Ah Receptor-Mediated Disruption of the Cardiac Embryogenesis Program Alvaro Puga Department of Environmental Health University of Cincinnati College

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1 Ah Receptor-Mediated Disruption of the Cardiac Embryogenesis Program Alvaro Puga Department of Environmental Health University of Cincinnati College of Medicine, Ohio.

2 Source: texasheart.org Congenital Heart Disease Most prevalent form of human birth defect 30% of prenatal loss 0.4% 5% of live births Most common: VSD High economical burden $1.4 billion hospitalization cost per year (2013) Leading cause of adult cardiac insufficiency

3 Etiology of Congenital Heart Disease Genetic (explaining only ~15% of cases - CDC) Environmental Gene mutations» Cited2, Ets1, Foxh1, Gata4, Gata6, Hand1, Hand2, Hoxa1, Irx1, Nkx2-5, Nkx2-6, Pitx2 and Tbx1 Metabolic Infectious Xenobiotics» Persistent organic pollutants (POP)

4 At risk Gestation in areas with high industrial, natural, or accidental release of POPs (e.g. dioxins) Important Maternal lifelong dietary exposure to dioxins and dioxinlike compounds +18 other diseases VA Association

5 Dioxins are potent agonists of the aryl hydrocarbon receptor (AHR) Dioxin (TCDD/TCDF) and Dioxin-like (PCBs) At least toxic compounds Dioxins share: Structural relationship Environmental persistence and bioaccumulation Bind to and elicit AHR-dependent biochemical/toxic response

6 Biological processes regulated by the naïve AHR Positive regulation of biological process Organ development Positive regulation of cellular process Positive regulation of cellular metabolism Positive regulation of nucleobase, nucleoside, nucleotide, and nucleic acid metabolism Positive regulation of metabolism Brain development Anatomical structure formation Tube morphogenesis Anatomical structure morphogenesis Organ morphogenesis Blood vessel morphogenesis Positive regulation of transcription Branching morphogenesis of a tube Angiogenesis Positive regulation of transcription from RNA polii promoters Patterning of blood vessels

7 Outline AHR modulates lineage specification pathways during early differentiation Loss of pluripotency Cardiogenesis Neuroglia Dioxin exposure and Ahr ablation disrupt cardiogenesis Short and long-term effects of developmental dioxin exposure Gene-environment interactions in cardiac disease Ahr Nkx2.5 interactions in cardiac haploinsufficiency

8 DNA methylation Ahr expression The preimplantation Ahr gene is silent Fertilization Cleavage Implantation E11.5 E13.5 E E1.5 E2.0 E2.5 E3.0 E3.5 E4.0 E E12.5 E15.5 Birth Puberty A Z 2C 4C 8C EB LB Cell type- and tissue- specific Ahr expression B Imprinting DMR Soma Paternal genome Maternal genome PGCs Spermatogenesis Oogenesis

9 The Ahr gene is repressed in ES cells Distal silencer Repressed state ES cells SOX2 OCT3/4 NANOG PRC2 TxG-containing EZH2 complexes SUZ12 Active state Differentiated cells PRC1 RING1B Differentiation KDM6A RNAPII-complex Ahr short transcripts Proximal promoter Full-length Ahr mrna Ko, et al. Stem Cell Res. 12: (2013) polycomb/trithorax

10 12-15% of the ES cell population expresses AHR

11 egfp - egfp + Fluctuating state of AHR expression in ES cells Day 1 Day 2 Day 3 Day 4 AE cells AH cells

12 Pluripotency factors are down-regulated in AE cells

13 Doubling time (hours) Cell count Cell count Percent cells Cell count AE cells proliferate more slowly and accumulate in G2/M AH E E E * * E+06 AE 40 * E E+05 * * * * E Days in culture AH AE 0 7AAD AH AE

14 AHR modulates mitotic progression and pluripotency Ko, et al. Stem Cells Jul 4. doi: /stem [Epub ahead of print]

15 AE cells promote unsustained neuroglia differentiation Differentiation of neuroglia Differentiation of astrocytes Endothelial cell development Development of endothelial tissue Formation of connective tissue Central nervous system diff. Self-renewal of stem cells Generation of stem cells Self-renewal of cells Development of sensory organ Respiratory system development Development of abdomen Development of body trunk Midline defect Cardiogenesis Pluripotency of cells Formation of muscle AE vs AH day 3 day 6 day Activation z-score

16 Percent beating EBs Mouse ES cells form embryoid bodies Not the embryoid bodies formed by AE cells that differentiate into beating cardiomyocytes * * * * * Days of differentiation AH AE

