Transcriptome profile of the sinoatrial ring reveals conserved and novel genetic programs of the zebrafish pacemaker

Abstract

Background Sinoatrial Node (SAN) is part of the cardiac conduction system, which controls the rhythmic contraction of the vertebrate heart. The SAN consists of a specialized pacemaker cell population that has the potential to generate electrical impulses. Although the SAN pacemaker has been extensively studied in mammalian and teleost models, including the zebrafish, their molecular nature remains inadequately comprehended. Results To characterize the molecular profile of the zebrafish sinoatrial ring (SAR) and elucidate the mechanism of pacemaker function, we utilized the transgenic line sqet33mi59BEt to isolate cells of the SAR of developing zebrafish embryos and profiled their transcriptome. Our analyses identified novel candidate genes and well-known conserved signaling pathways involved in pacemaker development. We show that, compared to the rest of the heart, the zebrafish SAR overexpresses several mammalian SAN pacemaker signature genes, which include hcn4 as well as those encoding calcium- and potassium-gated channels. Moreover, genes encoding components of the BMP and Wnt signaling pathways, as well as members of the Tbx family, which have previously been implicated in pacemaker development, were also overexpressed in the SAR. Among SAR-overexpressed genes, 24 had human homologues implicated in 104 different ClinVar phenotype entries related to various forms of congenital heart diseases, which suggest the relevance of our transcriptomics resource to studying human heart conditions. Finally, functional analyses of three SAR-overexpressed genes, pard6a, prom2, and atp1a1a.2, uncovered their novel role in heart development and physiology. Conclusion Our results established conserved aspects between zebrafish and mammalian pacemaker function and revealed novel factors implicated in maintaining cardiac rhythm. The transcriptome data generated in this study represents a unique and valuable resource for the study of pacemaker function and associated heart diseases.

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

“…It has been shown that compared to other regions of the heart, the zebrafish SAN overexpresses several signature mammalian pacemaker genes, including those encoding HCN and Ca 2+ - and K + -gated channels ( von der Heyde et al, 2020 ; Minhas et al, 2021 ). In the context of SAN function, it has also been shown that knock down of the large-conductance Ca 2+ -activated K + channel (K Ca 1.1) in zebrafish, which is strongly expressed in the human SAN, results in sinus bradycardia ( Pineda et al, 2021 ), so it may play an important role in zebrafish SAN automaticity through its contribution to repolarisation.…”

“…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, 2015Stoyek et al, , 2016Abu 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). The first indication of the currents involved in zebrafish SAN automaticity came from the discovery of a mutation (slow mo) that caused a reduction in HR by affecting a hyperpolarisation-activated inward current with similar properties to I f (Baker et al, 1997;Warren et al, 2001).…”

RA signaling pathway combined with Wnt signaling pathway regulates human-induced pluripotent stem cells (hiPSCs) differentiation to sinus node-like cells

“…Many signaling pathways (including bone morphogenetic protein (BMP) signaling pathway, Wnt signaling pathway and Retinoic acid (RA) signaling) regulate embryonic sinoatrial node (SAN) development, which is instructive for generating biological pacemakers [ 1 – 4 ]. With the establishment of human-induced pluripotent stem cell (hiPSC) lines and the tracer technology, many scientists have imitated SAN development to induce sinus node-like cells in vitro by adding several small molecular substances [ 5 – 8 ].…”

Two HCN4 Channels Play Functional Roles in the Zebrafish Heart

“…To discriminate possible physiological functions and roles of DrHCN4 and DrHCN4L in the heart, we tried to suppress the expression of each gene. In the present study, we used antisense morpholino (MO) to knock down DrHCN4 and DrHCN4L genes because MO knockdown is commonly used in zebrafish ( Blum et al, 2015 ; Fujii et al, 2020 ; Minhas et al, 2021 ).…”