NFAT Transcription Factors Are Critical Survival Factors That Inhibit Cardiomyocyte Apoptosis During Phenylephrine Stimulation In Vitro
Biomechanical stress on the heart results in activation of numerous signaling cascades, leading to cardiomyocyte hypertrophy, apoptosis, and ultimately, heart failure. The Ca2+-dependent phosphatase calcineurin is an essential mediator of cardiac hypertrophy, and in most but not all studies, calcineurin inhibition attenuated cardiac hypertrophy in vivo. However, calcineurin inhibition has been reported to have adverse effects on cardiac remodeling and cardiomyocyte apoptosis. Calcineurin regulates the activity of a number of downstream targets, including the transcription factors NFAT, MEF2, and NF-κB, and the apoptotic factor Bad. To evaluate the contribution of NFAT activation by calcineurin to cardiomyocyte responses to hypertrophic stimulation, we used adenovirus to express VIVIT, a selective peptide inhibitor of calcineurin-mediated NFAT activation. We found that selective NFAT inhibition during phenylephrine stimulation inhibited hypertrophy but resulted in increased cardiomyocyte apoptosis. In contrast, nonselective inhibition of calcineurin by cyclosporin A did not cause cardiomyocyte apoptosis after phenylephrine stimulation. Cyclosporin A suppressed the effect of VIVIT on cardiomyocyte apoptosis. These results demonstrate that during phenylephrine stimulation calcineurin activates both pro- and antiapoptotic pathways in cardiomyocytes, and that NFAT activity is a critical component of the antiapoptotic pathway that regulates whether the outcome of calcineurin activation is cardiomyocyte apoptosis or survival.
Imposition of hemodynamic loads on the heart results in the activation of numerous signaling pathways in cardiomyocytes, leading to increased cardiomyocyte size. However, chronic activation of these growth pathways results in cardiomyocyte dysfunction, apoptosis, and ultimately heart failure. Recently, the calcium/calmodulin-dependent protein phosphatase calcineurin was found to be an important mediator of the hypertrophic response. Transgenic overexpression of activated calcineurin results in marked cardiac hypertrophy, indicating that activation of calcineurin is sufficient to elicit the hypertrophic response.1 Calcineurin inhibition with pharmacological agents (CsA or FK506) or dominant-negative protein expression, and genetic knockout of calcineurin, have demonstrated that calcineurin is essential for the development of cardiac hypertrophy (reviewed in Reference 2).
In addition to its necessary role in cardiac hypertrophy, calcineurin also regulates cardiomyocyte apoptosis. In cultured cardiomyocytes, calcineurin has been found to promote both programmed cell death and cell survival, depending on the experimental context.R3-127880 R4-127880 R5-127880 3–6 In animal models, calcineurin inhibition with CsA during hypertrophic stimulation has been found to adversely affect cardiac remodeling and increase cardiomyocyte apoptosis.R3-127880 3,7 The mechanisms that determine whether the consequences of calcineurin stimulation are pro- or antiapoptotic have not yet been identified.
In response to sustained increases in intracellular calcium, calcineurin activates a number of downstream targets, including the apoptotic factor Bad, and the transcription factors NFATc1-c4, MEF2, and NF-κB. The NFAT transcription factors are prototypical effectors of calcineurin signaling (reviewed in Reference 8). In the basal state, NFAT transcription factors are excluded from the nucleus by phosphorylation of a regulatory domain that is conserved among NFAT isoforms. Calcineurin directly interacts with NFAT through a conserved docking motif, and in the presence of calcium/calmodulin, calcineurin dephosphorylates NFAT, unmasking a nuclear localization signal. NFAT subsequently enters the nucleus and activates transcription.8
Overexpression of a constitutively activated NFATc4 mutant protein resulted in cardiac hypertrophy in transgenic mice, suggesting that NFAT activation contributes to the hypertrophic response of the heart.1 NFAT proteins translocate to the nucleus after cardiomyocyte stimulation with phenylephrine (PE) and other hypertrophic agonists,R9-127880 R10-127880 9–11 and an NFAT binding site has been found to be essential for upregulation of the BNP promoter during PE stimulation.1 However, an essential role for NFAT in the hypertrophic response has been more difficult to demonstrate, due the absence of selective pharmacological antagonists and genetic redundancy of NFAT isoforms in the heart (Figure 1 and Reference 10). Recent data using expression of a dominant-negative NFAT mutant protein in cultured cardiomyocytes10 and genetic ablation of NFATc3 in mice12 have indicated that NFAT proteins are essential mediators of cardiac hypertrophy.
