Generation of VCS-specific Tbx3:Tbx5 double-conditional knockout mice.

(A) Strategy to generate VCS-specific Tbx3:Tbx5 double-conditional knockout mouse line. A new Tbx3:Tbx5 double-conditional knockout mouse line (Tbx3fl/fl;Tbx5fl/fl) was generated using the CRISPR-Cas9 system (31, 32) which allowed for the targeting ofTbx5 in the background of the previously validated Tbx3 floxed allele (26). A newly engineered Tbx5 floxed allele has been developed to mirror a previously published allele (20). This design has enabled the utilization of the previously published individual Tbx3 floxed allele (26) and individual Tbx5 floxed allele (20) as controls. To conditionally delete Tbx3 and Tbx5 genes specifically from the adult VCS and generate the experimental animals, the Tbx3:Tbx5 double-floxed mouse line (Tbx3fl/fl;Tbx5fl/fl) was combined with a VCS-specific tamoxifen inducible Cre transgenic mouse line (MinKCreERT2 [Tg(RP23-276I20-MinkCreERT2) (8). All allelic combinations were generated and evaluated as littermates in a mixed genetic background. The experimental mice employed in all studies were administered tamoxifen at 6 weeks of age and subsequently evaluated at 8 weeks of age (2 weeks post-tamoxifen administration). (B, C) The loss of Tbx3 and Tbx5 expression, on both the protein and mRNA levels, assessed by immunohistochemistry (B) and qPCR (C), respectively, was observed in the VCS of adult Tbx3:Tbx5 double-conditional mutant mice (Tbx3fl/fl;Tbx5fl/fl;R26EYFP/+;MinKCreERT2/+), but not in their littermate controls (Tbx3+/+;Tbx5+/+;R26EYFP/+;MinKCreERT2/+) (B and C). (C) QPCR analysis showed a partial loss of Tbx3 and Tbx5 expression in the adult VCS of Tbx3:Tbx5 double-conditional heterozygous mice (Tbx3fl/+;Tbx5fl/+;R26EYFP/+; MinKCreERT2/+) compared to their littermate controls (Tbx3+/+;Tbx5+/+;R26EYFP/+; MinKCreERT2/+). Additionally, qPCR analysis confirmed the specificity of the Tbx3:Tbx5 double knockout for the VCS by assessing Tbx3 and Tbx5 expression levels in the atria and ventricles of tamoxifen-treated experimental mice. Consistent with the VCS selectivity of Cre activity in the MinKCreERT2 mice (8), Tbx3 and Tbx5 expression remained similar in the atrial and ventricular myocardium across all allelic combinations, including Tbx3:Tbx5 double-conditional knockout mice (Tbx3fl/fl;Tbx5fl/fl;R26EYFP/+;MinKCreERT2/+). (D) Conducted longitudinal studies revealed a significantly increased mortality rate in VCS-specific Tbx3:Tbx5-deficient mice compared to their littermate controls (***P < 0.0001, log-rank test), suggesting a requirement for both Tbx3 and Tbx5 in the mature VCS. All allelic combinations of experimental and control mice (n=40 biological replicates/genotype) were followed longitudinally after tamoxifen administration at 6 weeks of age. Tbx3:Tbx5 double-conditional knockout mice began to die suddenly at 3 to 4 weeks post-tamoxifen administration. Within the 3 months post-tamoxifen administration, all tamoxifen-treated Tbx3fl/fl;Tbx5fl/fl;R26EYFP/+;MinKCreERT2/+ mice had died suddenly (n=40) without previous signs of illness. In contrast, no mortality was observed among the tamoxifen-treated Tbx3+/+;Tbx5+/+;R26EYFP/+;MinKCreERT2/+ and Tbx3fl/+;Tbx5fl/+;R26EYFP/+;MinKCreERT2/+ littermates (each cohort n=40) during this period. TBX3 and TBX5 protein expression was evaluated by immunohistochemistry (green and red signals, respectively) on serial sections from hearts of all allelic combinations (n=3 biological replicates/genotype). Nuclei were stained with DAPI (blue signal). IHC original magnification: 40x. QPCR data are presented as mean±SD normalized to Gapdh and relative to Tbx3+/+;Tbx5+/+;R26EYFP/+;MinKCreERT2/+ mice (set as 1). Welch t test: *P<0.05; **P<0.005; *** P<0.001; n=3 biological replicates/genotype (VCS cardiomyocytes pooled from 30 mice per each biological replicate); multiple testing correction using Benjamini & Hochberg procedure; *false discovery rate ≤0.05.

