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The G4 resolvase RHAU regulates ventricular trabeculation and compaction through transcriptional and post-transcriptional mechanisms

  • Xinyi Huang
    Affiliations
    State Key Laboratory of Pharmaceutical Biotechnology, MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center, and Jiangsu Key Laboratory of Molecular Medicine, Nanjing University Medical School, Nanjing, China
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  • Ke Zhao
    Affiliations
    State Key Laboratory of Pharmaceutical Biotechnology, MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center, and Jiangsu Key Laboratory of Molecular Medicine, Nanjing University Medical School, Nanjing, China
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  • Mingyang Jiang
    Affiliations
    State Key Laboratory of Pharmaceutical Biotechnology, MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center, and Jiangsu Key Laboratory of Molecular Medicine, Nanjing University Medical School, Nanjing, China
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  • Dehui Qiu
    Affiliations
    State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry & Chemical Engineering, Nanjing University, Nanjing, China
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  • Jun Zhou
    Affiliations
    State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry & Chemical Engineering, Nanjing University, Nanjing, China
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  • Zhongzhou Yang
    Correspondence
    For correspondence: Zhongzhou Yang
    Affiliations
    State Key Laboratory of Pharmaceutical Biotechnology, MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center, and Jiangsu Key Laboratory of Molecular Medicine, Nanjing University Medical School, Nanjing, China
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Open AccessPublished:November 24, 2021DOI:https://doi.org/10.1016/j.jbc.2021.101449
      The G-quadruplex (G4) resolvase RNA helicase associated with AU-rich element (RHAU) possesses the ability to unwind G4 structures in both DNA and RNA molecules. Previously, we revealed that RHAU plays a critical role in embryonic heart development and postnatal heart function through modulating mRNA translation and stability. However, whether RHAU functions to resolve DNA G4 in the regulation of cardiac physiology is still elusive. Here, we identified a phenotype of noncompaction cardiomyopathy in cardiomyocyte-specific Rhau deletion mice, including such symptoms as spongiform cardiomyopathy, heart dilation, and death at young ages. We also observed reduced cardiomyocyte proliferation and advanced sarcomere maturation in Rhau mutant mice. Further studies demonstrated that RHAU regulates the expression levels of several genes associated with ventricular trabeculation and compaction, including the Nkx2-5 and Hey2 that encode cardiac transcription factors of NKX2-5 and Hey2, and the myosin heavy chain 7 (Myh7) whose protein product is MYH7. While RHAU modulates Nkx2-5 mRNA and Hey2 mRNA at the post-transcriptional level, we uncovered that RHAU facilitates the transcription of Myh7 through unwinding of the G4 structures in its promoter. These findings demonstrated that RHAU regulates ventricular chamber development through both transcriptional and post-transcriptional mechanisms. These results contribute to a knowledge base that will help to understand the pathogenesis of diseases such as noncompaction cardiomyopathy.

      Keywords

      Abbreviations:

      EGFP (enhanced GFP), G4 (G-quadruplex), IF (immunofluorescence), LV (left ventricle), MI (myocardial infarction), NCC (noncompaction cardiomyopathy), NMM (N-methyl mesoporphyrin IX), qPCR (quantitative PCR), RHAU (RNA helicase associated with AU-rich element), RV (right ventricle), ThT (thioflavin T)
      Noncompaction cardiomyopathy (NCC) is a genetically heterogeneous disorder with diverse clinical phenotypes, which has gained increased awareness and prevalence in both children and adults, perhaps because of advances in cardiovascular imaging (
      • Oechslin E.
      • Jenni R.
      • Klaassen S.
      Left ventricular noncompaction is a myocardial phenotype: Cardiomyopathy-yes or no?.
      ,
      • Towbin J.A.
      • Lorts A.
      • Jefferies J.L.
      Left ventricular non-compaction cardiomyopathy.
      ). Clinically, NCC manifests nine distinct subtypes, and all of them show the common characteristic of abnormal trabeculations in the left ventricle (LV) (
      • Towbin J.A.
      • Lorts A.
      • Jefferies J.L.
      Left ventricular non-compaction cardiomyopathy.
      ,
      • Jefferies J.L.
      Left ventricular noncompaction cardiomyopathy: New clues in a not so new disease?.
      ).
      NCC emerges from myocardial developmental defects as a consequence of impaired trabeculation or disrupted compaction or both of them during embryonic heart development (
      • Liu Y.
      • Chen H.
      • Shou W.
      Potential common pathogenic pathways for the left ventricular noncompaction cardiomyopathy (LVNC).
      ). Initiation of ventricular trabeculation distinguishes the trabecular zone from adjacent compact zone (
      • Choquet C.
      • Kelly R.G.
      • Miquerol L.
      Defects in trabecular development contribute to left ventricular noncompaction.
      ,
      • Chen H.
      • Zhang W.
      • Li D.
      • Cordes T.M.
      • Mark Payne R.
      • Shou W.
      Analysis of ventricular hypertrabeculation and noncompaction using genetically engineered mouse models.
      ), and these two zones demonstrate regional-specific properties in cell proliferation and gene expression patterns (
      • Tian X.
      • Li Y.
      • He L.
      • Zhang H.
      • Huang X.
      • Liu Q.
      • Pu W.
      • Zhang L.
      • Li Y.
      • Zhao H.
      • Wang Z.
      • Zhu J.
      • Nie Y.
      • Hu S.
      • Sedmera D.
      • et al.
      Identification of a hybrid myocardial zone in the mammalian heart after birth.
      ,
      • Yue Y.
      • Zong W.
      • Li X.
      • Li J.
      • Zhang Y.
      • Wu R.
      • Liu Y.
      • Cui J.
      • Wang Q.
      • Bian Y.
      • Yu X.
      • Liu Y.
      • Tan G.
      • Zhang Y.
      • Zhao G.
      • et al.
      Long-term, in toto live imaging of cardiomyocyte behaviour during mouse ventricle chamber formation at single-cell resolution.
      ). For instance, the trabecular zone is featured by the expression of Irx3, Bmp10, Cx40, Slit2, Etv1, and Sema3a, whereas in the compact zone, Hey2, Tbx20, and N-Myc are specifically expressed (
      • Choquet C.
      • Kelly R.G.
      • Miquerol L.
      Defects in trabecular development contribute to left ventricular noncompaction.
      ,
      • Luxán G.
      • D'Amato G.
      • MacGrogan D.
      • de la Pompa J.L.
      Endocardial Notch signaling in cardiac development and disease.
      ). In this respect, endocardial Notch signaling is required for the formation and maturation of both trabecular and compact myocardium (
      • Luxán G.
      • D'Amato G.
      • MacGrogan D.
      • de la Pompa J.L.
      Endocardial Notch signaling in cardiac development and disease.
      ,
      • D'Amato G.
      • Luxán G.
      • de la Pompa J.L.
      Notch signalling in ventricular chamber development and cardiomyopathy.
      ). Mutation of Mib1, a positive regulator of Notch signaling, has been identified in NCC patients, and animal model has confirmed Mib1 function in ventricular chamber development (
      • Luxán G.
      • Casanova J.C.
      • Martínez-Poveda B.
      • Prados B.
      • D'Amato G.
      • MacGrogan D.
      • Gonzalez-Rajal A.
      • Dobarro D.
      • Torroja C.
      • Martinez F.
      • Izquierdo-García J.L.
      • Fernández-Friera L.
      • Sabater-Molina M.
      • Kong Y.Y.
      • Pizarro G.
      • et al.
      Mutations in the NOTCH pathway regulator MIB1 cause left ventricular noncompaction cardiomyopathy.
      ).
      On the other hand, defective myofibrillogenesis (myofibril assembly) was proposed as a key pathogenic factor for NCC because mutation of the sarcomere genes of Myh7, Mybpc3, and TTN was frequently identified in NCC patients (
      • Oechslin E.
      • Jenni R.
      Nosology of noncompaction cardiomyopathy: The emperor still wears clothes!.
      ,
      • Richard P.
      • Ader F.
      • Roux M.
      • Donal E.
      • Eicher J.C.
      • Aoutil N.
      • Huttin O.
      • Selton-Suty C.
      • Coisne D.
      • Jondeau G.
      • Damy T.
      • Mansencal N.
      • Casalta A.C.
      • Michel N.
      • Haentjens J.
      • et al.
      Targeted panel sequencing in adult patients with left ventricular non-compaction reveals a large genetic heterogeneity.
      ). Interestingly, a recent study of compound heterozygosity for the single-nucleotide variants of Myh7, Mkl2, and Nkx2-5 in mice recapitulated the human NCC phenotype (
      • Gifford C.A.
      • Ranade S.S.
      • Samarakoon R.
      • Salunga H.T.
      • de Soysa T.Y.
      • Huang Y.
      • Zhou P.
      • Elfenbein A.
      • Wyman S.K.
      • Bui Y.K.
      • Cordes Metzler K.R.
      • Ursell P.
      • Ivey K.N.
      • Srivastava D.
      Oligogenic inheritance of a human heart disease involving a genetic modifier.
      ). This study affirmed the contribution of Myh7 mutation to incidence of NCC and revealed Nkx2-5 variant as a genetic modifier (
      • Gifford C.A.
      • Ranade S.S.
      • Samarakoon R.
      • Salunga H.T.
      • de Soysa T.Y.
      • Huang Y.
      • Zhou P.
      • Elfenbein A.
      • Wyman S.K.
      • Bui Y.K.
      • Cordes Metzler K.R.
      • Ursell P.
      • Ivey K.N.
      • Srivastava D.
      Oligogenic inheritance of a human heart disease involving a genetic modifier.
      ). Nonetheless, one puzzling issue is that mutations of Myh7 and Nkx2-5 were also identified in other forms of cardiomyopathies including hypertrophic cardiomyopathy and dilated cardiomyopathy (
      • Richard P.
      • Ader F.
      • Roux M.
      • Donal E.
      • Eicher J.C.
      • Aoutil N.
      • Huttin O.
      • Selton-Suty C.
      • Coisne D.
      • Jondeau G.
      • Damy T.
      • Mansencal N.
      • Casalta A.C.
      • Michel N.
      • Haentjens J.
      • et al.
      Targeted panel sequencing in adult patients with left ventricular non-compaction reveals a large genetic heterogeneity.
      ). Therefore, it is necessary and interesting to distinguish the action and contribution of Myh7 and the modifier genes such as Nkx2-5 in the occurrence of distinct cardiomyopathies.
      G-quadruplex (G4) is a four-stranded tetrad structure resulting from stacking of G-quartets formed by four guanines via Hoogsteen base paring in a planar arrangement (
      • Sauer M.
      • Paeschke K.
      G-quadruplex unwinding helicases and their function in vivo.
      ,
      • Varshney D.
      • Spiegel J.
      • Zyner K.
      • Tannahill D.
      • Balasubramanian S.
      The regulation and functions of DNA and RNA G-quadruplexes.
      ). G4 structures are abundantly distributed in the genome and mRNA molecules, with preference in the areas of regulatory domains like promoters and transcription start sites, replication hot spots, and telomeres in the genome, and are enriched in the UTRs of mRNAs (
      • Sauer M.
      • Paeschke K.
      G-quadruplex unwinding helicases and their function in vivo.
      ). Thus, G4 structures are involved in regulating transcription, DNA replication and telomere maintenance, and mRNA translation and stability (
      • Sauer M.
      • Paeschke K.
      G-quadruplex unwinding helicases and their function in vivo.
      ,
      • Varshney D.
      • Spiegel J.
      • Zyner K.
      • Tannahill D.
      • Balasubramanian S.
      The regulation and functions of DNA and RNA G-quadruplexes.
      ). RHAU (DHX36 and G4R1) is a resolvase/helicase to unwind the G4 structures in DNA and RNA (
      • Chen M.C.
      • Tippana R.
      • Demeshkina N.A.
      • Murat P.
      • Balasubramanian S.
      • Myong S.
      • Ferré-D'Amaré A.R.
      Structural basis of G-quadruplex unfolding by the DEAH/RHA helicase DHX36.
      ,
      • Sauer M.
      • Juranek S.A.
      • Marks J.
      • De Magis A.
      • Kazemier H.G.
      • Hilbig D.
      • Benhalevy D.
      • Wang X.
      • Hafner M.
      • Paeschke K.
      DHX36 prevents the accumulation of translationally inactive mRNAs with G4-structures in untranslated regions.
      ,
      • Tran H.
      • Schilling M.
      • Wirbelauer C.
      • Hess D.
      • Nagamine Y.
      Facilitation of mRNA deadenylation and decay by the exosome-bound, DExH protein RHAU.
      ). Previously, we demonstrated an essential function of RHAU in mouse embryonic heart development (
      • Nie J.
      • Jiang M.
      • Zhang X.
      • Tang H.
      • Jin H.
      • Huang X.
      • Yuan B.
      • Zhang C.
      • Lai J.C.
      • Nagamine Y.
      • Pan D.
      • Wang W.
      • Yang Z.
      Post-transcriptional regulation of Nkx2-5 by RHAU in heart development.
      ). Our recent work also revealed a pivotal role of RHAU in postnatal heart function (
      • Jiang M.
      • Hu H.
      • Zhao K.
      • Di R.
      • Huang X.
      • Shi Y.
      • Yue Y.
      • Nie J.
      • Yu S.
      • Wang W.
      • Yang Z.
      The G4 resolvase RHAU modulates mRNA translation and stability to sustain postnatal heart function and regeneration.
      ). In these two studies, we have identified the mRNAs of Nkx2-5, Yap1, and Hexim1 as RHAU regulatory targets, and RHAU promotes their translation through association with the G4 structures in the 5′-UTR of these mRNAs. Meanwhile, RHAU binds to the 3′-UTR of these mRNAs to facilitate mRNA decay (
      • Nie J.
      • Jiang M.
      • Zhang X.
      • Tang H.
      • Jin H.
      • Huang X.
      • Yuan B.
      • Zhang C.
      • Lai J.C.
      • Nagamine Y.
      • Pan D.
      • Wang W.
      • Yang Z.
      Post-transcriptional regulation of Nkx2-5 by RHAU in heart development.
      ,
      • Jiang M.
      • Hu H.
      • Zhao K.
      • Di R.
      • Huang X.
      • Shi Y.
      • Yue Y.
      • Nie J.
      • Yu S.
      • Wang W.
      • Yang Z.
      The G4 resolvase RHAU modulates mRNA translation and stability to sustain postnatal heart function and regeneration.
      ). While these results uncovered the critical function of RHAU as RNA G4 resolvase to modulate gene expression at post-transcriptional level, whether RHAU functions to resolve DNA G4 to regulate cardiac physiology is still elusive.
      Deletion of Rhau in cardiac progenitors (using Nkx2-5-Cre or Mesp1-Cre) caused severe heart defects and embryonic lethality by E14.5, excluding study of ventricular chamber development in midterm (
      • Nie J.
      • Jiang M.
      • Zhang X.
      • Tang H.
      • Jin H.
      • Huang X.
      • Yuan B.
      • Zhang C.
      • Lai J.C.
      • Nagamine Y.
      • Pan D.
      • Wang W.
      • Yang Z.
      Post-transcriptional regulation of Nkx2-5 by RHAU in heart development.
      ). In an effort to extend the study of RHAU in late heart development, particularly in ventricular chamber formation, we deleted Rhau in embryonic cardiomyocytes (via cTNT-Cre–mediated excision) and identified the NCC phenotype. Furthermore, we have figured out that RHAU modulated gene expression level of several genes involved in ventricular trabeculation and compaction. In addition to the post-transcriptional mechanisms, RHAU controlled gene transcription through unwinding G4 structures in the genome (DNA) to govern ventricular chamber development. These findings demonstrated that RHAU regulates ventricular chamber formation through transcriptional and post-transcriptional mechanisms. Meanwhile, this study provided a valuable mouse model for understanding NCC.

