The G Protein-Coupled Receptor Latrophilin-2, A Marker for Heart Development, Induces Myocardial Repair After Infarction

Figures

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  Figure 1.. Lphn2 expression in mouse embryonic heart tissues, adult cardiomyocytes, and adult heart tissues. (A): Before and after images of a cleared mouse embryo (E10.5) prepared by fixing in paraformaldehyde, embedding in a hydrogel, electrophoretic tissue clearing, and refractive-index matching. (B): Three-dimensional rendering of a clearing-processed whole mouse embryo (E9.5), imaged by confocal microscopy for Lphn2 (green), α-SA (red), and merged expression. Blue, nuclear counterstain (DAPI). Scale bar, 500 μm. (C): Immunostaining of a clearing-processed embryonic heart (E10.5) for Lphn2 (green) and α-SA (red). Blue, nuclear counterstain (DAPI). Scale bar, 200 μm. OFT, outflow tract; V, ventricle; A, atrium. (D) Cytospin/immunostaining for Lphn2 in adult cardiomyocytes extracted by the Langendorff perfusion method from wild–type mice and Myh6 promoter–driven EYFP mice. Blue, nuclear counterstain (DAPI).

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  Figure 2.. Expression of heart–specific genes by RNA sequencing method in Lphn2 KO embryos. (A): RNA sequencing of whole embryonic tissues obtained from six samples of wild-type and Lphn2-homozygous (−/−) knockout embryos at E9.5 and gene clustering using MeV 4.9.0. Hierarchical cluster analyses were carried out with Euclidean distance correlation as the distance measurement with average linkage. Downregulated and upregulated genes in Lphn2−/− KO embryos are shown in green and red, respectively. Dysregulated genes with a >2-fold change in expression and with a P-value < .05 are shown (n = 3). (B): Heat map showing mRNA expression of gene ontology representing heart development, embryonic heart tube development, atrioventricular valve morphogenesis, outflow tract morphogenesis, and ventricular cardiac muscle tissue morphogenesis in six samples of wild-type and Lphn2−/− KO embryos at E9.5. To perform gene–annotation enrichment analysis, the genes were selected and input using the DAVID functional annotation tool.

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  Figure 3.. Functional significance of Lphn2 in cardiac development. (A): Expression pattern of Lphn2 overlap with early cardiac markers in heart development. Immunostaining of Lphn2 (green), Gata4 (red), and α-SA (white) in the heart of wild-type, Lphn2+/−, and Lphn2−/− embryos at E11.5. Blue, nuclear counterstain (DAPI). Scale bar, 200 µm. LV, left ventricle; OFT, outflow tract; RA, right atrium; RV, right ventricle. (B): Representative H&E-stained transverse sections revealed the disrupted conotruncal septation of ventricles in Lphn2−/− KO embryos at E14.5. All embryos were from the same litter. Scale bars, 20 µm, LA, left atrium; LV, left ventricle; RA, right atrium; RV, right ventricle. (C): Immunostaining of a clearing-processed heart of wild-type and Lphn2−/− KO embryos at E14.5 for Lphn2 (green) and α-SA (red). Blue, nuclear counterstain (DAPI). LA, left atrium; RA, right atrium; LV, left ventricle; RV, right ventricle; OFT, outflow tract.

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  Figure 4.. Role of Lphn2 in mesodermal, ectodermal, and endodermal development of embryos. (A, B): Immunostaining of non-myogenic, vascular lineage markers SMA (A) and ILB4 (B) in wild-type and Lphn2–KO embryos at E11.5. Scale bars, 1000 µm (C): Expression of βIII-tubulin, an ectoderm lineage marker, in wild-type, Lphn2+/− and Lphn2−/– embryos at E11.5. (D): Expression of HepPar1, a hepatic endoderm marker, in wild-type, Lphn2+/− and Lphn2−/− embryos at E11.5. Scale bar, 20 µm.

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  Figure 5.. Therapeutic potential of Lphn2 to repair the heart after infarction. (A): Experimental scheme to assess the therapeutic efficacy of iPSC-derived Lphn2+ cells using a mouse myocardial infarction model. iPSCs were generated from primary skin fibroblasts isolated from Actin-GFP transgenic mice. iPSC-derived GFP+/Lphn2+ or GFP+/Lphn2− cells were sorted at day 7 after cardiac differentiation and transplanted into the peri-infarct area. (B): Masson’s trichrome staining was performed two weeks after the injection of PBS, iPSC-derived Lphn2+ cells, and Lphn2− cells into the peri-infarct zone of the heart after ligation of the left coronary artery (n = 15 per group). (C): Echocardiographic parameters at 14 days after cell transplantation (∗∗P < .01, ANOVA test and post hoc Bonferroni test, n = 10 per group). LVESD, LV end-systolic dimension; LVEDD, LV end-diastolic dimension; LVFS, LV fractional shortening; LVEF, LV ejection fraction. (D): Immunostaining of mouse myocardial infarction heart tissues injected with PBS, iPSC-derived Lphn2+ cells, and Lphn2− cells. Injected GFP+ cells were traced and immunostained for GFP (green), Lphn2 (red), and α-SA (white). Blue, nuclear counterstain…