Cooperative Binding of ETS2 and NFAT Links Erk1/2 and Calcineurin Signaling in the Pathogenesis of Cardiac Hypertrophy

Abstract

Background: Cardiac hypertrophy is an independent risk factor for heart failure, a leading cause of morbidity and mortality globally. The calcineurin/NFAT (nuclear factor of activated T cells) pathway and the MAPK (mitogen-activated protein kinase)/Erk (extracellular signal-regulated kinase) pathway contribute to the pathogenesis of cardiac hypertrophy as an interdependent network of signaling cascades. How these pathways interact remains unclear and few direct targets responsible for the prohypertrophic role of NFAT have been described.

Methods: By engineering cardiomyocyte-specific ETS2 (a member of the E26 transformation-specific sequence [ETS] domain family) knockout mice, we investigated the role of ETS2 in cardiac hypertrophy. Primary cardiomyocytes were used to evaluate ETS2 function in cell growth.

Results: ETS2 is phosphorylated and activated by Erk1/2 on hypertrophic stimulation in both mouse (n=3) and human heart samples (n=8 to 19). Conditional deletion of ETS2 in mouse cardiomyocytes protects against pressure overload-induced cardiac hypertrophy (n=6 to 11). Silencing of ETS2 in the hearts of calcineurin transgenic mice significantly attenuates hypertrophic growth and contractile dysfunction (n=8). As a transcription factor, ETS2 is capable of binding to the promoters of hypertrophic marker genes, such as ANP, BNP, and Rcan1.4 (n=4). We report that ETS2 forms a complex with NFAT to stimulate transcriptional activity through increased NFAT binding to the promoters of at least 2 hypertrophy-stimulated genes: Rcan1.4 and microRNA-223 (=n4 to 6). Suppression of microRNA-223 in cardiomyocytes inhibits calcineurin-mediated cardiac hypertrophy (n=6), revealing microRNA-223 as a novel prohypertrophic target of the calcineurin/NFAT and Erk1/2-ETS2 pathways.

Conclusions: Our findings point to a critical role for ETS2 in calcineurin/NFAT pathway-driven cardiac hypertrophy and unveil a previously unknown molecular connection between the Erk1/2 activation of ETS2 and expression of NFAT/ETS2 target genes.

Keywords: ETS2; MAPK/Erk pathway; calcineurin-NFAT pathway; cardiac hypertrophy; cardiomegaly; microRNA-223.

Figure 1.

ETS2 is activated in hypertrophic hearts. A, Western blot analyses and quantification showing ETS2 phosphorylation and protein levels in mouse hearts at 1 or 3 weeks after sham operation or severe thoracic aortic constriction (sTAC; n=3). B and C, Western blot analyses (B) and quantification (C) of ETS2 phosphorylation and protein levels in cytoplasmic and nuclear fractions extracted from sham-operated control hearts and sTAC-induced hypertrophied hearts (n=3). D, ETS2 phosphorylation is increased in hearts from patients with dilated cardiomyopathy (DCM; n=8 for normal controls, n=19 for DCM). **P<0.01; ***P<0.001. ETS2 indicates a member of the E26 transformation-specific sequence (ETS) domain family; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; ns, not significant; and sTAC, severe transverse aortic constriction.

Figure 2.

Cardiomyocyte-specific ETS2 (a member of the E26 transformation-specific sequence [ETS] domain family) knockout (KO) mice are protected from severe thoracic aortic constriction (sTAC)–induced cardiac hypertrophy. A, The ratio of heart weight/tibia length (HW/TL) in α-myosin heavy chain–Cre (Cre), ETS2-floxed (F/F), and ETS2 knockout (KO) mice after 3 weeks of sham or sTAC (n=6 to 11). B, ETS2 KO mice showed enhanced contractile function after sTAC. Left, M-mode echocardiography images; right, fractional shortening (n=6 to 11). C and D, Histologic analyses of heart sections from Cre, F/F, and KO mice subjected to sham or sTAC for 3 weeks. Heart sections were stained with hematoxylin and eosin to analyze hypertrophic growth (C; scale bar, 2 mm) or stained with wheat germ agglutinin to demarcate cell boundaries (D; scale bar, 20 μm). E, Quantification of the relative cross-sectional area of the indicated groups (n=6 to 8). F, mRNA levels of hypertrophic marker genes (ANP [atrial natriuretic peptide], BNP [brain natriuretic peptide], βMHC [β-myosin heavy chain], and Rcan1.4) were decreased in ETS2 KO mice after sTAC (n=6 to 8). *P<0.05; **P<0.01; ***P<0.001. ns indicates not significant.

