Fig 1: Cardiac hypertrophy in neonatal LOI mice is mediated by circulating IGF2 activation of AKT/mTOR signaling in cardiomyocytes and is independent of H19 gene function.(A, B) Heart morphology in wild-type, LOI, and LOI+ H19 BAC littermates (A) or in wild-type and H19-deficient littermates (B) Top panels, transverse sections were taken from fixed hearts at 200 mm from the apex. Bottom panels, Ki-67 (brown stain) is a marker for cell proliferation. LOI, H19?ICR /H19+; LOI+ BAC, H19?ICR/H19+ that also carry a 140 kb Bacterial Artificial Chromosome transgene that restores normal H19 expression (Figure 2—figure supplement 2). Notice the thickened walls, misshaped right ventricles, and high levels of Ki-67 expression in LOI and in LOI+ BAC transgenic neonates. (C) Quantitation of Ki-67 expression as assayed in panel A. (N = 5). (D) Immunoblot analyses of heart extracts prepared from wild-type and LOI littermates. LOI hearts show increased levels of proliferation markers, Ki-67, Cyclin EI, and Cyclin D1 and also increased levels of phosphorylated AKT and S6K1 (a target of mTORC1). See Figure 2—figure supplement 1A for quantifed results. (E, F) Cardiomyocyte cellular hypertrophy in LOI animals is cell non-autonomous. Primary cardiomyocyte cultures were prepared from wild type, LOI, and from littermates carrying an ICR deletion only in cardiomyocytes (see below). Cells were cultured overnight, stained for MYH6 (to identify cardiomyocytes) and Phalloidin (to facilitate measurement of surface areas). For each culture (N = 5 per genotype), at least 30 cells were measured. (G, H) Exogenous IGF2 peptide induces cellular hypertrophy in wild-type cardiomyocytes through mTOR pathways. Primary cardiomyocytes were prepared from wild-type neonates and cultured overnight with IGF2 before measurement of cell surface area (G) or preparation of protein extracts for immunoblotting. (H) The effect of increased IGF2 is prevented by treatment with BMS 754807 or with Rapamycin. BMS inhibits IgfR1 and Ins2 receptor kinases (Carboni et al., 2009). Rapamcyin blocks a subset of mTOR activities (Li et al., 2014). See Figure 2—figure supplement 1B for quantified results. (I, J) LOI phenotypes in cardiomyocytes are cell non-autonomous. H19ICRflox/H19?ICR females were crossed with males carrying the Myh6-Cre transgene to generate four kinds of pups: H19?ICR/H19+ (#1) and H19?ICR/H19+ Myh6 Cre (#3) will display LOI in all cell types; H19ICRflox/H19+ (#2) will display wild-type expression patterns for Igf2 and H19; and H19ICRflox/H19+ Myh6 Cre mice will show LOI only in cardiomyocytes. Hearts were analyzed for cellular hypertrophy (E), megacardia and hyperplasia (I), and protein expression (J). See Figure 2—figure supplement 1C for quantified results of protein expression. In all assays, H19ICRflox/H19+ Myh6 Cre mice were highly similar to their wild-type littermates and distinct from the congenital LOI littermates. *p<0.05; ***p<0.001 (Student’s t-test). LOI, loss of imprinting (H19?ICR/H19+). Figure 2—source data 1.Analyses of LOI phenotype in neonatal mice.
Fig 2: Restoring monoallelic expression of Igf2 in LOI cells rescues their differentiation defects. (A) Genetic scheme to restore monoallelic expression of Igf2 in LOI cells. In mouse genetic nomenclature, maternal alleles are indicated first. For example, ΔICR/Igf2- means that the maternal chromosome carries the ICR deletion and the paternal chromosome carries the Igf2 deletion described in the text. Here we crossed ΔICR/ΔICR mice with Igf2+/- mice to obtain two genotypes of interest: ΔICR/Igf2+, which phenocopies ΔICR/ΔICR (extra Igf2, low H19) and ΔICR/Igf2- which genetically rescues Igf2 expression while maintaining LOI levels of H19. (B, C) Ablation of the paternal Igf2 allele in LOI cells restores expression of IGF2 to wild type levels. (B) Immunoblot analyses. (C) ELISA analyses. (D) H19 expression relative to GAPDH was determined by qRT-PCR. Expression in wild type cells is set at 1. The ΔICR/Igf2- and ΔICR/Igf2+ cells have significantly downregulated H19 levels, similar to ΔICR/ΔICR. (E, F) Ablation of paternal Igf2 in LOI cells rescues differentiation defects. ΔICR/Igf2- myotubes have improved morphology with near-normal fusion, while ΔICR/Igf2+ cells have aberrant myotube morphology similar to LOI cells. (E) DAPI (blue) and Myh3 (green) staining. (F) Quantitation of cell fusion. In all experiments, cells were grown for 72 h in differentiation media and at least 3 independent cultures were analyzed. Statistical significance was evaluated by t-test, comparing to wild type controls. **P < 0.01; ***P < 0.001.
