Transcriptome profile of the zebrafish atrioventricular canal reveals molecular signatures of pacemaker and valve mesenchyme

Abstract

The atrioventricular canal (AVC) is an essential feature of the heart, which separates the atrium from the ventricle. During heart morphogenesis, it is a hub of molecular processes necessary for distinguishing heart regions; most importantly, for the formation of the AV conduction system and cardiac valves. To better understand the molecular processes underlying AVC development and function, we utilized the transgenic zebrafish line sqet31Et with EGFP expression in the AVC region to isolate this cell population by FACS and profiled its transcriptome by RNA-seq at 48 and 72 hours post fertilization (hpf). Compared to the rest of the heart, the AVC is enriched for the expression of molecular markers associated with mammalian AVC and AV node, including cx36.7 and cx45 which encode connexins forming low conductance gap junctions. Using the transgenic line Tg(myl7:mermaid) encoding the voltage-sensitive fluorescent protein, we showed that loss of function of Isl1 abolished the pacemaker-containing sinoatrial ring (SAR) and resulted in an erratic spread of excitation pattern from the SAR to AVC, indicating the dysfunction of the primary pacemaker. Concurrently, ectopic excitation in the AVC region was observed, suggesting that the zebrafish AVC possesses inherent automaticity although insufficient to replace the primary pacemaking activity of the SAR. Comparisons between the SAR and AVC transcriptomes revealed partially overlapping expression profiles of various ion channels and gap junction proteins which reflects their diversified functions. Lastly, we observed dynamic expression of epithelial-to-mesenchymal transition markers, as well as components of TGF-{beta}, Notch, and Wnt signaling pathways, which have been implicated in the formation of AVC conduction and cardiac valves. Our results uncovered the molecular hallmarks of the developing AVC region and demonstrated its role in the structural and electrophysiological separation between the atrium and ventricle. Author summaryThe atrioventricular canal is a structure in the embryonic heart which separates the atrium from the ventricle. It gives rise to the AV node and cardiac valves - two important structures which ensure unidirectional blood flow between heart chambers. The AV node serves to regulate the propagation of electrical impulses between the two chambers, such that they contract consecutively. Using the zebrafish as model organism, we performed gene expression profiling and characterized electrical conduction patterns between the sinoatrial primary pacemaker and AVC. We discovered that the zebrafish AVC possesses similar features to the mammalian AV node, including slow conduction, inherent pacemaking activity, and the expression of conserved developmental genes. The molecular profile of the AVC is distinct from that of the sinoatrial pacemaker, which reflects their distinct roles. In addition, we found that genes regulating cardiac valve development were also expressed in the AVC, illustrating the importance of this region for establishing both electrophysiological and structural separation between the heart chambers. Besides establishing conserved aspects between zebrafish and mammalian conduction system, the data generated in this study constitutes a valuable resource for studying AVC development and discovery of novel candidate genes implicated in regulating cardiac rhythm and cardiac valve formation.