17 Outline AHR modulates lineage specification pathways during early differentiation Loss of pluripotency Cardiogenesis Neuroglia Dioxin exposure and Ahr ablation disrupt cardiogenesis Short and long-term effects of developmental dioxin exposure Gene-environment interactions in cardiac disease Ahr Nkx2.5 interactions in cardiac haploinsufficiency

18 Relative mrna level (log 2 ) Ahr expression is up-regulated as ES cells differentiate * * * * * AHR bactin Days of differentiation 11

19 Cardiomyocyte differentiation has a narrow window of TCDD sensitivity

20 TGFβ1 restores contractility blocked by TCDD

21 The AHR/TCDD axis interferes at multiple developmental stages with the differentiation of contractile cardiomyocytes Noggin TGFβ1 BMP Nodal WNT Activin BMP WNT AHR/TCDD WNT WNT Activin TGFβ1 WNT AHR/TCDD Pluripotent ESCs Panmesoderm Cardiac mesoderm Cardiac progenitors Cardiomyocyte NANOG OCT4 SOX2 Brachyury MESP1 MESP2 NKX2-5 TBX GATA4 MEF2c HAND1/2 Hcn4 MHC MLC2a/v ANP Shox2 CX40 Day0 Day2-3 Day3-4 Day5-6 Day7+

22 First organ Dosing scheme in vivo analyses Organogenesis TCDD exposure Developmental Samples E7.5 E9.5 E11.5 E13.5 E15.5 E18.5 De Boer et al. 2012

23 What are the gene expression effects of AHR disruption?

24 AHR disruption affects the expression of key networks involved in cardiogenesis, cardiac homeostasis, and mitochondrial function TCDD at E E11.5 RNA.seq right atrium at E15.5 TCDD Ahr -/- Common

25 Gene networks common and not-common to Ahr KO and TCDD exposure A AHR-KO E15.5 B AHR-WT-TCDD E15.5

26 Genes deregulated at E15.5 IDH3B DLST OGDH SDHC ACO2 MDH1 FH CS SUCLA2 DLD SDHA SUCLG1 MDH2 SDHD TCA Cycle Genes TCA Cycle Genes at E15.5 SAN IDH3A SDHB TCDD Log Ratio KO Log Ratio Fold change (log2ratio)

27 Developmental AHR disruption globally downregulates mitochondrial complexes Mitochondrial Dysfunction at E15.5 Ahr -/-

28 Developmental AHR disruption globally downregulates mitochondrial complexes Mitochondrial Dysfunction at E15.5 Ahr +/+ + TCDD

29 Developmental AHR disruption affects mitochondria structure and abundance Naïve Ahr +/+ Naïve Ahr -/- Ahr +/+ TCDD E13.5 * * * * E15.5 * E18.5

30 Decline of cardiovascular performance in TCDD-exposed E15.5 embryos determined by very high frequency echosonography % µl/min mm Oil TCDD Oil TCDD Oil TCDD 0 EF FS 0 CO 0 Diameter;s Diameter;d

31 Gene expression changes remain 9 months later COX6B2 GPX7 NDUFB2 COX7A2 COX6A2 PRX3 NDUFA6 NDUFA3 COX7B CAT Caspase 3 COX7A1 ATP5L ATP5G1 NDUFB6 CYTC NDUFA4 Parkin PINK1 ATP5J COX5A LRRK2 NDUFS3 BCL2 NDUFA7 GPD2 MAOA mtsod COX8A NDUFS1 NDUFB4 UQCR10 MAOB MKK4 UQCRFS1 COX4I2 ATP5F1 NDUFS8 Xanthineoxidase KGDH UQCRC2 ATP5G3 SDHD ATP5C1 COX6A1 NDUFA4L2 Caspase 9 NCT DHOH PDHA NDUFB11 NDUFA1 NDUFAF1 TRXR2 NDUFV3 ATP5J2 NDUFA5 ABAD UQCR11 HtrA2 APP GRX2 NDUFA9 NDUFB9 NDUFS6 SDHB ATP5A1 PSEN-1 FURIN GSR NDUFV2 PEN-2 NDUFS4 Caspase 8 COX4I1 TRAK1 UQCRB SDHC NDUFA10 NDUFB8 NDUFA8 ATP5G2 NDUFB5 Synuclein SDHA COX6B1 ATP5B DJ-1 NDUFA2 ATP5D NDUFB7 NDUFS2 NDUFB3 NDUFAB1 MIRO2 NDUFB10 NDUFS7 GPX4 TRX2 NDUFA12 PRX5 NDUFV1 UQCRC1 UCP2 FIS1 AIF ATP5H NDUFA13 ATP5O Mitochondrial Pathways Genes Mitochondrial Pathways Genes - Ventricles High Dose Female Fold change (log2ratio) COX6B2 NDUFB2 COX7A2 COX6A2 NDUFA6 NDUFA3 COX7B COX7A1 NDUFA1 ATP5L ATP5G1 NDUFB6 NDUFA4 ATP5J COX5A NDUFS3 NDUFA7 COX8A NDUFS1 NDUFB4 UQCR10 UQCRFS1 COX4I2 ATP5F1 NDUFS8 UQCRQ UQCRC2 ATP5G3 SDHD ATP5C1 COX6A1 NDUFB11 NDUFA11 NDUFV3 ATP5J2 NDUFA5 UQCR11 NDUFA9 NDUFB9 NDUFS6 SDHB ATP5A1 NDUFV2 NDUFS4 COX4I1 UQCRB SDHC NDUFA10 NDUFB8 NDUFA8 ATP5G2 NDUFB5 SDHA COX6B1 ATP5B NDUFA2 ATP5D NDUFB7 NDUFS2 NDUFB3 NDUFAB1 NDUFB10 NDUFS7 NDUFA12 NDUFV1 UQCRC1 ATP5H NDUFA13 ATP5O Oxidative Phosphorylation Genes Oxidative Phosphorylation Genes - Ventricles Ahr +/+ High TCDD Females Fold change (log2ratio)

