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First published online August 2, 2004
doi: 10.1242/10.1242/dev.01265

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The T-Box transcription factor Tbx5 is required for the patterning and maturation of the murine cardiac conduction system

¹ Department of Genetics, Harvard Medical School and Howard Hughes Medical Institute, Boston, MA 02115, USA
² Department of Pathology and Cardiac Registry, Children's Hospital and Harvard Medical School, Boston, MA 02115, USA
³ Molecular Cardiology Research Center and Section of Cardiac Electrophysiology, University of Pennsylvania, Philadelphia, PA 19104, USA
⁴ Department of Cardiology, Children's Hospital and Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA
⁵ Programs in Cardiovascular Research and Developmental Biology, The Hospital for Sick Children, Toronto, ON M5G 1X8, Canada
⁶ Department of Molecular and Medical Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada
⁷ Departments of Anesthesiology and Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232-6602, USA
⁸ Departments of Medicine and Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232-6602, USA
⁹ Division of Cardiology, Brigham and Women's Hospital, and Howard Hughes Medical Institute, Boston, MA 02115,USA

View larger version (84K): [in a new window]   Fig. 1. Tbx5 is expressed in the murine cardiac conduction system. (A) Schematic representation of the atrioventricular canal conduction system, showing the plane of dissection (dashed line) and specimen orientation (arrow) for images in (B,C). Atria were removed and the atrioventricular canal was viewed from the superior/posterior. The atrioventricular canal from newborn minKlacZ/+ (B) and wild-type (C) mouse hearts. X-gal staining of minKlacZ/+ hearts (B) showed two rings of specialized conduction cells, surrounding the tricuspid and mitral annulus. Scale bar: 200 µm. Whole-mount in-situ hybridization with a Tbx5 probe of wild-type hearts (C) demonstrated rings of Tbx5 expression that overlap minK expression. (D) Schematic representation of dissection of the interventricular septum for images in (E-H). Sagital sections of the muscular interventricular septum of newborn minKlacZ/+ (E) and wild-type (F-H) mouse hearts. A minKlacZ/+ heart (E) revealed ß-galactosidase activity in the atrioventricular bundle (arrow), and the ventricular bundle branches (arrowheads). Scale bar: 100 µm. In-situ hybridization with a Tbx5 probe in wild-type hearts (F) demonstrated expression in the atrioventricular bundle (arrow) and bundle branch (arrowheads) conduction system. In-situ hybridization with a connexin 40 probe (G) and Tbx5 probe (H) in sequential sections from the same wild-type heart demonstrates overlapping expression in the atrioventricular bundle (arrow) and bundle branch (arrowheads) conduction system.

View larger version (112K): [in a new window]   Fig. 2. Conduction system maturation failure in Tbx5del/+ mice. The atrioventricular canal conduction system in minKlacZ/+ (A,B) and Tbx5del/+/minKlacZ/+ (C,D) hearts was studied in newborn (A,C) and adult (B,D) mice. The atria were removed and the atrioventricular canal was viewed from the superior/posterior, with the tricuspid annulus on the right and the mitral annulus on the left. Rings of specialized conduction cells observed in the atrioventricular canal of newborn minKlacZ/+ mouse hearts (A) matured into a well-defined atrioventricular node (arrow) and atrioventricular bundle in adult minKlacZ/+ mouse hearts (B). (A) Scale bar: 200 µm. (B) Scale bar: 800 µm. Rings of specialized conduction cells in the atrioventricular canal of newborn Tbx5del/+/minKlacZ/+ mouse hearts (C) failed to mature into a discrete atrioventricular node or atrioventricular bundle in adult Tbx5del/+/minKlacZ/+ mouse hearts (D). Instead the neonatal pattern (rings of specialized conduction tissue) was maintained. Arrow denotes expected location of the atrioventricular node.

View larger version (14K): [in a new window]   Fig. 3. PQ maturation defect and QRS prolongation in Tbx5del/+ mice. (A) Schematic representation of electrical impulse propagation through the mammalian heart correlated with surface ECG and in-vivo electrophysiology intervals. PQ intervals include impulse propagation throughout the atria and atrioventricular node (proximal AH interval) and the atrioventricular bundle and proximal bundle branches (distal HV interval). P-wave duration represents atrial depolarization. QRS intervals represent ventricular activation, and include bundle branch and Purkinje conduction. Bundle-branch block causes QRS prolongation with characteristic ECG wave front morphology. SAN, sinoatrial node; AVN, atrioventricular node; AVB, atrioventricular bundle; RBB, right bundle branch; LBB, left bundle branch. (B) Representative ECGs from wild-type and Tbx5del/+ newborns and adult mice. Note comparable PQ intervals (atrial plus atrioventricular canal conduction time) in neonatal wild-type and Tbx5del/+ mice. Adult wild-type mice had significantly shorter PQ intervals than those of Tbx5del/+ mice. QRS intervals of newborn and adult Tbx5del/+ mice were longer than in wild-type mice (Table 1). (C) Representative ECG recordings from right precordial leads (V1) in wild-type and Tbx5del/+ adult mouse. Wild-type mice had normal QRS complexes. Tbx5del/+ mice had QRS prolongation with a RSR' wave front pattern indicative of RBB. RBB occurred in 9 of 11 of adult Tbx5del/+ mice versus 3 of 27 adult wild-type mice (P<0.001).

