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The piRNA CHAPIR regulates cardiac hypertrophy by controlling METTL3-dependent N6-methyladenosine methylation of Parp10 mRNA

Abstract

PIWI-interacting RNAs (piRNAs) are abundantly expressed during cardiac hypertrophy. However, their functions and molecular mechanisms remain unknown. Here, we identified a cardiac-hypertrophy-associated piRNA (CHAPIR) that promotes pathological hypertrophy and cardiac remodelling by targeting METTL3-mediated N6-methyladenosine (m6A) methylation of Parp10 mRNA transcripts. CHAPIR deletion markedly attenuates cardiac hypertrophy and restores heart function, while administration of a CHAPIR mimic enhances the pathological hypertrophic response in pressure-overloaded mice. Mechanistically, CHAPIR–PIWIL4 complexes directly interact with METTL3 and block the m6A methylation of Parp10 mRNA transcripts, which upregulates PARP10 expression. The CHAPIR-dependent increase in PARP10 promotes the mono-ADP-ribosylation of GSK3β and inhibits its kinase activity, which results in the accumulation of nuclear NFATC4 and the progression of pathological hypertrophy. Hence, our findings reveal that a piRNA-mediated RNA epigenetic mechanism is involved in the regulation of cardiac hypertrophy and that the CHAPIR–METTL3–PARP10–NFATC4 signalling axis could be therapeutically targeted for treating pathological hypertrophy and maladaptive cardiac remodelling.

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Fig. 1: Identification of CHAPIR in cardiac-hypertrophy-induced hearts of mice.
Fig. 2: CHAPIR deletion inhibits cardiac hypertrophy and remodelling.
Fig. 3: Knockdown of CHAPIR attenuates Ang-II-induced hypertrophic responses in cardiomyocytes.
Fig. 4: CHAPIR interacts with METTL3 and blocks its activity.
Fig. 5: Identification of potential CHAPIR targets using transcriptome-wide m6A-seq and RNA-seq assays.
Fig. 6: CHAPIR promotes Parp10 gene expression by targeting METTL3.
Fig. 7: CHAPIR regulates cardiac hypertrophy by targeting METTL3 and PARP10.
Fig. 8: GSK3β and NFATC4 function as downstream molecules of PARP10 during cardiac hypertrophy.

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Data availability

Microarray, MeRIP-seq and RNA-seq data that support the findings of this study have been deposited in the Gene Expression Omnibus (GEO) under accession codes GSE153445, GSE154699 and GSE154781. The MS proteomics data have been deposited into the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD020098. Source data for Figs. 18 and Extended Data Figs. 19 are available online. All other data supporting the findings of this study are available from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (81770275, 81828002, 91849209, 31671447, 81800272, 81870236 and 81660046); the Chinese Academy of Medical Sciences—Innovation Fund for Medical Sciences (2016-12M-1-006); the Taishan Scholar Program of Shandong Province and Qingdao public domain science and technology support plan project (19-6-1-6-nsh); and the Natural Science Foundation of Shandong Province (2017GSF18127 and 2016JQB01015).

Author information

Authors and Affiliations

Authors

Contributions

Kun Wang, P.-F.L. and X.-Q.G. conceived this project. Kun Wang, X.-Q.G., Y.-H.Z., F.L. and M.P. designed and supervised the experiments. X.-Q.G., Y.-H.Z., F.L., M.P., X.-M.Z., L.-Y.Z., M.Z., C.-Y.L., X.-M.L., M.W., C.S., P.-P.S., Y.-H.W., Y.-H.D., L.-L.Q., T.Y., J.J., T.W., Kai Wang and X.-Z.C. performed experiments. Kun Wang, X.-Q.G., Y.-H.Z., F.L., M.P., X.-M. Z., Y.W. and J.Z. analysed the data. Kun Wang, X.-Q.G., Y.-H.Z. and M.P. wrote the manuscript.

Corresponding author

Correspondence to Kun Wang.

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Extended data

Extended Data Fig. 1 Identification of dysregulated piRNAs in cardiac hypertrophy hearts of mice.

a, piRNA microarray result shows the length distribution of piRNAs in hearts collected at 4 weeks after TAC and sham operated control mice. b, qPCR analysis of 10 highly downregulated piRNAs in TAC-induced cardiac hypertrophy mice hearts selected from piRNA microarray data (n=4 independent experiments). c,d, Cardiac stress marker β-MHC expression in cardiomyocytes transfected with DQ566911 (c, n=4 independent experiments) or DQ691332 (d, n=5 independent experiments) and their negative controls (NC). e,f, Relative expression level of DQ695228 (e) and DQ545263 (f) in different tissues of normal adult mice as determined by qPCR (n=6 mice per group). Data are presented as Mean ± SD. Two-sided Student’s t-test (b) or one-way ANOVA (cf). Statistical source data are available in Source Data Extended Data Fig. 1.

