PRMT7 is declined in cardiac hypertrophy evoked by chronic Ang II treatment
To characterize the role of PRMT7 in cardiac function, the immunostaining against PRMT7 in human and mouse hearts were examined for the expression of PRMT7. Immunostaining result displayed that PRMT7 is expressed in cardiomyocytes (Fig. S1a, b). Next, we have analyzed the datasets (GDS4310, GDS3661, GDS3684, GDS3228 and GDS3465) obtained from hearts of mouse disease models. The expression of Prmt7 was significantly reduced in disease models, implicating the potential involvement of PRMT7 in cardiac function (Fig. 1a). To understand the role of PRMT7 in cardiac function, the expression pattern of PRMT7 was examined in cardiac hypertrophy. As previously reported [4], the chronic treatment of angiotensin II (Ang II) for 2 weeks elicited cardiac hypertrophy and myocardial fibrosis (Fig. 1b, c). Consistently, the expressions of myocardial fibrosis gene such as Atrial Natriuretic Peptide (ANP) and Collagen 1a1 (COL1A1) were elevated, while PRMT7 proteins were greatly reduced in hearts infused with Ang II for 2 weeks, relative to control hearts (Fig. 1d). Besides, newborn rat ventricular cardiomyocytes (NRVMs) were treated with Ang II for 3 days. Similar to the in vivo effect, Ang II treatment decreased PRMT7 proteins, while elevated ANP and COL1A1, compared to the control (Fig. 1e). Collectively, these data suggest that PRMT7 might play the pivotal role in cardiac function.
PRMT7 deletion in cardiomyocytes induces cardiac hypertrophy
To examine the effect of acute PRMT7 depletion in cardiomyocytes, NRVM cells transfected with 2 different PRMT7 shRNAs. PRMT7 proteins were decreased by tested shRNAs with variable efficiencies (Fig. 2a). PRMT7 depletion by shRNA#1 in NRVM cells evoked hypertrophic responses, as seen in about 2-fold enlargement of cell surface area (Fig. 2b, c). The capacitance analysis confirmed this increased size of PRMT7-depleted NRVMs (Fig. S2a). Furthermore, the expression of fibrotic response genes, ANP, Brain Natriuretic Peptide (BNP), and b-Myosin Heavy Chain (b-MHC) was significantly elevated in PRMT7-depleted NRVM cells with shRNA#1 or 2, compared to control shRNA-transfected cells (Fig. 2d). In addition, PRMT7+/+ (WT) and PRMT7-/- (KO) cardiomyocytes were isolated from hearts and subjected to analysis for cell size and capacitance. PRMT7-deficient cardiomyocytes were morphologically enlarged, compared to wildtype cardiomyocytes (Fig. S2b, c). Also, the capacitance analysis revealed a dramatically increased size of KO atrial and ventricular cardiomyocytes, compared to wildtype cells (Fig. S2d). In addition, the hematoxylin and eosin (H&E) staining showed cardiac hypertrophy in KO hearts and the fibrosis staining revealed increased fibrotic area around enlarged cardiomyocytes (Fig. 2e, f). Furthermore, PRMT7-deficient hearts exhibited substantially increased levels of ANP and COL1A1, markers for hypertrophy and fibrosis (Fig. 2g). However, the expression of other PRMTs including PRMT1, PRMT4, and PRMT5 was not significantly altered. These data suggest that PRMT7 deficiency causes cardiomyocyte hypertrophy.
PRMT7 deficiency exacerbates the Ang II-induced cardiomyopathy
Next, we examined the effect of PRMT7 deficiency in Ang II-induced cardiomyopathy by using cardiac-specific PRMT7 deleted mice. We have generated mice lacking cardiomyocyte-specific PRMT7 by breeding PRMT7Tm1C/Tm1c (PRMT7f/f, WT) mice with PRMT7f/f mice carrying a single copy of cardiac-specific myosin-heavy chain (Myh6)-Cre recombinase. Resulting littermates of PRMT7f/f and PRMT7f/f;Myh6-cre (cKO) were born in the ratio of 53.7% to 46.3%, respectively. Vehicle or Ang II-infused WT and cKO mice for 2 weeks were examined by echocardiographic analysis. Chronic Ang II infusion impaired cardiac function with decreased the ejection fraction (EF) and the fractional shortening (FS) in both WT and cKO mice, however PRMT7 deficiency exacerbated cardiac dysfunction caused by Ang II (Fig. 3a, b). In addition, PRMT7 deficiency aggravated Ang II-induced cardiac hypertrophy and myocardial fibrosis (Fig. 3c, d, and e). Consistently, the protein level of ANP was markedly elevated in Ang II-infused WT and cKO hearts (Fig. 3f). Taken together, these data suggest that PRMT7 might play a protective role against cardiomyopathy triggered by Ang II.
