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A long noncoding RNA protects the heart from pathological hypertrophy

Nature volume 514, pages 102–106 (2014)Cite this article

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Abstract

The role of long noncoding RNA (lncRNA) in adult hearts is unknown; also unclear is how lncRNA modulates nucleosome remodelling. An estimated 70% of mouse genes undergo antisense transcription1, including myosin heavy chain 7 (Myh7), which encodes molecular motor proteins for heart contraction2. Here we identify a cluster of lncRNA transcripts from Myh7 loci and demonstrate a new lncRNA–chromatin mechanism for heart failure. In mice, these transcripts, which we named myosin heavy-chain-associated RNA transcripts (Myheart, or Mhrt), are cardiac-specific and abundant in adult hearts. Pathological stress activates the Brg1–Hdac–Parp chromatin repressor complex3 to inhibit Mhrt transcription in the heart. Such stress-induced Mhrt repression is essential for cardiomyopathy to develop: restoring Mhrt to the pre-stress level protects the heart from hypertrophy and failure. Mhrt antagonizes the function of Brg1, a chromatin-remodelling factor that is activated by stress to trigger aberrant gene expression and cardiac myopathy3. Mhrt prevents Brg1 from recognizing its genomic DNA targets, thus inhibiting chromatin targeting and gene regulation by Brg1. It does so by binding to the helicase domain of Brg1, a domain that is crucial for tethering Brg1 to chromatinized DNA targets. Brg1 helicase has dual nucleic-acid-binding specificities: it is capable of binding lncRNA (Mhrt) and chromatinized—but not naked—DNA. This dual-binding feature of helicase enables a competitive inhibition mechanism by which Mhrt sequesters Brg1 from its genomic DNA targets to prevent chromatin remodelling. A Mhrt–Brg1 feedback circuit is thus crucial for heart function. Human MHRT also originates from MYH7 loci and is repressed in various types of myopathic hearts, suggesting a conserved lncRNA mechanism in human cardiomyopathy. Our studies identify a cardioprotective lncRNA, define a new targeting mechanism for ATP-dependent chromatin-remodelling factors, and establish a new paradigm for lncRNA–chromatin interaction.

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Figure 1: Profile of the noncoding RNA Mhrt.
Figure 2: Mhrt inhibits cardiac hypertrophy and failure.
Figure 3: Mhrt complexes with Brg1 through the helicase domain.
Figure 4: Mhrt inhibits chromatin targeting and gene regulation by Brg1.

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Gene Expression Omnibus

Data deposits

Data have been deposited in the Gene Expression Omnibus under accession number GSE49716.

Change history

  • 01 October 2014

    Minor changes were made to author names and affiliations.

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Acknowledgements

We thank C.-H. Chen for assisting with echocardiography; L. Chen, A. Kuo and G. Crabtree for transgene injection and northern blot; M. Ecarkt and E. Zuo for ribosome analysis. C.-P.C. was supported by the American Heart Association (AHA; Established Investigator Award 12EIA8960018), National Institutes of Health (NIH; HL118087, HL121197), March of Dimes Foundation (#6-FY11-260), California Institute of Regenerative Medicine (CIRM; RN2-00909), Oak Foundation, Baxter Foundation, Stanford Heart Center Research Program, Indiana University (IU) School of Medicine—IU Health Strategic Research Initiative, and the IU Physician-Scientist Initiative, endowed by Lilly Endowment. W.L. and Y.X. were supported by the Oak Foundation; Y.X. by the AHA and Lucile Packard Children’s Foundation; C.S. by an NIH fellowship; T.Q. by NIH (HL109512); H.-S.V.C. by CIRM (RB2-01512, RB4-06276) and NIH (HL105194); P.-S.C. by NIH (HL78931, HL71140); B.Z. by NIH (HL116997, HL111770).