In this study, we used a previously characterized peptide that selectively antagonizes NFAT activation by calcineurin to probe the role of NFAT proteins in cardiomyocyte hypertrophy. We found that in cultured cardiomyocytes, NFAT activation is essential for hypertrophy in response to stimulation by the α-adrenergic agonist phenylephrine (PE). Surprisingly, we also found that inhibition of NFAT activation during PE stimulation led to increased cardiomyocyte apoptosis, and this effect could be blocked by nonselective calcineurin inhibition with CsA. These results indicate that NFATs act as critical survival factors in the setting of calcineurin stimulation and explain at least in part the disappointing effect of calcineurin inhibition on apoptosis and cardiac remodeling despite its demonstrated activity in blocking cardiac hypertrophy.
Materials and Methods
VIVIT-GFP and human NFATc4 were provided by A. Rao.13 HA-tagged NFATc4 was obtained from G. Crabtree. Nc4, a constitutively activated mutant of NFATc4, was generated from human NFATc4 by using PCR mutagenesis to add an N-terminal FLAG tag and to delete the N-terminal 317 amino acids, which include the regulatory domain of NFATc4.1 Nc4 mt, a point mutant of Nc4 in which Arg430 of NFATc4 has been changed to alanine, was constructed by site-directed mutagenesis (QuikChange, Stratagene) based on mutagenesis data for NFATc1.14 The inability of this mutant to bind DNA was confirmed by gel-shift analysis and by NFAT reporter assays (data not shown). The NFAT-luciferase reporter was previously described.15 All PCR-generated constructs were verified by DNA sequencing.
Reverse transcription from DNase-treated total RNA was performed using SuperScript II (Invitrogen). PCR was performed under nonsaturating conditions using NFAT isoform-specific primer pairs for 20 cycles in the presence of 32Pα-dATP. PCR products were resolved on a nondenaturing polyacrylamide gel and visualized by autoradiography.
Adenoviruses were generated using the AdEasy system (Stratagene) and purified on CsCl gradients. Viruses were titered by the toxic infectious dose method and used at a multiplicity of infection of 5 to 10.
Neonatal rat ventricular cardiomyocytes were dissociated using collagenase and trypsin and purified from nonmyocytes by centrifugation on a Percoll gradient as previously described.16 Cardiomyocytes were plated at a density of 0.5 to 1×105/cm2 in 5% horse serum, and switched to serum-free media 16 hours after plating. Cells were infected with adenovirus on the day after plating, proteins were allowed to express for 48 hours, and cells were then stimulated with 10 μmol/L PE. Where indicated, antagonists CsA (1 μg/mL; Sigma) or z-VAD-fmk (100 μmol/L; Calbiochem) were added 30 minutes before PE addition. Adult cardiomyocytes were dissociated by retrograde perfusion with collagenase as previously described.17
Cell viability was determined 48 hours after PE stimulation by staining with the membrane permeable nucleic acid stain Syto 13 (Molecular Probes) and the membrane impermeable DNA stain propidium iodide. Randomly selected fields were acquired using an MDS290 digital imaging system (Kodak) for later analysis. At least 10 randomly selected 200× fields were counted per sample. To determine cell surface area, myocytes were fixed with 10% buffered formalin followed by staining with α-actinin antibody (Sigma) and DAPI. Cells were imaged as above using at 400× magnification and measured using NIH Image. At least 100 cells were measured per sample. Nuclear translocation was measured 8 hours after stimulation with 10 μmol/L PE. These conditions were chosen to circumvent cell death seen at longer time points. After fixation with 4% PFA, cells were stained with an affinity purified NFATc2 antibody (gift from Dr A. Rao, Harvard Medical School, Boston, Mass) or monoclonal anti-HA antibody (Covance) and imaged using a confocal microscope (BioRad). To quantitate the fraction of cells with nuclear HA-NFATc4 nuclear localization, over 100 consecutive cardiomyocytes transfected with both HA-NFATc4 and VIVIT-GFP (or GFP) were counted. TUNEL staining was performed per the manufacturer’s recommendations (Roche). DNA internucleosomal fragmentation was measured as described previously.18
Statistical analysis was performed using StatView (SAS Institute). Unless otherwise noted, means derived from 3 independent samples were compared by ANOVA with the Scheffé’s F procedure for post-hoc comparisons. Values are displayed as mean±SEM.
