The histone trimethyllysine demethylase JMJD2A promotes cardiac hypertrophy in response to hypertrophic stimuli in mice (original) (raw)
Generation and characterization of Jmjd2a hKO. JMJD2A is expressed ubiquitously and higher in the heart, the skeletal muscle, and the liver in mice (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI46277DS1). To study the biological function of JMJD2A in the heart, we generated mice with conditional alleles for Jmjd2a and mice with deleted Jmjd2a in the heart using α-MHC–Cre (ref. 23 and Figure 1, A–D). The deletion of exon 3 in the Jmjd2afl/fl_×α_-MHC–Cre mouse (Jmjd2a hKO) resulted in a significant reduction of JMJD2A protein in the heart compared with control mice (Jmjd2afl/fl) (Figure 1D). The residual JMJD2A in hKO hearts may come from the contribution of noncardiomyocytes in the heart and/or incomplete deletion of Jmjd2a in the cardiomyocyte by the α-MHC–Cre transgene. Jmjd2a hKO mice are viable and no overt baseline cardiac phenotypes were observed for mice up to 6 months of age. The cardiac function of adult Jmjd2a hKO mice was indistinguishable from that of control mice as assayed by echocardiograph (data not shown).
Generation and assessment of a cardiac-specific Jmjd2a KO mouse model. (A) Strategy used to delete Jmjd2a in mouse cardiomyocytes and generate the Jmjd2a hKO model. Schematic protein structure of JMJD2A (14) and maps of the WT Jmjd2a locus, the targeting vector, the floxed allele, and the excised allele are shown. Exons are shown in bars. Exon 3 is flanked by loxP sites. (B) Southern blot of tail DNA from a WT (+/+) and a heterozygous (fl/+) mouse using 5′ probe shown in A after digestion with BclI. (C) PCR genotyping on genomic DNA isolated from a WT (+/+), a heterozygous (fl/+), and a homozygous floxed (fl/fl) mouse using primers p1/p2 indicated in A. (D) Western blot demonstrating significantly reduced JMJD2A protein in the heart homogenate of Jmjd2a hKO mice at 4 weeks. (E) H&E stains of representative paraffin sections of control and hKO hearts 3 weeks after sham and TAC operation. (F) HW/BW ratios of control and hKO mice 3 weeks after sham and TAC operation (n = 10–14 per group). The HW/BW ratio after TAC operation was increased 79% compared with sham operation in control mice, whereas it was only increased 42% in hKO mice. (G) Myocyte cross-sectional areas from control and hKO mice (>200 myocytes per heart in randomly selected filed) were assessed (n = 6–8 per group). (H) mRNA transcripts of ANP, BNP, and myh7 in control and hKO mouse hearts after 3 weeks sham and TAC operation. RNAs were normalized to internal GAPDH expression and presented as the fold change relative to control sham samples (n = 4–6 per group). Values are mean ± SEM. *P < 0.05.
Jmjd2a hKO mice have attenuated hypertrophic responses. To determine whether Jmjd2a hKO mice have altered cardiac responses under pathological conditions, we subjected hKO and control (Jmjd2afl/fl) littermates to transverse aortic constriction (TAC), which causes pathological cardiac hypertrophy due to increased afterload (24). Three weeks after TAC, Jmjd2a hKO hearts were significantly smaller than those of control mice (Figure 1E). This is reflected in a significantly smaller increase in heart weight/body weight (HW/BW) ratio (Figure 1F) and cross-sectional cardiomyocyte areas (Figure 1G) in hKO mice compared with those of controls. hKO mice also showed better preserved cardiac function after TAC compared with TAC-operated control mice, as indicated by echocardiograph (Table 1). A blunted hypertrophic response in hKO mice was further indicated by significantly decreased expression of the “fetal gene” markers ANP, BNP, and myh7 in response to TAC-induced pressure overload compared with that of control mice (Figure 1H). To address the possible effect of Cre-transgene on the cardiac phenotype, we also subjected _Jmjd2a+/+_×α-MHC–Cre mice to sham and TAC operation and compared the phenotype with those of Jmjd2afl/fl littermates. No significant differences of hypertrophic responses were observed between the 2 genotypes (Supplemental Figure 2), suggesting that the hypertrophic response we observed in hKO mice is not due to the effect of the α-MHC–Cre transgene.