Fig 3: Differentiation defects in LOI myoblasts. (A) Cartoon depiction of the gene architecture and expression patterns in wild type and in ?ICR/?ICR cells. Igf2 and H19 are about 80 kb apart on distal mouse chromosome 7. Parent-of-origin transcription depends upon the 2.4 kb H19ICR located just upstream of the H19 promoter. On the maternal chromosome, the H19ICR binds the CTCF genome organizing protein which positions the region into loop structures that prevent interaction of the Igf2 promoter with the shared muscle specific enhancer (E, unfilled circle) located 26 kb upstream of the H19 transcriptional start site. At the same time, these loop structures facilitate H19 promoter-enhancer interactions. Upon paternal inheritance, methylation of ICR CpGs prevents CTCF binding, thus enabling alternative loop structures that promote interactions between the paternal Igf2 promoter and the shared enhancers (60,61). In addition, developmentally programmed ‘spread’ of heterochromatin from the H19ICR to the adjacent H19 promoter prevents H19 transcription (62). (B) Primary myoblasts derived from wild type or ?ICR/?ICR neonates were cultured in growth medium (GM) or in serum-depleted differentiation medium (DM) for 72 h. DAPI staining (blue) identifies nuclei and staining for Myh3 (green) identifies differentiating cells. The ?ICR/?ICR cells in DM show highly aberrant morphology, indicating a severe differentiation defect. (C) Quantitation of Myogenin RNA by qRT-PCR (n = 3). Expression relative to GAPDH is reported. (D) Immunoblot analyses of cell extracts prepared from myoblasts (0 timepoint) and from cells grown in differentiating media for 24, 48 or 72 h. Differentiation markers, Myh3 and Myogenin, are significantly downregulated in LOI cells. Phosphorylated forms of Erk1/Erk2 peptides (also commonly referred to as MAPK3/1 or p44/p42) are also reduced in these cells, although total protein levels are unchanged. H3 is the loading control.
Fig 4: The H19/Igf2 locus.(A) Schematic of maternal (mat) and paternal (pat) chromosomes in wild-type and in loss of imprinting (LOI) mice. Gene expression is indicated by horizontal arrows. In wild-type mice, the paternal copy of the imprinting control region (ICR) is inactivated by DNA methylation (filled lollipops). In LOI patients, inappropriate inactivation of the maternal ICR typically occurs due to microdeletion or to inappropriate DNA methylation. In the mouse LOI model, the maternal ICR is inactivated by deletion. (B) Schematic of wild-type, ?Ex1, and ?Let7 H19 alleles. H19 exons 1–5 are shown as filled rectangles. ?Ex1 is a 700 bp deletion at the 5’ end of exon 1. ?Let7 was constructed for this study by simultaneous deletion of Mirlet7 binding sites in H19 exons 1 and 4. The blue oval identifies coding sequences for Mir675-3p and -5 p. Arrowheads show the direction of transcription. (C–E). Maternal loss of imprinting results in reduced H19 lncRNA and 2× doses of Igf2. (C, D) Hearts were isolated from wild type (WT) or from H19?ICR/H19+ (LOI) littermates at postnatal day 2 or at 2 months. RNAs were extracted, analyzed by qRT-PCR, normalized to GAPDH, and then normalized to levels observed in wild-type neonates. Despite the dramatic postnatal repression, H19 expression in adults remains substantial as H19 lncRNA is in the top 10-percentile of all RNAs. (C) IGF2 peptide levels in serum were measured by ELISA. Statistical significance was evaluated with Student’s t-test type 2. Figure 1—source data 1.Maternal Loss of Imprinting (LOI) results in decreased Igf2 and increased H19 expression.
Fig 5: H19’s Mirlet7 binding domains are essential for normal function.(A) The H19?Let7 allele was generated by deleting 25 and 48 bp sequences within exons 1 and 4 to eliminate binding sites for Mirlet7g, Mirlet7i, and Mirlet7e miRNAs. (B) The H19?Let7 allele is expressed at wild-type levels. RNAs were isolated from hearts from H19?Let7/H19+ and quantitated by qRT-PCR, normalizing first to GAPDH and then to the levels of H19 observed in H19+/H19+. Similarly, Igf2 is expressed at equivalent levels in wild-type and mutant mice. Thus phenotypes associated with the H19?let7 allele should be ascribed to changes in H19 function and not to changes in H19 levels or in Igf2 expression. See Figure 6—figure supplement 1 for experiments demonstrating that the H19?let7 lncRNA cannot interact with Mirlet7 miRNAs. (C) Transverse sections were collected midway along the longitudinal axis from hearts collected from 12-month-old wild-type (N = 4) and mutant (N = 3) littermates and stained with hematoxylin and eosin. (D) Fiber diameters were quantitated using three sections per mouse. (E, F). Masson’s trichrome staining of sections described in panel (C) Red, muscle fibers; blue, collagen. Sections from three wild-type and four mutant littermates were used to calculate fibrosis. (G) Immunoblot analyses of whole heart extracts prepared from 12 month WT (N = 3) and mutant littermates (N = 3). Altered expression of ANP, Myh7, Cleaved Caspase-3, Cleaved Caspase-7, and Cleaved PARP. ß-tubulin is a loading control. Quantified data is presented in Figure 6—figure supplement 2. Statistical significance was evaluated with Student’s t-test type 2. Figure 6—source data 1.Mirlet7 binding sites on the H19lncRNA are essential to prevent cardiomyopathy.
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