(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, H19ΔICR /H19+; LOI+ BAC, H19ΔICR/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/H19ΔICR females were crossed with males carrying the Myh6-Cre transgene to generate four kinds of pups: H19ΔICR/H19+ (#1) and H19ΔICR/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 (H19ΔICR/H19+).