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Circadian REV-ERBs repress E4bp4 to activate NAMPT-dependent NAD+ biosynthesis and sustain cardiac function

Nature Cardiovascular Research volume 1, pages 45–58 (2022)Cite this article

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Abstract

The heart is a highly metabolic organ that uses multiple energy sources to meet its demand for ATP production. Diurnal feeding–fasting cycles result in fluctuations in substrate availability, which, together with increased energetic demand during the active period, impose a need for rhythmic cardiac metabolism. The nuclear receptors REV-ERBα and REV-ERBβ are essential repressive components of the molecular circadian clock and major regulators of metabolism. To investigate their role in the heart, here we generated mice with cardiomyocyte (CM)-specific deletion of both Rev-erb genes (Nr1d1/2), which died prematurely due to dilated cardiomyopathy. Loss of REV-ERBs markedly downregulated expression of fatty acid-oxidation genes before overt pathology, which was mediated by induction of the transcriptional repressor E4BP4, a direct target of cardiac REV-ERBs. E4BP4 directly controls circadian expression of Nampt and its biosynthetic product NAD+ via distal cis regulatory elements. Thus, REV-ERB-mediated E4BP4 repression is required for Nampt expression and NAD+ production by the salvage pathway. Together, these results highlight the indispensable role of circadian REV-ERBs in cardiac gene expression, metabolic homeostasis and function.

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Fig. 1: CM-RevDKO mice die from dilated cardiomyopathy.
Fig. 2: REV-ERBs control metabolic gene expression in the heart.
Fig. 3: Enhancers of downregulated genes are enriched for E4BP4, a direct REV-ERB target that is induced in CM-RevDKO hearts.
Fig. 4: Constitutive binding of E4BP4 contributes to downregulated gene expression in CM-RevDKO hearts.
Fig. 5: REV-ERBs control cardiac NAD+ biosynthesis via E4BP4-mediated repression of Nampt.
Fig. 6: Scheme of metabolic deregulation in hearts of CM-RevDKO mice.

Data availability

The authors declare that the data supporting the findings of this study are available within the paper and its Supplementary Information. The RNA-seq, Cut&Run and ChIP–seq datasets reported in this paper have been deposited in the Gene Expression Omnibus under the accession number GSE153014. Source data are provided with this paper.

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Acknowledgements

We thank L. Cheng for help with histology. We thank the Functional Genomics Core of the Penn Diabetes Research Center (DK19525), the Mouse Cardiovascular Phenotyping Core (S. Shewale) at the Penn Cardiovascular Institute and the Electron Microscopy Resource Laboratory Core. This work was supported by NIH R01DK45586, the JPB Foundation and the Cox Institute for Medical Research (M.A.L.); RO1 DK111495 and the Department of Defense (W81XWH20-1-0042 and W81XWH20-1-0089) (L.P.); NIH R01HL058493, R01HL128349 and R01HL151345 (D.P.K.); NIH R01 DK098656 (J.A.B.); and R01HL149159 (M.E.Y.). P.D. was supported by a Netherlands Heart Institute postdoctoral fellowship and an American Heart Association postdoctoral fellowship (20POST35210738); M.W.V. was supported by an EMBO long-term fellowship (ALTF 540-2018) and an American Heart Association postdoctoral fellowship (836074); Y.X. was supported by an American Heart Association postdoctoral fellowship (827529); T.S.L. was supported by F32HL145923; and A.K.H. was supported by F32DK122684-01A1.

Author information

Authors and Affiliations

  1. Institute for Diabetes, Obesity, and Metabolism, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA

    Pieterjan Dierickx, Kun Zhu, Bryce J. Carpenter, Chunjie Jiang, Yang Xiao, Timothy S. Luongo, Amy K. Hauck, Brianna Krusen, Hoang C. B. Nguyen, Daniel P. Kelly, Liming Pei, Joseph A. Baur & Mitchell A. Lazar

  2. Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA

    Pieterjan Dierickx, Kun Zhu, Bryce J. Carpenter, Chunjie Jiang, Yang Xiao, Amy K. Hauck, Brianna Krusen, Hoang C. B. Nguyen & Mitchell A. Lazar

