TRANSCRIPTOMIC HETEROGENEITY OF THE POSTNATAL ATRIOVENTRICULAR CONDUCTION SYSTEM AT A SINGLE-CELL RESOLUTION

Abstract

BACKGROUND The atrioventricular conduction system (AVCS) is a network of specialized cardiomyocytes responsible for coordinated spreading of electrical impulses throughout the ventricles to allow synchronized contractions. Specifically, it is composed of the AV node (AVN), which delays the electrical signal, and the ventricular conduction system (VCS) (i.e. His bundle, bundle branches and Purkinje fibers), which rapidly propagates the signal throughout the ventricles. Different AVCS regions possess different electrophysiological properties, such as conduction velocity. Overlapping and nonoverlapping expression patterns of some key developmental transcription factors (TFs), including Irx3, Tbx3, Etv1 and Nkx2.5 within the AVCS, suggest heterogeneous gene expression profiles. Although these structures of the AVCS were discovered >100 years ago, our understanding about its molecular constituents remains largely limited. METHODS AND RESULTS AVCS cells, which make up ∼0.05% of myocytes (∼500 cells/1 million cardiomyocytes), were isolated from early postnatal hearts (P1 to P4) of fluorescent reporter mice (Cntn2-Cre;Rosa26TdTomato), purified using fluorescence-activated cell sorting, and were subjected to single-cell RNA-sequencing. Unbiased cluster analysis on ∼7000 AVCS cells demonstrated distinct transcriptomic profiles in the AVN, proximal-VCS (i.e. His bundle and bundle branches) and distal-VCS (i.e. Purkinje fibers). Conforming to previous studies, well-known conduction markers, such as Etv1 and Kcne1, were present throughout the entire AVCS, whereas a VCS-specific TF, Irx3, was expressed throughout only the VCS. Additionally, Tbx3, Pcp4, Nppa and Lyz2 were enriched in the proximal, not distal, VCS. Sub-clustering analysis of AVN and proximal-VCS further identified unique expression patterns of molecular markers, such as Shox2 (exclusively in compact AVN) and Rspo3 (likely in lower nodal region connecting AVN and proximal-VCS). Moreover, we uncovered a potential regulatory relationship between Irx3 and Tbx3 in mediating their downstream targets, gap junction Gja5 (Cx40) and Gja1 (Cx43). In Irx3- and Tbx3-positive proximal-VCS, Gja5 and Gja1 expressions were lower compared to Irx3-positive/Tbx3-negative distal-VCS. In only Irx3-negative/Tbx3-positive AVN, both Gja5 and Gja1 expressions were undetected. Importantly, this regulatory relationship was recapitulated in cultured neonatal mouse atrial and ventricular myocytes overexpressing Irx3 and/or Tbx3. We also identified a cluster of cells which co-expressed conductive genes (i.e. Irx3, Kcne1, Gja5, Etv1) as well as G2/M cell cycle genes (i.e. Birc5, Ube2c, Cdk1, Ccnb1/2), suggesting the presence of committed conduction cells undergoing proliferation after birth. CONCLUSION Our study provides a comprehensive view of the cellular and molecular heterogeneity within the postnatal AVCS at a high-resolution and identifies potential novel markers with unique and overlapping transcriptomic patterns. The atrioventricular conduction system (AVCS) is a network of specialized cardiomyocytes responsible for coordinated spreading of electrical impulses throughout the ventricles to allow synchronized contractions. Specifically, it is composed of the AV node (AVN), which delays the electrical signal, and the ventricular conduction system (VCS) (i.e. His bundle, bundle branches and Purkinje fibers), which rapidly propagates the signal throughout the ventricles. Different AVCS regions possess different electrophysiological properties, such as conduction velocity. Overlapping and nonoverlapping expression patterns of some key developmental transcription factors (TFs), including Irx3, Tbx3, Etv1 and Nkx2.5 within the AVCS, suggest heterogeneous gene expression profiles. Although these structures of the AVCS were discovered >100 years ago, our understanding about its molecular constituents remains largely limited. AVCS cells, which make up ∼0.05% of myocytes (∼500 cells/1 million cardiomyocytes), were isolated from early postnatal hearts (P1 to P4) of fluorescent reporter mice (Cntn2-Cre;Rosa26TdTomato), purified using fluorescence-activated cell sorting, and were subjected to single-cell RNA-sequencing. Unbiased cluster analysis on ∼7000 AVCS cells demonstrated distinct transcriptomic profiles in the AVN, proximal-VCS (i.e. His bundle and bundle branches) and distal-VCS (i.e. Purkinje fibers). Conforming to previous studies, well-known conduction markers, such as Etv1 and Kcne1, were present throughout the entire AVCS, whereas a VCS-specific TF, Irx3, was expressed throughout only the VCS. Additionally, Tbx3, Pcp4, Nppa and Lyz2 were enriched in the proximal, not distal, VCS. Sub-clustering analysis of AVN and proximal-VCS further identified unique expression patterns of molecular markers, such as Shox2 (exclusively in compact AVN) and Rspo3 (likely in lower nodal region connecting AVN and proximal-VCS). Moreover, we uncovered a potential regulatory relationship between Irx3 and Tbx3 in mediating their downstream targets, gap junction Gja5 (Cx40) and Gja1 (Cx43). In Irx3- and Tbx3-positive proximal-VCS, Gja5 and Gja1 expressions were lower compared to Irx3-positive/Tbx3-negative distal-VCS. In only Irx3-negative/Tbx3-positive AVN, both Gja5 and Gja1 expressions were undetected. Importantly, this regulatory relationship was recapitulated in cultured neonatal mouse atrial and ventricular myocytes overexpressing Irx3 and/or Tbx3. We also identified a cluster of cells which co-expressed conductive genes (i.e. Irx3, Kcne1, Gja5, Etv1) as well as G2/M cell cycle genes (i.e. Birc5, Ube2c, Cdk1, Ccnb1/2), suggesting the presence of committed conduction cells undergoing proliferation after birth. Our study provides a comprehensive view of the cellular and molecular heterogeneity within the postnatal AVCS at a high-resolution and identifies potential novel markers with unique and overlapping transcriptomic patterns.