ERp44 is required for endocardial cushion development by regulating VEGFA secretion in myocardium

Figures

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  FIGURE 1. Generation and characterization of ERp44 knockout mice. (A) Strategy used to generate conventional ERp44 knockout mice. (B) Genotyping of WT, heterozygote (HZ) and KO mice. (C) Western blot detection of ERp44 expression in newborn WT, HZ and KO hearts. (D) Photograph of adult WT and KO mice (8 weeks). (E) Body weights of WT, HZ and KO mice of different ages. The statistical data are represented as means ± s. ** p < 0.01; *** p < 0.001. (F) Survival curve of WT (n = 96), HZ (n = 146) and KO (n = 20) mice from the newborn stage to adulthood. (G–I) Photographs of WT and KO mice at postnatal day (G), E9.5 (H) and E10.5 (I)

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  FIGURE 2. ERp44 deletion causes septal defects in the heart. (A) Photographs of the hearts of WT and KO newborn mice. (B) Schematic map of (A). (C–F) H&E staining of WT (C) and KO (D–F) hearts within 24 h of P0 hearts. Asterisks indicate a defective septum. (D) Primary aortic septal defects (PASDs). (E) Perimembranous ventricular septal defects (PVSDs). (F) Atrioventricular septal defects (AVSDs). (G–H) Pulmonary congestion and oedema in neonatal WT (G) and KO (H) mice. (I–J) Transverse sections of WT (I) and KO (J) hearts at E14.5. (K–L) Transverse sections of adult WT (K) and KO (L) hearts. (M) Frequency of cardiovascular abnormalities found in KO mice at E14.5 and P0. RA, right atrium; LA, left atrium; RV, right ventricle; LV, left ventricle; AV, aortic valve; MV, mitral valve; AS, atrial septum

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  FIGURE 3. Dysplasia of the endocardial cushion in ERp44 knockout embryos. (A) Histological examination of the atrioventricular canals in WT and KO embryos at E10.5. (B) Quantitation of total endocardial cushion cells. (C and E) Immunostaining of the proliferation marker Ki67 (C) and apoptosis marker TUNEL (E) in WT and KO embryos at E10.5 to E11.5, respectively. (D and F) Quantitation of positive signals (arrows pointed) in Ki67 (D) and TUNEL (F) staining. Arabic numerals in columns represent the experiment number for each genotype. All statistical data are represented as means ± s. ** p < 0.01; *** p < 0.001

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  FIGURE 4. ERp44 is required for EndMT. (A) Schematic diagram to describe the culture of endocardial cushion in vitro. (B) Outgrowths of mesenchymal cells 48 h after endocardial cushion explantation from WT or KO E9.5 embryos as observed by light‐field microscopy. (C) Immunostaining with antibody for the mesenchymal cell marker (αSMA) and for nuclei with DAPI from E9.5 explants. (D) EndMT of adjacently cultured WT and KO cushion explants in E9.5 for 48 h. (E) Statistics of endocardial cushion explantation trial in (B) and (D). The statistical data are represented as means ± s. * p < 0.05

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  FIGURE 5. Myocardial‐specific deletion of ERp44 leads to AV cushion defects. (A) Photograph of ERp44fl / fl and cTNT‐Cre +/−; ERp44fl / fl newborn mice. (B) Quantitation of ERp44fl / fl and cTNT‐Cre +/−; ERp44fl / fl body weight. All statistical data are represented as means ± s. *** p < 0.001. (C) Genotyping results of litters at different stages. (D–E) H&E staining of ERp44fl / fl and cTNT‐Cre +/−; ERp44fl / fl at E14.5 (D) and P0 (E). Asterisks indicate defective septum. (F) Frequency of cardiovascular abnormalities found in KO mice at E14.5 and P0. (G) Photograph of ERp44fl / fl and Mck‐Cre +/−; ERp44fl / fl newborn mice. (H) Quantitation of ERp44fl / fl and Mck‐Cre +/−; ERp44fl / fl body weight. (I) H&E staining of ERp44fl / fl and Mck‐Cre +/−; ERp44fl / fl at P0. (J) Genotyping results of litters at P0

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  FIGURE 6. ERp44 KO suppresses VEGFA expression. (A) Volcano description of differential genes (KO vs WT). Sig‐up, significant upregulation; NoDiff, no difference; Sig‐Down, significant downregulation. Top 20 differently expressed genes are listed in the diagram. (B) GO and KEGG analyses depicted the functional classification and pathways of the differentially expressed genes. (C) Real‐time PCR analysis of EndMT‐related genes selected from (A). (D) Western blot detection of multiple crucial proteins that affect the development of mouse endocardial cushion targeting WT and KO AVC tissue in E9.5. β‐Actin was used as an internal reference. (E) Immunohistochemical analysis of VEGF expression in E9.5 and E12.5. (F) Quantification of protein expression in (D). (G) VEGF detection of WT or KO heart in E9.5, E12.5 and P0. A, atrium; VW, ventricular wall; S, septum. (H) Quantification of protein expression in (C). In particular, the atrium, ventricular wall and septum of neonatal heart tissue were separated as previous description. 59 All statistical data are represented as means ± s. * p < 0.05, ** p < 0.01 and *** p < 0.001

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  FIGURE 7. ERp44 regulates the maturation and secretion of VEGF. (A) Micrographs described the expression of VEGFA in WT and ERp44 KO H9C2 cells. The cells were stained with mouse anti‐ERp44 (red) and rabbit anti‐VEGF (green) antibody. The nuclei were stained with DAPI (blue). (B) Relative fluorescence intensity of VEGF in (A). 50 (WT) and 41(KO) fields were taken into consideration. The statistical data are represented as means ± s. *** p < 0.001. (C) VEGF expression is positively correlated with ERp44 level. OE and KO represent ERp44 overexpression H9C2 and ERp44 knockout H9C2, respectively. (D) Detection of interaction between ERp44 and VEGF in H9C2 by co‐immunoprecipitation assay. (E) Detection of intracellular and extracellular VEGF‐myc expression in HEK‐293T cells stably expressing VEGF‐myc after transfecting with ERp44‐WT and mutants. β‐Actin was used as an internal reference. CON, pcDNA3.1; VEGF165‐myc, pcDNA4‐hVEGFA165‐myc; HA‐WT, pcDNA3.1‐HA‐ERp44; HA‐C29S, pcDNA3.1‐HA‐C29S (C29S mutant of ERp44); HA‐△T, pcDNA3.1‐HA‐△T (C‐terminal truncation of ERp44. (F) C29 site of ERp44 contributes to the interaction between ERp44 and VEGF in 293T cells. (G) Glycosylation site N101 of VEGF165…