Arrhythmias and conduction abnormalities in mice with VCS-specific Tbx3:Tbx5 double-conditional knockout.

(A-F) VCS-specific Tbx3:Tbx5 double-conditional knockout causes significant VCS conduction slowing in adult Tbx3fl/fl;Tbx5fl/fl;R26EYFP/+;MinKCreERT2/+ mice. (A) PR (left graph) and QRS (right graph) intervals calculated from ambulatory telemetry ECG recordings in (B-F). Tbx3:Tbx5 double-conditional adult mice (Tbx3fl/fl;Tbx5fl/fl;R26EYFP/+;MinKCreERT2/+) displayed significant PR and QRS intervals prolongation compared to littermate controls (Tbx3+/+;Tbx5+/+;R26EYFP/+;MinKCreERT2/+) (A left and right graphs, respectively). Data are presented as mean±SD. Welch t test: *P<0.05; n=7 biological replicates/genotype, multiple testing correction using Benjamini & Hochberg procedure; *false discovery rate ≤0.05. (B-F) Representative ambulatory telemetry ECG of Tbx3+/+;Tbx5+/+; R26EYFP/+;MinKCreERT2/+ (B), Tbx3fl/+;Tbx5fl/+;R26EYFP/+;MinKCreERT2/+ (C), Tbx3fl/fl;Tbx5fl/fl; R26EYFP/+;MinKCreERT2/+ (D), Tbx3fl/fl;Tbx5+/+;R26EYFP/+;MinKCreERT2/+ (E), Tbx3+/+;Tbx5fl/fl; R26EYFP/+;MinKCreERT2/+ (F) mice. (G) Simultaneous genetic removal of Tbx3 and Tbx5 from the adult VCS resulted in significantly increased episodes of spontaneous ventricular tachycardia. Episodes of spontaneous ventricular tachycardia were observed in 4 of 7 Tbx3fl/fl;Tbx5fl/fl;R26EYFP/+;MinKCreERT2/+ mice versus 0 of 7 littermate controls (Tbx3+/+;Tbx5+/+;R26EYFP/+;MinKCreERT2/+) in ambulatory studies. Welch t test: *P<0.05; n=7 biological replicates/genotype; multiple testing correction using Benjamini & Hochberg procedure; *false discovery rate ≤0.05. (H) Tbx3:Tbx5 double-conditional knockout mice (Tbx3fl/fl;Tbx5fl/fl;R26EYFP/+;MinKCreERT2/+) showed significantly increased susceptibility to ventricular tachycardia following burst stimulation in invasive electrophysiology studies (3 of 3 Tbx3fl/fl;Tbx5fl/fl;MinKCreERT2/+ mice versus 0 of 7 control Tbx3+/+;Tbx5+/+;R26EYFP/+;MinKCreERT2/+.mice. Fisher’s exact test: *P<0.05; n=7 biological replicates/genotype). (I) Intracardiac electrophysiology detected no significant changes in SNRT, AERP, AVERP, and VERP recorded from experimental and control animals (Welch t test: *P<0.05; n=7 biological replicates/genotype; multiple testing correction using Benjamini & Hochberg procedure; *false discovery rate ≤0.05). (J) Graphical summary of conduction defects observed in adult, VCS-specific Tbx3:Tbx5-deficient mice. Simultaneous genetic deletion of Tbx3 and Tbx5 from the mature VCS results in conduction slowing, prolonged PR and QRS intervals, as well as ventricular tachycardia.