PRMT7 overexpression attenuates Ang II-induced cardiomyocyte hypertrophy
Next, we have examined the protective effects of PRMT7 overexpression on cardiomyocyte hypertrophy. NRVM cells were transfected with control pcDNA or HA-tagged PRMT7 (PRMT7-HA), followed by the treatment with Ang II. In contrast to the hypertrophic response caused by PRMT7 depletion, PRMT7 overexpression attenuated ANP induction triggered by Ang II treatment (Fig. 4a). Additionally, PRMT7-overexpressing cardiomyocytes exhibited smaller cell size in both vehicle and Ang II treatment, compared to control-transfected cells (Fig. 4b, c). Taken together, these data suggest that PRMT7 suppresses cardiomyocyte hypertrophy elicited by Ang II.
PRMT7 deficient hearts exhibited the alteration in gene expression profile related to Wnt signaling
To investigate the molecular mechanisms of PRMT7 in cardiac hypertrophy, we performed RNA sequencing with WT and KO hearts followed by the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis (> 1.3fold, an average of normalized RC log2 > 2, p value < 0.05). The RNA sequencing data revealed that KO hearts had 450 downregulated and 489 upregulated genes, compared to the WT hearts (Fig. 5a). Genes involved in pathways related to Wnt, TLR, and JAK/STAT signaling were altered in KO hearts (Fig. 5b). Recent studies reported the implication of Wnt signaling in cardiac hypertrophy and fibrosis [2, 27]. The heatmap analysis shows the alteration in genes related to Wnt signaling in KO hearts (Fig. 5c). To analyze the relationship between PRMT7 and Wnt signaling, we examined 112 sets (n=1103) of cardiac transcriptomes from 6 organisms (Homo sapiens, Mus musculus, Rattus norvegicus, Canis lupus, Sus scrofa, and Danio rerio) as an unbiased survey (Fig. S3a). We found an inverse correlation between PRMT7 and major Wnt signaling targets, Ctnnb1, Axin2, and Isl1 (Fig. S3b). Furthermore, the negative correlation between Prmt7 and Ctnnb1 was observed in 4 different species (Homo sapiens, Mus musculus, Canis lupus, and Danio rerio) (Fig. S3c). In addition, GSO dataset GDS4310 from hypertrophic cardiomyopathies induced by Ang II-infusion [28] revealed a negative correlation between Prmt7 and Ctnnb1, the hierarchical cluster analysis divided into two opposing groups (Fig. 5d). Ctnnb1, Axin2, and Isl1 were clustered as one group which was separated from the PRMT7 group. To verify this inverse correlation, we have examined the levels of b-Catenin in hearts infused with Ang II. Ang II-infused hearts displayed downregulated levels of PRMT7, while b-Catenin was increased in Ang II-infused hearts (Fig. 1d and 5e). Consistently, PRMT7-depleted NRVMs and KO hearts exhibited elevated b-Catenin proteins, compared to controls (Fig. 5f, g). Taken together, these data suggest that PRMT7 deficiency activates the Wnt signaling pathway.
PRMT7 suppresses β-Catenin activation
To further assess the relationship between PRMT7 and b-Catenin, NRVM cells were treated with an inhibitor of PRMT7, DS437. DS437 treatment enhanced the level of active b-Catenin (b-Catenin*) and total b-Catenin proteins without affecting Prmt5 or PRMT7 levels (Fig. 6a). Consistently, immunostaining against b-Catenin revealed that NRVMs treated only with DS437 exhibited a significant increase in nuclear b-Catenin accumulation which was further elevated by Wnt3a treatment (Fig. 6b, c). To verify the role of PRMT7 in b-Catenin activation, PRMT7 overexpression in 10T1/2 MEFs attenuated the activity of a Topflash Wnt-reporter, compared to control (Fig. 6d). In a converse experiment, control or PRMT7-depleted NRVM cells were treated with DMSO or a Wnt signaling inhibitor XAV939. PRMT7 depletion in NRVM cells enhanced the expression of Axin2 and ANP, compared to control cells. XAV939 treatment resulted in decreased Axin2 and ANP expression in both control and PRMT7-depleted NRVM cells. However, the expression of Axin2 and ANP was still higher in PRMT7-depleted NRVM cells (Fig. 6e). To further assess, H9c2 cardiomyocytes were transfected with PRMT7-HA expression vectors and treated with Wnt3a, followed by immunoblot analysis and immunostaining for b-Catenin and HA. PRMT7 overexpression attenuated the accumulation of active and total b-Catenin proteins induced by Wnt3a (Fig. 6f). Wnt3a treatment elicited readily detectable nuclear b-Catenin staining in HA-negative cardiomyocytes, while PRMT7-HA-transfected H9c2 cells showed no or weak staining (Fig. 6g, h). Taken together, these data suggest that PRMT7 suppresses b-Catenin nuclear accumulation and activity.