NFAT Isoform Expression in Cardiomyocytes
Five NFAT isoforms have been identified. NFATc1 (also known as NFAT2), NFATc2 (NFAT1), NFATc3 (NFAT4), and NFATc4 (NFAT3) share a common calcineurin docking motif and are regulated by calcium-calmodulin, whereas NFAT5 is not regulated by calcium-calmodulin or calcineurin. In order to determine the distribution of NFAT isoforms in cardiomyocytes, we generated isoform-specific primers and amplified NFATc1 to 4 and NFAT5 by RT-PCR. All five NFAT isoforms were detected in total RNA from whole heart and could be amplified with comparable efficiency under nonsaturating conditions from total RNA from a dissociated adult mouse cardiomyocyte preparation that was composed of at least 90% cardiomyocytes (Figure 1). These results indicate that all known NFAT isoforms are expressed at the RNA level in cardiomyocytes.
NFAT Inhibition With VIVIT Peptide
The redundancy of NFAT isoform expression in cardiomyocytes suggested that analysis of a necessary role for NFATs in cardiac hypertrophy using traditional knockout approaches might be difficult. To circumvent these potential problems, we chose to use a dominant-negative approach using a previously described peptide inhibitor of NFAT activation.13 This 16 residue peptide (Figure 2A), named VIVIT, was selected from a combinatorial peptide library based on its high affinity for the NFAT docking site of calcineurin.13 The VIVIT peptide was found to be 25 times more effective at inhibiting NFAT binding to calcineurin than a peptide spanning the naturally occurring calcineurin binding site of NFATc2.13 To test the effectiveness of VIVIT-GFP in blocking NFAT activation, we cotransfected Jurkat cells with VIVIT-GFP or GFP and an NFAT-dependent reporter. In this system, activation of the reporter requires activation of AP-1 as well as calcineurin; therefore, cells were stimulated by the calcium ionophore ionomycin as well as the PKC agonist phorbol 12-myristate 13-acetate (PMA). As described previously,13 VIVIT-GFP blocked the activation of the NFAT reporter as effectively as the calcineurin antagonist cyclosporin A (CsA), whereas GFP alone had no effect (Figure 2B).13 In comparison to CsA, which nonselectively antagonizes calcineurin catalytic activity, VIVIT peptide is more selective, because it did not interfere with the activation of other calcineurin targets such as NF-κB.13
We used adenovirus to express VIVIT-GFP or GFP in cultured cardiomyocytes (Figure 2C). In response to stimulation with PE and other hypertrophic agonists, NFAT isoforms translocate to the nucleus in cultured neonatal cardiomyocytes.R9-127880 R10-127880 9–11 To measure the effect of VIVIT-GFP expression on NFAT activation, we cotransfected VIVIT-GFP and HA-NFATc4 and monitored the subcellular localization of NFATc4 after PE treatment. In unstimulated cardiomyocytes expressing either GFP or VIVIT-GFP, NFATc4 was excluded from the nucleus (Figure 2D, top left). After PE stimulation, NFATc4 entered the nucleus in some GFP-expressing cardiomyocytes (Figure 2D, top right two panels). We determined the fraction of cardiomyocytes with nuclear localization of NFATc4 by counting more than 100 consecutive transfected cardiomyocytes. Consistent with previously reported data,R9-127880 9,11 PE stimulation increased the fraction of cardiomyocytes with nuclear NFATc4 localization in cardiomyocytes expressing GFP (Figure 2D, bottom). PE-induced nuclear translocation of NFATc4 was inhibited by VIVIT-GFP (Figure 2D, bottom).
We also examined the effect of VIVIT-GFP expression on PE-induced nuclear import of endogenous NFATc2 (Figure 2E). Endogenous NFATc2 was excluded from the nucleus in unstimulated GFP- or VIVIT-GFP–expressing cells (Figure 2E, top). After PE stimulation, NFATc2 translocated into the nucleus in some GFP-expressing cells, but not in VIVIT-GFP–expressing cells (Figure 2E, bottom).