Source data

Extended Data Fig. 2 CHAPIR overexpression aggravates pressure-overload induced cardiac hypertrophy.

a, Quantitative analysis of the mRNA level of β-MHC in WT and CHAPIR KO mice subjected to Sham or TAC for 4 weeks (n=6 mice per group). b, Heart function measured by left ventricle fractional shortening (FS) in WT and CHAPIR KO mice subjected to Sham or TAC for 4 weeks using echocardiography (n=8 mice per group). cg, CHAPIR mimic (CHAPIR) or negative control (NC) were injected into 8-weeks old mice and Sham or TAC surgery was performed. Heart samples were collected 4 weeks post-TAC surgery. c, CHAPIR level was analyzed by qPCR (n=5 mice per group). d, Representative images of transverse sections of hearts stained with hematoxylin and eosin (H&E) (Bar=2 mm; upper row). Representative images of heart tissues stained with H&E and evaluated with light microscopy (Bar=20μm; bottom row). Images are representative of six independent experiments. e, Heart weight (HW) to body weight (BW) ratio measured after 4 weeks of Sham or TAC surgery in CHAPIR or NC injected mice (n=8 mice per group). f, QPCR analysis of ANP mRNA level in CHAPIR or NC injected mice subjected to Sham or TAC for 4 weeks (n=6 mice per group). g, Heart function measured by left ventricle fractional shortening (FS) in CHAPIR or NC injected mice with sham or TAC using echocardiography (n=7 mice per group). Data are presented as Mean ± SD. Two-way ANOVA (ag). Statistical source data are available in Source Data Extended Data Fig. 2.

Source data

Extended Data Fig. 3 PIWIL4 directly binds to CHAPIR.

a, Scheme of biotinylated CHAPIR pull-down assay. b, Pull-down of proteins from the whole cell extract of cardiomyocytes using biotinylated CHAPIR (Bio-CHAPIR) or scrambled RNA (Bio-control) and then separated by electrophoresis followed by coomassie blue staining. c, LC-MS/MS identification of PIWIL4 in cardiomyocytes using biotinylated Bio-CHAPIR. d, Detection of specific binding of CHAPIR with PIWIL4 using biotin-based pull-down assay. Cardiomyocytes were harvested and RNA pulldown assay was performed using Bio-CHAPIR or Bio-NC. Associated proteins were pulled down using streptavidin beads and bound levels of PIWIL2 and PIWIL4 were analyzed by western blot. Images are representative of four independent experiments. e, RNA immunoprecipitation (RIP) was performed in isolated cardiomyocytes using antibodies against PIWIL2 or PIWIL4. The binding of CHAPIR to PIWIL4 was confirmed by qPCR (n=5 independent experiments). f,g, METTL3 binds with PIWIL4. Immunoprecipitation (IP) of PIWIL4 or METTL3 were performed in the whole cell lysate of mouse cardiomyocytes using specific antibodies and resulting complex were purified and subjected to immunoblot analysis using METTL3 or PIWIL4 antibodies. The whole cell lysate used as an input reference. Images are representative of three independent experiments. h, RNA pull down assay showing the binding ability of purified PIWIL4 protein or METTL3 protein with biotin-labelled CHAPIR. Images are representative of four independent experiments. Data are presented as Mean ± SD. One-way ANOVA (e). Statistical source data and unprocessed blots are available in Source Data Extended Data Fig. 3.

Source data

Extended Data Fig. 4 Epitranscriptome and transcriptome analyses in CHAPIR mimic treated mice hearts.

Mice were administrated with CHAPIR mimic or negative control, and hearts were collected for MeRIP-seq and RNA-seq. a, The number of representative coding and non-coding RNAs containing m6A peaks. b, Percentage of mRNAs with different numbers of m6A peaks. c,d, Gene ontology (GO) analysis of enriched terms in biological processes associated with CHAPIR target genes. e, Scatter plot of differential expression of mRNAs assessed from RNA-seq data. Red dots denote up-regulated genes and green dots denote down-regulated genes. f, Gene ontology (GO) analysis of biological processes enriched in hypomethylated genes.

