of NFATc1, -c2, -c3, or -c4 mRNA in adult C57BL/6 murine ventriculocytes was analyzed by RT-PCR using primers specific for the individual NFAT isoforms as described before (21). Northern blot hybridizations on size-fractionated total RNA (10 g) from indicated tissues were performed as described previously (22). To obtain probes specific for the NFAT isoforms, the mRNA sequences of murine NFATc1 through c4 were aligned (using ClustalW software) and primers designed for the 3Ј-untranslated regions showing no or minimal overlap (primers: NFATc1 fw, 5Ј-GATGCTGAACCTGAGACGCC and rv, 5Ј-GCCACCAGCCAGTCTGGTGT; NFATc2 fw, 5Ј-ATTGCTATCT-TAGTAAAATCAAGG and rv, 5Ј-TAATCTGAAAGCAAGA; NFATc3 fw, 5Ј-GGTGATGAGAGACACTCCTCTCCC and rv, 5Ј-ATCATATAAAAG-TACCTA; NFATc4 fw, 5Ј-CCGCACAGCCTCACTGATGT and rv 5Ј-GC-CACCGCTCCTTCCTCC). The isoform-specific probes were randomly labeled with [ 32 P]dCTP (E. I. du Pont de Nemours & Co. NV, Brussels, Belgium), added to the blots and incubated in Rapid Hyb hybridization solution (Amersham Biosciences) at 58°C. Stringent post-hybridization wash conditions were used. Filters were exposed to phosphorimaging screens (Bio-Rad) and analyzed using Quantity 1 (Bio-Rad) and Adobe Photoshop 6.0 software. The intensity of the18 S ribosomal RNA band detected with a radiolabeled 18 S probe was used as a quantitative control.
Immunocytochemistry-Fixed cultured cardiomyocytes underwent immunocytochemistry as previously described in detail (11,20,24). To visualize the subcellular localization of NFATc1, -c2, and -c3, primary, isoform-specific antibodies (see Western blots) were used at a dilution of 1:400 followed by corresponding anti-mouse or anti-rabbit Oregon green-labeled secondary antibody incubation (Molecular Probes) at a dilution of 1:400. Cells were washed with phosphate-buffered saline/ 0.1% Nonidet P-40, including bisbenzimide (Sigma) to visualize nuclei. For visualization of cardiomyocyte size, sarcomeric organization, and perinuclear ANF expression, the primary antibody included polyclonal anti-rat ANF (Peninsula laboratories), followed by secondary anti-rabbit Oregon green (Molecular Probes)-conjugated antibody and a phalloidin Texas Red-conjugated antibody (Molecular Probes), all used at a dilution of 1:400. An epifluorescence microscope (Eclipse E800, Nikon) was used to visualize the cells at a 400ϫ magnification. Quantitation of cardiomyocyte cell surface area was performed on digitized images using NIH Image software. At least 50 cardiomyocytes in 10 -20 fields were examined in three independent experiments.
Statistical Analysis-The results are presented as mean values Ϯ S.E. Statistical analyses were performed using InStat 3.0 software (GraphPad Software Inc., San Diego, CA) and analysis of variance followed by Bonferroni's post-test when appropriate.

Presence of Four NFAT Members in Ventricular
Cardiomyocytes-RT-PCR analysis was employed to investigate which members of the NFAT transcription factor family are present in ventricular cardiomyocytes, and hence, might function as calcineurin effectors. Using RNA from adult mouse ventricular cardiomyocytes, transcripts of the expected size for NFATc2, -c3, and -c4 were easily detected following a limited number of amplifications (Fig. 1A). NFATc1 was also detectable albeit at lower levels than the other isoforms (Fig. 1A, top left panel, lane 2). As a control for RT quality, a glyceraldehyde-3-phosphate dehydrogenase RT-PCR was performed, which resulted in robust product in RT material (data not shown). These data suggest that transcripts of all NFAT isoforms are present in the ventricular muscle cell lineage.