  3. Division of Hematology, the Children’s Hospital of Philadelphia, Philadelphia, PA, USA

    Marit W. Vermunt & Gerd A. Blobel

  4. Department of Physiology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA

    Timothy S. Luongo & Joseph A. Baur

  5. Cardiovascular Institute and Department of Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA

    Tsunehisa Yamamoto, Íngrid Martí-Pàmies & Daniel P. Kelly

  6. Division of Cardiovascular Diseases, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA

    Sobuj Mia, Mary Latimer & Martin E. Young

  7. Division of Cardiology, Washington University School of Medicine, St. Louis, MO, USA

    Abhinav Diwan

  8. Center for Mitochondrial and Epigenomic Medicine, Children’s Hospital of Philadelphia, Philadelphia, PA, USA

    Juanjuan Zhao & Liming Pei

  9. Department of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia, Philadelphia, PA, USA

    Juanjuan Zhao & Liming Pei

Authors
  1. Pieterjan Dierickx
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Contributions

P.D. and M.A.L. conceived and designed the overall study. P.D., K.Z., Y.X., C.J., M.W.V., under the supervision of G.A.B., J.Z., under the supervision of L.P., and H.C.B.N. contributed to next-generation sequencing experiments and bioinformatic analyses. T.S.L. performed NAD+ measurements under the supervision of J.A.B. P.D., B.J.C., A.K.H. and B.K. helped with circadian sampling. P.D. and B.J.C. performed gene expression measurements, western blotting, cell culture experiments, animal caretaking, reporter assays and metabolic experiments. T.Y., under the supervision of D.P.K., and I.M.-P. assisted with echocardiography experiments. S.M. and M.L. (under the supervision of M.E.Y.) performed RT–qPCR and western blots on CM-specific Bmal1-KO, E4bp4-KO and Bmal1;E4bp4-DKO hearts. A.D. assisted with the generation of CM-specific E4bp4-KO mice. P.D. and M.A.L. wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to Pieterjan Dierickx or Mitchell A. Lazar.

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Competing interests

M.A.L. receives research support from Pfizer for unrelated work; serves as an advisory board member for Pfizer and Flare Therapeutics; has consulted for Novartis, Madrigal, Calico and Third Rock; and holds equity in KDAC Therapeutics and Flare Therapeutics. D.P.K. serves as an advisory board member for Pfizer and Amgen. J.A.B. is an inventor on a patent for using NAD+ precursors in liver injury, is a consultant for Pfizer and Cytokinetics and has received research funding and materials from Elysium Health and Metro International Biotech, both of which have an interest in NAD+ precursors. No funds or materials from Elysium Health and Metro International Biotech were used to generate any data for this study. The remaining authors declare no competing interests.

Additional information

Peer review information Nature Cardiovascular Research thanks Junichi Sadoshima and Li Wang for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Additional validation of CM-RevDKO.

a, Immunoblot for BMAL1 with quantification from control (αMHC-Cre-) vs CM-RevDKO (αMHC-Cre + ) hearts from 2-month-old male mice (n = 2/timepoint/genotype). b, Relative mRNA expression in control vs CM-RevDKO hearts from 2-month-old male mice (n = 3 hearts/genotype/timepoint except for n = 2 for ZT2 and n = 4 for ZT22 in CM-RevDKO) and c, young and old WT mice (n = 3/genotype). n represents biologically independent replicates. Data are presented as mean ± SEM, except for a. ns, non significant; * P < 0.05; ** P < 0.01; *** P < 0.001; ****P < 0.0001 by 2-way ANOVA (exact P values are provided in the Source Data).

Source data

Extended Data Fig. 2 Age-dependent impairment of cardiac structure and function in CM-RevDKO mice.