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Author notes

  1. Chiou-Hong Lin: These authors contributed equally to this work.

Authors and Affiliations

  1. Krannert Institute of Cardiology and Division of Cardiology, Department of Medicine, Indiana University School of Medicine, Indianapolis, 46202, Indiana, USA

    Pei Han, Wei Li, Jin Yang, Peng-Sheng Chen & Ching-Pin Chang

  2. Division of Cardiovascular Medicine, Cardiovascular Institute, Stanford University School of Medicine, Stanford, 94305, California, USA

    Pei Han, Wei Li, Chiou-Hong Lin, Ching Shang, Sylvia T. Nurnberg, Kevin Kai Jin, Chieh-Yu Lin, Chien-Jung Lin, Yiqin Xiong, Huan-Chieh Chien, Euan Ashley & Thomas Quertermous

  3. Stanford Genome Technology Center, Stanford University School of Medicine, Stanford, 94305, California, USA

    Weihong Xu

  4. Department of Genetics, Pediatrics, and Medicine (Cardiology), Albert Einstein College of Medicine of Yeshiva University, 1301 Morris Park Avenue, Price Center 420, Bronx, 10461, New York, USA

    Bin Zhou

  5. Department of Pediatrics, Stanford University School of Medicine, Stanford, 94305, California, USA

    Daniel Bernstein

  6. Del E. Webb Neuroscience, Aging & Stem Cell Research Center, Sanford/Burnham Medical Research Institute, La Jolla, 92037, California, USA

    Huei-Sheng Vincent Chen

  7. Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, 46202, Indiana, USA

    Ching-Pin Chang

  8. Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, 46202, Indiana, USA

    Ching-Pin Chang

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Contributions

C.-P.C. and P.H. were responsible for the original concepts, design and manuscript preparation. W.L. and C.-H.L. contributed equally to the work. P.H. conducted most experiments; W.L. and J.Y. assisted with TAC, echo and reporter analyses; C.-H.L. assisted with protein purification; S.T.N. assisted with ribosome data analysis; K.K.J. assisted with protein sequence and motif analysis; C.S. assisted with western blot studies; W.X. assisted with CSF scoring; Y.X. assisted with RNA/protein staining; C.-J.L. and C.-Y.L. assisted with Brg1-null tissue preparation and H-10 antibody-ChIP optimization; H.-C.C. assisted with cloning; H.-S.V.C. assisted with tissue collection; E.A. assisted with tissue collection/rat tissue supply; B.Z. assisted with driver line generation; D.B., P.-S.C. and T.Q. assisted with data analysis.

Corresponding author

Correspondence to Ching-Pin Chang.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Mhrt RNAs have no coding potential.

a, RNA in situ analysis of Mhrt (blue) in a mouse E12 heart. The RNA probe targets all Mhrt species. Red: nuclear fast red. Black arrowheads indicate nuclei of endothelial, endocardial or epicardial cells. Inset shows magnified region from the boxed area. endo, endocardium; epi, epicardium; IVS, interventricular septum; LV, left ventricle; RA and RV, right atrium and ventricle, respectively. Scale bars = 100 μm. b, Codon substitution frequency (CSF) scores of TfIIb and Hprt1 mRNA, as well as full-length Mhrt species. c, In vitro translation of control Mhrt species (709, 779, 826, 828, 857, 1147) and luciferase (Luc). Arrow points to the protein product of luciferase. d, Biotin-labelling of Mhrt species (709, 779, 826, 828, 857, 1147) and luciferase in the in vitro translation reactions. Arrow points to the RNA product of luciferase. e, Ribosome profiling relative to whole transcriptome RNA sequencing. x-axis: genomic position at the human GAPDH and the murine Myh7 loci. y-axis: mapped reads. f, Scatter plot of RNA in fragments per kilobase per million reads (FPKM). Noncoding RNAs (purple) cluster towards the x-axis; coding RNAs (orange) towards the y-axis. Mhrt779 is found below both the identity line (dashed, slope = 1, intercept = 0) and the smooth-fit regression line (in blue). RNA examples are endogenous except that HOTAIR was co-transfected with Mhrt779.

Extended Data Figure 2 Quantification of Myh6/Myh7, northern blot, and Mhrt779 characterization.

a, Quantification of cardiac Myh6/Myh7 ratio 2–42 days after sham or TAC operation. b, Northern blot analysis of Mhrt, Myh6 and Myh7. Negative: control RNA from 293T cells. Size control: 826 is recombinant Mhrt826; 643 (not a distinct Mhrt species) contains the 5′ common region of Mhrt. Heart: adult heart ventricles. c, Un-cropped northern blots of Mhrt, Myh6 and Myh7. d, RNA in situ hybridization of Mhrt779 of adult heart ventricles. White arrowheads indicate nuclei of myocardial cells. Black arrowheads indicate nuclei of endothelial, endocardial or epicardial cells. Blue: Mhrt779; Red: nuclear fast red. Epi, epicardium. The dashed line separates the epicardium from myocardium. Scale bars = 50 μm. e, Quantification of TfIIb, Hprt1, 28S rRNA, Neat1 and Mhrt779 in the nuclear and cytoplasmic fraction of adult heart ventricle extracts. The nuclear/cytoplasmic ratio of TfIIb is set as 1. P values: Student’s t-test. Error bars show s.e.m.