To extend these results, Northern blot analyses for the different NFAT isoforms were performed on RNA isolated from several murine muscle types, including the individual cardiac chambers (right and left ventricle and atria), three skeletal muscle types (diaphragm, quadriceps, and gastrocnemius), and visceral smooth muscle (stomach) using NFAT isoform-specific probes. The NFATc4-specific probe yielded the most intense signal in both cardiac chambers, displaying two prominent transcripts, one of 6.0 and one of about 4.5 kb, with the latter giving the most intense signal. The same two transcripts were present throughout all muscle types, albeit at lower levels ( Fig.  1B, lower panel). NFATc3 gave two transcript sizes, with a smaller one of about 4.8 kb being the most intense in all muscle types, but most prominently expressed in the cardiac ventricular chambers. Two transcripts for NFATc2 were also detected, which were each expressed at comparable levels in all tissues tested. NFATc1 showed three different transcripts, with the smallest transcript (2.0 kb) being expressed in the left ventricle and the two larger transcripts being expressed at roughly equivalent levels in most other tissues analyzed (Fig. 1B, upper  panel). All blots were probed for 18 S to verify quality and equal loading of RNA (data not shown). Collectively, these data indicate that transcripts for all four calcineurin-regulated NFAT factors are present in cardiomyocytes, with those for NFATc3 and NFATc4 being present at the highest levels.
To verify whether these transcripts were also efficiently translated into their respective protein products, a series of Western blots were performed on total protein lysate from cultured neonatal rat ventriculocytes using isoform-specific antibodies. Tissue extracts of rat brain and rat thymus (data not shown) served as positive controls. Fig. 1C demonstrates that signals for all four calcineurin-regulated NFAT factors were obtained in cardiomyocytes, albeit at differing intensity, and with products ranging in mass from 70 to 200 kDa. For NFATc1 and NFATc2, discrete protein signals were observed, whereas for NFATc3 and NFATc4 multiple bands in the range of 100 to 200 kDa were observed. This may reflect generation of proteins by alternatively spliced transcripts (see Fig. 1B) and/or by differential phosphorylation states of the NFATc3 and NFATc4 proteins. Although the different affinities of the separate antibodies used do not allow for direct comparison of signal intensity, it is interesting to note that the relatively higher intensity of the protein signals for NFATc3 (Fig. 1C) correlates with its relatively high signal in the Northern blot analysis (Fig. 1B). Conclusively, RT-PCR, Northern blot, and Western blot analyses all point toward the existence of all four calcineurin-regulated NFAT isoforms in the ventricular cardiomyocyte.
Nuclear Translocation of All Cardiac NFAT Isoforms upon Calcineurin Activation-NFAT transcription factors are dephosphorylated upon activation of the Ca 2ϩ /CaM-dependent phosphatase calcineurin, resulting in unmasking of their nuclear localization signals permitting nuclear import. To verify that NFATc isoforms could be activated by calcineurin in cardiac myocytes, we performed immunocytochemistry for each NFAT factor at baseline or after infection with an adenovirus expressing a constitutively activated form of calcineurin (AdCnA) or after stimulation with Endo-1. Cardiomyocytes were 4Ј,6-diamidino-2-phenylindole-stained to visualize the nuclei and facilitate observation of nuclear localization (Fig. 2, B, D, F, and H, left and right panels). NFATc1, -c2, and -c3 were easily detectable using their respective antibodies and displayed a predominant cytosolic localization in unstimulated cardiomyocytes ( Fig. 2A, left and right panels, NFATc1 and -c3, and data not shown for NFATc2). AdCnA infection resulted in nuclear accumulation of each NFAT isoform in nearly 100% of the myocytes evaluated. Stimulation with the agonist Endo-1 for 12 h resulted in efficient NFATc1 and c3 nuclear translocation in about 70% of cardiomyocytes (Fig. 2E, left and right panels). Similar findings were obtained for NFATc2 (data not shown). To control for the specificity of the antibodies used, the primary isoform-specific antibody was omitted, which resulted in background fluorescence (Fig. 2G, left and right panels). All cells examined were also positive for sarcomeric actin using phalloidin staining, thereby confirming their identity as cardiomyocytes (data not shown). These results indicate that NFATc1, -c2, and -c3 are equally sensitive to endogenous calcineurin activation as demonstrated previously for NFATc4 (10) and point toward a potential contri-bution for all NFAT members in calcineurin signaling in the ventricular cardiomyocyte.