      Results

      RHAU deficiency led to the phenotypes of NCC

      We previously unveiled the essential function of RHAU in embryonic heart development of mouse (
      • Nie J.
      • Jiang M.
      • Zhang X.
      • Tang H.
      • Jin H.
      • Huang X.
      • Yuan B.
      • Zhang C.
      • Lai J.C.
      • Nagamine Y.
      • Pan D.
      • Wang W.
      • Yang Z.
      Post-transcriptional regulation of Nkx2-5 by RHAU in heart development.
      ). However, these cardiac progenitor-specific Rhau-deletion mice manifested severe heart defects and embryonic lethality by E14.5 with complete penetrance, which made it impossible to investigate the ventricular chamber formation during the period from midterm of gestation to birth.
      To study the function of RHAU in the ventricular chamber formation, we first removed Rhau using Mef2c-AHF-Cre that showed Cre activity in both outflow tract and the right ventricle (RV) (
      • Verzi M.P.
      • McCulley D.J.
      • De Val S.
      • Dodou E.
      • Black B.L.
      The right ventricle, outflow tract, and ventricular septum comprise a restricted expression domain within the secondary/anterior heart field.
      ). The Rhauf/f;Mef2c-AHF-Cre embryos could develop to term and be born, and the majority of these mice could survive up to 6 months (Fig. 1A). Gross analysis of Rhauf/f;Mef2c-AHF-Cre and control mice (Rhauf/f or Rhauf/+ littermates in this study) at postnatal 21 days (P21) displayed prominently enlarged hearts in Rhauf/f;Mef2c-AHF-Cre mice compared with control (Fig. 1, B and C). Histological examination of heart tissues revealed dilated ventricle chamber, thinner myocardium wall, and sponge-like trabecula in the RV of Rhauf/f;Mef2c-AHF-Cre mice, indicative of ventricular noncompaction (NCC) (Figs. 1, DG and S1, A and A').
      Figure thumbnail gr1
      Figure 1RHAU deficiency recapitulated the phenotypes of human NCC. A, survival curves of control and Rhauf/f;Mef2c-AHF-Cre mice. N = 22 for control mice and N = 22 for Rhauf/f;Mef2c-AHF-Cre mice. p < 0.001 (∗∗∗). B and C, gross analysis of the hearts from control and Rhauf/f;Mef2c-AHF-Cre mice at P21. The RV was remarkably enlarged in the Rhau mutant mice. The scale bars represent 1 mm. D and E, histological analysis of the heart at P21. The RV displayed spongiform cardiomyopathy in the Rhau mutant mice. The scale bars represent 1 mm. F and G, higher magnification of the boxed areas in (D) and (E). The scale bars represent 500 μm. H, survival curves of control and Rhauf/f;cTNT-Cre mice. N = 15 for control mice and N = 15 for Rhauf/f;cTNT-Cre mice. p < 0.001 (∗∗∗). I and J, gross analysis of heart from control and Rhauf/f;cTNT-Cre mice at P13. The scale bars represent 1 mm. K and L, histological analysis of the heart at P13. The scale bars represent 1 mm. M and N, higher magnification of the boxed areas in (K) and (L). The LV displayed spongiform cardiomyopathy in the Rhau mutant mice. The scale bars represent 1 mm. O and P, histological analysis of heart in control and Rhauf/f;Tie2-Cre mice at P3. The scale bars represent 200 μm. Control mice were Rhauf/f or Rhauf/+ littermates. LV, left ventricle; NCC, noncompaction cardiomyopathy; RHAU, RNA helicase associated with AU-rich element; RV, right ventricle.
      Ventricular noncompaction is more frequent and harmful in the LV. To see whether RHAU deficiency in the LV could cause noncompaction as in the RV, we crossed Rhau floxed mice with cTNT-Cre (Tnnt2-Cre) mice that have Cre activity in the cardiomyocytes of both ventricles (
      • Jiao K.
      • Kulessa H.
      • Tompkins K.
      • Zhou Y.
      • Batts L.
      • Baldwin H.S.
      • Hogan B.L.
      An essential role of Bmp4 in the atrioventricular septation of the mouse heart.
      ) to generate Rhauf/f;cTNT-Cre mice. These mice could also develop to term but started to die several days after birth (Fig. 1H). We observed loss of nearly 80% of Rhauf/f;cTNT-Cre mice by 2 months (Fig. 1H). Gross assessment of the hearts from P13 mice exhibited bigger size in Rhauf/f;cTNT-Cre mice than in control (Fig. 1, I and J). Histological inspection disclosed spongiform cardiomyopathy, thinned ventricular wall, and chamber dilation in the LV of Rhauf/f;cTNT-Cre mice (Figs. 1, KN and S1, B and B').
      In addition, we asked question of whether losing RHAU activity in the endocardium had a contribution to ventricular noncompaction. We removed RHAU in the endocardial and endothelial cells of Rhauf/f;Tie2-Cre mice and found normal embryonic development. A thorough investigation failed to observe a difference between the hearts of Rhauf/f;Tie2-Cre and control mice (Fig. 1, O and P), suggesting that RHAU function was dispensable in the endocardium and endothelium.
      Taken together, these data demonstrated that mice with RHAU deficiency in the embryonic cardiomyocytes truthfully recapitulated typical human NCC phenotypes.

      Defective trabeculation and compaction in RHAU-deficient mice

      To understand the NCC phenotype observed in postnatal Rhauf/f;cTNT-Cre mice, we performed gross and histological analysis of embryonic heart. At E12.5, the hearts appeared comparable between the two groups (Fig. 2, A and B). However, substantially enlarged ventricles were noticed in the Rhauf/f;cTNT-Cre mice compared with control at E14.5 (Fig. 2, C and D). Histological study of these hearts not only confirmed the ventricular dilation but also recognized thinned ventricular wall in the heart of Rhauf/f;cTNT-Cre mice at E14.5 (Fig. 2, E, F, E', and F'). The ventricles were more dilated in the Rhauf/f;cTNT-Cre mice at E16.5 than at E14.5 (Fig. 2, G, H, G', H', and I).
      Figure thumbnail gr2
      Figure 2Defective trabeculation and compaction in RHAU-deficient mice. AD, gross analysis of the hearts from control and Rhauf/f;cTNT-Cre mice at E12.5 and E14.5. The ventricles were larger in the Rhau mutant mice than in control at E14.5. The scale bars represent 500 μm. EH, histological analysis of the hearts in control and Rhauf/f;cTNT-Cre mice at E14.5 and E16.5. The scale bars represent 200 μm. E′–H′, higher magnification of the boxed areas in (EH). Thicker trabeculae and thinner ventricular compact zones were observed in the Rhau mutant mice. The scale bars represent 50 μm. I, quantification of the diameter of ventricles in (AD). The Rhau mutants showed increased diameter of ventricles at E12.5, E14.5, and E16.5. Control mice: E12.5 (N = 8), E14.5 (N = 6), and E16.5 (N = 6). Rhauf/f;cTNT-Cre mice: E12.5 (N = 4), E14.5 (N = 5), and E16.5 (N = 3). p > 0.05 (ns), p < 0.01 (∗∗), and p < 0.0001 (∗∗∗∗). J, ratio of trabecular area to compact area in (EH). The ratio was increased in Rhauf/f;cTNT-Cre mice at E12.5, E14.5, and E16.5 compared with control. Control mice: E12.5 (N = 3), E14.5 (N = 5), and E16.5 (N = 3). Rhauf/f;cTNT-Cre mice: E12.5 (N = 3), E14.5 (N = 4), and E16.5 (N = 3). p > 0.05 (ns), p < 0.01 (∗∗). K, quantification of the trabeculae thickness at E14.5. N = 4 for control mice and N = 4 for Rhauf/f;cTNT-Cre mice. p < 0.05 (∗). Control mice were Rhauf/f or Rhauf/+ littermates. LV, left ventricle; ns, nonsignificant; RHAU, RNA helicase associated with AU-rich element; RV, right ventricle.
      In addition, significantly elevated trabeculation was detected in the ventricles of Rhauf/f;cTNT-Cre mice at E14.5 (Fig. 2, E, F, E', and F'). Quantitative examination of the area of trabecular and compact zone showed an increased ratio of trabecular area/compact area in the Rhauf/f;cTNT-Cre mice compared with control at E14.5, and the number of this ratio was more than doubled in the Rhauf/f;cTNT-Cre mice compared with control at E16.5 (Fig. 2J). Moreover, the trabeculae of Rhauf/f;cTNT-Cre mice was thicker than control mice at E14.5 (Fig. 2K).
      Gross and histological analysis were also performed with the embryonic heart of Rhauf/f;Mef2c-AHF-Cre mice. Ventricular dilation and thickened trabeculae in the RV were noticed (Fig. S1, CN).
      Collectively, these results demonstrated enhanced trabeculation with thickened trabeculae in the Rhau mutant mice.

      Reduced cardiomyocyte proliferation and advanced sarcomere maturation in Rhau mutant mice

