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

The cardiac conduction system (CCS) constitutes a highly specialized network of cardiomyocytes that initiate and propagate the electrical impulses required for synchronized contractions of the heart. In the mature mammalian heart, the functional components of the CCS can be broadly divided into the slowly propagating atrial nodes (~5 cm/s), containing the sinoatrial node (SAN) and atrioventricular node (AVN), and the rapidly propagating ventricular conduction system (VCS) (~200 cm/s), including the AV (His) bundle and the right and left bundle branches (BBs). The VCS is responsible for rapid propagation of the electrical impulse from the AVN to the ventricular apex to enable synchronous ventricular contraction and effective ejection of blood from the ventricles (Arnolds et al., 2012; Park and Fishman, 2011; Moskowitz et al., 2007). Defects of CCS can occur in normally formed hearts as well as in patients with structural congenital heart disease and are a major source of morbidity and mortality (Arnolds et al., 2012; Moskowitz et al., 2007; Munshi, 2012; Rubart and Zipes, 2005; Huikuri et al., 2001). The VCS specifically has been recognized as a substrate for life-threatening ventricular arrhythmias, including bundle branch reentry tachycardia, idiopathic fascicular tachycardia, short-coupled torsade de pointes, and ventricular fibrillation (Arnolds et al., 2012; Huikuri et al., 2001; van Duijvenboden et al., 2014; Arnolds and Moskowitz, 2011b; Scheinman, 2009). Despite the severe clinical consequences of CCS disorders, the molecular mechanisms that establish and maintain regional functionality of the mature CCS domains require further study.

Human genetic studies have identified numerous loci associated with adult human CCS function, including the developmentally important factors Tbx3 and Tbx5 (reviewed in Arnolds et al., 2012; Arnolds et al., 2011a). Tbx3 and Tbx5 play crucial roles in adult CCS development and function (Arnolds et al., 2012; Moskowitz et al., 2007; Burnicka-Turek et al., 2020; Moskowitz et al., 2004; van Weerd and Christoffels, 2016; van den Boogaard et al., 2012; Bakker et al., 2012; Hatcher and Basson, 2009; Bakker et al., 2008; Hoogaars et al., 2007b; Mori et al., 2006; Bruneau et al., 2001). Tbx5 encodes a T-box transcriptional activator required for structural and conduction system cardiac development (Moskowitz et al., 2007; Moskowitz et al., 2004; Bruneau et al., 2001; Hoogaars et al., 2007a). Dominant mutations in human TBX5 cause Holt–Oram syndrome (HOS, OMIM:142900), an autosomal dominant disorder characterized by upper limb malformations, congenital heart defects, and CCS abnormalities (Basson et al., 1997; Li et al., 1997; Basson et al., 1994). The cardiac phenotype of HOS, including atrioventricular conduction delay, has been recapitulated in the Tbx5 heterozygous mice (Bruneau et al., 2001). Moreover, VCS-specific Tbx5 knockout caused slowed VCS function and ventricular tachycardia (VT) resulting in sudden death in mice (Arnolds et al., 2012), emphasizing the importance of Tbx5 in VCS conduction. Tbx5 is strongly expressed in the atria and VCS (Arnolds et al., 2012; Moskowitz et al., 2007; Moskowitz et al., 2004; Bakker et al., 2008), and directly regulates several targets required for VCS function (Moskowitz et al., 2007; Bruneau et al., 2001; Hiroi et al., 2001), including Gja5 (Cx40) (Bruneau et al., 2001) and Scn5a (Nav1.5) (Arnolds et al., 2012). Tbx3 encodes a T-box transcriptional repressor which is critical for cardiac development (Bakker et al., 2008; Frank et al., 2011). Dominant mutations in human TBX3 cause Ulnar–Mammary syndrome (OMIM:181450), a developmental disorder (Meneghini et al., 2006; Bamshad et al., 1997), that includes functional conduction system defects (Linden et al., 2009). In the heart, Tbx3 is specifically expressed within CCS (Bakker et al., 2008; Hoogaars et al., 2004), and its deficiency below critical level leads to lethal arrhythmias (Frank et al., 2011). Furthermore, Tbx3 is required for the molecular identity but not the function of the VCS (Bakker et al., 2008). In contrast, Tbx3 in SAN and AVN is required for their proper function (Hoogaars et al., 2004), emphasizing its critical role in maintaining proper cardiac rhythm.