PRMT7 depletion reduced symmetric arginine methylation of β-Catenin
We then investigated the mechanism by which PRMT7 controls b-Catenin activity. Hereof, a possible interaction between PRMT7 and b-Catenin was examined by immunoprecipitation with control IgG or b-Catenin antibodies in heart lysates. PRMT7 was immunoprecipitated with b-Catenin (Fig. 7a). Next, in vitro methylation assay was performed by using GST-b-Catenin protein and the immunoprecipitated PRMT7 in the presence of the methyl group donor, S-adenosyl methionine (SAM). Then the immunoblotting analysis was performed by utilizing the MMA and Sym10 antibodies that recognize the mono-methylated and symmetric methylated arginine residues, respectively. PRMT7 induced the mono- and symmetric arginine demethylation of b-Catenin (Fig. 7b). To further assess, we asked whether b-Catenin is methylated in NRVM cardiomyocytes by utilizing the MMA and Sym10 antibodies. Both MMA and Sym10-positive b-Catenin proteins were decreased in NRVM cells by DS437 treatment (Fig. 7c, e). Similarly, the basal MMA and Sym10-positive b-Catenin levels were mildly but significantly reduced in KO hearts which correlated well with the elevated b-Catenin level in the lysates, compared to WT hearts (Fig. 7d, f). These data suggest that PRMT7 might regulate b-Catenin through methylation.
β-Catenin activity is regulated through methylation at arginine residue 93 by PRMT7
As previously study suggests that arginine residue in RXR and arginine-glycine (RG) motif are favorable targets for PRMT7-mediated methylation [29]. The sequence analysis predicted arginine 93 (R93) and R591 as potential methylation sites by PRMT7 (Fig. 8a). Next, we have performed LC-MS/MS analysis by using purified flag-tagged human b-Catenin expressed in 293T cells that express PRMT7. In agreement with the sequence prediction, peptides containing dimethylated arginine residue 93 (R93) and R591 were detected with over 96% and 92% of peptide identification probability, respectively (Fig. 8b). Thus, the arginine-alanine mutants of b-Catenin at R93 and R591 (R93A and R591A; RA) were generated for functional analysis. The symmetric arginine dimethylation (SRM) levels of R93A or R591A mutants were decreased to 40% or 70%, related to WT/b-Catenin, respectively (Fig. 8c, d). Thus, b-Catenin is regulated by methylation at arginine residue 93.
To elucidate whether the methylation of b-Catenin has an effect on its activity, the transcriptional activities of WT/b-Catenin and RA/b-Catenin mutants were analyzed by utilizing the Top-flash luciferase and TCF/LEF;H2B-GFP reporter. The luciferase assay revealed that WT/b-Catenin and R591A mutants augmented comparable levels of luciferase activities, while R93A mutants significant elevated luciferase activities (Fig. 8e). Furthermore, WT/b-Catenin increased H2B-GFP-positive cells, reflecting the level of b-Catenin activity, compared to the control-transfected cells. The R93A mutant further enhanced the percentile of H2B-GFP-positive cells, while the R591A mutants exhibited a similar activity relative to WT/b-Catenin (Fig. 8f, g). In agreement with these results, H2B-GFP levels were elevated in all forms of b-Catenin expression cells, compared to control cells. WT/b-Catenin and R591A mutant induced similar levels of H2B-GFP proteins, whereas R93A mutant-expressing cells further increased H2B-GFP levels (Fig. 8h). Taken together, these data suggest that PRMT7 negatively regulates b-Catenin activity through methylation at R93.