      Cell proliferation of the ventricular cardiomyocytes was considered as a crucial element contributing to NCC, and both reduced and enhanced Bmp10 expression level were recorded in impacting cardiomyocyte proliferation in NCC mouse models (
      • Luxán G.
      • Casanova J.C.
      • Martínez-Poveda B.
      • Prados B.
      • D'Amato G.
      • MacGrogan D.
      • Gonzalez-Rajal A.
      • Dobarro D.
      • Torroja C.
      • Martinez F.
      • Izquierdo-García J.L.
      • Fernández-Friera L.
      • Sabater-Molina M.
      • Kong Y.Y.
      • Pizarro G.
      • et al.
      Mutations in the NOTCH pathway regulator MIB1 cause left ventricular noncompaction cardiomyopathy.
      ,
      • Chen H.
      • Shi S.
      • Acosta L.
      • Li W.
      • Lu J.
      • Bao S.
      • Chen Z.
      • Yang Z.
      • Schneider M.D.
      • Chien K.R.
      • Conway S.J.
      • Yoder M.C.
      • Haneline L.S.
      • Franco D.
      • Shou W.
      BMP10 is essential for maintaining cardiac growth during murine cardiogenesis.
      ,
      • Pashmforoush M.
      • Lu J.T.
      • Chen H.
      • Amand T.S.
      • Kondo R.
      • Pradervand S.
      • Evans S.M.
      • Clark B.
      • Feramisco J.R.
      • Giles W.
      • Ho S.Y.
      • Benson D.W.
      • Silberbach M.
      • Shou W.
      • Chien K.R.
      Nkx2-5 pathways and congenital heart disease; loss of ventricular myocyte lineage specification leads to progressive cardiomyopathy and complete heart block.
      ). We found that the proliferation activity of the cardiomyocytes was reduced in Rhauf/f;Mef2c-AHF-Cre mice at both E12.5 and E14.5 compared with control (Fig. S2, A and B). We therefore examined the cell proliferation status of the ventricular cardiomyocytes by immunofluorescence (IF) staining. The results demonstrated that the number of Ki-67+ cardiomyocytes was dramatically decreased in the ventricles of Rhauf/f;cTNT-Cre mice at both E12.5 and E14.5, and there was no big difference between the two ventricles (Figs. 3, AE and S2, C and D). In addition, Rhau deletion had little effect on the proliferative activity of endocardial cells (Fig. S2, E and F). Examination of Bmp10 expression level disclosed a slight reduction in the heart tissues of Rhauf/f;cTNT-Cre mice (Fig. S3A).
      Figure thumbnail gr3
      Figure 3Reduced cardiomyocyte proliferation in Rhau mutant mice. AD, IF staining for Ki-67 in heart sections at E12.5 and E14.5. MF20 stained cardiomyocytes (in red), and Ki-67 stained proliferative cells (in green). The nuclei were stained by DAPI (in blue). MF20+ and Ki-67+ double positive cells represented proliferative cardiomyocytes. The scale bars represent 100 μm. E, the percentage of proliferative cardiomyocytes. Significantly reduced proliferative activity of cardiomyocytes was detected in the Rhau mutant heart at E12.5 and E14.5. Control mice: E12.5 (N = 9) and E14.5 (N = 4). Rhauf/f;cTNT-Cre mice: E12.5 (N = 8) and E14.5 (N = 4). p < 0.01 (∗∗), p < 0.0001 (∗∗∗∗). FH, TUNEL staining in the heart sections at E12.5. The section in (F) was treated with DNase I as the positive control. TUNEL signal was in red, and nucleus was stained by DAPI (in blue). The scale bars represent 200 μm. Control mice were Rhauf/f or Rhauf/+ littermates. DAPI, 4′,6-diamidino-2-phenylindol; IF, immunofluorescence.
      In addition, cell apoptosis of cardiomyocytes was assessed by TUNEL staining, and no statistically significant difference was found between the control and Rhauf/f;cTNT-Cre mice at E12.5 and E14.5 (Figs. 3, FH and S2, GI). However, quantification analysis of the cardiomyocyte numbers in the ventricle at E14.5 revealed a significant reduction in the Rhauf/f;cTNT-Cre mice compared with control mice at E14.5 (Fig. S2J).
      From E12.5, ventricular cardiomyocytes start the process of maturation, and sarcomere growth is one of the features of cardiomyocyte maturation (
      • Guo Y.
      • Pu W.T.
      Cardiomyocyte maturation: New phase in development.
      ). We first checked the sarcomere structure in the cardiomyocytes of control and Rhauf/f;cTNT-Cre mice at E12.5 and E14.5 by IF staining for α-actinin, and the results showed comparable patterns between the two groups at these developmental stages (Fig. 4, AD). We also checked the structure of myofibrils of the LV and RV at both E12.5 and E14.5 through IF staining and failed to detect an obvious difference between control and Rhau mutant mice (Fig. S3, B and C). However, ultrastructural examination of the sarcomere by transmission electron microscopy displayed alterations with sarcomere length and thickness between these two groups (Fig. 4, EL). The distance of two Z-lines was defined as sarcomere length, and the length of single Z-line was regarded as sarcomere thickness. From E12.5 to E14.5, the average sarcomere length in the cardiomyocytes of both trabecular and compact zone increased prominently in the control mice, indicating sarcomere growth (Fig. 4, M and N). In the Rhauf/f;cTNT-Cre mice, we observed that the average sarcomere length in the trabecular cardiomyocytes at E12.5 was remarkably increased and nearly reached the sarcomere length in the cardiomyocytes of control mice at E14.5 (Fig. 4M). The average sarcomere length in the cardiomyocytes of compact zone of the Rhauf/f;cTNT-Cre mice was also enlarged compared with control at E12.5 but exhibited a slightly shorter status than control at E14.5 (Fig. 4N). As for the sarcomere thickness, it was bigger in the trabecular cardiomyocytes of the Rhauf/f;cTNT-Cre mice at E12.5 than control (Fig. 4O). At both E12.5 and E14.5, the sarcomeres in the compact cardiomyocytes of the Rhauf/f;cTNT-Cre mice were thicker than control (Fig. 4P). To summarize these changes, the sarcomeres in the trabecular zone of Rhau mutant mice increased fast in length and thickness by E12.5 and kept in a quiescent state by E14.5 when they were thicker than those in control mice. While in the compact zone, the sarcomeres became longer and thinner from E12.5 to E14.5 in control mice, those in the Rhau mutant mice stopped changes and were shorter and thicker than control by E14.5. In addition, the sarcomeres in the Rhau mutant mice failed to change (length and thickness) in either trabecular or compact zone between E12.5 and E14.5, suggesting a terminal maturation status. These changes with the sarcomeres, especially the increased sarcomere length and thickness by E12.5, indicated advanced maturation in the cardiomyocytes of Rhau mutant mice.
      Figure thumbnail gr4
      Figure 4Advanced sarcomere maturation in Rhau mutant mice. AD, IF staining to display the thin filament in sagittal heart sections at E12.5 and E14.5. Thin filaments were stained by α-actinin (in white). The scale bars represent 10 μm. EL, transmission electron microscopy of sarcomere structures of the cardiomyocytes from E12.5 and E14.5 embryos. The scale bars represent 2.5 μm. M and N, quantification of the sarcomere length in trabecular and compact zone at E12.5 and E14.5. Control mice: E12.5 (N = 3) and E14.5 (N = 4). Rhauf/f;cTNT-Cre mice: E12.5 (N = 3) and E14.5 (N = 4). p > 0.05 (ns), p < 0.01 (∗∗), p < 0.001 (∗∗∗), and p < 0.0001 (∗∗∗∗). O and P, quantification of the sarcomere thickness in trabecular and compact zone at E12.5 and E14.5. Control mice: E12.5 (N = 3) and E14.5 (N = 4). Rhauf/f;cTNT-Cre mice: E12.5 (N = 3) and E14.5 (N = 4). p > 0.05 (ns), p < 0.05 (∗), p < 0.01 (∗∗), and p < 0.0001 (∗∗∗∗). Control mice were Rhauf/f or Rhauf/+ littermates. IF, immunofluorescence.

      Altered expression pattern of NCC-associated genes in Rhau mutant heart

      Our previous studies have uncovered a post-transcriptional regulatory mechanism by RHAU in modulating mRNA stability and translation. Through binding to the 3′-UTR of target mRNAs, RHAU facilitated mRNA degradation. Upon removal of RHAU, the levels of the target mRNAs were enhanced tremendously. Therefore, we performed RNA-Seq analysis to search for potential target genes with increased levels in the Rhau mutant heart, and the initial RNA-Seq results were shown in the heat map (Fig. S3D). Examination of the core components (Jag1, Jag2, and Mib1) in the Notch signaling pathway failed to find a big difference between control and Rhauf/f;cTNT-Cre mice (Fig. S3E).
      A detailed analysis of the RNA-Seq data identified the NCC-associated genes with upregulated or downregulated expression levels (Fig. 5A). In the upregulated genes, the cardiac-specific transcription factor Nkx2-5 mRNA was thoroughly studied for post-transcriptional regulation in our previous reports. We found that RHAU bound and resolved the G4 structure in the 5′-UTR of Nkx2-5 mRNA for translation, and RHAU association with the 3′-UTR of Nkx2-5 mRNA promoted the mRNA degradation. Accordingly, RHAU deficiency resulted in substantially increased level of Nkx2-5 mRNA that could not be translated because of inhibitory effects of the G4 structure in its 5′-UTR, leading to remarkably reduced NKX2-5 protein level (
      • Nie J.
      • Jiang M.
      • Zhang X.
      • Tang H.
      • Jin H.
      • Huang X.
      • Yuan B.
      • Zhang C.
      • Lai J.C.
      • Nagamine Y.
      • Pan D.
      • Wang W.
      • Yang Z.
      Post-transcriptional regulation of Nkx2-5 by RHAU in heart development.
      ). Here, we disclosed a similar pattern of significantly enhanced Nkx2-5 mRNA level but greatly reduced NKX2-5 protein level in the heart of Rhauf/f;cTNT-Cre mice (Fig. 5, B and C), declaring a post-transcriptional regulation of Nkx2-5 mRNA by RHAU. In addition, we found that Hey2 exhibited a much higher mRNA level in the Rhau mutant heart than in control, but its protein level was markedly lower in the ventricular compact zone of Rhau mutant mice than control (Figs. 5, D and F and S4A). A G4-forming sequence was identified in the 5′-UTR of Hey2 mRNA (Fig. 5G). The G4-forming fragment was inserted upstream of GFP coding sequence (Hey2-5′UTR-pEGFPN1), and it suppressed GFP translation (Fig. S4, B and C), and overexpression of Rhau substantially enhances GFP translation (Fig. S4, D and E). In the meantime, the cells transfected with G4 mutant vectors (G to A, Hey2-5′UTR-G4mut-pEGFPN1) showed comparable GFP expression level to the control group (Fig. S4, B and C), and overexpression of Rhau did not affect GFP translation anymore (Fig. S4, F and G). These data verified that RHAU modulated the Nkx2-5 and Hey2 mRNA through the post-transcriptional regulatory mechanism and identified Hey2 mRNA as a new regulatory target of RHAU.
      Figure thumbnail gr5
      Figure 5Altered expression pattern of NCC-associated genes in Rhau mutant heart. A, heat map showed the upregulated and downregulated genes associated with NCC in Rhau mutant mice at E15.5. B and C, quantitative RT–PCR and Western blot analysis for Nkx2-5 mRNA and NKX2-5 protein in the heart from E14.5 embryos. N = 4 for control mice and N = 4 for Rhauf/f;cTNT-Cre mice. p < 0.001 (∗∗∗). D, quantitative RT–PCR for Hey2 mRNA expression level in the heart from E14.5 mice. N = 4 for control mice and N = 4 for Rhauf/f;cTNT-Cre mice. p < 0.0001 (∗∗∗∗). E and F, IF staining for Hey2 in sagittal heart sections at E14.5. Cardiomyocytes were stained by MF20 (in red) and Hey2 (in green). Hey2 was abundant in the cardiomyocytes of compact zone, and an obvious reduced amount of Hey2 was shown in the Rhau mutant heart. The scale bars represent 100 μm. G, the predicted G4 sequence in the 5′-UTR of Hey2 mRNA. H and I, quantitative RT–PCR of ventricular Myh7 and Myh6 expression levels at E10.5, E12.5, and E14.5. Control mice: E10.5 (N = 3), E12.5 (N = 3), and E14.5 (N = 3). Rhauf/f;cTNT-Cre mice: E10.5 (N = 3), E12.5 (N = 3), and E14.5 (N = 3). p > 0.05 (ns), p < 0.05 (∗), p < 0.01 (∗∗), and p < 0.0001 (∗∗∗∗). J, ratio of Myh6/Myh7 mRNA. A switch from fetal Myh7 to adult Myh6 was remarkable. Control mice: E10.5 (N = 3), E12.5 (N = 3), and E14.5 (N = 3). Rhauf/f;cTNT-Cre mice: E10.5 (N = 3), E12.5 (N = 3), and E14.5 (N = 3). p < 0.05 (∗), p < 0.01 (∗∗), and p < 0.001 (∗∗∗). K, Western blot analysis of RHAU, MYH7, MYH6, and BRG1 in the heart of control and Rhau mutant mice at E14.5. L, quantification of (K). N = 3 for control mice and N = 3 for Rhauf/f;cTNT-Cre mice. p > 0.05 (ns), p < 0.05 (∗). Control mice were Rhauf/f or Rhauf/+ littermates. IF, immunofluorescence; NCC, noncompaction cardiomyopathy; RHAU, RNA helicase associated with AU-rich element; TF, transcription factor.
      Defective myofibrillogenesis (sarcomere assembly) was proposed as a key pathogenic factor for NCC because mutation of the sarcomere genes was frequently identified in NCC patients. Therefore, we paid special attention to the sarcomere genes and noticed a considerable change of Myh6 and Myh7 mRNA level (Fig. 5A). Quantitative PCR (qPCR) and Western blot confirmed reduced Myh7 mRNA and MYH7 protein level but enhanced Myh6 mRNA and MYH6 protein level at different developmental stages (Fig. 5, HL). These results revealed a switch from fetal form of MYH7 to adult form of MYH6 in the heart of Rhauf/f;cTNT-Cre mice. BRG1 was reported to regulate the Myh6/7 expression, but we did not detect a change of BRG1 protein level in the heart of Rhauf/f;cTNT-Cre mice (Fig. 5, K and L).

