Visualization and functional characterization of the developing murine cardiac conduction system

Abstract

The cardiac conduction system is a complex network of cells that together orchestrate the rhythmic and coordinated depolarization of the heart. The molecular mechanisms regulating the specification and patterning of cells that form this conductive network are largely unknown. Studies in avian models have suggested that components of the cardiac conduction system arise from progressive recruitment of cardiomyogenic progenitors, potentially influenced by inductive effects from the neighboring coronary vasculature. However, relatively little is known about the process of conduction system development in mammalian species, especially in the mouse, where even the histological identification of the conductive network remains problematic. We have identified a line of transgenic mice where lacZ reporter gene expression delineates the developing and mature murine cardiac conduction system, extending proximally from the sinoatrial node to the distal Purkinje fibers. Optical mapping of cardiac electrical activity using a voltage-sensitive dye confirms that cells identified by the lacZ reporter gene are indeed components of the specialized conduction system. Analysis of lacZ expression during sequential stages of cardiogenesis provides a detailed view of the maturation of the conductive network and demonstrates that patterning occurs surprisingly early in embryogenesis. Moreover, optical mapping studies of embryonic hearts demonstrate that a murine His-Purkinje system is functioning well before septation has completed. Thus, these studies describe a novel marker of the murine cardiac conduction system that identifies this specialized network of cells throughout cardiac development. Analysis of lacZ expression and optical mapping data highlight important differences between murine and avian conduction system development. Finally, this line of transgenic mice provides a novel tool for exploring the molecular circuitry controlling mammalian conduction system development and should be invaluable in studies of developmental mutants with potential structural or functional conduction system defects.

Fig. 1

LacZ expression in the CCS of neonatal hearts. (A) Low magnification view of lacZ expression within the heart, which delineates components of the entire cardiac conduction system. (B) Higher magnification of the region of the His bundle (H) and bundle branches. Several fibers split off from the His bundle and travel along the right side of the interventricular septum (IVS) giving rise to the right bundle branch (RBB), which is out of the plane of focus. The termination of the His bundle gives rise to the fibers of the left bundle branch (LBB), which has a characteristic fan-shaped appearance. Fibers coursing directly from left atrium (LA) to left ventricle (LV) are indicated (arrowhead). (C) Higher magnification of the extensive Purkinje fiber network within the LV. (D–F) Analysis of Eosin-stained sections demonstrates preferential transgene expression within specific regions of the right atrium (RA), including the SA node (SAN), the right (R) and left (L) venous valves and the AV node (AVN). Ventricular transgene expression delineated the His bundle, located beneath the tricuspid annulus, and the bundle branches. M, mitral valve; T, tricuspid valve. Scale bar for D–F is shown.

Fig. 2

Dual imaging of electrical activity and lacZ expression in the RBB. Maps of electrical activity along the right septal surface that correspond to activation of the RBB were recorded in two CCS-lacZ transgenic hearts. The color-coded scheme in each panel describes progressive activation of the RBB, red cells depolarizing first and purple cells depolarizing either (A) 3.9 milliseconds or (D) 4.2 milliseconds later. The general direction of impulse propagation in both panels is from the AV nodal region (upper left corner, out of the field of view) towards the apex of the heart (lower right corner, out of the field of view). Additionally in D, branches can be seen coursing posteriorly toward a papillary muscle (to the left of the panel), as well as towards the anterior surface of the septum. (B,E) Following optical mapping, hearts were reacted with X-gal to reveal the pattern of lacZ expressing cells. (C,F) Merged image of electrical activity and lacZ expression in individuals hearts reveals overlapping patterns.

Fig. 3

CCS-lacZ expression delineates progressive maturation of the conduction network within the embryonic heart. To visualize formation and patterning of the CCS, β - galactosidase activity in CCS-lacZ transgenic embryos was analyzed at several developmental stages. (A) Initiation of expression within the heart occurred at 8.5 d.p.c. predominantly in CCS precursors along the dorsal wall of the AV canal. (B) By 9.5 d.p.c., expression could now be seen in precursors of the SA node (SAN) within the right sinus horn (RSH) as well as in precursors of the ventricular CCS located in the region between the undivided left and right ventricles. At later stages, hearts were dissected from the embryos following the X-gal reaction and chemically treated to better visualize the pattern of conduction cells. The labeled illustration in (D) corresponds to the 10.5 d.p.c. heart shown in (C), where the transparent outflow tract (OFT) cannot be visualized. At 10.5 d.p.c., the location of the CCS is similar to 9.5 d.p.c.; however, discrete fibers within the ventricles can now clearly be seen, as well as a group of cells along the right AV canal (AVC), where the developing AV node (AVN) is located. (E) An Eosinstained section through the ventricular region of a 10.5 d.p.c. embryo revealed that the location of trabecular CCS cells is predominantly subendocardial. (F) ln the 12.5 d.p.c. heart, the SA node and the presumptive SA ring, a bundle in the posterior right atrial wall leading towards the AV node, were detected. The AV node, located in the posterior AV canal, was continuous with the His bundle and bundle branches developing on top and astride the budding IVS. Components of the AV ring (AVR) were also detected. (G) The CCS of the 13.5 d.p.c. heart appears nearly mature, including the distal Purkinje networks within right and left ventricles. Fibers coursing from LA to LV are indicated (arrowhead). RA, right atrium; LA, left atrium.

Fig. 4

Functional maturation of the murine CCS. Maps of electrical activity viewed from the anteroapical surface of developing hearts were recorded. Representative activation maps and X-gal stained images from (A) 10.5 d.p.c. and (B) 11.5 d.p.c. embryos are shown, with isochrones drawn every 0.5 millisecond. Activation maps from hearts with double breakthroughs, with the right ventricle always preceding the left ventricle, are shown at (C) 11.5 d.p.c., (D) 12.5 d.p.c. and (E) 15.5 d.p.c. The second breakthrough is indicated (asterisk). Isochrones are drawn every 0.25 millisecond.