A model for regional CCS specialization suggests that the adult CCS is organized entirely as a slow conduction system ground state by Tbx3 with a T-box-dependent, physiologically dominant fast conduction system network driven specifically in the VCS by Tbx5 (Burnicka-Turek et al., 2020). The adult VCS-specific removal of TBX5 or overexpression of TBX3 shifted the fast VCS into a slow nodal-like system, indicating that the Tbx3/Tbx5 ratio determines nodal versus VCS function (Burnicka-Turek et al., 2020). However, a comprehensive assessment of the coordinated requirements for Tbx3 and Tbx5 has been hindered by the inability to achieve their compound deletion due to their genomic proximity. Tbx3 and Tbx5 are situated in cis within 0.6 Mb on chromosome 5 in mice, rendering their simultaneous deletion unattainable with the available single allele conditional alleles. To investigate the consequences of Tbx3 and Tbx5 compound removal from the mature VCS, we generated a novel compound Tbx3:Tbx5 double-conditional allele. We found that VCS-specific genetic removal of both TBX3 and TBX5 transformed fast-conducting, adult VCS into working myocardium-like cardiomyocytes, shifting them from conduction to non-conduction myocytes. These results demonstrated the coordinated requirements of both Tbx3 and Tbx5 for maintained specification of the mature VCS.

We generated a novel Tbx3:Tbx5 double-floxed allele to enable the simultaneous conditional deletion of Tbx3 and Tbx5 genes specifically from the adult VCS. Tbx3 and Tbx5 reside in cis on mouse chromosome 5 (Tbx3 mm39 chr5:119808734–119822789; Tbx5 mm39 chr5:119970733–120023284). Therefore, to generate a double-conditional knockout, we targeted Tbx5 in the background of a previously validated Tbx3 floxed allele (Frank et al., 2011) using the CRISPR–Cas9 system (Gurumurthy et al., 2021; Dow et al., 2015; Figure 1A). We engineered a Tbx5 floxed allele mirroring a previously published allele (Bruneau et al., 2001). This design enabled us to utilize the previously published individual Tbx3 floxed allele (Frank et al., 2011) and individual Tbx5 floxed allele (Bruneau et al., 2001) to serve as controls (Figure 1A, Figure 1—figure supplements 1 and 2).

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We assessed the impact of removing Tbx3 and Tbx5 from the mature VCS by combining the Tbx3:Tbx5 double-floxed mouse line (Tbx3^fl/fl;Tbx5^fl/fl) with a VCS-specific, tamoxifen (TM)-inducible Cre transgenic mouse line, Kcne1^CreERT2 [Tg(RP23-276I20-MinKCreERT2)] – hereafter referred to as MinK^CreERT2, in accordance with existing literature (Figure 1A; Arnolds et al., 2012; Arnolds and Moskowitz, 2011b; Burnicka-Turek et al., 2020). Individual Tbx3 floxed and Tbx5 floxed mouse lines combined with MinK^CreERT2 transgenic mouse lines (Tbx3^fl/fl;Tbx5^+/+; R26R^eYFP/+;MinK^CreERT2/+ and Tbx3^+/+;Tbx5^fl/fl;R26R^eYFP/+;MinK^CreERT2/+, respectively) were generated as controls, and all allelic combinations were evaluated in a mixed genetic background. We compared VCS-specific Tbx3:Tbx5 double-conditional mutants (Tbx3^fl/fl;Tbx5^fl/fl;R26R^eYFP/+;MinK^CreERT2/+) with control littermates (Tbx3^+/+;Tbx5^+/+; R26R^eYFP/+;MinK^CreERT2/+) and VCS-specific Tbx3:Tbx5 double-conditional heterozygous littermates (Tbx3^fl/+;Tbx5^fl/+;R26R^eYFP/+;MinK^CreERT2/+). Additionally, we validated the newly created Tbx5 floxed allele demonstrating that it is efficiently converted to the Tbx5 null allele through Cre recombinase, causing a phenotype consistent with that observed from conversion of the previously published Tbx5 floxed allele (Arnolds et al., 2012; Bruneau et al., 2001; Figure 1—figure supplements 1 and 2, Methods section).