NFAT Activation Is Necessary for Cardiac Hypertrophy in Response to PE
We examined the response of cardiomyocytes to PE stimulation in the presence of VIVIT-GFP. Control cardiomyocytes expressing GFP hypertrophied normally after PE stimulation, as assessed by the increase in cell surface area (Figure 3A, left panels). In contrast, PE stimulation did not increase the cell surface area of VIVIT-GFP–expressing cardiomyocytes (Figures 3A, right, and 3B). VIVIT-GFP–expressing cardiomyocytes remained competent to hypertrophy, because hypertrophy in response to the protein kinase C agonist PMA was not affected by expression of VIVIT-GFP (data not shown). The inhibition of cardiomyocyte hypertrophy in response to PE by VIVIT-GFP is consistent with prior findings that overexpression of calcineurin inhibitory proteins or a dominant-negative NFAT mutant, or treatment with CsA, blocked cardiomyocyte hypertrophy,R1-127880 R10-127880 R19-127880 R20-127880 R21-127880 1,10,19–22 and demonstrates an essential role for NFAT in cardiac hypertrophy induced by PE but not by PMA.
NFAT Activation Promotes Cellular Survival During PE Stimulation
While studying the effect of VIVIT on cardiac hypertrophy, we noticed that PE stimulation resulted in more than a 2-fold increase in the prevalence of dead cells in VIVIT-GFP–expressing cardiomyocytes (Figures 4A and 4B). This effect was not seen in unstimulated cells expressing VIVIT-GFP, and was unrelated to GFP overexpression or adenoviral infection because control cells infected with GFP-expressing adenovirus showed baseline levels of cell death in the presence or absence of PE (Figures 4A and 4B). In addition, we obtained similar results using an adenovirus that expresses a tandemly repeated VIVIT peptide without a GFP tag (data not shown).
To confirm that the decrease in cell viability in PE-treated, VIVIT-GFP–expressing cells was due to inhibition of NFAT signaling, we asked whether or not expression of a constitutively activated NFATc4 mutant protein (Nc4)1 would rescue cells from the effects of VIVIT-GFP plus PE. We found that compared with expression of β-galactosidase in control cells, expression of Nc4 significantly reduced the effect of VIVIT-GFP on cell death (Figure 4C). In contrast, in the context of VIVIT-GFP plus PE treatment, cells expressing an NFATc4 mutant deficient in DNA-binding (Nc4 mt) had an incidence of cell death that was indistinguishable from control cells expressing β-galactosidase (Figure 4C). These results demonstrate that the effect of VIVIT-GFP on cell viability was due to NFAT inhibition, and that cell survival after PE stimulation requires NFATc4 activation and DNA-binding. Consistent with an antiapoptotic role for NFAT proteins, expression of Nc4 significantly reduced cardiomyocyte cell death due to apoptotic stress by H2O2 or staurosporine (Figure 4D).
Calcineurin Inhibition Blocks VIVIT and PE-Induced Cell Death
Prior studies using nonselective calcineurin inhibitors in PE-stimulated cardiomyocytes did not describe an alteration in cellular viability. We found that CsA treatment of unstimulated or PE stimulated cells had no effect on cell death in GFP-expressing control cardiomyocytes (Figure 4E). In contrast, the increased cell death due to PE plus VIVIT-GFP was blocked by CsA (Figure 4E). This result indicates that calcineurin activation of other downstream targets is necessary for cell death due to NFAT inhibition.
Cell Death in the Presence of VIVIT Plus PE Is Due to Apoptosis
We asked if the increased cell death seen in cardiomyocytes treated with VIVIT-GFP and PE was due to apoptosis. Terminal dUTP nick end-labeling (TUNEL) of VIVIT-GFP– and PE-treated cardiomyocytes demonstrated increased TUNEL-positive nuclei compared with controls (Figure 5A). Analysis of DNA isolated from cardiomyocytes after treatment with VIVIT-GFP and PE showed increased internucleosomal DNA fragmentation (DNA laddering), consistent with increased apoptosis (Figure 5B). Treatment of cardiomyocyte cultures with the caspase inhibitor z-VAD-fmk blocked the increase in cell death observed after VIVIT-GFP and PE treatment (Figures 5C and 5D). Collectively, these results demonstrate that PE stimulation in the setting of NFAT inhibition by VIVIT-GFP resulted in increased cardiomyocyte apoptosis, and that apoptosis was the principal mechanism of increased cell death.
