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An essential role for connexin43 gap junctions in mouse coronary artery development

¹ Biology Department, Goddard Laboratories, University of Pennsylvania, Philadelphia, PA, USA
² Division of Neonatology, Department of Pediatrics, Duke University, Durham, NC, USA
³ Laboratory of Developmental Biology, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA
⁴ Department of Cell Biology and Anatomy, Medical University of South Carolina, Charleston, SC, USA
⁵ Department of Medicine, University of Pennsylvania Medical School, Philadelphia, PA, USA

View larger version (120K): [in a new window]   Fig. 1. Coronary artery defects in Cx431 knockout mouse hearts. (A) Transverse sections through the base of the heart of an E17.5 Cx431–/– embryo stained with an antibody to -cardiac myosin. A large pouch (Po) breached the pulmonary valve ring. Note the myosin-free subendocardial layer in the wall of the pouch. The right coronary artery stem was absent in this animal (not shown). (B) In this E14.5 Cx431–/– mouse heart, the mouth of the coronary artery (arrowhead) is narrowed and the stem of the coronary artery is continuous with a large thin-walled sinus (s) that empties into numerous intertrabecular sinusoids. Note the base of the right ventricle is filled with trabecula that obscures the right ventricular outflow. The lacZ-positive cells (blue) represent neural crest cells labeled with a Cx431 promoter driven lacZ transgene (Lo et al., 1997). (C-E) A caudal-to-cranial sequence of an E17.5 Cx431–/– heart stained with an antibody to -cardiac myosin. The left coronary artery exits the aorta normally (arrow in E) but the right coronary artery exits a sinus of the pulmonary trunk rather than the aorta. In C, the mouth of the right coronary artery (RCA) is just opening into the coronary sinus of the pulmonary trunk, seen more clearly in D (asterisk). (F-H) E17.5 Cx431–/– heart stained with an antibody to myosin. In a caudal-to-cranial sequence, a branch of the left coronary artery (arrow) empties into and becomes continuous with an enlarged intertrabecular sinusoid that is in the early stages of forming a pouch in the right ventricle. Ao, aorta; Av, aortic valve; PI, pulmonary infundibulum (right ventricular outflow tract); Po, pouch; Pv, pulmonary valve; OFS, remnant of the outflow tract septum; RV, right ventricle; RCA, right coronary artery. Scale bars: 100 µm.

View larger version (164K): [in a new window]   Fig. 2. Reduction of vascular smooth muscle myosin expression in the coronary arteries of Cx431 knockout hearts. Transverse sections of neonatal mouse hearts stained with anti-vascular smooth muscle myosin antibody. (A,B) As the coronary artery exits the aorta (LCA), the coronary artery stem and its mouth (double-headed arrow) are greatly reduced in the Cx431–/– (B) versus wild-type (A) heart. Ao, aorta; PI, pulmonary infundibulum (right ventricular outflow tract); RCA, right coronary artery; LCA, left coronary artery. (C,D) A reduction in vascular smooth muscle myosin expression in large branches of the right and left coronary arteries (BrRCA or BrLCA) of the Cx431–/– (D) versus wild-type (C) heart. (E-J) A similar reduction in smooth muscle labeling is seen in the mural arteries of the right (E,F) and left ventricular walls (I,J), and in arteries of the ventricular septum (G,H). (E,G,I) Wild-type vessels; (F,H, J) Cx431–/– vessels. Note the reduction in size of the Cx431 knockout vessels and the interruption of vascular smooth muscle myosin immunolabeling in the walls of the Cx431 knockout vessels.

View larger version (29K): [in a new window]   Fig. 3. Perturbation in major coronary artery patterning in the Cx431 knockout mouse. Major coronary arteries in wild type (Cx431+/+) littermates: the coronary arterial pattern of Cx431 wild-type hearts at E16.5-17.5 diagrammed from sectioned hearts. The aorta is viewed as if looking caudally. The three aortic sinuses are labeled ‘R’ and ‘L’ for right and left, and ‘N’ for the third non-coronary sinus. Coronary artery branches feed the ventricular septum (S), the free walls of the heart (M) and the base of the heart (C). The mural branch of the left coronary artery divides into a circumflex artery, which feeds the dorsal side of the heart, and the anterior descending branch (AD) that follows the interventricular groove on the ventral side of the heart. In hearts sectioned favorably, the mural branch of the right coronary artery gives off a slender artery that feeds the base of the atrial septum (AVS). Coronary artery patterns in the hearts of heterozygous (+/–) and homozygous (–/–) Cx431 knockout mice. Some of the changes in coronary artery pattern observed in the E16.5-17.5 heterozygous and homozygous Cx431 knockout mouse hearts are shown: small accessory coronary arteries (Ac) exiting the non-coronary aortic sinus (8462, 8463, 8065 and 8464) or the right or left coronary sinus (8065 and 8048), tunneling of the right coronary artery caudally through the wall of the aorta (8048, 8462, 8463 and 8065), main branches of a coronary artery exiting the aorta separately rather than branching from a main stem (8065), and in one case, the coronary artery stem divided in the wall (8064). In several hearts, a branch of the coronary artery became sinusoidal with thin walls (8489 and 8488).

