A long noncoding RNA protects the heart from pathological hypertrophy

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 transcription, including myosin heavy chain 7 (Myh7), which encodes molecular motor proteins for heart contraction. 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 complex 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 myopathy. 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.

Figure 1. Profile of the non-coding RNA Mhrt

a, Schematic illustration of Mhrts originating from the intergenic region between Myh6 and Myh7 and transcribed into Myh7. Myh7 exons and introns are indicated. F1 and R1, targeting 5' and 3′ Mhrt common sequences, are the primers used for subsequent PCR. b, RT-qPCR of Mhrts using primers targeting common regions of Mhrts in tissues from 2-month-old mice. P-value: Student’s t-test. Error bar: standard error of the mean (SEM). c, RT-qPCR of Mhrt, Myh6 and Myh7 in mouse hearts at different ages. Mhrt and Myh6/Myh7 ratio of E11 hearts are set as 1. Error bar: SEM. d, RNA in situ analysis of Mhrt (blue) in adult hearts. The RNA probe targets all Mhrt species. Red: nuclear fast red. White arrowheads: myocardial nuclei. Black arrowheads: nuclei of endothelial, endocardial or epicardial cells. Dashed lines demarcate the myocardium from endocardium (endo) or from epicardium (epi). Scale= 50 μm. e, RT-qPCR of nuclear/cytoplasmic RNA in adult hearts. TfIIb, Hprt1, and 28SrRNA are primarily cytoplasmic RNAs; Neat1, nuclear lncRNA. TfIIb ratio is set as 1. P-value: Student’s t-test. Error bar: SEM. f, Ribosome profiling: ribosome density on coding RNAs and lncRNAs.

Figure 2. Mhrt inhibits cardiac hypertrophy and failure

a, Quantitation of cardiac Mhrts 2–42 days after TAC operation. P-value: Student’s t-test. Error bar: SEM. b, RT-PCR of Mhrts in adult heart ventricles. Primers (F1 and R1, Fig. 1a) target Mhrt common regions. Size controls 779, 826, 709 are PCR products of recombinant Mhrt779, Mhrt826, and Mhrt709, respectively. c, Northern blot of Mhrts in adult heart ventricles. The probe targets common regions of Mhrts. 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. d Quantitation of Mhrt779 in control or Tg779 mice with/without doxycycline (Dox) or TAC operation. Mhrt779-specific PCR primers were used. Ctrl: control mice. Tg779: Tnnt2-rtTA;Tre-Mhrt779 mice. P-value: Student’s t-test. Error bar: standard error of the mean (SEM). e, Ventricle-body weight ratio of hearts 6 weeks after TAC. P-value: Student’s t-test. Error bar: SEM. Scale=1 mm. f, Quantitation of cardiomyocyte size in control and Tg779 mice 6 weeks after TAC by wheat-germ agglutinin staining. P-value: Student’s t-test. Error bar: SEM. g, Trichrome staining in control and Tg779 hearts 6 weeks after TAC. Red: cardiomyocytes. Blue: fibrosis. h, i, Echocardiographic measurement of left ventricular fractional shortening (h) and internal dimensions at end-diastole (LVIDd) and end-systole (LVIDs) (i) 6 weeks after TAC. P-value: Student’s t-test. Error bar: SEM.