We assessed experimental mice at 8–9 weeks of age following tamoxifen administration at 6 weeks of age (Figure 1A, Methods section). We observed loss of both Tbx3 and Tbx5 expression, on both the mRNA and protein levels, in the VCS of adult Tbx3:Tbx5 double-conditional mutant mice (Tbx3^fl/fl;Tbx5^fl/fl;R26R^eYFP/+;MinK^CreERT2/+) but not in their littermate controls (Tbx3^+/+;Tbx5^+/+;R26R^eYFP/+;MinK^CreERT2/+) (Figure 1B, C). Partial loss of Tbx3 and Tbx5 expression in the adult VCS of Tbx3:Tbx5 double-conditional heterozygous mice (Tbx3^fl/+;Tbx5^fl/+;R26R^eYFP/+;MinK^CreERT2/+) was observed compared to littermate controls (Tbx3^+/+;Tbx5^+/+;R26R^eYFP/+;MinK^CreERT2/+) (Figure 1C). We 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 MinK^CreERT2 mice (Arnolds and Moskowitz, 2011b), Tbx3 and Tbx5 expression remained similar in the atrial and ventricular myocardium of all allelic combinations, including Tbx3:Tbx5 double-conditional knockout mice (Tbx3^fl/fl;Tbx5^fl/fl;R26R^eYFP/+;MinK^CreERT2/+) (Figure 1C).

Tbx3:Tbx5 double-conditional knockout mice (Tbx3^fl/fl;Tbx5^fl/fl;R26R^eYFP/+; MinK^CreERT2/+) appeared morphologically and functionally normal and indistinguishable from control littermates (Tbx3^+/+;Tbx5^+/+;R26R^eYFP/+;MinK^CreERT2/+) at 2 weeks post-tamoxifen. However, longitudinal analysis demonstrated sudden death of VCS-specific double-knockout mice beginning 3 weeks post-tamoxifen administration (Figure 1D). Within the 3 months post-tamoxifen administration, all tamoxifen-treated Tbx3^fl/fl;Tbx5^fl/fl; R26R^eYFP/+;MinK^CreERT2/+ mice had died suddenly (n = 40). In contrast, no mortality was observed among the tamoxifen-treated Tbx3^+/+;Tbx5^+/+;R26R^eYFP/+;MinK^CreERT2/+ or Tbx3^fl/+;Tbx5^fl/+;R26R^eYFP/+;MinK^CreERT2/+ littermates (each cohort n = 40) during this period (Tbx3:Tbx5 double-conditional knockout mice vs. control mice p < 0.0001; Tbx3:Tbx5 double-conditional knockout mice vs. Tbx3:Tbx5 double-conditional heterozygous mice p < 0.0001, log-rank test; Figure 1D). These results revealed that double deletion of Tbx3 and Tbx5 from the adult VCS causes lethality beginning at 3 weeks post-tamoxifen.

The onset of mortality observed in VCS-specific Tbx3:Tbx5-deficient mice starting at 3 weeks post-tamoxifen prompted us to investigate the electrophysiologic consequences of VCS-specific Tbx3:Tbx5 double-knockout at 2–3 weeks post-tamoxifen, prior to the onset of lethality (Figure 2). VCS-specific Tbx3:Tbx5 deficiency caused profound conduction slowing in Tbx3^fl/fl;Tbx5^fl/fl;R26R^eYFP/+;MinK^CreERT2/+ mice by ambulatory telemetry electrocardiography (ECG) analysis compared to Tbx3:Tbx5 double-conditional heterozygous mice (Tbx3^fl/+;Tbx5^fl/+;R26R^eYFP/+;MinK^CreERT2/+) and littermate controls (Tbx3^+/+;Tbx5^+/+;R26R^eYFP/+;MinK^CreERT2/+) (Figure 2A–F). Specifically, the PR interval, representing the period between atrial and ventricular depolarization, and the QRS duration, indicating the length of ventricular depolarization and early repolarization in mice, were both significantly prolonged (Figure 2A, B, D; PR: Tbx3:Tbx5 double-conditional knockout mice vs. control mice p < 0.05, n = 7, Welch t-test; QRS: Tbx3:Tbx5 double-conditional knockout mice vs. control mice p < 0.05, n = 7, Welch t-test). Removal of both Tbx3 and Tbx5 from the adult VCS resulted in increased episodes of spontaneous VT. Ambulatory studies revealed episodes of spontaneous VT in four out of seven Tbx3^fl/fl;Tbx5^fl/fl;R26R^eYFP/+;MinK^CreERT2/+ mice, in contrast to none observed in seven littermate controls (Figure 2G, p < 0.05, n = 7, Welch t-test). Furthermore, Tbx3:Tbx5 double-conditional knockout mice (Tbx3^fl/fl;Tbx5^fl/fl;R26R^eYFP/+;MinK^CreERT2/+) showed significantly increased susceptibility to VT following burst stimulation in invasive electrophysiology (EP) studies (three of three Tbx3^fl/fl;Tbx5^fl/fl;R26R^eYFP/+;MinK^CreERT2/+ mice vs. zero of seven littermate controls; Figure 2H, p < 0.05, Welch t-test). In contrast, VCS-specific Tbx3:Tbx5 double-conditional heterozygous mice (n = 7) showed neither conduction nor electrophysiological defects (Figure 2). Consistent with the use of a VCS-specific Cre (MinK^CreERT2), no changes in the refractory/recovery periods of atrium, ventricle, or nodes (atrial effective refractory period, ventricular effective refractory period (VERP), atrioventricular nodal effective refractory period, or sinus node recovery time) were detected by intracardiac EP conducted on experimental and control mice (Figure 2I, p < 0.05, Welch t-test).