Cardiac function is preserved following double-conditional loss of Tbx3 and Tbx5 in the adult ventricular conduction system (VCS).

(A) Left ventricular (LV) fractional shortening (left graph) and left ventricular (LV) ejection fraction (right graph) calculated from the M-mode ECGs in (B-F) revealed no contractile dysfunction in VCS-specific Tbx3:Tbx5 double-conditional mutant mice (Tbx3fl/fl;Tbx5fl/fl;R26EYFP/+; MinKCreERT2/+). Data are presented as mean±SD. Welch t test: *P<0.05; ns, not significant; n=7 biological replicates/genotype. (B-F) Cardiac function, assessed by M-mode echocardiography from Tbx3+/+;Tbx5+/+;R26EYFP/+;MinKCreERT2/+ (B), Tbx3fl/+;Tbx5fl/+; R26EYFP/+;MinKCreERT2/+ (C), Tbx3fl/fl;Tbx5fl/fl;R26EYFP/+;MinKCreERT2/+ (D), Tbx3fl/fl;Tbx5+/+; R26EYFP/+;MinKCreERT2/+ (E), Tbx3+/+;Tbx5fl/fl;R26EYFP/+;MinKCreERT2/+ (F) mice shown above surface ECGs. No functional differences between mutant and control mice were detected. The most representative images for each genotype were utilized in the figure. n=7 biological replicates/genotype. (G-K) Histological examination of all four-chambers from Tbx3+/+;Tbx5+/+;R26EYFP/+;MinKCreERT2/+ (G), Tbx3fl/+;Tbx5fl/+;R26EYFP/+;MinKCreERT2/+ (H), Tbx3fl/fl;Tbx5fl/fl;R26EYFP/+;MinKCreERT2/+ (I), Tbx3fl/fl;Tbx5+/+;R26EYFP/+;MinKCreERT2/+ (J), Tbx3+/+;Tbx5fl/fl;R26EYFP/+;MinKCreERT2/+ (K) mice showed no histological abnormalities. The most representative images for each genotype were utilized in the figure. n=3-4 biological replicates/genotype. Boxed areas in (G-K) have been shown at higher magnification at their right sides. (L) qRT-PCR analysis for fibrosis genes Col1a1 and Postn confirmed that there was no increase in expression of fibrosis markers in the VCS of Tbx3:Tbx5-deficient mice. Data are presented as mean±SD normalized to Gapdh and relative to Tbx3+/+;Tbx5+/+;R26EYFP/+;MinKCreERT2/+ mice (set as 1). Welch t test: *P<0.05; ns, not significant; n=3 biological replicates/genotype (VCS cardiomyocytes pooled from 30 mice per each biological replicate). Histological examination original magnification: 2.5x, boxed area showed at the higher magnification: 40x.

In the adult murine heart, Tbx3 and Tbx5 cooperatively promote ventricular conduction system (VCS) versus working myocardium (WM) phenotype.

