Epicardial Abnormalities and Mesenchymal/Hematopoietic Cell Expansion in Plakophilin 2-Null Mouse Embryonic Hearts

Figures

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  Figure 1. Pkp2mt/mt myocardium lacks bona fide desmosomes. Visualization of desmosome formation by immunofluorescence microscopy of desmosomal components (A,B) and by transmission electron microscopy (C) in ventricular myocardium. (A) The fluorescence images show that Pkp2 localizes to distinct puncta at plasma membrane sites of adjacent cardiomyocytes in E10.5 wild-type myocardium. Reduced expression is observed in heterozygous Pkp2mt/wt samples and a complete loss of Pkp2 expression in homozygous Pkp2mt/mt hearts. Nuclei are stained with DAPI. Arrows indicate membrane-localized signals. Yellow dashed lines marks epicardium and white dashed line marks endocardial cells. N = 3–4 hearts per group. At least six heart sections and nine images were analyzed per individual heart. (B) The fluorescence micrographs show that anti-desmoplakin (Dsp) and anti-desmoglein 2 (Dsg2) immunofluorescence colocalize at defined plasma membrane sites in the wildtype. In contrast, Dsp staining looks diffuse while retaining some of the puncta, and Dsg2 does not enrich with Dsp in the remaining Dsp puncta and appears diffuse in Pkp2mt/mt mutant myocardium. N = 3 heart samples per group, at least six heart…

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  Figure 2. Pkp2mt/mt hearts are characterized by hemopericardium, abnormal epicardium, and myocardial hypoplasia. (A) Gross anatomy of embryos shows a reduction in body size and the presence of hemopericardium (arrows) in Pkp2mt/mt at E10.5. The border of the left ventricle (LV) is marked by a dashed line. Samples are imaged at the same magnification. (B–D) Images show comparisons of hematoxylin-eosin-stained tissue sections of wildtype, Pkp2mt/mt and Pkp2mt/mt embryonic hearts at E9.5, E10.5, and E11.5. (B) Short arrows point to circulating cells in the pericardial cavity of E9.5 embryos. They are rare in wildtypes and excessive in heterozygous and homozygous mutants. The curved arrows point to the epicardium (Epi). (C) Blood cells are consistently present in the pericardial cavity of E10.5 Pkp2mt/mt embryos (arrows). Signs of abnormal epicardial development are visible in the mutant embryos and marked by curved arrows. (D) The myocardial wall is hypoplastic in Pkp2mt/mt E11.5 hearts, and the tissue structure is disrupted (black dashed line). The organization and arrangement of epicardial cells are massively perturbed in the mutant heart (note curved arrows). Boxed areas in (B–D) are…

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  Figure 3. Non-cardiomyocyte cell clusters occur in the myocardium of E11.5 Pkp2mt/mt embryos. The fluorescence micrographs show the immunoreaction of anti-desmin antibodies detecting cardiomyocytes together with anti-CD31 antibodies reacting with endothelial cells (Endo) and nuclear DAPI staining. Desmin marks cardiomyocytes in wild-type (A) and Pkp2mt/mt hearts (B). CD31 marks endocardial cells in (B′). (B,B′) are parts of the same heart but in different sections. A yellow dashed line marks the basal aspect of epicardial cells. The white dashed line marks non-cardiomyocyte cluster in the myocardium. Blood cells (Ery, erythrocytes) are seen between the epicardial and endocardial cells within the myocardium of Pkp2mt/mt hearts. Arrows in B point to the deformed myocardial wall. Curved arrows point to epicardial cells (Epi) with loss of squamous morphology. LV: Left ventricle; CM: Cardiomyocytes. N = 2 hearts per group, at least 6 heart sections were analyzed per sample.

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  Figure 4. Floating erythropoietic and hematopoietic stem cells accumulate in the pericardial cavity of Pkp2mt/mt hearts at E10.5. (A) The microscopy images show cardiac tissue sections after immunohistochemical detection of Ter-119 and hematoxylin counterstaining. Note the abundance of Ter-119+ erythroid cells in the circulating blood of the left and right atrium (LA and RA, respectively) in the wild-type and mutant heart, whereas Ter-119+ cells are also present in the pericardial space of the Pkp2mt/mt hearts (arrows). (B) The fluorescence micrographs show immunodetection of the hematopoietic stem cell markers RUNX1 (arrow) and CD44 (arrowheads) in the pericardiac cavity of Pkp2mt/mt hearts. Desmin marks the myocardium. Epicardium and pericardial wall are marked by dashed yellow and white lines, respectively. Nuclei are stained with DAPI. N = 3–4 hearts per group, 3–6 heart sections were analyzed per sample. Epi, epicardium; CM, cardiomyocytes; Peri, Pericardium; RA, right atrium; LA, left atrium.