Extended Data Figure 3 Wheat germ agglutinin staining, time course and molecular marker studies of the stressed Tg779 mice.

a, Wheat germ agglutinin (WGA) immunostaining 6 weeks after the sham or TAC operation. Green: WGA stain, outlining cell borders of cardiomyocytes. Blue: 4′,6-diamidino-2-phenylindole (DAPI). Ctrl, control mice. Scale bars = 50 μm. b, Time course of fractional shortening (FS) in control and Tg779 mice. c, Quantification of Anf, Bnp, Serca2 and Tgfb1 in control and Tg779 mice 2 weeks after sham or TAC operation. d, Experimental design for treatment study and time course of left ventricular fractional shortening changes. e, Fractional shortening of the left ventricle (LV) 8 weeks after the operation. f, Ventricular weight/body weight ratio of hearts harvested 8 weeks after sham or TAC operation. P values: Student’s t-test. Error bars show s.e.m.

Extended Data Figure 4 Regulation of the Mhrt promoter.

a, Sequence alignment of Mhrt promoter loci from mouse, human and rat. Peak heights indicate degree of sequence homology. Black boxes (a1–a4) are sequences of high homology, which were used for further ChIP analysis. Green box region between Myh6 and Mhrt is the putative Mhrt promoter. Red, promoter regions; salmon, introns; yellow, untranslated regions. b–d, ChIP–qPCR analysis of Mhrt promoter using antibodies against Pol II (b), H3K4me3 (c), and H3K36me3 (d) in tissues of adult mice. e, RT–qPCR quantification of Mhrt in control and Brg1-null hearts after 7 days of TAC. Ctrl, control. Brg1-null, Tnnt2-rtTA;Tre-Cre;Brg1fl/fl. f, Luciferase reporter assay of Mhrt promoter in SW13 cells. Ctrl: dimethylsulphoxide (DMSO). PJ-34, PARP inhibitor; TSA, trichostatin (HDAC inhibitor). g, ChIP analysis of BRG1, HDAC2, HDAC9 and PARP1 in SW13 cells. The cells were transfected with episomal Mhrt promoter cloned in pREP4. h, Deletional analyses of the Mhrt promoter in luciferase reporter assays in SW13 cells. Luciferase activity of full-length Mhrt promoter was set up as 1. P values: Student’s t-test. Error bars show s.e.m.

Extended Data Figure 5 Mhrt does not affect Myh expression by direct RNA sequence interference.

a, qPCR analysis of Mhrt779, Myh6 and Myh7 in mice without TAC operation. Expression levels were normalized to TfIIb, and the control is set as 1. Ctrl, control mice. b, c, RNA quantification of Mhrt (b) and HOTAIR (c) in SW13 cells transfected with Vector (pAdd2), HOTAIR (pAdd2-HOTAIR) or Mhrt (pAdd2-Mhrt779). Expression in vector-transfected cells is set as 1. Constructs containing Myh6 or Myh7 were co-transfected into SW13 cells used for Fig. 2b–i. d, e, RNA quantification of Myh6 (d) and Myh7 (e) in SW13 cells relative to GAPDH. f, g, Western blot analysis of Myh6 (f) and Myh7 (g) in SW13 cells. Constructs containing Myh6- and Myh7-coding sequences were tagged with Flag and co-transfected with vector, HOTAIR or Mhrt779. GAPDH was used as the loading control. Flag–D1 was used as a positive control for the Flag antibody. h, i, Protein quantification of Myh6 (h) and Myh7 (i) in control and transfected SW13 cells relative to GAPDH. Signals of Myh6 and Myh7 from major bands or the entire lanes were quantified.WB, western blot. j, Luciferase reporter assay of Mhy6 and Myh7 promoters in SW13 cells transfected with vector (pAdd2) or Mhrt (pAdd2-Mhrt779). P values: Student’s t-test. Error bars show s.e.m.

