Figure 1.
Expression of VCAM-1 surface marker in mouse embryonic myocardium.
Immunofluorescence images of tissue cryo-sections showing that VCAM-1 is expressed by the myocardium in embryonic hearts (A–J) and by vascular endothelium in adult hearts (K–M). VCAM-1 co-localizes with cardiac specific cTropT (A) and is situated at the cell surface (E, H). VCAM-1 does not co-localize with endocardial/endothelial specific PECAM-1 in embryonic hearts (A, E). PECAM-1, VCAM-1 or cTropT in separate channels (B–D, F–G, I–J and L–M). Abbreviations: (atr) atria (hep) hepatic primordia (end) endocardium (myo) myocardium (ven) ventricle. Tissue sections: E10.5 (A–D, H–J); E12.5 (E–G); Adult (K–M). Optical sections: E–J.
Figure 2.
Purification of embryonic cardiomyocytes by Flow Cytometry.
Flow Cytometry plots of E10.5–E11.5 cardiac cells labeled with specific antibodies to VCAM-1 and PECAM-1 (A–C). Cells are gated and sorted based on doublet discrimination (A), viability (B) and VCAM-1 positive PECAM-1 negative population (C). Flow Cytometry plot of sorted fixed cells, stained with cTropT antibodies to verify cardiac identity (D). Flow Cytometry histograms (overlays) showing control cTropT staining of neonatal hearts (E). Un-stained cells (black), isotype control (red outline) and neonatal heart cells (green outline). The percentage of gated cells through each step of the sort is indicated in each plot. Immunofluorescence staining (cTropT) of sorted cells cultured on gelatin coated slides for two days to verify cardiac identity and cell viability (F). F-actin and cTropT in separate channels (G–H). A low frequent cTropT-negative cell is indicated (arrow).
Figure 3.
Protein expression profile of FACS-isolated VCAM-1+ embryonic cardiomyocytes.
Phase contrast image of FACS-isolated cells grown on gelatin for 48 hours (A). Immunofluorescence images of the gelatin-cultured cells (B–S). Sorted cells positive for embryonic cardiac markers cTropT (B–D), αlpha-SMA and GATA4 (H–J) and Nkx2.5 and alpha-actinin (K–M). Confocal analysis verifying expression of VCAM-1 (E–G), expression of alpha-actinin (Q–S) and MEF2C (N–P).
Figure 4.
RNA expression profile of isolated VCAM-1+ embryonic cardiomyocytes.
Gene expression, analyzed by real-time quantitative PCR, in E10.5–E11.5 un-sorted cardiac cells and sorted VCAM-1 positive cells compared to whole embryos. The fold change in gene expression is indicated by the Y-axis with standard deviation error-bars. Variations in RNA input were normalized through expression of the housekeeping gene GAPDH. Verification of cardiac-specific lineage genes alpha-MHC, beta-MHC BNP and MLC-2v (A). Differential expression of cardiac progenitor markers c-KIT, Flk-1 and Isl-1 (B) and gene markers for non-myocyte lineages, including hematopoietic (CD45), endothelial (VE-Cadherin (VE-Cad) and Endoglin (Eng)), fibroblasts (Ddr2) and mesenchymal cells (Vimentin (Vim)) (C). * Below detection level.
Figure 5.
Functional analysis of embryonic cardiomyocytes purified by FACS.
(A) Current clamp recordings revealed differentiation of FACS-isolated cells into cardiac subtypes: atrial and ventricle-like cells. (B) Representative voltage ramp protocol showing activation of inward and outward currents of a FACS-isolated cardiomyocyte. (C) APs recorded from a representative FACS-isolated cardiomyocyte, perfusion with the β-adrenergic agonist Isoprenalin evoked a positive chronotropic effect, this could be reversed upon wash-out. (D) APs recorded from a representative FACS-isolated cardiomyocyte, perfusion with the muscarinic agonist Carbachol induced a strong negative chronotropic effect, this could be reversed upon wash-out. (E) Statistics of the hormonal modulation of AP as % of frequency variation after the agonist application respect to the NS. Abbreviations: (NS) normal solution, (ISO) Isoprenalin, (CCh) Carbachol.
Figure 6.
In vitro culture of primary embryonic cardiomyocytes after FACS.
Phase contrast images of FACS-isolated cells grown on irradiated embryonic cardiac fibroblasts for two (A) or six (B) days. Rounded cells attached to the fibroblasts (A) and beating clusters of cardiomyocytes (B, C). Immunofluorescence images of the co-cultured cells (C–J). The FACS-isolated cells form a tight meshwork of beating cardiomyocytes in close contact with the surrounding fibroblasts as well as expressing cTropT (C) and Connexin43 (D). Co-cultures labeled with BrdU after five days for 24 h (E–G). Incorporation of BrdU is detected in cardiomyocytes but not in the surrounding irradiated fibroblasts. A optical section of a dividing cardiomyocyte in co-culture with fibroblasts for five days visualized by Ki-67 expression (H–J).