Coordinated Tbx3/Tbx5 transcriptional control of the adult ventricular conduction system

Figures

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  Figure 1.. 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 (Gurumurthy et al., 2021; Dow et al., 2015) which allowed for the targeting of Tbx5 in the background of the previously validated Tbx3 floxed allele (Frank et al., 2011). A newly engineered Tbx5 floxed allele has been developed to mirror a previously published allele (Bruneau et al., 2001). This design has enabled the utilization of the previously published individual Tbx3 floxed allele (Frank et al., 2011) and individual Tbx5 floxed allele (Bruneau et al., 2001) 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) Arnolds and Moskowitz, 2011b]). All allelic combinations were generated and evaluated as littermates in a mixed genetic background. The…

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  Figure 1—figure supplement 1.. Generation of a novel Tbx5 floxed allele in aTbx3 floxed background. (A) Strategy to generate a new Tbx5 floxed allele using the CRISPR/Cas9 system with long single-strand DNA donor (lssDNA donor) composed of the targeted exon 3 flanked by two lox2272 sites. The schematic shows the Cas9/sgRNA-targeting sites in the second and third introns of murine Tbx5, the lssDNA donor, and the targeted allele. (B) PCR analysis of Tbx5 and Tbx3 loci in G0 founders. Mice #35 to #41 are shown. Upper left panel: PCR genotyping of the Tbx5 locus using primers specific for the 5′ arm of the lssDNA donor with the lox2272 insertion. Upper right panel: PCR genotyping of the Tbx5 locus using primers specific for the 3′ arm of the lssDNA donor with the lox2272 insertion. Green labels indicate founders positive for lox2272 insertion in one arm. Red asterisks indicate founders positive for lox2272 insertion in both arms. Expected fragment sizes: for the 5′ arm with lox2272 insertion, positive band = 269 bp, WT = 229 bp; for the 3′ arm with insertion, positive band = 320 bp, WT = 280 bp. Lower panel: PCR genotyping of the Tbx3 locus using primers indicating Tbx3 floxed allele. Expected fragment sizes: Tbx3…

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  Figure 1—figure supplement 2.. Validation of the newly generated Tbx5 floxed allele. (A) Strategy to generate a Tbx5 null allele through recombination of the newly generated Tbx5 floxed allele in the presence of the Cre recombinase. (B) Strategy to generate germline Tbx5 null mice in presence of Mef2CCre allele. To verify the ability of the newly generated Tbx5 floxed allele to recombine into the Tbx5 KO (null) allele, three rounds of breeding were conducted (left panel), and a PCR assay (right panels) was utilized to distinguish the recombined Tbx5 null allele (Tbx5−) from the unrecombined floxed allele (Tbx5fl). Initially, homozygous Tbx5 floxed males (Tbx5fl/fl) were bred with homozygous Mef2CCre/Cre females, which express Cre recombinase at the zygote stage, to produce germline Tbx5+/−:Mef2Cre/+ double heterozygous mice. PCR analysis confirmed that 100% of the offspring were Tbx5+/−:Mef2CCre/+ double heterozygous mice (right upper and middle panels), demonstrating complete recombination of the Tbx5 floxed allele into the Tbx5 null allele in the presence of the Mef2CCre allele. Subsequently, Tbx5+/−:Mef2CCre/+ double heterozygous mice were backcrossed with wild-type CD1 IGS mice to obtain a germline Tbx5+/−…

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  Figure 1—figure supplement 3.. WT sequence of the targeted region at the mouse Tbx5 locus. The partial intron 2 (black, lowercase letters), entire exon 3 (black, bold, uppercase letters), and partial intron 3 (black, lowercase letters) are shown. The sgRNA-targeting sequence at the 5′ of exon 3 (Tbx5-I2-S22) is labeled in bold, light green, while the sgRNA-targeting sequence at the 3′ of exon 3 (Tbx5-I2-S31) is labeled in bold, dark green. The cleavage sites at the sgRNA-targeting sequences are underlined. The protospacer-adjacent motif (PAM) sequences are labeled in bold, light blue.

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  Figure 1—figure supplement 4.. Sequence of long ssDNA donor designed to target exon 3 of the mouse Tbx5 locus. It is composed of targeted exon 3 flanked by two lox2272 sites, along with 100/150 bp 5′/3′ homology arms. The partial intron 2 (black, lowercase letters), entire exon 3 (bold, black, uppercase letters), partial intron 3 (black, lowercase letters), as well as 5′ (purple, lowercase letters) and 3′ (pink, lowercase letters) homology arms are displayed. The lox2272 sites, indicated in bold red, have been inserted at the cleavage sites of the sgRNA-binding sequences. Unique restriction enzyme sites used for RFLP analysis are shown as bold yellow for EcoRI and bold navy blue for SphI. T7 promoter is labeled in bold orange.

