Figure 1. Tbx5/Tbx3 balance determines the regional functional output within the specialized mature CCS.

(A-C) Relative Tbx3/Tbx5 expression ratio in wild-type AVN and VCS cardiomyocytes isolated from adult mouse hearts. The qRT-PCR (A) and western blot (B) demonstrated that Tbx3 expression predominates over Tbx5 in the slow AV-nodal cardiomyocytes and that Tbx5 predominates over Tbx3 expression in the fast VCS cardiomyocytes. The AVN cardiomyocytes used in these studies were marked by EGFP expression and isolated from Tbx3BAC-Egfp mice (27) at 10 weeks of age. VCS cardiomyocytes, used in these studies were marked by EYFP expression using a VCS-specific tamoxifen (TM) inducible Cre transgenic mouse line (MinK^CreERT2) (14) in combination with conditional EYFP expression from the ROSA locus (R26^EYFP/+; MinK^CreERT2/+) (7) and microdissected at 10 weeks of age. (C) Model of Tbx3/Tbx5 expression ratio in cardiac conduction system. (D-G) VCS-specific Tbx3 haploinsufficiency in Tbx3:Tbx5 compound heterozygous mice (Tbx3^fl/+;Tbx5^fl/+;R26^EYFP/+;MinKCre^ERT2/+) rescues VCS defects caused by VCS-specific Tbx5 haploinsufficiency, in Tbx5 heterozygous mice (Tbx5^fl/+;R26^EYFP/+;MinK^CreERT2/+). PR (D, left graph), QRS (D, right graph) and H-V (E, right graph) intervals prolongation, widening of the His duration (E, middle graph), as well as ventricular arrhythmias including reproducible pacing into ventricular tachycardia (VT) (F), observed in VCS-specific Tbx5 heterozygous mice (Tbx5^fl/+;R26^EYFP/+; MinK^CreERT2/+), all got rescued in VCS-specific Tbx3:Tbx5 compound heterozygous mice (Tbx3^fl/+;Tbx5^fl/+;R26^EYFP/+; MinK^CreERT2/+). Control VCS-specific Tbx3 heterozygous mice (Tbx3^fl/+;R26^EYFP/+;MinK^CreERT2/+) showed neither conduction nor electrophysiological defects (D-F). Note, ventricular pacing at 100ms S1 drive train followed by 50ms S2 single extrastimulus induced VT in 5/9 Tbx5^fl/+;R26^EYFP/+; MinK^CreERT2/+mice (F, upper panel, representative image), but failed to induced VT in all Tbx3/Tbx5 compound Tbx3^fl/+;Tbx5^fl/+;R26^EYFP/+; MinK^CreERT2/+ mice (F, lower panel, representative image). (G) Summary of genetic interactions between Tbx5 and Tbx3 in the adult VCS. Data are presented as mean±SD. For (A), n=3 biological replicates/ analyzed tissue sample (AVN or VCS cardiomyocytes pooled from 30 mice per each biological replicate). (B), n=2 biological replicates/ analyzed tissue sample (AVN or VCS cardiomyocytes pooled from 50 mice per each biological replicate). For (D), n=4 animals/ genotype. For (E, F), n=4–9 animals/ genotype. For (A, B, D – QRP, F), Welch’s t-test; *P<0.05, ***P<0.01. For (D-PR, E), Mann-Whitney U test; *P<0.05. Abbreviations: PR, PR-interval duration; QRS, QRS complex, H-V, Hisio-Ventricular interval; Hd, His-duration.