All Cardiac NFAT Isoforms Participate in MCIP1 Induction-To explore whether the calcineurin-mediated, nuclear import of the cardiac NFAT isoforms was associated with their ability to participate in transcriptional activity of cardiac-specific, calcineurin-responsive promoters, a series of transient cotransfection assays were carried out. Recently, a novel gene was characterized that is present at low levels under physiological conditions in the heart but undergoes dramatic upregulation following calcineurin activation in the heart (15,18,25). Remarkably, the gene product itself is a highly specific inhibitor of calcineurin, and the gene was therefore designated myocyte-enriched calcineurin inhibitory protein-1 (MCIP1). It is thought that MCIP1 participates in a negative feedback loop to prevent the deleterious effects of unrestrained activation of the enzyme in the ventricular myocyte (15,18,25). Analysis of the gene structure revealed intron 3 to harbor multiple NFAT consensus sites and was found to be uniquely sensitive to calcineurin-NFAT activation (18). Cotransfection of hMCIP1-(Int3)Luc with expression vectors for the individual NFAT isoforms only slightly induced transcriptional activity (Fig. 3). Addition of a construct expressing a constitutively activated mutant of calcineurin (⌬CnA) increased this induction several- fold for all isoforms, ranging from 3.5-fold for NFATc4 to over 20-fold for NFATc2 (Fig. 3). Taken together, these results suggest that all myocardial NFAT members are able to induce transcriptional activation of the human MCIP1 calcineurinresponsive enhancer region.
Synergistic Activation of the BNP Gene by NFAT and GATA4 -It was previously demonstrated that the BNP gene promoter is regulated by a distal NFAT sequence element in cooperation with calcineurin and GATA4 (10). To assess whether the additional cardiac NFAT members are also capa-ble of synergizing with GATA4 in regulating this promoter, the hBNP(1800)Luc reporter was tested in the presence or absence of GATA4, ⌬CnA, and expression vectors for the individual NFAT isoforms. GATA4 alone markedly up-regulated hBNP(1800)Luc to about 17-fold over baseline (Fig. 4A), confirming the previously documented sensitivity of this gene to GATA factors (26,27). Each individual NFAT isoform demonstrated relatively weak activation in the presence of ⌬CnA (Fig.  4A). In contrast, cotransfection of any single NFAT isoform in the presence of ⌬CnA and GATA4 resulted in robust up-regulation of hBNP(1800)Luc, ranging from 25-to 50-fold induction depending upon the NFAT member studied (Fig. 4A). These results indicate that all NFAT members can participate in synergistic activation of the BNP gene in conjunction with GATA4.
To test the specificity of this interaction and whether functional binding sites for either factor are required in the synergistic activation of the BNP reporter, a series of hBNP(1800) promoter-luciferase mutants were generated and examined. As a control, the activity of the wild-type hBNP(1800)Luc is shown at the left panel in Fig. 4B. The two GATA binding sites centered around Ϫ116 bp were mutated, which rendered the BNP promoter construct insensitive to GATA4 but not to activation by NFATc3 or -c4 (Fig. 4B, middle panel). Next, in the context of the GATA-mutated reporter (⌬GATA)hBNP(1800)Luc, the distal NFAT site at Ϫ927 was mutated, which showed no activation in the presence of NFAT and/or GATA4 (Fig. 4B,  right panel). These data indicate that the synergistic activation pattern initially described for NFATc4 with GATA4 in control of the BNP gene is fully conserved among each myocardialexpressed NFAT and that this activation profile is critically dependent upon the presence of both intact NFAT and GATA4 binding sites.