Extended Data Fig. 5 MeRIP-qPCR assay and qPCR assay of m6A hypomethylated genes in CHAPIR mimic treated cardiomyocytes.

a,b, MeRIP-qPCR validation of m6A-hypomethylated-downregulated genes and m6A-hypomethylated-upregulated genes from CHAPIR mimic (CHAPIR) or mimic negative control (NC) treated cardiomyocytes (n=5 independent experiments). c,d, qPCR validation of m6A-hypomethylated-downregulated genes (c, n=4 independent experiments) and m6A-hypomethylated-upregulated genes (d, n=4 independent experiments) in CHAPIR mimic (CHAPIR) or mimic negative control (NC) treated cardiomyocytes. Data are presented as Mean ± SD. Two-sided Student’s t-test (ad). Statistical source data are available in Source Data Extended Data Fig. 5.

Source data

Extended Data Fig. 6 IGV tracks of MeRIP-seq and RNA-seq read distribution.

a-e, Integrative Genomics Viewer (IGV) tracks displaying results of MeRIP-seq (upper panels) and RNA-seq (lower panels) read distribution of Smc6 mRNA (a), Per1 mRNA (b), Rnf6 mRNA (c), Rab7b mRNA (d), Trim63 mRNA (e) in CHAPIR mimic (CHAPIR) treated and non-treated (NC) mice hearts. f, Cardiomyocytes were transfected with CHAPIR mimic or mimic negative control (NC), and then cells were treated with actinomycin D and collected at the indicated time. Parp10 mRNA level was analyzed by qPCR (n=5 independent experiments). g, Cardiomyocytes were infected with adenovirus harboring YTHDF2 or β-gal, and then cells were treated with actinomycin D and collected at the indicated time points. Parp10 mRNA level was analyzed by qPCR (n=5 independent experiments). Data are presented as Mean ± SD. Two-way ANOVA (f,g). Statistical source data are available in Source Data Extended Data Fig. 6.

Source data

Extended Data Fig. 7 Parp10 promotes cardiomyocyte hypertrophy.

Cardiomyocytes were transfected with Parp10-siRNA or scrambled control (Parp10-sc), and then were treated with Ang II. a, qPCR analysis shows Parp10 mRNA level (n=5 independent experiments). b, qPCR shows the level of ANP mRNA in Parp10-siRNA or Parp10-sc transfected cells treated with or without Ang II (n=4 independent experiments). c, Schematic diagram of AAV9 injection and the experimental procedure. Echo, echocardiography. df, AAV9-Parp10-shRNA (shRNA) or AAV9-scrambled control (shCTRL) were injected into 8-weeks old mice and TAC surgery was performed after 2 weeks to induce cardiac hypertrophy. Heart samples were collected 4 weeks post-TAC surgery. d, Representative western blots (top) and statistical data (bottom, n=6 mice per group) showing the expression of Parp10. e, Representative images of Masson’s trichrome-stained histological sections (Bar=20 μm). f, Semi-quantitative analysis of collagen contents in left ventricle samples (n=8 mice per group). g, Heart function measured by left ventricle fractional shortening (FS) using echocardiography (n=7 mice per group). h, AAV9 harboring METTL3-shRNA (MET3-shRNA) or scrambled negative control (MET3-shCTRL) were injected into wild type mice. The analysis of cardiac morphology. Bar=2mm (top row). Representative images of left ventricular muscle sections stained with wheat germ agglutinin (WGA) to demarcate cell boundaries. Bar=20μm (bottom row). i, Heart weight to body weight ratio (Saline, n=10 mice; MET3-shCTRL or MET3-shRNA, n=8 mice). j, Analysis of the cardiomyocyte sizes in histological sections (Saline, n=10 mice; MET3-shCTRL or MET3-shRNA, n=8 mice). k, Cardiac function measured by left ventricle fractional shortening (FS) using echocardiography (Saline, n=10 mice; MET3-shCTRL or MET3-shRNA, n=8 mice). Data are presented as Mean ± SD. One-way ANOVA (a,b,d,f,g,i,j,k). Statistical source data and unprocessed blots are available in Source Data Extended Data Fig. 7.