Because PE stimulation of VIVIT-GFP–expressing cardiomyocytes increased apoptosis, we asked whether or not the failure of VIVIT-GFP–expressing cells to hypertrophy in response to PE was due to activation of apoptotic pathways. We found that addition of z-VAD-fmk to VIVIT-GFP–expressing cardiomyocytes did not restore the capacity of these cells to hypertrophy in response to PE. The surface area of VIVIT-GFP–expressing cells treated with PE and z-VAD-fmk was 1003±53 μm2, compared with 973±55 μm2 in the absence of z-VAD-fmk (P>0.05; unpaired t test). Thus, increased apoptosis and attenuation of hypertrophic responses are distinct phenotypes that result from selective NFAT inhibition.
Essential Role for NFAT in Cardiomyocyte Hypertrophy
Multiple lines of evidence indicate that calcineurin activation is essential for cardiomyocyte hypertrophy (reviewed in Reference 2). However, a necessary role for NFAT in cardiac hypertrophy is less well established. By overexpressing a dominant-negative peptide that selectively antagonizes NFAT activation by calcineurin, we demonstrate that NFAT activation is necessary for hypertrophy in response to PE in cultured cardiomyocytes. These results are consistent with recently published data showing that expression of a dominant-negative protein consisting of the N-terminus of NFATc4 blocked hypertrophy of cultured cardiomyocytes treated with cardiotropin-1 or endothelin-1.10 In addition, genetic knockout of NFATc3 but not NFATc4 attenuated the hypertrophic response to angiotensin II and pressure-overload.12
Antiapoptotic Function of NFAT During Cardiomyocyte Hypertrophy
While inhibition of NFAT activation did not alter the survival of unstimulated cardiomyocytes, we found that PE stimulation of cardiomyocytes overexpressing the dominant-negative NFAT inhibitory peptide underwent apoptosis at an increased rate. This effect could be blocked by expression of constitutively activated NFATc4, but not by a similar NFATc4 mutant protein that failed to bind DNA. These results indicate that NFAT activation protects cardiomyocytes from apoptosis during stimulation by PE.
PE stimulates the hypertrophic response in cardiomyocytes by activating the heterotrimeric G protein Gq. Gq activation has been associated with cardiomyocyte apoptosis both in vitro and in vivo. Whereas physiological levels of Gq activation protected cardiomyocytes from apoptotic stress,6 high levels of Gq stimulation induced by adenoviral overexpression of activated Gq led to cardiomyocyte apoptosis in cultured cardiomyocytes.23 In transgenic mice, overexpression of Gq resulted in compensated cardiac hypertrophy, and imposition of additional hemodynamic loads (pregnancy or pressure overload) resulted in decompensated heart failure associated with increased cardiomyocyte apoptosis.24 The calcineurin-NFAT pathway is activated by Gq stimulation, and our results indicate that NFAT activation may account at least in part for the protective effects of physiological levels of Gq activation.6
Pro- and Antiapoptotic Consequences of Calcineurin Activation
We found that treatment of cardiomyocytes with the nonselective calcineurin antagonist CsA did not alter cardiomyocyte cell death during PE stimulation. In addition, CsA rescued cardiomyocytes from increased cell death associated with selective NFAT inhibition during PE stimulation. These results are consistent with a model in which calcineurin activates both pro- and antiapoptotic pathways in cardiomyocytes, and NFAT is a critical component of the antiapoptotic pathway (Figure 6). This model is consistent with data from other cell types, in which calcineurin activation has been found to have both proapoptotic and antiapoptotic consequences,R25-127880 R26-127880 R27-127880 25–28 sometimes in the same cell type.29 In cardiomyocytes, calcineurin likewise has been found previously to both promote and inhibit apoptosis.R3-127880 R4-127880 R5-127880 3–6
Our results suggest that NFAT is a critical regulator of the balance between pro- and antiapoptotic consequences of calcineurin activation. Importantly, NFAT transcriptional activity is also regulated by its interaction with a number of other transcription factors such as AP-1,8 MEF2,30 and GATA4,1 each of which is regulated by additional signaling pathways. In addition, mitogen-activated protein kinases directly phosphorylate NFAT and modify its activity.R31-127880 31,32 Thus, NFAT may integrate inputs from multiple signaling pathways to regulate cardiomyocyte apoptosis during the hypertrophic response.
