YIA1 TBX18 Biopacemaking Improves Beating Rate and Alters Gene Expression in Bradycardic Subsidiary Right Atrial Pacemaker Tissue

Abstract

Introduction Biopacemaker research aims to faithfully recapitulate robust pacemaking seen from the heart's natural pacemaker, the sinoatrial node (SAN). The transcription factor Tbx18, involved in the development of the SAN core, has previously been used to reprogramme adult ventricular myocytes into SAN-like myocytes. Subsidiary atrial pacemaker (SAP) tissue in the infero-posterior wall of the right atrium has been shown to share molecular characteristics with the SAN but is bradycardic functionally. We therefore used this SAP tissue as a model for SAN dysfunction and hypothesised that overexpressing Tbx18 in this region ectopically would increase beating rate and alter key pacemaker genes. Methods We isolated beating SAN and SAP tissues from rat right atria in vitro . We compared SAN tissue (n = 6), uninfected SAP tissue (n = 8) and SAP tissue infected with the recombinant adenovirus Ad-Tbx18 (n = 7). Beating rates were monitored during 48 h of tissue culture. We then measured mRNA levels of 14 key genes using qPCR in Ad-Tbx18 infected (n = 8) and uninfected (n = 8) SAP tissue. Data were analysed using one-way ANOVA. Results After 48 h, the mean beating rate of Ad-Tbx18 infected SAP tissue was significantly higher than uninfected SAP tissue (Ad-Tbx18: 194 ± 15.6 bpm, uninfected SAP: 142 ± 8.2 bpm; p 0.05). qPCR showed a 99.7-fold increase in Tbx18 mRNA as a result of Ad-Tbx18 infection (p < 0.001). HCN1, HCN2, RYR2, and Kv1.5 levels were significantly higher in Ad-Tbx18 infected SAP tissue than uninfected SAP tissue (p < 0.01), however HCN4 was significantly lower (p < 0.01). No significant changes were seen with Cx43, Cx45, Kir2.1, Nav1.5, NCX1, Cav1.2, Cav3.1 or Tbx3. Implications Ad-Tbx18 infection overexpressed Tbx18 in SAP tissue and increased beating rate in vitro . This led to HCN4 downregulation, but HCN1, HCN2 and RYR2 were all upregulated and this could explain the observed increase in rate. Our data raises interesting questions about a potential application of Tbx18 upregulation to the dysfunctional SAN as a biopacemaking strategy in sick sinus syndrome. ![Abstract YIA1 Figure 1][1] Abstract YIA1 Figure 1 Beating rates (mean ±SEM) were monitored between 20 and 48 h of tissue culture for isolatedsinoatrial node tissue (n = 6), uninfected inferior subsidiary pacemaker tissue(n = 8) and inferior subsidiary pacemaker tissue infected with the adenovirusAd-Tbx18 (n = 7). Rates at 48 h were significantly higher in Ad-Tbx18 infectedpreparations than uninfected preparations (p < 0.01). RT-qPCR was used toquantify mRNA for key pacemaker genes isolated after 48 h of tissue culturefrom uninfected subsidiary pacemaker tissue (n = 8) and subsidiary pacemakertissue infected with Ad-Tbx18 (n = 8). HCN1, HCN2, RYR2 and Kv1.5 were upregulatedbut HCN4 was downregulated (p < 0.05) [1]: pending:yes