      RHAU regulated the expression of Myh7 via its DNA resolvase activity

      The concordantly altered mRNA and protein levels of Myh6/7 suggested that RHAU might regulate the transcription of these genes through DNA G4 resolvase activity. We first checked whether there were G4 structures in the promoter region of these genes using QGRS mapper, a program to predict potential G4s in the DNA sequence (
      • Kikin O.
      • D'Antonio L.
      • Bagga P.S.
      QGRS mapper: A web-based server for predicting G-quadruplexes in nucleotide sequences.
      ), and the analysis uncovered three G4-forming sequences (termed G4-1, G4-2, and G4-3) in the promoter region of the Myh7 genome (Fig. S5A). Next, we ordered synthesized DNA fragments based on these G4-forming sequences and performed in vitro G4 structure assay using G4-specific ligands of N-methyl mesoporphyrin IX (NMM) and thioflavin T (ThT) (
      • Kreig A.
      • Calvert J.
      • Sanoica J.
      • Cullum E.
      • Tipanna R.
      • Myong S.
      G-quadruplex formation in double strand DNA probed by NMM and CV fluorescence.
      ,
      • Xu S.
      • Li Q.
      • Xiang J.
      • Yang Q.
      • Sun H.
      • Guan A.
      • Wang L.
      • Liu Y.
      • Yu L.
      • Shi Y.
      • Chen H.
      • Tang Y.
      Thioflavin T as an efficient fluorescence sensor for selective recognition of RNA G-quadruplexes.
      ). Upon the ligand binding to G4 DNA sequence, it would turn on the fluorescence, and the assay showed dramatically enhanced fluorescence intensity of NMM and ThT (Fig. S5, B and C). Thermal difference spectra and CD spectra exhibited typical profiles of G4 for G4-1, G4-2, and G4-3 fragments (Fig. S5, D and E). The CD profiles revealed that the G4-1 and G4-3 DNA fragments formed parallel-stranded G4, whereas the G4-2 DNA fragment formed a hybrid quadruplex structure (Fig. S5E). Furthermore, the thermal melting assays showed that these three DNA G4 structures were relatively stable (Fig. S5F). These data indicated potential G4 structures in the promoter region of the Myh7 gene.
      Afterward, we performed CUT&Tag-Seq, a new generation of ChIP-Seq with high repeatability and fine signal-to-noise ratio, by use of cardiomyocytes from control mice at E14.5 and RHAU antibody. Data from the CUT&Tag-Seq showed that the sites for RHAU binding were predominantly distributed near the transcription start sites (Fig. 6A), and motif analysis of these RHAU-binding regions revealed that majority of these motifs were G-enriched sequences (Fig. 6B). Peak calling analysis identified several peaks in the genome of Myh7, and one of them was located at the promoter region (Fig. 6C).
      Figure thumbnail gr6
      Figure 6RHAU regulated the expression of Myh7 via its DNA resolvase activity. A, transcription start site (TSS) analysis of RHAU-binding sequences. B, RHAU-binding motifs. C, peak calling analysis showed RHAU could bind to the promoter of Myh7. Black arrow indicated the RHAU-binding peak that was positioned in the Myh7 promoter. D, Western blot analysis of GFP expression level with or without RHAU overexpression in 293T cells. E, quantification of (D). N = 12 for control group and N = 12 for Rhau overexpression group. p < 0.0001 (∗∗∗∗). F, quantitative RT–PCR of GFP mRNA expression level with or without RHAU overexpression in 293T cells. N = 3 for control group and N = 3 for Rhau overexpression group. p < 0.05 (∗). G, Western blot analysis of GFP expression level with or without RHAU overexpression in 293T cells. H, quantification of (G). N = 9 for control group and N = 9 for Rhau overexpression group. p < 0.01 (∗∗). I, quantitative RT–PCR of GFP mRNA expression level with or without RHAU overexpression in 293T cells. N = 3 for control group and N = 3 for Rhau overexpression group. p < 0.001 (∗∗∗). RHAU, RNA helicase associated with AU-rich element.
      To examine whether RHAU regulated the expression of Myh7 through resolving the DNA G4 structures in promoter region, we constructed enhanced GFP (EGFP)-derived reporters containing the G4-1 and G4-2 sequences (G4-1/2-pEGFPN1) and G4-3 sequence (G4-3-pEGFPN1). We transfected these reporters into 293T cells and collected cells after 72 h. Western blotting showed that the GFP protein level in the cells transfected with G4-1/2-pEGFPN1 and G4-3-pEGFPN1 were dramatically reduced compared with control (transfected with pEGFPN1) (Fig. S6A), indicating that the G4 sequences in the Myh7 promoter region could suppress GFP expression. Then, we cotransfected mouse RHAU (mRhau-pCMV7.1) and G4-1/2-pEGFPN1 or G4-3-pEGFPN1 to the cells for detection of the protein and mRNA levels of GFP. We found that overexpression of RHAU improved the GFP expression level (Fig. 6, DI). Multimutation of G to A in the G4 sequences of G4-1/2-pEGFPN1 and G4-3-pEGFPN1 reporters was generated, and the mutated reporters were used to transfect cells. Afterward, RHAU could no longer elevate the GFP expression level (Fig. S6, BE).
      One of the hallmarks of stress-induced cardiac pathological remodeling is the upregulation of Myh7 mRNA and protein levels. Our recent study showed that Rhau knockout impaired neonatal heart regeneration in mice (
      • Jiang M.
      • Hu H.
      • Zhao K.
      • Di R.
      • Huang X.
      • Shi Y.
      • Yue Y.
      • Nie J.
      • Yu S.
      • Wang W.
      • Yang Z.
      The G4 resolvase RHAU modulates mRNA translation and stability to sustain postnatal heart function and regeneration.
      ). To investigate whether RHAU regulates Myh7 expression in postnatal heart regeneration, we performed a myocardial infarction (MI) surgery on control and Rhauf/f;α-MHC-Cre mice at P5 and collected heart tissues at P19 for analysis of MYH7 protein level. In the control mice, MI injury robustly induced the expression of Myh7 in the infarct zone (I) (Fig. S7). However, this kind of induction of Myh7 was greatly suppressed in the Rhauf/f;α-MHC-Cre mice.
      Put together, these data demonstrated that RHAU facilitated transcription of Myh7 by unwinding the G4 structures in the Myh7 promoter.