Figure 3.

ETS2 (a member of the E26 transformation-specific sequence [ETS] domain family) knockout (KO) suppresses calcineurin-induced cardiac hypertrophy. A, Heart weight/tibia length (HW/TL) ratio of ETS2^F/F and KO mice crossed with calcineurin transgenic mice (CnA; n=8). B, Echocardiographic analyses of cardiac function of ETS2^F/F and KO mice crossed with calcineurin transgenic mice. Left, M-mode echocardiographic images; right, fractional shortening (n=8). C and D, Histologic analyses of heart sections from ETS2^F/F and KO mice crossed with calcineurin transgenic mice. Heart sections were stained with hematoxylin and eosin (C; scale bar, 2 mm) or wheat germ agglutinin (D; scale bar, 20 μm). E, Quantification of the relative cross-sectional area of the indicated groups (n=8). F, mRNA levels of hypertrophic marker genes (n=8). G, Western blot analyses and quantification of Rcan1.4 protein levels in indicated groups (n=6). *P<0.05; **P<0.01; ***P<0.001. ANP indicates atrial natriuretic peptide; βMHC, β-myosin heavy chain; BNP, brain natriuretic peptide; and Ctrl, control.

Figure 4.

ETS2 (a member of the E26 transformation-specific sequence [ETS] domain family) knockdown attenuates phenylephrine-induced hypertrophy in vitro. A, Representative Western blots and quantification showing ETS2 phosphorylation and protein levels in isolated neonatal rat ventricular myocytes (NRVMs) treated with 50 μmol/L phenylephrine (PE) for 6 or 24 hours (n=6). B, Western blot analyses and quantification of ETS2 phosphorylation and expression in cytoplasmic and nuclear fractions extracted from PE-treated NRVMs (n=4). C, ETS2 phosphorylation and protein levels in NRVMs treated with PE or MEK inhibitor U0126, or both, for 24 hours (n=6). D, ETS2 was silenced in NRVMs by small interfering RNA (siRNA) transfection. E, Representative immunofluorescence images of α-actinin staining in NRVMs transfected with siRNA control (siNC) or siRNA targeting ETS2 (siETS2) and then treated with PE for 48 hours. Scale bar, 20 μm. F, Quantification of relative cardiomyocyte surface area (n=50). G, ETS2 knockdown inhibits PE-induced protein synthesis in NRVMs (n=6). H, mRNA levels of ANP (atrial natriuretic peptide), BNP (brain natriuretic peptide), and βMHC (β-myosin heavy chain) in NRVMs transfected with siNC or siETS2 and then treated with PE for 24 hours (n=6). I, Rcan1.4 mRNA levels in NRVMs treated as in H (n=6). J, Chromatin immunoprecipitation (ChIP) analysis to detect ETS2 binding to the promoters of the indicated genes. ChIP was performed with an ETS2-specific antibody or immunoglobulin G control antibody in control NRVMs or NRVMs treated with PE or U0126, or both, for 6 hours. The occupancy of ETS2 at promoters is shown relative to background signals with immunoglobulin G control antibody (n=4). *P<0.05; **P<0.01; ***P<0.001. βMHC indicates β-myosin heavy chain; ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; Ctrl, control; Cyto, cytoplasm; ETS2, a member of the E26 transformation-specific sequence (ETS) domain family; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; ns, not significant; and nucl, nucleus.

Figure 5.