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  Figure 1—figure supplement 5.. Sequence of mouse new Tbx5 floxed allele generated using CRISPR/Cas9 system with lssDNA composed of targeted exon 3 flanked by two lox2272 sites. The partial intron 2 (black, lowercase letters), entire exon 3 (bold, black, uppercase letters), and partial intron 3 (black, lowercase letters) are displayed. The lox2272 sites, indicated in bold red, have been inserted at the cleavage sites of the sgRNA-binding sequences. Unique restriction enzyme sites used for RFLP analysis are shown as bold yellow for EcoRI and bold navy blue for SphI. PCR primers specific for the 5′ arm of the lssDNA donor with the lox2272 insertion (displayed as green box) as well as primers specific for the 3′ arm of the lssDNA donor with the lox2272 insertion (displayed as pink box) are shown.

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  Figure 2.. 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;R26ReYFP/+;MinKCreERT2/+ mice. (A) PR (left graph) and QRS (right graph) intervals calculated from ambulatory telemetry electrocardiography (ECG) recordings in (B–F). Tbx3:Tbx5 double-conditional adult mice (Tbx3fl/fl;Tbx5fl/fl;R26ReYFP/+;MinKCreERT2/+) displayed significant PR and QRS intervals prolongation compared to littermate controls (Tbx3+/+;Tbx5+/+;R26ReYFP/+;MinKCreERT2/+) (A left and right graphs, respectively). Data are presented as mean ± SD. N = 7 biological replicates/genotype, multiple testing correction using Benjamini and Hochberg procedure; *Welch t-test p < 0.05 and FDR <0.05; **Welch t-test p < 0.005 and FDR <0.05; ***Welch t-test p < 0.001 and FDR <0.05. Representative ambulatory telemetry ECG of Tbx3+/+;Tbx5+/+; R26ReYFP/+;MinKCreERT2/+ (B), Tbx3fl/+;Tbx5fl/+;R26ReYFP/+;MinKCreERT2/+ (C), Tbx3fl/fl;Tbx5fl/fl; R26ReYFP/+;MinKCreERT2/+ (D), Tbx3fl/fl;Tbx5+/+;R26ReYFP/+;MinKCreERT2/+ (E), Tbx3+/+;Tbx5fl/fl;R26ReYFP/+;MinKCreERT2/+…

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  Figure 3.. 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 LV ejection fraction (right graph) calculated from the M-mode electrocardiographies (ECGs) in (B–F) revealed no contractile dysfunction in VCS-specific Tbx3:Tbx5 double-conditional mutant mice (Tbx3fl/fl;Tbx5fl/fl;R26ReYFP/+; MinKCreERT2/+). Data are presented as mean ± SD. Welch t-test: ns, not significant (p > 0.05) ; n = 7 biological replicates/genotype. (B–F) Cardiac function, assessed by M-mode echocardiography from Tbx3+/+;Tbx5+/+;R26ReYFP/+;MinKCreERT2/+ (B), Tbx3fl/+;Tbx5fl/+; R26ReYFP/+;MinKCreERT2/+ (C), Tbx3fl/fl;Tbx5fl/fl;R26ReYFP/+;MinKCreERT2/+ (D), Tbx3fl/fl;Tbx5+/+; R26ReYFP/+;MinKCreERT2/+ (E), Tbx3+/+;Tbx5fl/fl;R26ReYFP/+;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+/+;R26ReYFP/+;MinKCreERT2/+ (G),…

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  Figure 4.. In the adult murine heart, Tbx3 and Tbx5 collectively 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;R26ReYFP/+;MinKCreERT2/+ mutant mice, compared to their Tbx3+/+;Tbx5+/+;R26ReYFP/+;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)…

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  Figure 5.. 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, highlighting 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. The 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. For (C-F), n=10 or 8 animals/ genotype.

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  Figure 5—figure supplement 1.. Tbx3:Tbx5 double-conditional knockout mice exhibit QRS prolongation. (A) Representative electrocardiography (ECG) traces recorded from Tbx3:Tbx5 double-conditional knockout mice (red) and control littermates (black). (B) Quantification of P, PR, QRS, QT, and RR intervals. For (A and B), n=10 or 8 animals/ genotype.*p < 0.05 denotes significance.

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  Figure 6.. Ventricular optical action potentials (OAPs) distal from His bundle have only one 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. For (C and D), n=10 or 8 animals/ genotype.