a, Birth rates for CM-RevDKO and littermate control animals. b, Locomotor activity and c, food consumption of 2-month-old male control (n = 3) and CM-RevDKO (n = 5) mice housed under 12:12 light:dark conditions. d, Body weight (in grams) of male control and CM-RevDKO mice at 2 (n = 17 for control and n = 34 for CM-RevDKO)-4 (n = 8 for control and n = 14 for CM-RevDKO)-6 (n = 8 for control and n = 11 for CM-RevDKO) months of age. e, Blood glucose concentrations of 6-months old male control (n = 6) and CM-RevDKO (n = 11) mice that underwent glucose tolerance test (GTT) and f, insulin tolerance test (ITT) (n = 6 for control and n = 10 for CM-RevDKO). g, Biventricular to body weight (BVW/BW) ratios of from control (n = 5) and CM-RevDKO (n = 6) hearts. h, Cardiomyocyte size assesment of 6-month old control and CM-RevDKO hearts. Representative images (cardiac sarcolemma stained by WGA in red, nuclei by DAPI = blue) of 11 control and 9 CM-RevDKO hearts are shown. Scale bars, 50μM. i, Cardiac structure (LVIDd/s: Left ventricular internal diameter during diastole/systole) and function (EF: Ejection fraction and FS: Fractional shortening) data from 2-month-old control and CM-RevDKO mice obtained through echocardiagraphy (n = 4/genotype). j, Echocardiographic parameters from control vs CM-RevDKO mice age 2 months (n = 4/genotype) versus 6 months (n = 11 for control and n = 8 for CM-RevDKO). (HR) Heart rate; (LVPWs/d) Left ventricular posterior wall during systole/diastole; (IVSs/d) Interventricular septum thickness during systole/diastole; (RWT) Relative wall thickness. k, Masson’s trichrome and l, TUNEL staining (green) on hearts of 6-month-old control and CM-RevDKO mice. Nuclei are stained with DAPI (blue). Representative images of n = 11 hearts for control and n = 9 hearts for CM-RevDKO are shown. Scale bars, (k) 50μM and (l), 100μM. n represents biologically independent replicates unless otherwise indicated. Data are presented as mean ± SEM. ns, non significant; *P < 0.05; ***P < 0.001 by 2-sided Student’s t test (exact P values are provided in the Source Data).

Source data

Extended Data Fig. 3 CM-RevDKO causes tissue-specific deregulated expression of circadian genes.

a, Circadian Rev-erbα/β and Bmal1 mRNA expression in 2-month-old male control hearts (n = 5 timepoint, except for ZT7, n = 4 and ZT10, n = 6). b, Left: Venn diagram showing overlap between DEGs in CM-RevDKO hearts and hepatocyte-specific Rev-erb DKO (HepDKO) livers22 (at ZT10). Right: relative mRNA expression of commonly (in both CM-RevDKO and Hep-RevDKO) deregulated clock genes in CM-RevDKO vs control hearts (n = 3 hearts/genotype, harvested at ZT10). c, qRT-PCR validation of genes derepressed upon CM-RevDKO in the heart of 2-month-old male mice (n = 5 for control and n = 6 for CM-RevDKO). d, Left: Venn diagram showing overlap between cardiac oscillators published in24 and all DEGs in CM-RevDKO that were assessed in24. Right: phase plots of rhythmic, differentially expressed genes identified on the left. n represents biologically independent replicates. Data are presented as mean ± SEM. Adj. P values in b were calculated by DESeq2. ***P < 0.001; ****P < 0.0001 by 2-sided Student’s t-test (exact P values are provided in the Source Data).

Source data

Extended Data Fig. 4 Mitochondrial size is affected in CM-RevDKO cardiomyocytes.

a, Relative Ppp1r1b mRNA expression in CM-RevDKO (n = 6) vs control (n = 5) hearts from 2-month-old male mice. b, Immunoblot for DARPP-32 in CM-RevDKO vs control hearts. c, Scatter plot and histogram of mitochondria area for ventricular CMs in control and CM-RevDKO hearts from 6-month-old male mice based on electron microscopy images (n = 86 mitochondria for control, measured from 5 images, n = 90 mitochondria for CM-RevDKO, measured from 7 images). d, Left: scatter plot of mitochondria area for ventricular CMs in control and CM-RevDKO hearts from 2-month-old mice. Right: Electron micrographs of ventricular tissue from control and CM-RevDKO hearts at 2 months (n = 108 mitochondria for control, measured from 7 images, n = 100 mitochondria for CM-RevDKO, measured from 9 images). Scale bars, 1μM. e, Relative levels of mitochondrial DNA quantified by RT-qPCR. mtCo1/2 and mtNd1 levels were normalized to nuclear genomic βActin (n = 3/genotype). n represents biologically independent replicates unless otherwise indicated. Data are presented as mean ± s.e.m. ns:, non significant; *P < 0.05; ***P < 0.001; ****P < 0.0001 by 2-sided Student’s t-test (exact P values are provided in the Source Data).