Source data

Extended Data Fig. 8 GSK3b inhibition rescues the effects of CHAPIR knockdown on cardiomyocyte hypertrophy.

a,b, Cardiomyocytes were transfected with GSK3β siRNA (GSK3β-siRNA) or scrambled negative control (GSK3β-sc), either transfected or not with CHAPIR antagomir (CHAPIR-anta) or negative control (anta-NC), and then were treated with Ang II for an additional 24 h. a, Analysis of cardiomyocyte size (n=5 independent experiments). b, Expression of hypertrophic marker genes ANP (n=5 independent experiments). c,d, Cardiomyocytes were transfected with GSK3β siRNA (GSK3β-siRNA) or scrambled negative control (GSK3β-sc), either transfected or not with Parp10 siRNA (Parp10-siRNA) or its control (Parp10-sc), and then were treated with Ang II for an additional 24 h. c, Analysis of cardiomyocyte size (n=5 independent experiments). d, Expression of hypertrophic marker genes ANP (n=5 independent experiments). e, Representative western blots showing the expression of β-catenin, c-Myc, phospho-GATA4 and phospho-CREB in cardiomyocytes with or without transfection of Parp10-siRNA and Ang II treatment. Data are presented as Mean ± SD. One-way ANOVA (ad). Statistical source data and unprocessed blots are available in Source Data Extended Data Fig. 8.

Source data

Extended Data Fig. 9 The effects of CHAPIR knockdown in established cardiac hypertrophy.

a, Schematic diagram of the CHAPIR antagomir injection and the experimental procedure. Echo, echocardiography. b, The analysis of cardiac morphology. Bar=2 mm (upper row). Representative images of left ventricular muscle sections stained with wheat germ agglutinin (WGA) to demarcate cell boundaries. Bar=20μm (bottom row). c, Heart weight (HW) to body weight (BW) ratio (Sham, n=9 mice; TAC, TAC+anta-NC and TAC+CHAPIR-anta, n=7 mice). d, The analysis of cardiomyocytes size (Sham, n=9 mice; TAC, TAC+anta-NC and TAC+CHAPIR-anta, n=7 mice). e, Cardiac function measured by left ventricle fractional shortening (FS) using echocardiography (Sham, n=9 mice; TAC, TAC+anta-NC and TAC+CHAPIR-anta, n=7 mice). f, Representative images of Masson’s trichrome-stained and semi-quantitative analysis histological sections of left ventricle. Bar=20μm (Sham, n=9 mice; TAC, TAC+anta-NC and TAC+CHAPIR-anta, n=7 mice). Data are presented as Mean ± SD. One-way ANOVA (cf). Statistical source data in Source Data Extended Data Fig. 9.

Source data

Supplementary information

Reporting Summary

Supplementary Tables

Supplementary Table 1. List of differentially expressed piRNAs in TAC-operated hearts compared with sham-operated hearts. Results of piRNA microarray analysis from TAC mouse hearts and sham-operated mouse hearts. Supplementary Table 2. List of biotinylated CHAPIR pull-down proteins identified by MS. Supplementary Table 3. List of differentially m6A-methylated RNA peaks in CHAPIR-overexpressing hearts compared with NC hearts. Results of MeRIP-seq analysis from CHAPIR mimic or mimic-NC-overexpressing hearts. Supplementary Table 4. List of differentially expressed mRNAs in CHAPIR-overexpressing hearts compared with NC hearts. Results of RNA-seq analysis from CHAPIR mimic or mimic-NC-overexpressing hearts. Supplementary Table 5. List of genes in the four quadrants graph of Fig. 5f. Correlation between the level of gene expression (overall transcript) and changes in m6A level in CHAPIR-treated mouse hearts compared with control. m6A peak data (fold change ≥ 1.5) was plotted against RNA-seq data of gene expression (fold change ≥ 3). Hyper-down: hypermethylated (increased m6A) and downregulated (decreased gene transcripts level); Hyper-up: hypermethylated (increased m6A) and upregulated (increased gene transcripts level); Hypo-down: hypomethylated (decreased m6A) and downregulated (decreased gene transcripts level); Hypo-up: hypomethylated (decreased m6A) and upregulated (increased gene transcripts level). Supplementary Table 6. The primer pairs used for RT–qPCR analysis of gene expression and RT–PCR primers for Gm12648.

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Gao, XQ., Zhang, YH., Liu, F. et al. The piRNA CHAPIR regulates cardiac hypertrophy by controlling METTL3-dependent N6-methyladenosine methylation of Parp10 mRNA. Nat Cell Biol 22, 1319–1331 (2020). https://doi.org/10.1038/s41556-020-0576-y

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