Echocardiographic analysis of control and Jmjd2a hKO mice after sham and TAC operations
Overexpression of Jmjd2a in postnatal hearts exacerbates the hypertrophic response to TAC-induced hypertrophy. To determine whether gain of function of JMJD2A promotes cardiac hypertrophy, we generated transgenic mice in FVB background with JMJD2A expressed specifically in the postnatal heart (Figure 2A). Four founder mice were obtained. Two transgenic lines (Tg-A and Tg-B) with modest JMJD2A expression shown by Western blot analysis (Figure 2A) were established. The results presented here are those obtained with the transgenic line B. In the absence of stress, _Jmjd2a_-Tg mice displayed normal cardiac morphologies and functions similar to those of WT littermates (data not shown). Three weeks after TAC, _Jmjd2a_-Tg mice manifested an exacerbated hypertrophic response (Figure 2B) compared with that of WT littermates with significantly increased HW/BW ratio (Figure 2C). Analysis of cross-sectional cardiomyocyte areas also revealed a further increase in cardiomyocyte size after TAC in Tg mice compared with that of WT littermates (Figure 2D) and more fibrosis (data not shown). TAC _Jmjd2a_-Tg mice also showed significant loss of cardiac function compared with TAC WT mice (Table 2). An increase in hypertrophic response in TAC _Jmjd2a_-Tg mice is further indicated by the significantly larger increases of the expression of cardiac fetal gene markers such as ANP, BNP, and myh7 compared with those of WT littermates (Figure 2E). Similar exacerbated hypertrophic responses to TAC were also observed with the transgenic line A (Supplemental Figure 3), suggesting that the observed phenotypes are specifically due to JMJD2A. We also noticed a difference in the TAC-induced hypertrophic response between the WT mice in Figure 2C and control/WT mice in Figure 1F. This may be due to the difference of the genetic background of the KO (129/C57BL6) and transgenic (FVB) mice.
JMJD2A promotes cardiac hypertrophy in response to TAC-induced pressure overload. (A) Schematic diagram of cDNA encoding a flag-tagged JMJD2A in an expression vector containing the α-MHC promoter (left panel). 4 founder mice were obtained. 2 transgenic lines (Tg-A and Tg-B) with modest JMJD2A expression shown by Western blot analysis with anti-JMJD2A antibody (right panel) were established. The exogenous JMJD2A protein level is about 2-fold higher in the Tg-A line and 8-fold higher in the Tg-B line compared with that of endogenous JMJD2A. (B) H&E stains of paraffin section of WT and _Jmjd2a_-Tg (Tg) mouse hearts 3 weeks after sham and TAC operations. (C) HW/BW ratio (n = 6–7 per group) and (D) relative areas of cardiomyocytes (n = 3–4 per group) in WT and _Jmjd2a_-Tg sham- and TAC-operated animals. (E) Transcript levels of fetal gene markers, including ANP, BNP, and myh7, were quantified by real-time qRT-PCR, normalized against levels of internal GAPDH, and expressed as the fold change relative to that of sham-operated WT animals (n = 3 per group). The HW/BW ratio after TAC operation was increased 50% compared with sham operation in WT mice, whereas it was increased 100% in Tg mice. Values are mean ± SEM. *P < 0.05.
Echocardiographic analysis of WT and _Jmjd2a_-Tg mice after sham and TAC operations
To determine whether JMJD2A is pathophysiologically relevant to human cardiac hypertrophy, we examined the protein levels of JMJD2A in the hearts of patients with HCM and non-HCM individuals by Western blot analysis. As shown in Figure 3, JMJD2A protein was significantly upregulated in HCM patients, and the upregulation was paralleled with that of BNP. An additional band was detected in human JMJD2A Western blot analysis. Whether this band is an alternative splice variant or degradation of JMJD2A remains to be determined. We also performed quantitative RT-PCR (qRT-PCR) analysis on the transcript of JMJD2A and BNP and observed similar upregulation of JMJD2A and BNP in HCM patients compared with controls. These data, together with those of loss-of- and gain-of-function studies of JMJD2A in mice, suggest that JMJD2A is a prohypertrophic factor under pathological conditions.
JMJD2A is upregulated in human hypertrophic hearts. (A) Heart tissue samples of human HCM patients and unmatched non-HCM controls (con) were subjected to Western blot analysis using antibodies against JMJD2A, BNP, and GAPDH. Consistent with the HCM phenotype, BNP was upregulated in HCM samples. The lanes were run on the same gel but were noncontiguous. (B) More importantly, JMJD2A was significantly upregulated in HCM samples (n = 7) compared with samples from the non-HCM patients (n = 4). GAPDH was used as the loading control. (C) Relative mRNA levels of JMJD2A and BNP in control (n = 3) and HCM patients (n = 7) measured by qRT-PCR. Values are mean ± SEM. *P < 0.05.