Figure 2

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To distinguish a primary conduction system abnormality from a secondary conduction abnormality resulting from cardiac dysfunction or remodeling, we evaluated cardiac form and function at the time of arrhythmia assessment, 2–3 weeks post-tamoxifen treatment (Figure 3). Transthoracic echocardiography revealed no significant differences in left ventricular ejection fraction (LVEF) and fractional shortening (FS) between VCS-specific Tbx3:Tbx5-deficient (Tbx3^fl/fl;Tbx5^fl/fl;R26R^eYFP/+;MinK^CreERT2/+) and control (Tbx3^+/+;Tbx5^+/+;R26R^eYFP/+;MinK^CreERT2/+) mice (LVEF: Tbx3:Tbx5 double-conditional knockout mice vs. control mice p < 0.05, n = 7, Welch t-test; and FS: Tbx3:Tbx5 double-conditional knockout mice vs. control mice p > 0.05, n = 7, Welch t-test; Figure 3A, B, D). Histological examination of all four chambers demonstrated no discernible differences between VCS-specific Tbx3:Tbx5 double-knockout (Tbx3^fl/fl;Tbx5^fl/fl; R26R^eYFP/+;MinK^CreERT2/+) and control (Tbx3^+/+;Tbx5^+/+;R26R^eYFP/+; MinK^CreERT2/+) mice, nor between the double-knockout (Tbx3^fl/fl;Tbx5^fl/fl;R26R^eYFP/+; MinK^CreERT2/+) and single-knockout models for either Tbx3 (Tbx3^fl/fl;Tbx5^+/+;R26R^eYFP/+;MinK^CreERT2/+) or Tbx5 (Tbx3^+/+;Tbx5^fl/fl;R26R^eYFP/+;MinK^CreERT2/+). Ventricular muscle appeared normal without hypertrophy or myofibrillar disarray, and no fibrosis was present (Figure 3G, I–K, respectively). qRT-PCR analysis for fibrosis genes Col1a1 (Pan et al., 2022; Hua et al., 2020; Zhao et al., 2018) and Postn (Zhao et al., 2018; Ackerman et al., 2024; Wu et al., 2024; Oka et al., 2007) further confirmed no fibrosis in VCS of Tbx3:Tbx5-deficient mice (Figure 3L). No contractile dysfunction, histological abnormalities, or increased expression of fibrosis genes were observed in VCS-specific Tbx3:Tbx5 double-conditional heterozygous mice (Tbx3^fl/+;Tbx5^fl/+;R26R^eYFP/+; MinK^CreERT2/+) (Figure 3A, C, H, L). Taken together, these data indicate that the conduction defect and VT observed in mice with VCS-specific Tbx3:Tbx5 deletion (Figure 2) occur prior to the onset of left ventricular (LV) dysfunction or evidence of remodeling (Figure 3), implying a primary origin.