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Extended Data Fig. 5 Characterization of the 3xHA-REV-ERBα cistrome in the heart.

a, Venn diagram showing the overlap between the downregulated differentially expressed genes (DEGs) in CM-RevDKO hearts and up/downregulated genes in cardiomyocyte-specific glucocorticoid receptor (GR) KO hearts32. b, Pearson correlation plots comparing 3xHA ChIP-seq replicate samples. c, Pie chart of annotated cardiac 3xHA-REV-ERBα ChIP-seq peaks. d, Results of motif search at 3xHA-REV-ERBα ChIP-seq peaks and enhancers that displayed increased H3K27ac Cut&Run signal (FC > 2) in CM-RevDKO vs control cardiomyocytes as reported by HOMER. e, Venn diagram showing overlap between DEGs in CM-RevDKO vs control hearts and annotated peaks from the cardiac 3xHA-REV-ERBα cistrome (at ZT10). f, ChIP-seq, Cut&Run and RNA-seq read distribution for REV-ERBα and H3K27ac near derepressed REV-ERBα canonical target genes Bmal1 and g, Cry1 and h, output genes p21 and Fbn2. i, Immunoblot and quantification for FLAG and REV-ERBα from DsRed (control) vs FLAG-Rev-erbα overexpressing plasmid transfected C2C12 cells (n = 2 independently transfected wells/condition). Significance of overlap in e is calculated via a hypergeometric test without multiple testing correction.

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Extended Data Fig. 6 E4BP4-based repression is a unifying mechanism to explain transcriptional changes and cardiomyopathy common to the cardiac-specific loss of BMAL1 or REV-ERBs.

a, Results of motif search at cardiac E4BP4 ChIP-seq peaks (Control at ZT22) as reported by HOMER. b, Pie chart of annotated cardiac E4BP4 ChIP-seq peaks (control ZT22). c, Venn diagram showing overlap between annotated peaks from the cardiac control (at ZT22) and CM-RevDKO (at ZT10 and ZT22) E4BP4 cistromes. d, ChIP-seq, Cut&Run and RNA-seq read distribution for E4BP4 and H3K27ac near identified E4BP4 target genes in control and CM-RevDKO hearts. e, Overlap between upregulated and g, downregulated genes in CM-RevDKO hearts and cardiomyocyte-specific Bmal1 KO (CBK) hearts. Proposed models for normal (Norm) and experimental (Exp.) conditions are depicted on the right. f, Overlap between commonly upregulated and h, downregulated genes in CBK/CM-RevDKO hearts (identified in e and g respectively) and cardiac REV-ERBα/E4BP4 cistromes. CBK data in (e,f,g,h) was obtained from35.

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Extended Data Fig. 7 Deletion of Rev-erb genes in cardiomyocytes derepresses key metabolic regulators leading to mitochondrial abnormalities and loss of normal heart function.

a, Relative Pgc-1α/β, Naprt1 and Nampt mRNA levels in CM-RevDKO and control hearts (n = 3/genotype) from 2-month-old male mice. b, Relative Bmal1, Rev-erbα/β, and E4bp4 mRNA levels in CBK (n = 7 for control and n = 8 for KO), CM-RevDKO (n = 5 for control and n n = 6 for KO), E4bp4 (n = 8 for control and n = 10 for KO) and CBK/E4bp4 (double) KO (n = 6 for control and n = 9 for DKO) and control hearts from 2-month-old male mice for CM-RevDKO and control at ZT10 and from 3-month-old male mice for the rest at ZT12. c, Representative immunoblots and relative protein quantification for NAMPT in hearts from 3-month-old male mice with the following genetic background: CBK (n = 6/genotype), E4bp4 (n = 7/genotype), and CBK/E4bp4 (double) KO (n = 6/genotype), harvested at ZT12. n represents biologically independent replicates. All data are presented as mean ± SEM. Adj. P values in a were calculated by DESeq2, while ns, non significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by 2-sided Student’s t-test in b and one-way ANOVA followed by a Tukey’s multiple comparisons test in c (exact P values are provided in the Source Data).

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Supplementary information

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Supplementary Table 1

DEGs in CM-RevDKO versus control hearts. Isolated cardiac mRNA from 2-month-old male CM-RevDKO versus control mice (n = 3 per genotype) was subjected to RNA-seq. For differential gene expression analysis, FC, P and adjusted P (FDR) values were calculated with DESeq254. Supplementary Table 2. Output list of IMAGE analysis integrating H3K27ac and RNA-seq changes in CM-RevDKO samples.

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Dierickx, P., Zhu, K., Carpenter, B.J. et al. Circadian REV-ERBs repress E4bp4 to activate NAMPT-dependent NAD+ biosynthesis and sustain cardiac function. Nat Cardiovasc Res 1, 45–58 (2022). https://doi.org/10.1038/s44161-021-00001-9

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