      Discussion

      In this study, we investigated the function of the G4 resolvase RHAU in ventricular chamber development through deletion of Rhau in ventricular cardiomyocytes of mice, which reliably recapitulated human NCC.
      Our mouse model supports the concept that defective myofibrillogenesis and sarcomere assembly are key pathogenic elements for NCC. Advanced sarcomere structure and disrupted MYH6/7 protein level were observed in the RHAU-deficient mice. The isoform switch of MYH protein from fetal MYH7 to adult MYH6 was generated ahead of developmental stage and might trigger pathological remodeling of embryonic myocardium, leading to abnormal trabeculation and compaction. The advanced sarcomere assembly was possibly a consequence of earlier MYH7 to MYH6 switch that was programmed after birth during normal developmental process.
      Our previous studies elucidated the function of RHAU in post-transcriptional regulation of gene expression via binding to mRNA. Here, we uncovered that RHAU could also modulate gene expression at transcriptional level through its DNA G4 resolvase activity and identified Myh7 as a regulatory target of RHAU. RHAU facilitated the transcription of Myh7, and in the absence of RHAU, a switch from Myh7 to Myh6 was provoked, suggesting that suppression of Myh7 could activate the transcription of Myh6. This finding added to our knowledge toward the regulation of Myh6/7 expression by BRG1. The Myh7 gene is located in tandem with the Myh6 gene, and numerous studies uncovered the intriguing phenomena that the expression level of these two genes often shows an opposite change. For instance, the expression level of Myh7 was greatly enhanced, whereas that of Myh6 was substantially suppressed during pathological heart remodeling.
      Mutations of Myh6, Myh7, Nkx2-5, and Hey2 were found in several forms of cardiomyopathies, including NCC, hypertrophic, and dilated cardiomyopathy. How mutations in the same panel of genes cause enormously distinct clinical phenotypes is puzzling. In a recent publication, Dr Srivastava et al. reported NKX2-5 variant as a genetic modifier in conjunction with the sarcomere MYH7 mutant and a transcription factor MKL2 mutant contributing to NCC, which helps understand this study. We detected a substantial reduction of NKX2-5 protein level in the RHAU mutant heart. Reduced NKX2-5 together with MYH isoform switch from MYH7 to MYH6 might triggered trabecular pathological remodeling, whereas NKX2-5 reduction in combination with decreased Hey2 level impaired compaction, leading to thinned ventricular wall. On the other hand, our study demonstrated that disrupted expression level, in addition to mutation, of these genes was pathogenic for NCC.
      The contribution of cardiomyocyte proliferation to NCC is of debate. Our finding of reduced cardiomyocyte proliferation in the RHAU mutant mice suggests that poor cardiomyocyte mitosis was one of the driving factors for NCC.
      In summary, our findings unveiled the synergistic effects of altered protein levels of NKX2-5, MYH6/7, and Hey2 in the pathogenesis of NCC. Especially, the fetal MYH7 to adult MYH6 switch-elicited pathological remodeling might contribute hugely to NCC (Fig. 7). Some of the RHAU mutant mice reported in this study could survive to 2 months, which makes this mouse model particularly useful to understand NCC.
      Figure thumbnail gr7
      Figure 7Working model of RHAU regulation of ventricular chamber development and RHAU deficiency–elicited NCC. Left panel (control mice), RHAU regulated gene expression through transcriptional (Myh7) and post-transcriptional (Nkx2-5 and Hey2) mechanisms. The G4 structures in the promoter region of Myh7 and the 5′-UTRs of Nkx2-5 and Hey2 mRNA could be resolved by RHAU for transcription and translation. Right panel (RHAU-deficient mice), under conditions of RHAU deficiency, mRNA levels of Nkx2-5 and Hey2 were markedly elevated, but their protein level was profoundly reduced. Meanwhile, the transcription of Myh7 was decreased leading to reduced MYH7 but enhanced MYH6 protein level. A synergistic effect of altered protein levels of NKX2-5, MYH6/7, and Hey2 in the pathogenesis of NCC was proposed. Especially, the fetal MYH7 to adult MYH6 switch-elicited pathological remodeling might contribute hugely to NCC. NCC, noncompaction cardiomyopathy; RHAU, RNA helicase associated with AU-rich element.

      Experimental procedures

      Mice

      Mouse strains used in this study are previously reported: Rhau floxed mice (
      • Nie J.
      • Jiang M.
      • Zhang X.
      • Tang H.
      • Jin H.
      • Huang X.
      • Yuan B.
      • Zhang C.
      • Lai J.C.
      • Nagamine Y.
      • Pan D.
      • Wang W.
      • Yang Z.
      Post-transcriptional regulation of Nkx2-5 by RHAU in heart development.
      ,
      • Jiang M.
      • Hu H.
      • Zhao K.
      • Di R.
      • Huang X.
      • Shi Y.
      • Yue Y.
      • Nie J.
      • Yu S.
      • Wang W.
      • Yang Z.
      The G4 resolvase RHAU modulates mRNA translation and stability to sustain postnatal heart function and regeneration.
      ); Mef2c-AHF-Cre mice (
      • Verzi M.P.
      • McCulley D.J.
      • De Val S.
      • Dodou E.
      • Black B.L.
      The right ventricle, outflow tract, and ventricular septum comprise a restricted expression domain within the secondary/anterior heart field.
      ); cTNT-Cre mice (
      • Jiao K.
      • Kulessa H.
      • Tompkins K.
      • Zhou Y.
      • Batts L.
      • Baldwin H.S.
      • Hogan B.L.
      An essential role of Bmp4 in the atrioventricular septation of the mouse heart.
      ); Tie2-Cre mice (
      • Feng Q.
      • Di R.
      • Tao F.
      • Chang Z.
      • Lu S.
      • Fan W.
      • Shan C.
      • Li X.
      • Yang Z.
      PDK1 regulates vascular remodeling and promotes epithelial-mesenchymal transition in cardiac development.
      ); and α-MHC-Cre mice (
      • Zhao X.
      • Lu S.
      • Nie J.
      • Hu X.
      • Luo W.
      • Wu X.
      • Liu H.
      • Feng Q.
      • Chang Z.
      • Liu Y.
      • Cao Y.
      • Sun H.
      • Li X.
      • Hu Y.
      • Yang Z.
      Phosphoinositide-dependent kinase 1 and mTORC2 synergistically maintain postnatal heart growth and heart function in mice.
      ). Genotyping of these mice was as reported before. All mouse strains were maintained on C57BL/6 genetic background. Mice were group-housed in accordance with the regulations on mouse welfare and ethics of Nanjing University, with 12 h/12 h light–dark cycles and had ad libitum access to food and water. The Institutional Animal Care and Use Committee of Model Animal Research Center of Nanjing University approved all animal procedures used in this study. Control mice used in this study were usually Rhau f/f littermates.

      Western blotting analysis

      Embryonic heart tissues were collected, snap frozen in liquid nitrogen, and then stored at −80 °C until use. Heart tissue lysates were prepared in radioimmunoprecipitation assay buffer (Beyotime Biotechnology; P0013B) with PMSF. To prepare cell lysates, the culture medium was discarded and the cells were washed with PBS. Afterward, radioimmunoprecipitation assay was added to the cells, and the cells were collected for centrifugation at 4 °C for 15 min. The heart tissue and cell lysate samples were resolved by 10% SDS-PAGE gels and transferred onto polyvinylidene fluoride membranes (Millipore). The membranes were blocked with 5% bovine serum albumin in Tris-buffered saline with Tween-20 for 1 h at room temperature and were then incubated with primary antibodies overnight at 4 °C. The next day, membranes were washed with Tris-buffered saline with Tween-20 and incubated with a secondary antibody for 2 h at room temperature. The results were visualized with the Tanon-5200 imaging system (Tanon). The antibodies are listed in Table S1.

      RNA-Seq and RT–qPCR

      Heart ventricles were dissected from control and Rhauf/f;cTNT-Cre mice at E13.5 and E15.5. Total RNA was extracted from these tissues using TRIzol Reagent (Life Technologies; 15596018) following the manufacturer's instructions. RNA-Seq was conducted by Vazyme Biotech. The RNA integrity was accurately detected using the Agilent 2100. After library construction, sequencing, and other steps, raw reads (150 bp read length) of corresponding samples were obtained. The reference sequences of the corresponding species downloaded from the National Center for Biotechnology Information, University of California, Santa Cruz, Ensemble, and other databases were used for biological information analysis. The expected number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced was used to show gene expression level. The cuffDiff analysis module of CuffLinks was used to analyze gene differential expression between samples.
      For qPCR, reverse transcription was performed using a HiScript II Q Select RT Supermix for qPCR (+gDNA wiper) kit (Vazyme Biotech; R223-01), and the products were applied to qPCR using an AceQ Universal SYBR Master Mix Kit (Vazyme Biotech; Q511-02) and an ABI QuantStudio 5. Data were normalized to control value as one, and the fold changes were presented. The primers for qPCR were provided in Table S2.