Dominant Negative NFAT Inhibits Calcineurin-mediated MCIP1 Gene Expression-Because all four members of the  3. Activation of the human MCIP1-promoter by cardiac NFAT isoforms. COS-7 cells were transiently transfected with a luciferase reporter gene linked to an intragenic segment proximal of exon 4 of the human MCIP1 gene (hMCIP1(Int3)Luc) with or without expression vectors present for NFATc1, -c2, -c3, -c4, or activated calcineurin (⌬CnA) as indicated. Forty-eight hours later, cells were harvested and luciferase activity was determined. The hMCIP1(Int3)Luc construct proved to be exceptionally sensitive to NFATc activation, independent of the isoform studied. The data represent the mean Ϯ S.E. of four independent experiments and are presented as -fold activation compared with a control with hMCIP(Int3)Luc alone.
NFAT transcription factor family are present in cardiomyocytes, a dominant inhibitory strategy was developed that targets NFAT activation. Such a strategy would bypass gene redundancy issues to permit evaluation of the role of NFAT as a calcineurin effector in the heart. Several independent groups have demonstrated that constructs consisting of only the Nterminal domain of NFAT can interfere with NFAT-mediated transcription in a dominant inhibitory fashion. This region includes the Ser-rich region and three conserved Ser-Pro repeats (Ser-Pro boxes A, B, and C) (Fig. 5A). The Ser-Pro repeat boxes represent major sites of interaction of NFAT with calcineurin in vitro (28), and sites of NFAT phosphorylation in vivo have been identified in the Ser-rich region (28 -30). Previous studies identified the conserved Pro-Xaa-Ile-Xaa-Ile-Thr (PXIXIT) box (residues 114 -119 in NFATc4) as the region that confers inhibitory NFAT transcriptional activity. To globally inactivate all NFAT factors, we generated a construct encoding amino acid residues 3-191 of the N terminus from NFATc4 that contains this calcineurin-interacting region (Fig. 5A). Cotransfection assays in COS-7 cells were performed using this construct, each of the NFATc isoforms, and the hMCIP1(Int3)luciferase reporter plasmid (Fig. 3B). Overexpression of ⌬CnA and NFATc1 through c4 each induced robust NFAT transcriptional activity (Fig. 5B). However, expression of the dominant negative NFATc4(PXIXIT) construct, the N-terminal NFATc4 homology domain (residues 3-191), dose-dependently inhibited transcription mediated by each NFAT isoforms (Fig. 5B). In the absence of full-length NFAT isoforms, transcriptional activity was not observed in either the absence or the presence of NFATc4(PXIXIT) (data not shown). These data indicated that the N-terminal NFAT homology domain interferes in a dominant inhibitory fashion with NFAT-mediated regulation of a cardiac responsive promoter, regardless of the NFAT isoform studied.
To test whether our dominant inhibitory NFATc4(PXIXIT) construct was dependent upon the presence of an intact PX-IXIT box and to exclude issues regarding cytotoxicity, a similar N-terminal NFATc4 construct, NFATc4(Ala mut), was included in cotransfection assays. In this construct, the conserved PXIXIT box residues were mutated to Ala residues to generate an AXAXAA box, which is now ineffective in blocking calcineurin interaction. As anticipated, coexpression of this mutant construct displayed no inhibitory effect on NFAT-mediated induction of the hMCIP1(Int3)Luc reporter (Fig. 5B). These data indicate that the PXIXIT box mediates the dominant negative action of our NFATc4(PXIXIT) construct.
Dominant Negative NFAT Inhibits Calcineurin-mediated Cardiomyocyte Hypertrophy-To investigate the requirement of NFAT activation in calcineurin-mediated cardiomyocyte hypertrophy, we generated two replication-deficient adenoviral vectors expressing either the dominant negative N-terminal NFAT construct NFATc4(PXIXIT) or the control construct NFATc4(Ala mut) under control of the CMV promoter (Fig. 6A). Infection of COS-7 cells with either AdNFATc4(PXIXIT) or AdNFATc4(Ala mut) at an m.o.i. of 100 resulted in robust expression of polypeptide fragments of ϳ19 and ϳ16 kDa, respectively, which were easily detectable on the basis of their FLAG-immunoreactivity (lanes 2 and 3, Fig. 6B). Conversely, Ad␤gal infection resulted in the absence of any proteins reactive for the anti-FLAG antibody (lane 1, Fig. 6B). Taken together, these results demonstrate that AdNFATc4(PXIXIT) and AdNFATc4(Ala mut) are correctly expressed and should represent an effective way to inhibit NFAT activity in cultured neonatal cardiomyocytes.