View larger version (76K): [in a new window]   Fig. 4. Pouches show elevated proliferation and express markers of vascular smooth muscle cells. (A,C,E) A pouch from an E17.5 Cx431 knockout heart was consecutively sectioned and stained with cardiac myosin antibody (A) and PCNA antibody (C), and TUNEL labeled to detect apoptosis (E). The walls of the pouches consist of a myosin-free subendocardial layer of tissue (between arrows in A,C,E) that exhibits increased PCNA staining and TUNEL labeling (see dark nuclei between arrows in C,E, respectively). (B) Ventricular myosin light chain 2 (MLC2V) is also absent in the subendocardial layer of a pouch from an E17.5 Cx431–/– mouse heart. (D,F) SM22 (D) is expressed subendocardially in the pouches of E16.5 hearts. After birth, vascular smooth muscle myosin (F) is also observed in this subendocardial layer. (G) The Masson-Goldner trichrome stain shows abnormal extracellular matrix deposition in the pouches of the Cx431 knockout mouse heart (pale blue staining denoted by white arrow). (H) A heterozygous E16.5 knockout mouse heart stained with anti-smooth muscle -actin exhibits a large pouch breaching the wall of the right ventricular outflow just beneath the pulmonary valve. AV, aortic valve; LV, left ventricle; Po, pouch; PV, pulmonary valve; RV, right ventricle. Scale bars in A,C,E,H, 100 µm; D,F, 50 µm.

View larger version (124K): [in a new window]   Fig. 5. Cx431 expression in the proepicardial organ. (A) E9.5 embryo whole mount immunostained with a Cx431 antibody was embedded and sectioned. (A) Phase contrast image of a section which includes the PEO. The boxed region is magnified in B,C. Note the dendritic mesenchymal cell morphology (B, phase contrast). Cx431 immunostaining (C, darkfield) show punctate and long profiles of gap junction contacts between cell processes (arrows in B,C).

View larger version (137K): [in a new window]   Fig. 6. Expression of Cx431 gap junctions in the epicardium of the fetal mouse heart. (A,B) Immunostaining shows Cx431 expression in the compact layer of the ventricular myocardium (myocard), in the ventricular trabeculae (trab) and in the epicardium (white arrows) in the interventricular sulcus of an E12.5 mouse heart. Cx431 expression is depicted in purple. The green background is autofluorescence recorded in the FITC (green) channel to delineate the myocardium and epicardium. Cx431 expression in the epicardium is comparable with that of the compact myocardium, while expression in the trabeculae is much higher. Cx431 expression in the ventricular myocardium and epicardium (white arrows) is shown at higher magnification in B. (C,D). Immunostaining with a cytokeratin antibody revealed abundant cytokeratin (blue) expression in the epicardium of Cx431+/– (C) and Cx431–/– (D) hearts.

View larger version (89K): [in a new window]   Fig. 7. Cx431 expression in a PEO explant. A 24 hour PEO explant culture (A) is comprised of a monolayer of cells with epithelial morphology. These cells express cytokeratin (B) and Cx431 (C). Punctate Cx431 immunolabeling is found in abundance along regions of cell-cell contact (arrows in C). In many cells, the Cx431 immunostaining occupied much of the peripheral cell membrane, outlining the polygonal shape of the proepicardial cells (C). By contrast, Cx431 immunolabeling is absent in proepicardial cells from a Cx431 knockout mouse embryo (D). Scale bars: 200 µm in A; 80 µm in B-D.

View larger version (57K): [in a new window]   Fig. 8. Proepicardial cells are functionally coupled by gap junctions. Single cells in a wild-type (A) and a Cx431 knockout (C) proepicardial explant culture were impaled with a microelectrode (arrow) and iontophoretically injected with carboxyfluorescein. The gap junction mediated spread of carboxyfluoescein to adjacent cells can be seen in the darkfield images of a wild-type PEO explant (B), but not in a Cx431 knockout explant (D). Scale bar: 60 µm.

View larger version (130K): [in a new window]   Fig. 9. The migratory paths of individual cells in the proepicardial explants. The colored lines represent the migratory paths of individual cells in the PEO explant as observed by time lapse videomicroscopy (images captured every 5 minutes for 20 hours). The Cx431-deficient proepicardial cells (left explant) exhibit longer migration paths, indicating a higher speed of cell locomotion.

View larger version (35K): [in a new window]   Fig. 10. Epicardial cell differentiation into smooth muscle-like cells. Epicardial cells are obtained from an E11.5 mouse heart explanted in culture on a collagen coated glass coverslip. After 5 days, small round or spindle-shaped like calponin positive cells are scattered between unstained epicardial cells from a wild-type heart (A). This is shown at higher magnification in B. These same cell types were also seen in the Cx431 knockout heart explants (C).