View larger version (53K): [in a new window]   Fig. 4. Bundle-branch patterning defects in Tbx5del/+ mice. (A) Schematic representation of dissection of left-bundle-branch conduction system showing the plane of dissection (dashed line) and specimen orientation (arrow). The left ventricular free wall and mitral valve were removed to view the interventricular septum from the left. ß-Galactosidase expression marked the left-bundle-branch conduction system in minKlacZ/+ (B,C) and Tbx5del/+/minKlacZ/+ (D,E) mouse hearts. The atrioventricular node (black arrow), atrioventricular bundle (red arrowhead) and left bundle branch (black arrowhead) were visible on the surface of the muscular interventricular septum. The broad left bundle branch in newborn minKlacZ/+ mice (B) matured into a narrow fascicle with a well-defined atrioventricular bundle (red arrowhead) in adult minKlacZ/+ mice (C). Comparable maturation did not occur in Tbx5del/+/minKlacZ/+ mice (D,E) and adult Tbx5del/+/minKlacZ/+ mice (E) retained the broad band of specialized conduction cells, without a discrete atrioventricular bundle. (F) Schematic representation of dissection of right-bundle-branch conduction system showing the plane of dissection (dashed line) and specimen orientation (arrow). The right ventricular free wall and tricuspid valve were removed and the interventricular septum viewed from the right. ß-Galactosidase expression marked the right bundle branch in minKlacZ/+ (G,H) and Tbx5del/+ minKlacZ/+ (I,J) mouse hearts. The atrioventricular node (black arrow) and right bundle branch (black arrowhead) were visible on the surface of the muscular interventricular septum. In newborn minKlacZ/+ mouse hearts (G), the right bundle branch was visible as a poorly defined band of cells loosely associated with the septal band and anterior papillary muscle of the right ventricle. In adult minKlacZ/+ mouse hearts (H), the right bundle branch was well defined by a thin band of cells running along the inferior aspect of the septal band and onto the anterior papillary muscle of the right ventricle. In 9/20 newborn (I) and 7/15 adult (J) Tbx5del/+/minKlacZ/+ mouse hearts, the right bundle branch was absent from the right ventricular septal surface, with conduction cells present only along the crest of the interventricular septum demarcating the atrioventricular bundle. (K) Schematic representation of dissection of the interventricular septum for images in (L,M). In-situ hybridization with a connexin 40 probe in wild-type (L) and Tbx5del/+ (M) mouse hearts. Connexin 40 expression was observed in the atrioventricular bundle (arrow), and left and right bundle branches (arrowheads) in the wild-type mouse heart (L) and in the atrioventricular bundle (arrow) and left bundle branch but not the right bundle branch in the Tbx5del/+ mouse heart (M). In 3/5 newborn Tbx5del/+/minKlacZ/+ mouse hearts, the right bundle branch could not be identified.

View larger version (100K): [in a new window]   Fig. 5. Normal conduction system morphology in Cx40–/– mice. ß-Galactosidase expression in minKlacZ/+ (A,C,E) and Cx40–/–/minKlacZ/+ (B,D,F) mouse hearts. In the atrioventricular canal, a well-defined atrioventricular node (arrow) and atrioventricular bundle were observed in both minKlacZ/+ (A) and Cx40–/–/minKlacZ/+ (B) mouse hearts. The atria were removed and the atrioventricular canal was viewed from the superior/posterior, with the tricuspid annulus on the right and the mitral annulus on the left. A well-formed left bundle branch was found in both minKlacZ/+ (C) and Cx40–/–/minKlacZ/+ (D) mouse hearts. The left ventricular free wall and mitral valve were removed and the left interventricular septum viewed from the left. A well-formed right bundle branch was present in minKlacZ/+ (E) and Cx40–/–/minKlacZ/+ (F) mouse hearts. The right ventricular free wall and tricuspid valve were removed and the right interventricular septum viewed from the right. Comparable ß-galactosidase expression and morphology were demonstrated in 12/12 minKlacZ/+ and 10/10 Cx40–/–/minKlacZ/+ mice.