Cardiomyocytes were first infected with the control adenovirus Ad␤gal (Fig. 6C, panels A-C), AdNFATc4(PXIXIT) (Fig.  6C, panels D-F), or AdNFATc4(Ala mut) (Fig. 6C, panels G-I). After 24 h, the cultured cells were then stimulated with the hypertrophic agonist cardiotrophin-1 (CT-1) or Endo-1 (data not shown), by infection with the activated calcineurinexpressing adenovirus, or left untreated for 24 h (Fig. 6C). The data demonstrate that only AdNFATc4(PXIXIT) infection prevented cardiomyocyte hypertrophy in response to Ad-CnA or CT-1 (Fig. 6C, panels E and F). Adenoviral infection with either Ad␤gal (Fig. 6C, panels B and C) or the control adenovirus AdNFATc4(Ala mut) (Fig. 6C, panels H and I) had no discernable effects on either AdCnA or agonist-induced sarcomeric deposition and cellular enlargement. Importantly, neither Ad␤gal, AdNFATc4(PXIXIT), or AdNFATc4(Ala mut) infection induced cardiomyocyte apoptosis nor did it affect the morphology (Fig. 6C,   FIG. 4. Cardiac NFAT and GATA-4 synergistically activate the human BNP promoter. A, COS-7 cells were transiently transfected with a luciferase reporter gene linked to an 1800-bp flanking region of the human BNP promoter (hBNP(1800)Luc) with or without expression vectors present encoding the individual NFAT isoforms, activated calcineurin (⌬CnA), or GATA4, as indicated. Forty-eight hours later, cells were harvested and luciferase activity was determined. As reported previously for NFATc4 (10), NFATc1, -c2, and -c3 were also able to transactivate the hBNP(1800)Luc construct synergistically with GATA-4. Luciferase values represent the mean Ϯ S.E. of three independent experiments and are presented as -fold activation compared with a control with hBNP(1800)Luc alone. B, COS-7 cells were transfected with either the wild-type hBNP(1800)Luc reporter (left panel), the GATA-mutated (⌬GATA)hBNP(1800)Luc reporter (middle panel), or the GATA and NFAT site mutated (⌬NFAT, ⌬GATA)hBNP(1800)Luc reporter with or without expression vectors present encoding the most cardiac NFAT isoforms NFATc3 or -c4, activated calcineurin (⌬CnA), and GATA4. Forty-eight hours later, cells were harvested and luciferase activity was determined. Luciferase values represent the mean Ϯ S.E. of three independent experiments and are presented as -fold activation compared with a control with either hBNP(1800)Luc reporter alone.
Increased ANF expression is a hallmark of cardiac hypertrophy and is readily detected by immunocytochemistry as perinuclear staining (31,32). Serum-free cultured cardiomyocytes infected with either Ad␤gal, AdNFATc4(PXIXIT), or AdNFATc4(Ala mut) were stimulated with agonist or the activated calcineurin-expressing adenovirus and scored for the numbers of cells with perinuclear ANF expression (Fig. 6C). The data demonstrate that only AdNFATc4(PXIXIT) infection blocked ANF expression in response to the prohypertrophic stimuli investigated (Fig. 6C, panels E and F). Taken together, the results indicate that dominant negative NFAT abrogated ANF expression following calcineurin activation or agonist stimulation in cultured cardiomyocytes.