(A-C) qRT-PCR analysis of molecular changes driven by VCS-specific Tbx3:Tbx5 double-conditional knockout in adult mice. Transcriptional characterization of the adult VCS in Tbx3fl/fl;Tbx5fl/fl;R26EYFP/+;MinKCreERT2/+ mutant mice, compared to their Tbx3+/+;Tbx5+/+;R26EYFP/+;MinKCreERT2/+ control littermates, was conducted using three distinct sets of molecular markers. (A) Genes expressed throughout the entire conduction system (Pan-CCS), implicated in the slow-conducting nodal phenotype. (B) Genes highly expressed in the fast-conducting VCS, critical for VCS function. (C) Markers specifically present in the working myocardium but absent in the CCS. VCS-specific Tbx3:Tbx5-deficient mice lost the VCS expression profile, including genes necessary for fast ventricular conduction (B) and those typically expressed in the entire CCS (Pan-CCS genes), essential for the slow conducting nodal phenotype (A). In contrast, they acquired VCS expression of working myocardium-specific molecular markers important for working myocardial function (C). (D) Immunoblotting analysis confirmed transcriptional changes indicated by qRT-PCR analysis (A-C) in VCS-specific Tbx3:Tbx5 double-conditional knockout in adult mice. (E) Graphical summary of transcriptional changes observed in VCS of VCS-specific Tbx3:Tbx5-deficient mice. Simultaneous genetic deletion of Tbx3 and Tbx5 from the mature VCS resulted in a transcriptional profile resembling that of ventricular working myocardium. Data are presented as mean±SD normalized to Gapdh and relative to Tbx3+/+;Tbx5+/+;R26EYFP/+;MinKCreERT2/+ mice (set as 1). Welch t test: *P<0.05; *P<0.005; *P<0.0005; n=3 biological replicates/genotype (VCS cardiomyocytes pooled from 30 mice per each biological replicate).

Loss of VCS optical action potential (OAP) morphology in Tbx3:Tbx5 double-conditional knockout mice.(A) Schematic of the posterior view of a mouse heart with right ventricle (RV) free wall removed, highlight the RV, septum, His bundle, and right atria (RA). (B) Representative 100×100 OAP map of OAP recorded during sinus rhythm from Tbx3:Tbx5 double-conditional knockout mice and control littermates with the free wall removed. Rge region of the His bundle is highlighted in red. (C) Representative 10×10 OAP map from the region of the His bundle. (D) Representative 10×10 map of the first derivative of the OAP from the region of the His bundle. (E) Representative ventricular OAP from whole heart intact preparation from Tbx3:Tbx5 double-conditional knockout mice (red) and control littermates (black). (F) Quantification of APD50 and APD80 at a basic cycle length of 125 ms.

Ventricular optical action potentials (OAPs) distal from His bundle have only 1 OAP upstroke.

(A) Schematic of the posterior view of mouse heart with right ventricle (RV) free wall removed. (B) Representative 100×100 pixel OAP map recorded during sinus rhythm from Tbx3:Tbx5 double-conditional knockout mice and control littermates with RV free wall removed. The region of the working ventricular myocardium distal from the His bundle is highlighted in red. (C) Representative 10×10 pixel OAP map from the region distal to the His bundle. (D) Representative 10×10 dOAP/dt map from the region distal to the His bundle.

Tbx3 and Tbx5 play distinct roles in the adult VCS while cooperatively promoting CCS regional specification—a model elucidating our hypothesis for Tbx5/Tbx3 dose-dependent CCS regional specification.

(A) The Tbx3/Tbx5 balance not only governs nodal versus ventricular conduction system (VCS) function but also collaboratively promotes the VCS versus working myocardium (WM) phenotype. Specifically, a high level of Tbx3 is linked to the specification to the nodal conduction system, while an elevated Tbx5 level in nodal cells activates local expression of the Tbx5-dependent fast conduction network, resulting in the generation of VCS. (B) CCS regional specialization is driven by local expression of Tbx5-dependent fast conduction network in the VCS, which overlaps underlying Pan-CCS expression of nodal, slow conduction network. (C) VCS-specific simultaneous genetic removal of both the Tbx3 and Tbx5 transcription factors transforms the fast-conducting, adult VCS into cells resembling working myocardium, thereby re-patterning them from conduction to non-conduction myocytes. Therefore, within the adult CCS, the Tbx3 and Tbx5 expression levels are crucial not only for normal fast versus slow patterning but also for maintaining the conduction versus contraction specification of the VCS.AVB indicates atrioventricular bundle; AVN, atrioventricular node; CCS, cardiac conduction system; CKO, conditional KO; LBB, left bundle brunch; RBB, right bundle brunch; VCS, ventricular conduction system.