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  Figure 5. Epicardial coverage and thickness of the myocardium are perturbed in Pkp2mt/mt hearts. (A–C) show analyses at E10.5, (D–F) at E11.5. The left panels (A,D) present Wt1 immunostaining counterstained with hematoxylin tissue sections of wild-type, Pkp2mt/wt, and Pkp2mt/mt hearts at low and high magnification. The black dashed lines mark areas without epicardial coverage, and the asterisk demarcates regions with disorganized cardiomyocytes in the Pkp2mt/mt samples. Arrows point to epicardial cells. (B,E) show the results of quantifying the number of Wt1+ epicardial cells per 100 µm of myocardial surface. (C,F) present measurements of compact myocardial thickness. Note the significantly reduced epicardial coverage of ventricles and the reduced myocardial thickness of atria and ventricles at E11.5 in Pkp2mt/mt hearts. Each data point on the graphs represents the average of measurements or counts normalized to the surface area obtained from one heart. N = 5–7 hearts per group in (B,C); at least three heart sections were analyzed to obtain one data point per animal. N = 2 embryos per group in (E,F); * p < 0.05 by one-way ANOVA and Tukey’s multiple comparison test.

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  Figure 6. Epicardial cell morphology and attachment to the myocardium are severely perturbed in E9.5 and E10.5 Pkp2mt/mt embryos. The fluorescence micrographs show the detection of integrin α4 (ITGα4; red) in epicardial cells and desmin (Des; green) in cardiomyocytes after immunofluorescence labeling. Nuclei are stained with DAPI. Note the squamous epithelial morphology of the wild-type epicardium (arrows), whereas epicardial cells are more rounded and form clusters in the Pkp2mt/mt hearts (curved arrows). Detached ITGα4+ cells are present in the pericardial cavity of mutant left ventricle (LV) and right atrium (RA) at E10.5 (curved arrows). Note disrupted cardiomyocyte arrangements in E10.5 ventricle adjacent to a cluster of cells with reduced ITGα4 expression. The dashed white line delineates a region of the myocardial surface that is not covered by epicardial cells. N = 3–5 hearts per group, at least 6 heart sections, and 10 images were analyzed per sample. CM, compact myocardium; TM, trabecular myocardium; Epi, epicardium; RA, right atrium; LV, left ventricle.

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  Figure 7. RUNX1+ hematopoietic cells located in epicardial and endocardial cell layer are increased in Pkp2mt/mt hearts at E10.5 and E11.5. The images show detection of RUNX1 by immunofluorescence (A–C) and immunohistology (F–H) in Pkp2wt/wt and Pkp2mt/mt hearts. N = 3–5 hearts per group at E10.5; N = 2 embryos per group at E11.5. At least 6 heart sections were analyzed per individual heart. Desmin marks the cardiomyocytes, and nuclei are stained with DAPI. The immunohistological sections are counterstained with hematoxylin. Arrows point to RUNX1+ cells. Note that the scarce RUNX1+ cells in wild-type hearts are attached to the epicardial and endocardial surface (A). In contrast, an increased number of RUNX1+ cells is detected in Pkp2mt/mt epicardium and in regions of lost epicardium at E10.5 (B,C), and among endocardial cells and within the myocardium at E11.5 (G,H). (D) The transmission electron micrograph of Pkp2mt/mt myocardium confirms the loss of epicardial cells and their replacement by rounded cells that differentiate into erythrocyte-like cells (arrows). Note the disarrangement of adjacent cardiomyocytes. The dashed yellow line marks the border between cardiomyocytes and epicardial…