DISCUSSION
Overlapping Expression of NFAT Isoforms in the Heart-One unexpected finding of the present study is that the ventricular cardiomyocyte population contains all four, calcineurin-sensitive NFAT isoforms described in the literature to date (reviewed in Refs. 6, 33, and 34). All NFAT members of the transcription factor family are expressed in multiple isoforms, generated by alternative splicing (35)(36)(37)(38). The results in the present study support this notion (Fig. 1B). The existence of multiple splice isoforms has been shown in detail for NFATc2 and -c4 in T lymphocytes and other cells (35)(36)(37), and it has been shown that all spliced isoforms elicit transactivation of NFAT-responsive promoters, albeit with slightly differing efficiencies (38). The observation that cardiomyocytes express each of the four calcineurin-regulated NFAT family members, which themselves undergo differential splicing, justifies the dominant negative strategy employed here to inhibit NFATmediated transcriptional activation.
Although initially characterized in T-cells, almost all tissues in the mammalian organism express one or more NFAT family member. For example, NFATc2 is somewhat restricted in expression to immune cells and skeletal muscle, whereas NFATc3 expression is enriched in thymocytes and skeletal muscle cells but also present at lower levels in various other tissues. NFATc1 and NFATc4 appear to be expressed in a more ubiquitous pattern (6, 39 -44), where they influence development, proliferation, and differentiation of a number of mammalian tissues (33,45). The data in the present study confirm this ubiquitous expression pattern of NFAT members throughout several muscle types, supporting the function of the calcineurin-NFAT signaling pathway regulating cardiac hypertrophy, skeletal muscle myogenesis, and fiber-type specification, and smooth muscle cell proliferation and vessel remodeling (33,(45)(46)(47)(48)(49)(50).
Crucial Role for NFAT Signaling in Cardiomyocyte Hypertrophy-The NFAT dominant inhibitory approach employed here specifically blocked the ability of calcineurin or agonist stimuli to promote nuclear accumulation and transcriptional activation of endogenous or overexpressed NFAT factors. Our approach utilized overexpression of the NFAT N-terminal calcineurin-docking domain containing the conserved sequence Pro-Xaa-Ile-Xaa-Ile-Thr (PXIXIT box) (51). It should be noted that NFATc4(PXIXIT), the dominant negative NFAT construct used in the present study, encompasses aa residues 3-191 of human NFATc4, whereas the "internal control" construct, NFATc4(Ala mut), was slightly shorter and encompasses residues 2-130 of human NFATc4. Although it would have been formally more correct to use dominant negative and control constructs of the same length, it is highly unlikely that the differing phenotypic effects observed between the dominant negative construct and the control construct may be due to this slight difference in length. Indeed, Chow et al. (28) have clearly demonstrated that the dominant inhibitory action of N-terminal portions of NFAT only depends upon the presence of the PXIXIT box, which encompasses residues 114 -119 in human NFATc4, rendering the relative length of truncated NFAT constructs beyond residue 119 in this particular context irrelevant.
To address whether NFAT signaling is required for (calcineurin-mediated) cardiomyocyte hypertrophy, two model systems are routinely employed: cultured cardiomyocytes and genetically altered mice. In this study we employed adenoviralmediated gene transfer in cultured cardiomyocytes to circumvent potential difficulties associated with gene targeting such as isoform redundancy or compensatory changes in gene ex- pression. For example, gene targeting of individual NFAT family members did not reveal a widespread defect in the ability of T cells to proliferate or generate cytokines such as interleukin-2 (28,(52)(53)(54)(55), even though the calcineurin-NFAT paradigm was established as a regulator of interleukin-2 gene transcription. Germane to our study, Chow et al. (28) established the involvement of NFAT activity in regulating interleukin-2 expression using a similar dnNFAT molecule. This dnNFAT molecule selectively inhibited NFAT transcription activity by interfering with the activation-induced nuclear import of NFAT, and the active component of this inhibitor corresponds to the PXIXIT box located in the conserved N-terminal homology region of NFAT (28).