32 Treadmill endurance test of Ahr-/- and Ahr+/+ mice exposed or not to TCDD in utero Time to Failure

33 Outline AHR modulates lineage specification pathways during early differentiation Loss of pluripotency Cardiogenesis Neuroglia Dioxin exposure and Ahr ablation disrupt cardiogenesis Short and long-term effects of developmental dioxin exposure Gene-environment interactions in cardiac disease Ahr Nkx2.5 interactions in cardiac haploinsufficiency

34 The molecular signature of developmental AHR disruption is similar to the Nkx2-5 -/- heart transcriptome Nkx2-5 -/- gene expression (Prall et al 2007) Ahr +/+ + TCDD Naïve Ahr -/- Downregulated Direction Agrees Nkx2-5 mutant Direction Agrees Nkx2-5 mutant Ankrd1 up no unchanged no Nppb (Bnp) down yes down yes Nr2f2 (coup TFII) up no up no Myl2 down yes down yes Irx4 down yes down yes Smpx (Chisel) unchanged no down yes Nppa (Anf) up no up no Wnt11 up no up no Upregulated IGFBP5 up yes up yes Pdgfra up yes up yes Tnc up yes up yes Isl1 up yes up yes Tbx5 up yes up yes Bmp2 unchanged no unchanged no Fgf10 up yes up yes Hhex unchanged no unchanged no Unaltered Cer1 unchanged yes unchanged yes Dan unchanged yes unchanged yes NOG unchanged yes unchanged yes Bmp4 up no up no Bmp7 down no down no Dkk1 unchanged yes unchanged yes

35 Loss of Ahr or TCDD exposure repress NKX2-5 A B LV C * * TCDD dose (µk/kg) Compartment independent

36 Heart-specific Ahr knockout with a Nkx2-5 +/cre knockin Excised Ahr fx allele (180bp) H L K Sp Th P St Co fx/+ H In M Lu B E Bm Fx/+ Unexcised Ahr fx allele (140bp) Ahr wild type allele (106bp) The genotype of these mice is Nkx2-5 +/cre Ahr fx/fx Because they have only one functional Nkx2-5 allele These mice are haploinsufficient

37 Ahr ablation rescues NKX2.5-dependent cardiac insufficiency Nkx2.5 haploinsufficient Ahr wild-type Nkx2.5 wild-type Ahr floxed (wild-type) Nkx2.5 haploinsufficient Ahr excised

38 Congenital Cardiac Disease NKX2.5 AHR Environment Congenital cardiac malformations Valvular stenosis Hypoplastic left heart syndrome 8 per 1000 live births 25 30% of cardiac disease Deadly if not surgically repaired at birth

39 I hope that I have convinced you that The Ahr gene is positively silenced in ES cells Ahr modulates lineage specification pathways during early differentiation Ahr ablation and dioxin exposure disrupt cardiomyocyte differentiation Gene networks, Mitochondria, OXPHOS, Heart function, Endurance Ahr reinforces Nkx2.5-dependent haploinsufficiency

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41 Ligand mediated toxicity

42 Many transcription factors are disrupted by TCDD in ES cells

43 % Positive Area at 40x AHR disruption during development alters the structure of the adult myocardium Male Female 1.5 Myocardial Fibrosis * Fibrosis * * * 0.0 M F M F M F M F G1M G1F G2M G2F G3M G3F G5M G5F Naïve Ahr +/+ Naïve Ahr -/- Ahr +/+ + High TCDD Ahr +/+ + Low TCDD Masson s trichrome