      Plasmid construction

      5′-UTR (135 bp) of Hey2 was amplified via PCR from mice genomic DNA and inserted into the pEGFPN1 vectors to generate Hey2-5′UTR-pEGFPN1 constructs. Two 320 bp fragments containing the three G4s (one with G4-1/2 sequence and the other with G4-3 sequence) of the mouse Myh7 were amplified via PCR from mice genomic DNA and subsequently cloned into the pEGFPN1 vectors to generate G4-1/2-pEGFPN1 and G4-3-pEGFPN1 constructs.
      Hey2-5′UTR-pEGFPN1 was used as template to generate the mutant form of Hey2-5′UTR-G4mut-pEGFPN1 plasmids by PCR with 5′- and 3′- complementary primers containing the mutation sequence (GGG to GAG). PCR was carried out using the 5′- and 3′- complementary primers for 35 cycles, respectively. Then, the two PCR products were denatured at 95 °C for 5 min and annealed at room temperature overnight. The next day, Dpn I (11001ES) was added to destroy the template DNA, and the products were used to transform competent cells (C502-02). Single colonies were picked up for sequencing. G4-1/2mut-pEGFPN1 and G4-3mut-pEGFPN1 plasmids were obtained by the same method. The primers for plasmid construction are listed in Table S3.

      Cell culture and transfection

      Human embryonic kidney 293T cells were maintained in Dulbecco's modified Eagle's medium, supplemented with 10% fetal calf serum and 1% penicillin–streptomycin, in the presence of 5% CO2 at 37 °C incubators. Transient transfection was performed using Lipofectamine 2000 (Life Technologies; 11668-027) following the manufacturer's instructions. Fresh medium was added to the cells 4 to 6 h after transfection. The cells were collected 48 to 72 h after transfection for further analysis. The siRNA sequences were showed in Table S4.

      Histological and IF staining analysis

      Hearts were dissected and fixed in 4% paraformaldehyde overnight at 4 °C. The samples were dehydrated (30% ethanol, 50% ethanol, 75% ethanol, 85% ethanol, 95% ethanol twice, and butanol three times) and embedded in paraffin. For H&E staining, 8 μm sections were prepared.
      For IF staining, hearts were dissected and fixed in 4% paraformaldehyde overnight at 4 °C, then rinsed in PBS for three times, and incubated in 30% sucrose at 4 °C overnight. Afterward, the heart samples were embedded in optical coherence tomography and stored at −20 °C for frozen sections (10 μm). For staining, the frozen sections were rinsed three times in PBS and blocked in goat serum for 1 h at room temperature. Then, sections were incubated with primary antibodies overnight at 4 °C and washed in PBS and incubated with secondary antibodies for 2 h at room temperature. Primary antibodies were listed in Table S2. IF staining for TUNEL assay was carried out using standard protocols (Vazyme Biotech; A113-01). Imaging were processed with Leica SP5 laser confocal microscope and Olympus FV3000 microscope.

      Cardiomyocytes isolation and CUT&Tag assay

      E14.5 cardiac ventricles were harvested and finely chopped to 1 to 2 mm3. Then these tissues were transferred into 15 ml centrifuge tube, and the enzyme buffer (0.1% trypsin and 0.25% collagenase type II) was added. The centrifuge tube was constantly shaken in a 37 °C water bath to digest the cardiac tissues. Fetal bovine serum was added 10 to 15 min later to terminate digestion, and the supernatant was collected and centrifuged. The enzyme buffer was added back into the tissue containing tubes for further digestion. Then, these procedures were repeated three and four times until no visible myocardial tissue was observed. The harvested cardiac cells were seeded into cell dishes. About 2 to 3 h later, the supernatant was transferred into centrifuge tube (differential attachment techniques) for further experiment.
      Isolated cardiomyocytes were used for the CUT&Tag assay. CUT&Tag assay were processed according to the manufacturer's protocol (TD901-02; Vazyme). And the libraries were generated using TruePrep Index Kit V2 for Illumina (TD202; Vazyme). CUT&Tag analysis was performed as previously described (
      • Xu M.
      • Yao J.
      • Shi Y.
      • Yi H.
      • Zhao W.
      • Lin X.
      • Yang Z.
      The SRCAP chromatin remodeling complex promotes oxidative metabolism during prenatal heart development.
      ).

      MI surgery

      MI surgery was performed as previously described (
      • Jiang M.
      • Hu H.
      • Zhao K.
      • Di R.
      • Huang X.
      • Shi Y.
      • Yue Y.
      • Nie J.
      • Yu S.
      • Wang W.
      • Yang Z.
      The G4 resolvase RHAU modulates mRNA translation and stability to sustain postnatal heart function and regeneration.
      ). Briefly, MI surgery was performed in control and Rhauf/f;α-MHC-Cre mice at P5. The mice were placed on ice for 3 to 5 min until they are unconscious. Cut the skin of mice with microscissors, and blunt separation was performed between the third and fourth ribs. The left anterior descending branch was ligated with a 10 to 0 suture, and then, the skin wound was closed. The whole procedure lasted within 10 min. After the operation, the mice were placed on a heating lamp at 37 °C until their body temperature and activity were restored. The mice were sacrificed 2 weeks after the MI surgery (P19) for Western blot analysis.

      Spectroscopy measurement and NMM/ThT fluorescence spectrum

      Preparation of DNA solution: oligonucleotide was diluted to 5 μM in a 10 mM Tris–HCl buffer containing 50 mM KCl at pH 7.0. The DNA solution was heated at 95 °C for 5 min, then slowly cooled to room temperature, and then stored at 4 °C for the subsequent experiment.
      The fluorescent ligand NMM/ThT can specifically bind to the G4 structures, thereby limiting the rotation of the fluorescent molecules and enhancing the fluorescence intensity. The NMM/ThT fluorescence experiment was measured by the FS980 fluorescence spectrometer (Edinburgh Technologies Ltd). The samples (10 μM NMM/ThT and 1 μM oligonucleotides in 10 mM Tris–HCl buffer, pH 7.0) were measured at excitation wavelength of 440 nm, emission wavelength of 475 to 570 nm, slit of 5 nm, photo multiplier tube of 600 V, and 25 °C.
      The thermal difference spectra were performed on a Cary 100UV/VIS spectrophotometer (Agilent Technologies). Absorption spectra were recorded in the range of 220 to 350 nm at the scanning speed of 600 nm/min and data interval of 1 nm at both temperatures.
      The CD spectrum was recorded by Chirascan (Applied Optical Physics), and the scanning speed is 2 nm/s, and the scanning wavelength ranges from 220 to 350 nm at 25 °C.
      Ultraviolet melting was measured using a Cary 100 UV/Visible Spectrophotometer (Agilent Technologies), and the absorbance at 295 nm was recorded. The temperature range was 5 to 95 °C, and the temperature gradient was 0.5 °C/min.

      Statistical analysis

      All data analyses were performed with GraphPad Prism software, version 8.0 (GraphPad Software). ImageJ (National Institutes of Health) was applied for quantification study. Statistical comparisons were carried out using the two-tailed Student's t tests. A value of p < 0.05 (∗) was considered statistically significant; p < 0.01 (∗∗), p < 0.001 (∗∗∗), and p < 0.0001 (∗∗∗∗) were considered very statistically significant. ns represented no significant difference. In all the figures, measurements were reported as the mean ± SD. Please see Table S5 for a list of abbreviations.

      Data availability

      All data are available in the main article or the supporting information.

      Supporting information

      This article contains supporting information.

      Conflict of interest

      The authors declare that they have no conflicts of interest with the contents of this article.

      Acknowledgments

      We thank Dr Bin Zhou (Albert Einstein College of Medicine, New York) for providing the cTNT-Cre mice.

      Author contributions

      X. H. and Z. Y. conceptualization; M. J. and J. Z. methodology; X. H., K. Z., and D. Q. investigation; X. H. writing–original draft; J. Z. and Z. Y. writing–review & editing; J. Z. and Z. Y. supervision.

      Funding and additional information

      This work was supported by grants from the National Key Research and Development Program of China (grant no.: 2019YFA0801601 ) and grants from the National Natural Science Foundation of China (grant nos.: 31930029 , 91854111 , and 31571490 ) (to Z. Y.).

