Left ventricular non-compaction (LVNC) is a rare cardiomyopathy associated with a hypertrabeculated phenotype and a large spectrum of symptoms. It is still unclear whether LVNC results from a defect of ventricular trabeculae development and the mechanistic basis that underlies the varying severity of this pathology is unknown. To investigate these issues, we inactivated the cardiac transcription factor Nkx2-5 in trabecular myocardium at different stages of trabecular morphogenesis using an inducible Cx40-creERT2 allele. Conditional deletion of Nkx2-5 at embryonic stages, during trabecular formation, provokes a severe hypertrabeculated phenotype associated with subendocardial fibrosis and Purkinje fiber hypoplasia. A milder phenotype was observed after Nkx2-5 deletion at fetal stages, during trabecular compaction. A longitudinal study of cardiac function in adult Nkx2-5 conditional mutant mice demonstrates that excessive trabeculation is associated with complex ventricular conduction defects, progressively leading to strain defects, and, in 50% of mutant mice, to heart failure. Progressive impaired cardiac function correlates with conduction and strain defects independently of the degree of hypertrabeculation. Transcriptomic analysis of molecular pathways reflects myocardial remodeling with a larger number of differentially expressed genes in the severe versus mild phenotype and identifies Six1 as being upregulated in hypertrabeculated hearts. Our results provide insights into the etiology of LVNC and link its pathogenicity with compromised trabecular development including compaction defects and ventricular conduction system hypoplasia. During fetal heart morphogenesis, formation of the mature ventricular wall requires coordinated compaction of the inner trabecular layer and growth of the outer layer of myocardium. Arrested trabecular development has been implicated in the pathogenesis of hypertrabeculation associated with ventricular non-compaction cardiomyopathy. However much uncertainty still exists among clinicians concerning the physiopathology of ventricular non-compaction cardiomyopathy, including its clinical characteristics, prognosis, classification and even the definition of hypertrabeculation. In particular, distinguishing between pathological and non-pathological subtypes of non-compaction is currently a major issue. Here we show that deletion of the gene encoding the transcription factor Nkx2-5 at critical steps during trabecular development recapitulates pathological features of hypertrabeculation, providing the first model of ventricular non-compaction cardiomyopathy in adult mice. We demonstrate that excessive trabeculation due to failure of trabecular compaction during fetal development is associated with Purkinje fiber hypoplasia and subendocardial fibrosis. Longitudinal functional studies reveal that these mice present all the clinical signs of symptomatic left ventricular non-compaction cardiomyopathy, including conduction defects, strain defects and progressive heart failure. Our results, including transcriptomic analysis, suggest that pathological features of non-compaction are primarily developmental defects. This study clarifies the origin of the pathological outcomes associated with LVNC and may provide helpful information for clinicians concerning the etiology of this rare cardiomyopathy.

A small network of spontaneously active Tbx3⁺ cardiomyocytes forms the cardiac conduction system (CCS) of the heart. Understanding the origin and mechanism of development of the CCS network are important steps towards disease modeling and biological pacemaker development to treat arrhythmias. We found that Tbx3 expression already in the embryo associated with automaticity. Genetic inducible fate mapping revealed that Tbx3⁺ cells in the early heart tube are fated to form the definitive CCS components, except the Purkinje fiber network. At mid-fetal stages contribution of Tbx3⁺ cells was restricted to the definitive CCS. We identified a Tbx3⁺ population in the outflow tract of the early heart tube that formed the atrioventricular bundle. While Tbx3⁺ cardiomyocytes also contributed to the adjacent Gja5⁺ atrial and ventricular chamber myocardium, embryonic Gja5⁺ chamber cardiomyocytes did not contribute to the Tbx3⁺ sinus node or atrioventricular ring bundles. In conclusion, the CCS is established by progressive fate-restriction of a Tbx3⁺ cell population in the early developing heart, and implicates Tbx3 as a useful tool to develop strategies to study and treat CCS diseases.

The ventricular conduction system coordinates heartbeats by rapid propagation of electrical activity through the Purkinje fiber (PF) network. PFs share common progenitors with contractile cardiomyocytes, yet the mechanisms of segregation and network morphogenesis are poorly understood. Here, we apply genetic fate mapping and temporal clonal analysis to identify murine cardiomyocytes committed to the PF lineage as early as E7.5. We find that a polyclonal PF network emerges by progressive recruitment of conductive precursors to this scaffold from a pool of bipotent progenitors. At late fetal stages, the segregation of conductive cells increases during a phase of rapid recruitment to build the definitive PF network through a non-cell autonomous mechanism. We also show that PF differentiation is impaired in Nkx2-5 haploinsufficient embryos leading to failure to extend the scaffold. In particular, late fetal recruitment fails, resulting in PF hypoplasia and persistence of bipotent progenitors. Our results identify how transcription factor dosage regulates cell fate divergence during distinct phases of PF network morphogenesis.

The rapid propagation of electrical activity through the ventricular conduction system (VCS) controls spatiotemporal contraction of the ventricles. Cardiac conduction defects or arrhythmias in humans are often associated with mutations in key cardiac transcription factors that have been shown to play important roles in VCS morphogenesis in mice. Understanding of the mechanisms of VCS development is thus crucial to decipher the etiology of conduction disturbances in adults. During embryogenesis, the VCS, consisting of the His bundle, bundle branches, and the distal Purkinje network, originates from two independent progenitor populations in the primary ring and the ventricular trabeculae. Differentiation into fast-conducting cardiomyocytes occurs progressively as ventricles develop to form a unique electrical pathway at late fetal stages. The objectives of this review are to highlight the structure–function relationship between VCS morphogenesis and conduction defects and to discuss recent data on the origin and development of the VCS with a focus on the distal Purkinje fiber network.

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.