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  Figure 8. The transition of epicardial cells to mesenchymal and erythropoietic cells is accompanied by changes in intermediate filament protein expression in Pkp2mt/mt hearts. The immunofluorescence images were recorded at E10.5 in Pkp2wt/wt (A) and Pkp2mt/mt hearts (B,C), detecting keratin 8 (Krt8) and vimentin (Vim). Nuclei were stained with DAPI. (A) Note that keratin 8 and vimentin are co-expressed in the squamous epicardial cells (Epi) of the wild-type heart (arrows), whereas endocardial cells (Endo) are only positive for vimentin (arrow). (B) shows an immunofluorescence recording of a region in a Pkp2mt/mt heart where epicardial cells are arranged in two layers. Note that some epicardial cells had migrated into the sub-epicardial space while others face the pericardial space (arrowheads). (C) The immunofluorescence micrographs depict a tangential section through the mesenchymally transformed epicardium of a Pkp2mt/mt heart at the interventricular groove, revealing rounded mesenchymal cells that are strongly positive for vimentin and less positive for keratin (arrowheads). Note also the presence of a cell cluster that is surrounded by these cells and lacks both keratin and vimentin…

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  Figure 9. Epicardial cells lose contact with the myocardium, transform into rounded mesenchymal-like cells, and differentiate into erythrocytes. The electron micrographs depict different stages of epicardial-to-hematopoietic transition in Pkp2mt/mt hearts (B–E) in comparison to wild-type control hearts (A–A″) at E10.5. (A–A″) The pictures (low magnification at left, high magnification of boxed area at right) illustrate the typical arrangement of epicardium (Epi) and myocardium in the Pkp2wt/wt heart, whereby the elongated flat epicardial cells are coupled to each other via junctional complexes and attach directly to cardiomyocytes at multiple foci, where the plasma membranes of both cell types juxtapose but lack cytoplasmic plaques (arrows; magnified view in (A″)). The dashed red line delineates the border between epicardium and myocardium. Note also the columnar arrangement of the junction-linked cardiomyocytes in the compact myocardium (CM). (B–E) The electron micrographs show that epicardial cells dissociate from the myocardium in Pkp2mt/mt hearts (bar lines, blue) and that the arrangement of cardiomyocytes is perturbed with increased intercellular spaces (bar lines, red). In some…

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  Figure 10. Intercellular epicardial junctions and epicardial–myocardial focal contact points are perturbed in Pkp2mt/mt hearts. The electron micrographs show details of epicardial and myocardial junctions in E10.5 Pkp2wt/wt and Pkp2mt/mt hearts. (A,A′) Focal contact sites between epicardial cells and cardiomyocytes are marked by short arrows, epicardial intercellular junctions by long arrows. (A′) is a magnification of the boxed area in (A). (B,B′) Adhesion of Pkp2mt/mt epicardial cells to cardiomyocytes is severely compromised, while intercellular junctions are still preserved in this region. Note the distance between epicardium and cardiomyocytes (bar lines). (B′) is a magnification of the boxed area in (B). (C,C′) The images depict a region where intercellular epicardial junctions are also compromised. Long arrows in (C′) mark junction-free membrane domains of adjacent epicardial cells. (C′) is a magnification of the boxed area. N = 2 hearts per group. About 200 images were captured from wildtype samples, and about 400 images were captured from the mutant samples. Epi, epicardium; CM, cardiomyocyte; Col, collagen fiber; Jun, intercellular junction; Li, lipid droplet; Ery, erythrocyte;…

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  Figure 11. A proposed model that depicts abnormal epicardial development, altered tissue organization, and the emergence of hematopoietic cells in Pkp2mt/mt hearts compared with wildtype. The scheme highlights the epicardium and compact myocardium interface and the expression of molecular factors in each cell type at E10.5. Wild-type epicardial cells form a squamous, one-layered epithelium that covers the underlying myocardium and is directly attached to cardiomyocytes through multiple focal contact points. In Pkp2mt/mt hearts, epicardial coverage is compromised. The remaining epicardial cells round up, lose contact with each other, and attain a migratory phenotype indicative of epithelial-to-mesenchymal transition, which is accompanied by downregulation of Wt1, keratin 8, and ITGα4. Subsequent mesenchymal-to-hematopoietic transition is reflected by the formation of hematopoietic stem cells, which are positive for RUNX1 and CD44 but lack keratin 8 altogether. These cells proliferate and give rise to nucleated red blood cells that become Ter-119 positive and completely lose vimentin.