      Supporting information

      References

        • Oechslin E.
        • Jenni R.
        • Klaassen S.
        Left ventricular noncompaction is a myocardial phenotype: Cardiomyopathy-yes or no?.
        Can. J. Cardiol. 2020; 37: 366-369
        • Towbin J.A.
        • Lorts A.
        • Jefferies J.L.
        Left ventricular non-compaction cardiomyopathy.
        Lancet. 2015; 386: 813-825
        • Jefferies J.L.
        Left ventricular noncompaction cardiomyopathy: New clues in a not so new disease?.
        J. Am. Heart Assoc. 2021; 10e018815
        • Liu Y.
        • Chen H.
        • Shou W.
        Potential common pathogenic pathways for the left ventricular noncompaction cardiomyopathy (LVNC).
        Pediatr. Cardiol. 2018; 39: 1099-1106
        • Choquet C.
        • Kelly R.G.
        • Miquerol L.
        Defects in trabecular development contribute to left ventricular noncompaction.
        Pediatr. Cardiol. 2019; 40: 1331-1338
        • Chen H.
        • Zhang W.
        • Li D.
        • Cordes T.M.
        • Mark Payne R.
        • Shou W.
        Analysis of ventricular hypertrabeculation and noncompaction using genetically engineered mouse models.
        Pediatr. Cardiol. 2009; 30: 626-634
        • Tian X.
        • Li Y.
        • He L.
        • Zhang H.
        • Huang X.
        • Liu Q.
        • Pu W.
        • Zhang L.
        • Li Y.
        • Zhao H.
        • Wang Z.
        • Zhu J.
        • Nie Y.
        • Hu S.
        • Sedmera D.
        • et al.
        Identification of a hybrid myocardial zone in the mammalian heart after birth.
        Nat. Commun. 2017; 8: 87
        • Yue Y.
        • Zong W.
        • Li X.
        • Li J.
        • Zhang Y.
        • Wu R.
        • Liu Y.
        • Cui J.
        • Wang Q.
        • Bian Y.
        • Yu X.
        • Liu Y.
        • Tan G.
        • Zhang Y.
        • Zhao G.
        • et al.
        Long-term, in toto live imaging of cardiomyocyte behaviour during mouse ventricle chamber formation at single-cell resolution.
        Nat. Cell Biol. 2020; 22: 332-340
        • Luxán G.
        • D'Amato G.
        • MacGrogan D.
        • de la Pompa J.L.
        Endocardial Notch signaling in cardiac development and disease.
        Circ. Res. 2016; 118: e1-e18
        • D'Amato G.
        • Luxán G.
        • de la Pompa J.L.
        Notch signalling in ventricular chamber development and cardiomyopathy.
        FEBS J. 2016; 283: 4223-4237
        • Luxán G.
        • Casanova J.C.
        • Martínez-Poveda B.
        • Prados B.
        • D'Amato G.
        • MacGrogan D.
        • Gonzalez-Rajal A.
        • Dobarro D.
        • Torroja C.
        • Martinez F.
        • Izquierdo-García J.L.
        • Fernández-Friera L.
        • Sabater-Molina M.
        • Kong Y.Y.
        • Pizarro G.
        • et al.
        Mutations in the NOTCH pathway regulator MIB1 cause left ventricular noncompaction cardiomyopathy.
        Nat. Med. 2013; 19: 193-201
        • Oechslin E.
        • Jenni R.
        Nosology of noncompaction cardiomyopathy: The emperor still wears clothes!.
        Can. J. Cardiol. 2017; 33: 701-704
        • Richard P.
        • Ader F.
        • Roux M.
        • Donal E.
        • Eicher J.C.
        • Aoutil N.
        • Huttin O.
        • Selton-Suty C.
        • Coisne D.
        • Jondeau G.
        • Damy T.
        • Mansencal N.
        • Casalta A.C.
        • Michel N.
        • Haentjens J.
        • et al.
        Targeted panel sequencing in adult patients with left ventricular non-compaction reveals a large genetic heterogeneity.
        Clin. Genet. 2019; 95: 356-367
        • Gifford C.A.
        • Ranade S.S.
        • Samarakoon R.
        • Salunga H.T.
        • de Soysa T.Y.
        • Huang Y.
        • Zhou P.
        • Elfenbein A.
        • Wyman S.K.
        • Bui Y.K.
        • Cordes Metzler K.R.
        • Ursell P.
        • Ivey K.N.
        • Srivastava D.
        Oligogenic inheritance of a human heart disease involving a genetic modifier.
        Science. 2019; 364: 865-870
        • Sauer M.
        • Paeschke K.
        G-quadruplex unwinding helicases and their function in vivo.
        Biochem. Soc. Trans. 2017; 45: 1173-1182
        • Varshney D.
        • Spiegel J.
        • Zyner K.
        • Tannahill D.
        • Balasubramanian S.
        The regulation and functions of DNA and RNA G-quadruplexes.
        Nat. Rev. Mol. Cell Biol. 2020; 21: 459-474
        • Chen M.C.
        • Tippana R.
        • Demeshkina N.A.
        • Murat P.
        • Balasubramanian S.
        • Myong S.
        • Ferré-D'Amaré A.R.
        Structural basis of G-quadruplex unfolding by the DEAH/RHA helicase DHX36.
        Nature. 2018; 558: 465-469
        • Sauer M.
        • Juranek S.A.
        • Marks J.
        • De Magis A.
        • Kazemier H.G.
        • Hilbig D.
        • Benhalevy D.
        • Wang X.
        • Hafner M.
        • Paeschke K.
        DHX36 prevents the accumulation of translationally inactive mRNAs with G4-structures in untranslated regions.
        Nat. Commun. 2019; 10: 2421
        • Tran H.
        • Schilling M.
        • Wirbelauer C.
        • Hess D.
        • Nagamine Y.
        Facilitation of mRNA deadenylation and decay by the exosome-bound, DExH protein RHAU.
        Mol. Cell. 2004; 13: 101-111
        • Nie J.
        • Jiang M.
        • Zhang X.
        • Tang H.
        • Jin H.
        • Huang X.
        • Yuan B.
        • Zhang C.
        • Lai J.C.
        • Nagamine Y.
        • Pan D.
        • Wang W.
        • Yang Z.
        Post-transcriptional regulation of Nkx2-5 by RHAU in heart development.
        Cell Rep. 2015; 13: 723-732
        • Jiang M.
        • Hu H.
        • Zhao K.
        • Di R.
        • Huang X.
        • Shi Y.
        • Yue Y.
        • Nie J.
        • Yu S.
        • Wang W.
        • Yang Z.
        The G4 resolvase RHAU modulates mRNA translation and stability to sustain postnatal heart function and regeneration.
        J. Biol. Chem. 2020; 296: 100080
        • Verzi M.P.
        • McCulley D.J.
        • De Val S.
        • Dodou E.
        • Black B.L.
        The right ventricle, outflow tract, and ventricular septum comprise a restricted expression domain within the secondary/anterior heart field.
        Dev. Biol. 2005; 287: 134-145
        • Jiao K.
        • Kulessa H.
        • Tompkins K.
        • Zhou Y.
        • Batts L.
        • Baldwin H.S.
        • Hogan B.L.
        An essential role of Bmp4 in the atrioventricular septation of the mouse heart.
        Genes Dev. 2003; 17: 2362-2367
        • Chen H.
        • Shi S.
        • Acosta L.
        • Li W.
        • Lu J.
        • Bao S.
        • Chen Z.
        • Yang Z.
        • Schneider M.D.
        • Chien K.R.
        • Conway S.J.
        • Yoder M.C.
        • Haneline L.S.
        • Franco D.
        • Shou W.
        BMP10 is essential for maintaining cardiac growth during murine cardiogenesis.
        Development. 2004; 131: 2219-2231
        • Pashmforoush M.
        • Lu J.T.
        • Chen H.
        • Amand T.S.
        • Kondo R.
        • Pradervand S.
        • Evans S.M.
        • Clark B.
        • Feramisco J.R.
        • Giles W.
        • Ho S.Y.
        • Benson D.W.
        • Silberbach M.
        • Shou W.
        • Chien K.R.
        Nkx2-5 pathways and congenital heart disease; loss of ventricular myocyte lineage specification leads to progressive cardiomyopathy and complete heart block.
        Cell. 2004; 117: 373-386
        • Guo Y.
        • Pu W.T.
        Cardiomyocyte maturation: New phase in development.
        Circ. Res. 2020; 126: 1086-1106
        • Kikin O.
        • D'Antonio L.
        • Bagga P.S.
        QGRS mapper: A web-based server for predicting G-quadruplexes in nucleotide sequences.
        Nucleic Acids Res. 2006; 34: W676-W682
        • Kreig A.
        • Calvert J.
        • Sanoica J.
        • Cullum E.
        • Tipanna R.
        • Myong S.
        G-quadruplex formation in double strand DNA probed by NMM and CV fluorescence.
        Nucleic Acids Res. 2015; 43: 7961-7970
        • Xu S.
        • Li Q.
        • Xiang J.
        • Yang Q.
        • Sun H.
        • Guan A.
        • Wang L.
        • Liu Y.
        • Yu L.
        • Shi Y.
        • Chen H.
        • Tang Y.
        Thioflavin T as an efficient fluorescence sensor for selective recognition of RNA G-quadruplexes.
        Sci. Rep. 2016; 6: 24793
        • Feng Q.
        • Di R.
        • Tao F.
        • Chang Z.
        • Lu S.
        • Fan W.
        • Shan C.
        • Li X.
        • Yang Z.
        PDK1 regulates vascular remodeling and promotes epithelial-mesenchymal transition in cardiac development.
        Mol. Cell. Biol. 2010; 30: 3711-3721
        • Zhao X.
        • Lu S.
        • Nie J.
        • Hu X.
        • Luo W.
        • Wu X.
        • Liu H.
        • Feng Q.
        • Chang Z.
        • Liu Y.
        • Cao Y.
        • Sun H.
        • Li X.
        • Hu Y.
        • Yang Z.
        Phosphoinositide-dependent kinase 1 and mTORC2 synergistically maintain postnatal heart growth and heart function in mice.
        Mol. Cell. Biol. 2014; 34: 1966-1975
        • Xu M.
        • Yao J.
        • Shi Y.
        • Yi H.
        • Zhao W.
        • Lin X.
        • Yang Z.
        The SRCAP chromatin remodeling complex promotes oxidative metabolism during prenatal heart development.
        Development. 2021; 148dev199026