Genomic and physiological analyses of the zebrafish atrioventricular canal reveal molecular building blocks of the secondary pacemaker region

Abstract

The atrioventricular canal (AVC) is the site where key structures responsible for functional division between heart regions are established, most importantly, the atrioventricular (AV) conduction system and cardiac valves. To elucidate the mechanism underlying AVC development and function, we utilized transgenic zebrafish line sqet31Et expressing EGFP in the AVC to isolate this cell population and profile its transcriptome at 48 and 72 hpf. The zebrafish AVC transcriptome exhibits hallmarks of mammalian AV node, including the expression of genes implicated in its development and those encoding connexins forming low conductance gap junctions. Transcriptome analysis uncovered protein-coding and noncoding transcripts enriched in AVC, which have not been previously associated with this structure, as well as dynamic expression of epithelial-to-mesenchymal transition markers and components of TGF-β, Notch, and Wnt signaling pathways likely reflecting ongoing AVC and valve development. Using transgenic line Tg(myl7:mermaid) encoding voltage-sensitive fluorescent protein, we show that abolishing the pacemaker-containing sinoatrial ring (SAR) through Isl1 loss of function resulted in spontaneous activation in the AVC region, suggesting that it possesses inherent automaticity although insufficient to replace the SAR. The SAR and AVC transcriptomes express partially overlapping species of ion channels and gap junction proteins, reflecting their distinct roles. Besides identifying conserved aspects between zebrafish and mammalian conduction systems, our results established molecular hallmarks of the developing AVC which underlies its role in structural and electrophysiological separation between heart chambers. This data constitutes a valuable resource for studying AVC development and function, and identification of novel candidate genes implicated in these processes.

Drivers of Sinoatrial Node Automaticity in Zebrafish: Comparison With Mechanisms of Mammalian Pacemaker Function

“…The function and genetics of the zebrafish SAN is similar to that of human. In zebrafish, the SAN is found in a ring-like structure at the venous pole of the heart (the border between the venous sinus and the atrium), embedded within the leaflets of the sinoatrial valve ( Arrenberg et al, 2010 ; Tessadori et al, 2012 ; Stoyek et al, 2015 , 2016 ; Abu Nahia et al, 2021 ). Its development parallels that of the mammalian SAN, occurring from isl1 -, tbx -, bmp4 -, and hcn4 -expressing cells under the control of shox2 and facilitated by the suppression of nkx2.5 through Wnt signalling ( Burkhard et al, 2017 ; Martin and Waxman, 2021 ; Minhas et al, 2021 ).…”

ERG potassium channels and T‐type calcium channels contribute to the pacemaker and atrioventricular conduction in zebrafish larvae

“…Since the AV node also possesses automaticity, inactivation of Ca v 3.1 channels in mice was found to slow AV node pacemaking and to produce an erratic HR 19 . In zebrafish larvae, transcripts encoding for TTCCs ( cacna1g ) are enriched in the SA ring and AV canal 23,24 . Here, alterations in the AV conduction with dose‐dependent severity were induced by blocking TTCCs in 3 dpf larvae: second‐degree AV blocks of Mobitz type 1, and 2:1 AV block (Figures 3 and 4).…”

“…19 In zebrafish larvae, transcripts encoding for TTCCs (cacna1g) are enriched the SA ring and AV canal. 23,24 Here, alterations in the AV conduction with dose-dependent severity were induced by blocking TTCCs in 3 dpf larvae: seconddegree AV blocks of Mobitz type 1, and 2:1 AV block (Figures 3 and 4).…”

“…In mammals, Ca v 3.1 (T-type) and Ca v 1.3 channels are involved in automaticity of the SAN and conduction in the AV node, [17][18][19][20] and alterations of these channels have been associated with congenital bradycardia and heart block disease in humans. 21,22 In zebrafish larvae, Ca v 3.1 (cacna1g) transcripts encoding TTCCs are enriched in the SA ring and AV canal, 23,24 although their functional relevance is yet unknown. TTCCs are also highly expressed in zebrafish atrial and ventricular myocytes, 5,8,25 in contrast with the adult human heart, in which LTCCs (Ca v 1.2 ∝subunit) represent 96%-98% of the transcripts, 8 although Ca v 3.1 channel protein has recently been detected in working cardiomyocytes from mice.…”

POPDC1 Variants Cause Atrioventricular Node Dysfunction and Arrhythmogenic Changes in Cardiac Electrophysiology and Intracellular Calcium Handling in Zebrafish

“…In vivo functional imaging of V m or Ca 2+ in zebrafish larvae was performed as previously described [ 41 ]. Larvae were epi-illuminated with a mercury arc lamp (U-HGLPS, Olympus, Tokyo, Japan) through a compound upright fluorescent microscope (BX63, Olympus) with a 0.80 NA, 26.5 FN, 40×, infinity-corrected water immersion objective (LUMPlanFLN, Olympus).…”