Our results have implications for therapeutic strategies designed to treat heart failure. Although calcineurin inhibition resulted in attenuation of cardiac hypertrophy in most animal models,33 recent studies have suggested that calcineurin inhibition by CsA may adversely affect cardiac remodeling and increase cardiomyocyte apoptosis in vivo.R3-127880 3,7 Our results indicate that the increased apoptosis may be due in part to decreased NFAT activity and suggest that therapeutic strategies of calcineurin inhibition that spare NFAT function may avert these deleterious consequences of nonselective calcineurin inhibition.
W.T.P. was supported by grants from the Hood Foundation and the National Heart, Lung, and Blood Institute (NHLBI). S.I. was supported by NHLBI grants RO1 AG61716 and HL65742. The authors thank A. Rao for reagents and advice.
- Received October 28, 2002.
- Revision received February 27, 2003.
- Accepted March 18, 2003.
Saito S, Hiroi Y, Zou Y, Aikawa R, Toko H, Shibasaki F, Yazaki Y, Nagai R, Komuro I. β-Adrenergic pathway induces apoptosis through calcineurin activation in cardiac myocytes. J Biol Chem. 2000; 275: 34528–34533.
Kakita T, Hasegawa K, Iwai-Kanai E, Adachi S, Morimoto T, Wada H, Kawamura T, Yanazume T, Sasayama S. Calcineurin pathway is required for endothelin-1–mediated protection against oxidant stress-induced apoptosis in cardiac myocytes. Circ Res. 2001; 88: 1239–1246.
De Windt LJ, Lim HW, Taigen T, Wencker D, Condorelli G, Dorn GW2nd, Kitsis RN, Molkentin JD. Calcineurin-mediated hypertrophy protects cardiomyocytes from apoptosis in vitro and in vivo: an apoptosis-independent model of dilated heart failure. Circ Res. 2000; 86: 255–263.
Oie E, Bjornerheim R, Clausen OP, Attramadal H. Cyclosporin A inhibits cardiac hypertrophy and enhances cardiac dysfunction during postinfarction failure in rats. Am J Physiol Heart Circ Physiol. 2000; 278: H2115–H2123.
Ichida M, Finkel T. Ras regulates NFAT3 activity in cardiac myocytes. J Biol Chem. 2001; 276: 3524–3530.
Van Rooij E, Doevendans PA, De Theije CC, Babiker FA, Molkentin JD, De Windt LJ. Requirement of nuclear factor of activated T-cells (NFAT) in calcineurin mediated cardiomyocyte hypertrophy. J Biol Chem. 2002; 277: 48617–48626.
Fiedler B, Lohmann SM, Smolenski A, Linnemuller S, Pieske B, Schroder F, Molkentin JD, Drexler H, Wollert KC. Inhibition of calcineurin-NFAT hypertrophy signaling by cGMP-dependent protein kinase type I in cardiac myocytes. Proc Natl Acad Sci U S A. 2002; 99: 11363–11368.
Wilkins BJ, De Windt LJ, Bueno OF, Braz JC, Glascock BJ, Kimball TF, Molkentin JD. Targeted disruption of NFATc3, but not NFATc4, reveals an intrinsic defect in calcineurin-mediated cardiac hypertrophic growth. Mol Cell Biol. 2002; 22: 7603–7613.
Aramburu J, Yaffe MB, Lopez-Rodriguez C, Cantley LC, Hogan PG, Rao A. Affinity-driven peptide selection of an NFAT inhibitor more selective than cyclosporin A. Science. 1999; 285: 2129–2133.
Sun LJ, Peterson BR, Verdine GL. Dual role of the nuclear factor of activated T cells insert region in DNA recognition and cooperative contacts to activator protein 1. Proc Natl Acad Sci U S A. 1997; 94: 4919–4924.
Hedin KE, Bell MP, Kalli KR, Huntoon CJ, Sharp BM, McKean DJ. δ-Opioid receptors expressed by Jurkat T cells enhance IL-2 secretion by increasing AP-1 complexes and activity of the NF-AT/AP-1-binding promoter element. J Immunol. 1997; 159: 5431–5440.
Aoki H, Richmond M, Izumo S, Sadoshima J. Specific role of the extracellular signal-regulated kinase pathway in angiotensin II–induced cardiac hypertrophy in vitro. Biochem J. 2000; 347: 275–284.