Jmjd2a upregulates the expression of FHL1 and binds to the FHL1 promoter in vivo in response to TAC. To understand the molecular mechanism or mechanisms that underlie the prohypertrophic function of JMJD2A, we performed gene profiling experiments with cDNAs from the hearts of WT, Jmjd2a hKO, and _Jmjd2a_-Tg mice. Of the genes whose expressions were altered in the hKO and transgenic mouse hearts in comparison with those of control/WT mice, we noticed that FHL1 was significantly changed (Figure 4). FHL1 was upregulated in response to TAC, which was significantly reduced in Jmjd2a hKO mice compared with controls (Figure 4, A and C) and exacerbated in transgenic mouse hearts (Figure 4, B and C). No change of expression for FHL2, a close member of the FHL protein family, was observed in either hKO or Tg mouse hearts compared with either control or WT mice, respectively (Figure 4C). We also noticed that, although both mRNA and protein levels of FHL1 were consistently altered in Jmjd2a hKO and/or in _Jmjd2a_-Tg mouse hearts when compared with their respective controls, the fold change in protein level was less than that of mRNA, suggesting an alternative posttranslational mechanism for FHL1 regulation independent of JMJD2A.
JMJD2A upregulates the expression of FHL1. qRT-PCR analysis of transcript levels of FHL1 from (A) control (Jmjd2afl/fl) and Jmjd2a hKO mouse hearts (n = 4–6) and (B) WT and _Jmjd2a_-Tg mouse hearts (n = 3–4) 21 days after sham and TAC surgery. Values are mean ± SEM. *P < 0.05. (C) Western blot of heart lysates of sham- and TAC-operated control and Jmjd2a hKO mice (left panel) and sham- and TAC-operated WT and _Jmjd2a_-Tg mice (right panel) using antibodies against FHL1, FHL2, phospho-ERK1/2, ERK1/2, phospho-AKT, and AKT. Representative of 4–6 experiments was shown. The relative fold changes for FHL1 and p-ERK were quantified by ImageJ. GAPDH was used as the loading control.
Sheikh et al. have shown that FHL1 is a biomechanical stress sensor that mediates the MAPK-activated signaling pathway. FHL1 is required for pressure overload–induced cardiac hypertrophy, as deletion of FHL1 blunts the TAC-induced hypertrophic response (25). Upregulation of FHL1 leads to activation of the MAPK pathway (25). Consistent with this notion, we observed upregulation of p-ERK1/2 at baseline (Figure 4C) and a further increase in transgenic mouse hearts upon TAC (Figure 4C). Upregulation of p-ERK1/2 in response to TAC was attenuated in TAC-operated Jmjd2a hKO mice (Figure 4C). We also observed upregulation of fetal gene expression in FHL1-transduced neonatal cardiomyocytes (Supplemental Figure 4), suggesting that FHL1 can promote cardiac hypertrophy under cardiac stresses. In light of the functional importance of FHL1 in promoting cardiac hypertrophy, we next focused on investigating whether and how FHL1 is regulated by JMJD2A.
We first determined whether JMJD2A regulates FHL1 transcription by binding to the FHL1 promoter in vivo and in response to pressure overload using ChIP assays. As shown in Figure 5A, JMJD2A binds the FHL1 promoter in response to TAC in either WT or control (Jmjd2afl/fl) mice (Figure 5A). Binding of JMJD2A to the FHL1 promoter is specific, as a JMJD2A ChIP assay on the GAPDH promoter (a negative control) showed very little binding of JMJD2A to the GAPDH promoter (data not shown). Furthermore, ChIP assays with anti-JMJD2A antibodies in Jmjd2a hKO hearts showed very little binding (Figure 5A), which was similar to the results obtained using IgG control (data not shown). Significant amounts of JMJD2A were also found to bind the FHL1 promoter in _Jmjd2a_-Tg mouse hearts at baseline and were further augmented in TAC-operated _Jmjd2a_-Tg mouse hearts compared with those of WT counterparts (Figure 5A). ChIP assays with anti-H3K9me3 antibody indicated that binding of JMJD2A to the FHL1 promoter was associated with decreased levels of H3K9me3 (Figure 5B). The H3K9me3 level was also significantly downregulated in _Jmjd2a_-Tg mice and further decreased upon TAC (Figure 5B). Consistent with the role of JMJD2A in regulating the level of H3K9me3, no significant reduction of H3K9me3 was observed in association with TAC operation in Jmjd2a hKO mice (Figure 5B).
