Segregation of Central Ventricular Conduction System Lineages in Early SMA+ Cardiomyocytes Occurs Prior to Heart Tube Formation.

The cardiac conduction system (CCS) transmits electrical activity from the atria to the ventricles to coordinate heartbeats. Atrioventricular conduction diseases are often associated with defects in the central ventricular conduction system comprising the atrioventricular bundle (AVB) and right and left branches (BBs). Conducting and contractile working myocytes share common cardiomyogenic progenitors, however the time at which the CCS lineage becomes specified is unclear. In order to study the fate and the contribution to the CCS of cardiomyocytes during early heart tube formation, we performed a genetic lineage analysis using a Sma-CreERT2 mouse line. Lineage tracing experiments reveal a sequential contribution of early Sma expressing cardiomyocytes to different cardiac compartments, labeling at embryonic day (E) 7.5 giving rise to the interventricular septum and apical left ventricular myocardium. Early Sma expressing cardiomyocytes contribute to the AVB, BBs and left ventricular Purkinje fibers. Clonal analysis using the R26-confetti reporter mouse crossed with Sma-CreERT2 demonstrates that early Sma expressing cardiomyocytes include cells exclusively fated to give rise to the AVB. In contrast, lineage segregation is still ongoing for the BBs at E7.5. Overall this study highlights the early segregation of the central ventricular conduction system lineage within cardiomyocytes at the onset of heart tube formation.

Resolving cell lineage contributions to the ventricular conduction system with a Cx40-GFP allele: a dual contribution of the first and second heart fields.

BACKGROUND: The ventricular conduction system (VCS) coordinates the heartbeat and is composed of central components (the atrioventricular node, bundle, and right and left bundle branches) and a peripheral Purkinje fiber network. Conductive myocytes develop from common progenitor cells with working myocytes in a bimodal process of lineage restriction followed by limited outgrowth. The lineage relationship between progenitor cells giving rise to different components of the VCS is unclear. RESULTS: Cell lineage contributions to different components of the VCS were analysed by a combination of retrospective clonal analysis, regionalized transgene expression studies, and genetic tracing experiments using Connexin40-GFP mice that precisely delineate the VCS. Analysis of a library of hearts containing rare large clusters of clonally related myocytes identifies two VCS lineages encompassing either the right Purkinje fiber network or left bundle branch. Both lineages contribute to the atrioventricular bundle and right bundle branch that segregate early from working myocytes. Right and left VCS lineages share the transcriptional program of the respective ventricular working myocytes and genetic tracing experiments discount alternate progenitor cell contributions to the VCS. CONCLUSIONS: The mammalian VCS is comprised of cells derived from two lineages, supporting a dual contribution of first and second heart field progenitor cells.

Establishment of the mouse ventricular conduction system.

The ventricular conduction system represents the electrical wiring responsible for the co-ordination of cardiac contraction. Defects in the circuit produce a delay or conduction block and induce cardiac arrhythmias. Understanding how this circuit forms and identification of the factors important for its development thus provide insights into the origin of cardiac arrhythmias. Recent advances, using genetically modified mouse models, have contributed to our understanding of how the ventricular conduction system is established during heart development. Transgenic mice carrying different reporter genes have highlighted the conservation of the anatomy and development of the ventricular conduction system between mice and humans. Lineage tracing and retrospective clonal analysis have established the myogenic origin of the ventricular conduction system and determined properties of conductive progenitor cells. Finally, gene knock-out models reproducing human cardiac defects have led to the identification of transcription factors important for the development of the ventricular conduction system. These transcription factors operate at the levels of both conduction system morphogenesis and differentiation by controlling the expression of genes responsible for the electrical activity of the heart. In summary, defects in the ventricular conduction system are a major cause of arrhythmias, and deciphering the molecular pathways responsible for conduction system morphogenesis and the differentiation of conductive myocytes furthers our understanding of the mechanisms underlying heart disease.

Inducible Cx40-Cre expression in the cardiac conduction system and arterial endothelial cells.

The Connexin-40 (Cx40) gene encodes a gap junction protein that plays an important role in cell-cell communication in cardiomyocytes of the atria and cardiac conduction system and endothelial cells of large arteries. During embryonic development, Cx40 expression is tightly regulated and correlates with progressive ventricular conduction system (VCS) differentiation and vessel function. We have generated Cx40(Cre) mice carrying a CreERT2-IRESmRFP cassette by targeted recombination. In Cx40(Cre) mice, the pattern of expression of RFP is identical to that of the endogenous Cx40 gene and a Cx40(GFP) allele. Using a LacZ-based Cre reporter mouse line, tamoxifen dependent Cre recombination was observed throughout the spatio-temporal profile of Cx40 expression in the VCS and arterial endothelial cells. Cx40(Cre) mice can therefore be used to direct inducible genetic modification in Cx40 expressing cells.

Biphasic development of the mammalian ventricular conduction system.

RATIONALE: The ventricular conduction system controls the propagation of electric activity through the heart to coordinate cardiac contraction. This system is composed of specialized cardiomyocytes organized in defined structures including central components and a peripheral Purkinje fiber network. How the mammalian ventricular conduction system is established during development remains controversial. OBJECTIVE: To define the lineage relationship between cells of the murine ventricular conduction system and surrounding working myocytes. METHODS AND RESULTS: A retrospective clonal analysis using the alpha-cardiac actin(nlaacZ/+) mouse line was carried out in three week old hearts. Clusters of clonally related myocytes were screened for conductive cells using connexin40-driven enhanced green fluorescent protein expression. Two classes of clusters containing conductive cells were obtained. Mixed clusters, composed of conductive and working myocytes, reveal that both cell types develop from common progenitor cells, whereas smaller unmixed clusters, composed exclusively of conductive cells, show that proliferation continues after lineage restriction to the conduction system lineage. Differences in the working component of mixed clusters between the right and left ventricles reveal distinct progenitor cell histories in these cardiac compartments. These results are supported by genetic fate mapping using Cre recombinase revealing progressive restriction of connexin40-positive myocytes to a conductive fate. CONCLUSIONS: A biphasic mode of development, lineage restriction followed by limited outgrowth, underlies establishment of the mammalian ventricular conduction system.