Shioi T, Kang PM, Douglas PS, Hampe J, Yballe CM, Lawitts J, Cantley LC, Izumo S. The conserved phosphoinositide 3-kinase pathway determines heart size in mice. EMBO J. 2000; 19: 2537–2548.
Khwaja A, Rodriguez-Viciana P, Wennstrom S, Warne PH, Downward J. Matrix adhesion and Ras transformation both activate a phosphoinositide 3-OH kinase and protein kinase B/Akt cellular survival pathway. EMBO J. 1997; 16: 2783–2793.
Taigen T, De Windt LJ, Lim HW, Molkentin JD. Targeted inhibition of calcineurin prevents agonist-induced cardiomyocyte hypertrophy. Proc Natl Acad Sci U S A. 2000; 97: 1196–1201.
De Windt LJ, Lim HW, Bueno OF, Liang Q, Delling U, Braz JC, Glascock BJ, Kimball TF, del Monte F, Hajjar RJ, Molkentin JD. Targeted inhibition of calcineurin attenuates cardiac hypertrophy in vivo. Proc Natl Acad Sci U S A. 2001; 98: 3322–3327.
Rothermel BA, McKinsey TA, Vega RB, Nicol RL, Mammen P, Yang J, Antos CL, Shelton JM, Bassel-Duby R, Olson EN, Williams RS. Myocyte-enriched calcineurin-interacting protein, MCIP1, inhibits cardiac hypertrophy in vivo. Proc Natl Acad Sci U S A. 2001; 98: 3328–3333.
Zou Y, Hiroi Y, Uozumi H, Takimoto E, Toko H, Zhu W, Kudoh S, Mizukami M, Shimoyama M, Shibasaki F, Nagai R, Yazaki Y, Komuro I. Calcineurin plays a critical role in the development of pressure overload-induced cardiac hypertrophy. Circulation. 2001; 104: 97–101.
Adams JW, Pagel AL, Means CK, Oksenberg D, Armstrong RC, Brown JH. Cardiomyocyte apoptosis induced by Gαq signaling is mediated by permeability transition pore formation and activation of the mitochondrial death pathway. Circ Res. 2000; 87: 1180–1187.
Adams JW, Sakata Y, Davis MG, Sah VP, Wang Y, Liggett SB, Chien KR, Brown JH, Dorn GW2nd. Enhanced Gαq signaling: a common pathway mediates cardiac hypertrophy and apoptotic heart failure. Proc Natl Acad Sci U S A. 1998; 95: 10140–10145.
Asada A, Zhao Y, Kondo S, Iwata M. Induction of thymocyte apoptosis by Ca2+-independent protein kinase C (nPKC) activation and its regulation by calcineurin activation. J Biol Chem. 1998; 273: 28392–28398.
Wang HG, Pathan N, Ethell IM, Krajewski S, Yamaguchi Y, Shibasaki F, McKeon F, Bobo T, Franke TF, Reed JC. Ca2+-induced apoptosis through calcineurin dephosphorylation of BAD. Science. 1999; 284: 339–343.
Jayaraman T, Marks AR. Calcineurin is downstream of the inositol 1,4,5-trisphosphate receptor in the apoptotic and cell growth pathways. J Biol Chem. 2000; 275: 6417–6420.
Zhao Y, Tozawa Y, Iseki R, Mukai M, Iwata M. Calcineurin activation protects T cells from glucocorticoid-induced apoptosis. J Immunol. 1995; 154: 6346–6354.
Lotem J, Kama R, Sachs L. Suppression or induction of apoptosis by opposing pathways downstream from calcium-activated calcineurin. Proc Natl Acad Sci U S A. 1999; 96: 12016–12020.
Blaeser F, Ho N, Prywes R, Chatila TA. Ca2+-dependent gene expression mediated by MEF2 transcription factors. J Biol Chem. 2000; 275: 197–209.
Yang TT, Xiong Q, Enslen H, Davis RJ, Chow CW. Phosphorylation of NFATc4 by p38 mitogen-activated protein kinases. Mol Cell Biol. 2002; 22: 3892–3904.
Gomez del Arco P, Martinez-Martinez S, Maldonado JL, Ortega-Perez I, Redondo JM. A role for the p38 MAP kinase pathway in the nuclear shuttling of NFATp. J Biol Chem. 2000; 275: 13872–13878.
Molkentin JD. Calcineurin and beyond: cardiac hypertrophic signaling. Circ Res. 2000; 87: 731–738.