JMJD2A binds to the FHL1 promoter in vivo. (A) JMJD2A-chromatin complexes were immunoprecipitated from lysates of sham- and TAC-operated WT, control (Jmjd2afl/fl), _Jmjd2a_-Tg, and Jmjd2a hKO mouse hearts, with anti-JMJD2A antibody and quantified by qPCR with primers in the FHL1 promoter. The amounts of immunoprecipitated complex were normalized against DNA purified from sonicated cell lysates (input) and expressed as relative to that of sham-operated WT/control. (B) ChIP was performed on aliquots of lysates from A using an anti-H3K9me3 antibody. The amounts of chromatin in the FHL1 promoter associated with H3K9me3 were measured by qPCR, normalized against input, and expressed relative to that of sham-operated WT/controls. JMJD2A binds the FHL1 promoter in response to TAC, and binding of JMJD2A is associated with decreased levels of H3K9me3. Values are expressed as mean ± SEM of 3 independent experiments. *P < 0.05.
Jmjd2a upregulates the transcription of FHL1 through SRF and myocardin. To understand how JMJD2A is recruited to the FHL1 promoter, we examined the 5′-upstream genomic sequences of the FHL1 promoter and identified a conserved serum responsive factor–binding (SRF-binding) site (Supplemental Figure 5), known as the CArG box (26). A gel mobility shift assay indicated that this CArG box is a functional SRF-binding site, as it formed a specific nucleotide-protein complex with SRF (Figure 6A). We next cloned the 1.9-kb promoter sequence containing this CArG box in front of a luciferase reporter plasmid and tested the ability of JMJD2A to transactivate the reporter. Myocardin (myocd) is a SRF cofactor and activates transcription of SRF-dependent genes in cardiac and smooth muscle cells (26–30). Myocardin activated the transcription of FHL1, which was further upregulated by JMJD2A (Figure 6B). The ability of JMJD2A to increase the transcription of FHL1 requires its demethylase activity, as a demethylase-inactive mutant of JMJD2A, 2A (H188A), failed to augment myocardin-activated FHL1 transcription (Figure 6B). Activation of FHL1 transcription by myocardin and JMJD2A was SRF-dependent, as mutation of the CArG box abolished the transactivation of FHL1 by myocardin or myocardin plus JMJD2A (Figure 6C). Furthermore, the ability of myocardin/JMJD2A to activate the transcription of FHL1 was abolished in _SRF_-null cells (Figure 6D). These results indicate that JMJD2A is a cofactor of SRF/myocardin and its catalytic activity is required for this coactivation. Since the cardiac “fetal” genes ANP and sm22 are known SRF/myocardin-targeted genes, we tested to determine whether JMJD2A can augment the transcription of these genes. As expected, JMJD2A upregulated myocardin-activated transcription of ANP and sm22 in a demethylase-dependent manner as well (Figure 6, E and F, respectively).
JMJD2A upregulates FHL1 transcription synergistically with SRF/myocardin. (A) A 32P-labeled oligonucleotide probe containing the CArG box from the FHL1 promoter was incubated with the in vitro–translated (ivt) SRF in the presence or absence of anti-SRF antibody or a 100-fold molar excess of unlabeled, WT, or mutant (mut) oligonucleotides. SRF forms a complex with the WT oligonucleotide probe (SRF-CArG) that can be super-shifted (ss) by an anti-SRF antibody. ns, nonspecific nucleoprotein complex. (B) A 1.9-kb FHL1 promoter–driven luciferase construct was transfected along with the plasmids indicated into QBI293A cells. FHL1-luciferase activities were measured 24 hours after transfection and normalized against cotransfected β-galactosidase. (C) Responsiveness of the deletion and site-specific mutants of the FHL1-luc reporter to myocardin and JMJD2A in QBI293A cells. Deletion or site-specific mutation of the SRF-binding site CArG box impairs the responsiveness of the promoter to myocardin and JMJD2A. (D) Relative activity of the FHL1-luc reporter in _SRF_-null and WT ES cells in the presence and absence of cotransfected expression plasmids as indicated. Representative of 3 independent experiments is shown. (E) Relative luciferase activity of the ANP-luc reporter and (F) the sm22-luc reporter in QBI293A cells transfected with the plasmids indicated. Myocardin-activated ANP- and sm22-luc reporters that can be further upregulated by WT but not mutant JMJD2A. Data shown are mean ± SEM of 3 independent experiments. *P < 0.05.