In addition to gene targeting, transgenesis in the mouse could be employed as a means of blocking NFAT activation through overexpression of the dominant negative NFAT protein domain in the heart. However, exhaustive attempts to generate dominant negative NFAT transgenic mice failed, presumably due to embryonic or early post-natal lethal effects associated with complete NFAT inhibition in the heart. 3 Indeed, NFATc3 ϫ NFATc4 double-null mice die during late embryogenesis with severe vascular abnormalities (47).
Another documented approach to abrogating NFAT signaling is the use of kinases that directly phosphorylate NFAT transcription factors, thus antagonizing nuclear accumulation. For example, glycogen synthase kinase-3 (GSK-3) is a serine/ threonine protein kinase with many targets, including at least two NFAT proteins (55). In addition, GSK-3 has been identified as a critical negative modulator of cardiomyocyte hypertrophy by directly antagonizing the prohypertrophic effects of activated calcineurin (52). Recently, GSK-3 was also proven to be capable of inhibiting hypertrophic signaling in the intact myocardium (53). Transgenic mice expressing a constitutively activated form of GSK-3␤ in cardiomyocytes displayed a severely blunted hypertrophic response to chronic ␤-adrenergic stimulation, pressure overload, and the actions of the calcineurin transgene (53). However, GSK-3␤ also inhibits GATA-4 function in cardiomyocytes (54), suggesting that GSK-3␤ likely also inhibits the hypertrophic response through NFAT-independent mechanisms. Nevertheless, we favor the interpretation that calcineurin-NFAT signaling is a dominant regulatory pathway for cardiac hypertrophy, and likely the germane target underlying the anti-hypertrophic effect of GSK-3␤. Indeed, cardiomyocytes infected with adenoviruses expressing truncated forms of either the calcineurin inhibitory protein Cain/cabin-1 or AKAP79 (20), which target and inhibit calcineurin itself (56 -58), also showed a severe attenuation of myocyte hypertrophy in vitro. The combined observations suggest a pivotal role for calcineurin-NFAT signaling in cardiomyocyte hypertrophy.
Cardiac NFAT Signaling: Functional Redundancy or Functional Specification?-Although this study establishes that NFAT activity is required for both calcineurin as agonist-induced cardiomyocyte hypertrophy, the present data await extrapolation to the in vivo situation. As discussed above, we were unsuccessful in generating cardiac-specific transgenic mice expressing this dominant negative NFAT protein. These observations suggest that NFAT factors are crucial during developmental maturation of the myocardium. However, it is not known if all NFAT factors contribute to the myocyte growth response through a generalized mechanism or if individual isoforms play specific functions. For example, NFATc1 genetargeted mice die during embryonic development due to defects in heart valve formation (59,60). With respect to the adult heart and the regulation of hypertrophic growth, we have recently targeted the NFATc4 gene in the mouse. Surprisingly, NFATc4-null mice did not show a defect in their ability to mount a hypertrophic response (10,61). By contrast, NFATc3null mice did show a significant attenuation of hypertrophy following diverse stimuli (61). Collectively, these observations suggest that several NFAT isoforms might play critical regulatory roles in the adult myocardium. Indeed, here we observed that NFATc3 is abundantly present in ventricular myocytes (Fig. 1).
Alternatively, it is possible that certain NFAT factors are specified to fulfill various pathophysiological roles in the heart, in addition to or even excluding hypertrophic signaling. For example, we have demonstrated that adenoviral expression of NFATc4 rendered cardiomyocytes less susceptible to staurosporine or oxidative stress-induced apoptosis (11). Moreover, Kakita et al. (2) demonstrated that NFATc1 plays a crucial role in endothelin-1-mediated protection against oxidative stressinduced apoptosis in cardiomyocytes. Because NFATc4 apparently plays a minor role in cardiac hypertrophic signaling (61), yet signals a pro-survival phenotype (11) it is possible that certain NFAT factors have highly specified functions in the heart. To more firmly establish the functional hierarchy or potential inter-isoform-specific roles between the individual myocardial NFAT members in the heart, generation of mouse models with loxP-flanked alleles for the four documented NFAT genes is warranted.