Extended Data Figure 6 RNA-IP controls; Opn is another target gene of Brg1 in stressed hearts.

a, Immunostaining of Brg1 in P1 heart. Red: Brg1. Green: WGA. Blue: DAPI. Ctrl, control. Scale bar = 50 μm. b, RNA-IP of Mhrt in P1 hearts using antibodies against Ezh2 and Suz12. Right panels show immunostaining of Ezh2 and Suz12 in P1 hearts. PRC2, polycomb repressor complex 2. Red: Ezh2 or Suz12. Green: WGA. Blue: DAPI. Scale bars = 50 μm. c, Quantification of Opn mRNA in control and Brg1-null (Tnnt2-rtTA;Tre-Cre;Brg1fl/fl) mice after sham or TAC operation. d, ChIP of Brg1 on Opn proximal promoter in control and transgenic (Tg779) mice after sham or TAC operation. e, Quantification of Opn in control and transgenic (Tg779) mice after sham or TAC operation. P values: Student’s t-test. Error bars show s.e.m.

Extended Data Figure 7 Induction of Mhrt779 is insufficient to change Brg1 mRNA or protein level.

a, qPCR analysis of Brg1 expression in hearts without TAC operation. Ctrl: control mice. b–e, Immunostaining of Brg1 (red) in adult heart ventricles 2 weeks after sham or TAC operation. Green: WGA. Blue: DAPI. Scale bars = 50 μm. f, Western blot analysis of Brg1 and Coomassie staining of total proteins in control or Tg779 hearts after 2 weeks of sham or TAC operation. g, Quantification of Myh6 and Myh7 in control (Ctrl) and Tg779 hearts after 2 weeks of sham or TAC operation. P values: Student’s t-test. Error bars show s.e.m.

Extended Data Figure 8 Brg1 sequence alignment and motif analysis.

Schematics of the architecture of mouse Brg1 and the sequence alignment of Brg1, Vasa (fruit fly), Rad54 (zebrafish, Sulfolobus solfataricus) and Chd1 (yeast). The motifs were outlined by blue boxes (D1 domain) and purple boxes (D2 domain).

Extended Data Figure 9 Purification of Brg1 helicase core domains, EMSA of naked Myh6 promoter, ChIP and reporter studies in SW13 cells.

a, Coomassie blue staining of purified MBP-tagged Brg1 helicase domains. Bovine serum albumin (BSA) was loaded as a control. b, EMSA assay of naked Myh6 promoter (−426 to +170) with helicase domains of Brg1. Probe: biotin-labelled Myh6 promoter. 50 μM of MBP, MBP–D1, MBP–D2 and MBP–D1D2 proteins were used for EMSA. c, d, ChIP (c) and luciferase reporter (d) analysis of Brg1 on chromatinized (episomal) and naked Myh6 promoter in SW13 cells. GFP, green fluorescent protein control. e, The luciferase reporter of helicase-deficient Brg1 on chromatinized (episomal) Myh6 promoter in SW13 cells. ΔD1: Brg1 lacking amino acids 774–913. ΔD2: Brg1 lacking amino acids 1086–1246. ChIP: H-10 antibody recognizing N terminus, non-disrupted region of Brg1. P values: Student’s t-test. Error bars show s.e.m.

Extended Data Figure 10 Brg1 outruns Mhrt to bind to its target Mhrt promoter.

a, Assembly of nucleosomes on the Mhrt promoter (a3/4). b, Amylose pull-down assay: amylose was used to pull down the chromatinized Mhrt promoter that was incubated with various doses of MBP and MBP–Brg1 D1D2. DNA precipitated by amylose was further quantified by qPCR. P values: Student’s t-test. Error bars show s.e.m.

Extended Data Figure 11 Sequence alignment and secondary structure prediction of human and mouse MHRT, and demography of heart transplantation donors.

a, Sequence alignment of human MHRT and mouse Mhrt779. b, Predicted secondary structure of mouse Mhrt779 and human MHRT, using minimal free energy (MFE) calculation of RNAfold WebServer. c, Demography of human subjects whose tissues were used for RT–qPCR analysis (Fig. 4l). ICM, ischaemic cardiomyopathy; IDCM, idiopathic cardiomyopathy; LVH, left ventricular hypertrophy.

Supplementary information

Supplementary Information

This file contains Supplementary Notes, Sequences of alternative spliced Mhrts, Sequences of human MHRT and additional references. (PDF 225 kb)

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Han, P., Li, W., Lin, CH. et al. A long noncoding RNA protects the heart from pathological hypertrophy. Nature 514, 102–106 (2014). https://doi.org/10.1038/nature13596

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