Hebbar and Archer have shown that all 4 core histones (H2A, H2B, H3, and H4) and the linker histone H1 are associated with transiently transfected DNA despite altered histone H1 stoichiometry and an absence of nucleosome positioning on transfected DNA (31). As demethylated H3K9me3 could provide binding sites to recruit transcription factors, the inability of the demethylase-inactive mutant JMJD2A (H188A) to augment myocardin-activated FHL1-luc reporter suggests that there may be less myocardin binding on the FHL1-luc promoter. We performed ChIP assays to test this hypothesis. As shown in Figure 7A, although the expression levels of myocardin in transfected cells were similar (data not shown), there was more myocardin bound on the FHL1 promoter in cells transfected with both JMJD2A and myocardin than in cells transfected with myocardin alone or myocardin with the catalytically inactive mutant JMJD2A (Figure 7A). An increased amount of SRF was also found to be associated with the endogenous FHL1 promoter chromatin in vivo in either _Jmjd2a_-Tg mouse heats or hypertrophic mouse hearts after TAC surgery (Figure 7B).
JMJD2A enhances the binding of SRF/myocardin to the FHL1 promoter. (A) QBI293A cells were transfected with the plasmids indicated. ChIP assays were performed 24 hours later using anti-myc and anti-JMJD2A antibodies as indicated. (B) ChIP assays were performed with lysates of sham- and TAC-operated WT mouse hearts and with WT and _Jmjd2a_-Tg mouse hearts using anti-SRF antibody. Chromatins associated with SRF on the FHL1 promoter were quantified using qPCR. Data are expressed as mean ± SEM of 3 independent experiments. *P < 0.05.
To test whether the demethylase activity of JMJD2A is required for activation of FHL1 transcription in vivo, we performed gene knockdown (KD) experiments in rat neonatal cardiomyocytes using JMJD2A-specific siRNA duplexes. The mRNA level of JMJD2A was significantly KD by JMJD2A-specific siRNAs compared with control nonspecific siRNA (80% reduction; Figure 8A). Consistent with what we observed in Jmjd2a hKO mice, downregulation of JMJD2A in cardiomyocytes resulted in a decrease in the FHL1 transcription at baseline (Figure 8B). Reexpression of JMJD2A in the JMJD2A KD cardiomyocytes using adenoviral transduction restored FHL1 expression (Figure 8B), whereas the mutant JMJD2A (H188A) did not (Figure 8B). We also determined whether JMJD2A is involved in phenylephrine-stimulated (PE-stimulated) myocyte hypertrophy. PE had no effects on the transcript level of JMJD2A (Figure 8A) and significantly upregulated FHL1 transcription (Figure 8C). Upregulation of FHL1 by PE was significantly attenuated in JMJD2A KD cells (Figure 8C). Upregulation of fetal genes by PE, including ANF, BNP, and myh7 (Figure 8C), and of the cardiomyocyte size (Figure 8D) in JMJD2A KD cells were significantly attenuated, suggesting that JMJD2A mediates the hypertrophic effect of PE on cardiomyocytes.
Downregulation of JMJD2A attenuates the expression of FHL1 and PE-activated fetal gene program in cardiomyocytes. Neonatal rat ventricular myocytes were transfected with nonspecific control siRNA (cRNAi) or JMJD2A-specific siRNA (2A-RNAi). Forty-eight hours after transfection, cells were treated with PBS (vehicle) or with PE (10 μM) (A, C, and D) or with various adenoviruses as indicated (B). Relative transcript levels of JMJD2A (A), FHL1 (B, lanes 1–5, and C, lanes 1–4), and fetal genes (C, lanes 5–16) were determined 24 hours after the treatment using qRT-PCR, normalized against internal GAPDH, and expressed relative to those of the vehicle-treated cRNAi transfected cells. (D) Cardiomyocytes were photographed, and areas were quantified using ImageJ and expressed relative to those of the vehicle-treated cRNAi transfected cells. Data are expressed as mean ± SEM of 3 independent experiments. *P < 0.05.