ETS2 (a member of the E26 transformation-specific sequence [ETS] domain family) cooperates with NFAT (nuclear factor of activated T cells) to regulate Rcan1.4 transcription. A, Luciferase activity in HEK-293 cells transfected with an Rcan1.4 luciferase reporter plasmid, along with expression plasmids of ETS2, ETS2 lacking DNA binding domain (ETS-△DBD), and NFATc1 (n=6). B, Chromatin immunoprecipitation (ChIP) analysis using an ETS2-specific antibody or a GFP antibody to detect ETS2 or NFATc1 binding to the Rcan1.4 promoter. HEK-293 cells were transfected with an ETS2 expression plasmid, a plasmid encoding full-length NFATc1 fused to green fluorescent protein (NFATc1-GFP), and an Rcan1.4 luciferase reporter plasmid. A total of 48 hours after transfection, cells were collected for ChIP analysis (n=4). C, ETS2 interacts with NFATc1. HEK-293 cells were transfected with expression plasmids of NFATc1-GFP and flag-tagged ETS2, then an ETS2-specific antibody and a GFP antibody were used for immunoprecipitation (IP) analysis, respectively. D, NFATc1 binds to ETS2 DBD. HEK-293 cells were transfected with expression plasmids of NFATc1-GFP and truncated ETS2, which has a myc tag. A total of 48 hours after transfection, cells were collected for IP analysis. E, ETS2 binds to NFATc1 DNA binding domain. HEK-293 cells were transfected with expression plasmids of flag-tagged ETS2 and truncated NFATc1, which has a myc tag, and then an IP assay was performed. **P<0.01; ***P<0.001. HLH indicates helix–loop–helix domain; IgG, immunoglobulin G; ns, not significant; SRD, serine-rich domain; and WB, Western blot.

Figure 6.

MicroRNA (MiR)–223 is required for calcineurin-NFAT (nuclear factor of activated T cells)/ETS2 (a member of the E26 transformation-specific sequence [ETS] domain family) pathway. A, MiR-223 levels in the hearts of Cre, F/F, and knockout (KO) mice subjected to sham or severe transverse aortic constriction (sTAC) for 3 weeks (n=6). B, MiR-223 levels in the hearts of ETS2^F/F and KO mice crossed with calcineurin transgenic mice (n=8). C, MiR-223 levels in neonatal rat ventricular myocytes (NRVMs) transfected with small interfering RNA (siRNA) control (siNC) or siRNA targeting ETS2 (siETS2) and then treated with phenylephrine (PE) for 24 hours (n=6). D, Chromatin immunoprecipitation (ChIP) analysis using an ETS2-specific antibody to detect ETS2 binding to miR-223 promoter in control NRVMs or NRVMs treated with PE for 6 hours (n=4). E, MiR-223 levels in NRVMs transfected with siNC or siRNA targeting NFATc1 (siNFATc1; n=6). F, Luciferase activity in HEK-293 cells transfected with a luciferase reporter plasmid harboring a full-length miR-223 promoter, along with expression plasmids of ETS2, ETS2 lacking DNA binding domain (ETS-△DBD), and NFATc1 (n=6). G, ChIP analysis using an ETS2-specific antibody or a GFP (green fluorescent protein) antibody to detect ETS2 or NFATc1 binding to the miR-223 promoter. HEK-293 cells were transfected with a miR-223 luciferase reporter plasmid, along with expression plasmids of ETS2 and NFATc1-GFP. A total of 48 hours after transfection, cells were collected for ChIP analysis (n=4). H, MiR-223 levels in indicated NRVMs. NRVMs were infected with an adenovirus expressing constitutively activated calcineurin (AdCnA) or a control adenovirus (AdLacZ). Before harvest, calcineurin-overexpressing NRVMs were treated with cyclosporin A (CsA) or U0126 for 6 hours (n=6). I, Quantification of the relative cardiomyocyte cross-sectional area. NRVMs were transfected with antagomir control (antagomir-NC) or miR-223 antagomir and then infected with AdCnA for 48 hours (n=50). J, Protein synthesis in NRVMs treated as in (I; n=6). *P<0.05; **P<0.01; ***P<0.001. Ctrl indicates control; IgG, immunoglobulin G; and ns, not significant.

Figure 7.

Schematic illustrating signaling pathways regulated by ETS2 (a member of the E26 transformation-specific sequence [ETS] domain family) in cardiac hypertrophy. Hypertrophic stress activates calcineurin and Erk1/2 Erk (extracellular signal-regulated kinase), which, in turn, activate nuclear factor of activated T cells (NFAT) and ETS2, respectively. ETS2 not only binds to the promoters of Rcan1.4 and microRNA (MiR)–223 in cardiomyocytes, it also synergizes with NFAT to transactivate Rcan1.4 and miR-223, and thus promotes cardiac hypertrophy. On the other hand, ETS2 binds to the MKP3 promoter to upregulate MKP3 expression in response to hypertrophic stress, thereby inhibiting Erk1/2 signaling. ANP indicates atrial natriuretic peptide; BNP, brain natriuretic peptide; Erk, extracellular signal-regulated kinase; ETS2, a member of the E26 transformation-specific sequence (ETS) domain family; MKP3, MAPK phosphatase 3; and NFAT, nuclear factor of activated T cells.