Figure 3. Mhrt complexes with Brg1 through the helicase domain

a, DNaseI digital footprinting of Myh6/Mhrt promoter loci from ENCODE. Myh6 and Mhrt are transcribed in opposite directions as indicated by arrows. Bars represent DNA fragments protected from DNaseI digestion. Black boxes (a1-a4) refer to promoter regions with high sequences homology (Extended Data Fig. 4a). b, Quantitation of Myh7/Myh6 ratio in control (Ctrl) and Tg779 (Tg) hearts 2 weeks after TAC. P-value: Student’s t-test. Error bar: standard error of the mean (SEM). c, RNA immunoprecipitation (RNA-IP) of Mhrt-Brg1 in ventricles from control hearts (Ctrl) with Sham/TAC operation; Tg779 hearts after TAC; Brg1-null (Tnnt2-rtTA;Tre-Cre;Brg1^f/f) hearts after TAC; P1 hearts. P-value: Student’s t-test. Error bar: SEM. d, ChIP analysis of Brg1 in control (Ctrl) and Tg779 hearts 2 weeks after TAC. Error bar: SEM. e, Luciferase reporter assay of Myh6 and Myh7 promoters in neonatal rat cardiomyocytes. Vector: pAdd2 empty vector. Mhrt: pAdd2-Mhrt779. P-value: Student’s t-test. Error bar: SEM. f, RNA-IP and EMSA of recombinant Brg1 proteins and in vitro transcribed Mhrt779. Biotin-labeled Mhrt779: 50 nM; unlabeled Mhrt779: 500 nM. P-value: Student’s t-test. Error bar: SEM. g, Schematics of mouse Brg1 protein. The helicase core includes DExx-c and HELIC-c domain. h, EMSA of Mhrt779 and Brg1 helicase. MBP: maltose binding protein. MBP-D1: MBP fused to Brg1 D1 (aa 774–913). MBP-D2: MBP fused to Brg1 D2 (aa 1086–1310). MBP-D1D2: MBP fused to Brg1 D1D2 (aa 774–1310). i, Binding affinity of Mhrt779 for MBP-tagged D1D2 determined by EMSA. Error bars represent the standard error from multiple independent measurements. Nonlinear regression curves were generated by GraphPad Prism.

Figure 4. Mhrt inhibits chromatin targeting and gene regulation by Brg1

a, Gel electrophoresis and quantitation of nucleosomal 5SrDNA, Myh6 promoter and Neo DNA. Arrowheads: DNA-histone complex. Arrows: naked DNA. Nucleosome assembly efficiency is defined as the fraction of DNA bound to histones (arrowheads). P-value: Student’s t-test. Error bar: standard error of the mean (SEM). b-d, Quantification of amylose pull-down of MBP-D1D2 (D1D2) with nucleosomal and naked Myh6 promoter DNA (b), with nucleosomal Myh6 promoter, Neo, and 5SrDNA (c), or with nucleosomal Myh6 promoter in the presence of Mhrt779 (d). P-value: Student’s t-test. Error bar: SEM. e, Amylose pull-down of MBP-D1D2 and histone 3. Anti-histone 3 and anti-MBP antibodies were used for western blot analysis. f, ChIP analysis of Brg1 on chromatinized and naked Myh6 promoter in rat ventricular cardiomyocytes. GFP: green fluorescence protein control. P-value: Student’s t-test. Error bar: SEM. g, h, Luciferase reporter activity of Brg1 on naked Myh6 promoter (g) or of helicase-deficient Brg1 on chromatinized Myh6 promoter (h) in rat ventricular cardiomyocytes. ΔD1: Brg1 lacking amino acid 774–913; ΔD2: Brg1 lacking 1086–1246. GFP: green fluorescence protein control. ChIP: H-10 antibody recognizing N-terminus, non-disrupted region of Brg1. P-value: Student’s t-test. Error bar: SEM. i, j, ChIP analysis in SW13 cells of chromatinized Myh6 promoter in the presence of Mhrt779 (i) or helicase-deficient Brg1 (j). Vector: pAdd2 empty vector. Mhrt: pAdd2-Mhrt779. P-value: Student’s t-test. Error bar: SEM. k, Schematic illustration and PCR of human MHRT. MHRT originates from MYH7 and is transcribed into MYH7. MYH7 exons and introns are indicated. R1 and R2 are strand-specific PCR primers; F1 and R1 target MHRT and MYH7; F2 and R2 are specific for MHRT. l, Quantification of MHRT in human heart tissues. Ctrl: control. LVH: left ventricular hypertrophy. ICM: ischemic cardiomyopathy. IDCM: idiopathic dilated cardiomyopathy. P-value: Student’s t-test. Error bar: SEM. m, Working model of a Brg1-Mhrt negative feedback circuit in the heart. Brg1 represses Mhrt transcription, whereas Mhrt prevents Brg1 from recognizing its chromatin targets. Brg1 functions through two distinct promoter elements to bidirectionally repress Myh6 and Mhrt expression. n, Molecular model of how Brg1 binds to its genomic DNA targets. Brg1 helicase (D1D2) binds chromatinized DNA, C-terminal extension (CTE) binds histone 3 (H3), and bromodomain binds acetylated (Ac) histone 3 or 4 (H4).

Figure 5