Figure 3

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To assess the hypothesis that Tbx3 and Tbx5 collectively promote VCS versus working myocardium phenotype, we conducted a transcriptional characterization of the adult VCS in Tbx3^fl/fl;Tbx5^fl/fl;R26R^eYFP/+;MinK^CreERT2/+ mutant mice compared to their Tbx3^+/+;Tbx5^+/+;R26R^eYFP/+;MinK^CreERT2/+ control littermates using three distinct sets of molecular markers by qRT-PCR (Figure 4A–C). The first set encompassed genes expressed throughout the entire conduction system (Pan-CCS) and implicated in slow-conducting nodal phenotype, such as Hcn1, Hcn4, Cacna1d (Cav1.3), Cacna1g (Cav3.1d), Cacna1h (Cav3.2), Gjd3 (Cx30.2), and Gjc1 (Cx45) (Bakker et al., 2012; Hoogaars et al., 2004; van Eif et al., 2020; Verheule and Kaese, 2013; Liang et al., 2013; Greener et al., 2011; Mangoni et al., 2006; Marionneau et al., 2005; Garcia-Frigola et al., 2003; Schram et al., 2002; Figure 4A). The second set included genes highly expressed in the fast-conducting VCS and important for VCS function, including Gja5 (Cx40), Scn5a (Nav1.5), Ryr2, Kcnk3 (Task-1), Kcnj2 (Kir2.1), Kcnj3 (Kir3.1), Kcnj4 (IRK3), and Kcnj12 (Kir2.2) (Figure 4B; van Eif et al., 2020; Verheule and Kaese, 2013; Schram et al., 2002; Donner et al., 2011; Miquerol et al., 2010; Remme et al., 2009; Graham et al., 2006). The third set contained markers specifically present in the working myocardium but absent in the CCS, such as Gja1 (Cx43) and Smpx (Figure 4C; Hoogaars et al., 2004; van Eif et al., 2020; Alcoléa et al., 1999; Kempen et al., 1996). VCS-specific Tbx3:Tbx5-deficient mice lost VCS expression profile of genes required for the fast ventricular conduction (Figure 4B) as well as genes normally expressed in whole CCS (Pan-CCS genes) (Figure 4A). In contrast, these mice obtained VCS expression of working myocardium-specific molecular markers (Figure 4C). Immunoblotting analysis confirmed transcriptional changes observed by qRT-PCR (Figure 4D). This molecular characterization indicated that the Tbx3:Tbx5 double mutant VCS adopted a gene expression profile similar to wild-type working myocardial-like cells (Figure 4).

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The impact of the Tbx3:Tbx5 double-conditional knockout on electrical impulse propagation in the VCS of the heart was assessed with optical mapping of the anterior epicardial surface of the ventricles and right septal preparations where VCS function should be observed (Figure 5). Optical mapping records changes in transmembrane potential from multiple cells in tissue preparations, where ventricular septal optical action potentials (OAPs) have two distinct action potential upstrokes. The first peak is a result of depolarization of the specialized fast VCS, followed by depolarization of ventricular working myocardium.

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To visualize electrical impulse propagation in Tbx3:Tbx5 double-conditional knockout mice, a 100 × 100 pixel data frame was plotted for the entire field of view of the right septal preparation (Figure 5A, B). For enhanced analysis of the VCS, the region encompassing the His bundle was distinguished in a red 10 × 10 area (Figure 5B). To specifically assess electrical impulse propagation within the His bundle, the same 10 × 10 pixel area representing this region was isolated from adult hearts of both control and Tbx3:Tbx5 double-conditional knockout mice (Figure 5C). We observed only one OAP upstroke in Tbx3:Tbx5 double-conditional knockout mice in contrast to control mice which showed two OAP upstrokes. The first derivative of the OAP (dOAP/dt) from the region of the His bundle was calculated and plotted in a dOAP/dt map further highlighting the number of temporally distinct upstrokes within the OAP (Figure 5D). In control mice, we observed two distinct depolarization upstrokes, one pertaining to the VCS and the second for working ventricular myocardium. However, in Tbx3:Tbx5 double-conditional knockout mice, only one maximum dOAP/dt was observed (Figure 5D). The OAP morphology observed in Tbx3:Tbx5 double-conditional knockout mice suggested a loss or reduction of the specialized fast VCS conduction (Figure 5C, D).

OAPs from the anterior epicardial surface of the ventricles were compared between control littermates and Tbx3:Tbx5 double-conditional knockout mice to assess changes in electrical impulse propagation in the ventricular working myocardium. Paced at a basic cycle length of 125 ms, control littermates and Tbx3:Tbx5 double-conditional knockout mice did not exhibit significant differences in action potential duration at 50% (APD50) or 80% (APD80) repolarization (Figure 5E, F). These observations suggested that the electrical activity of the ventricular working myocardium was not appreciably altered in double-knockout mice compared to controls. This finding is consistent with the lack of changes in the VERP observed in invasive EP studies of both control and Tbx3:Tbx5 double-conditional knockout mice (Figure 3I).

We further predicted that the double knockout would not affect action potentials or conduction properties distal to the VCS (Figure 6). OAP and dOAP/dt maps were created to observe the effects on electrical impulse propagation distal to the His bundle on the right septal preparation (Figure 6), similar to the methods applied to create OAP and dOAP/dt maps in the region of the His bundle (Figure 5), with the key difference being that the signals were recorded from the red 10 × 10 pixel regions plotted in the working ventricular myocardium distal to the His bundle instead of in the area of the His bundle (Figure 6A, B vs. Figure 5A, B, respectively). In both control littermates and Tbx3:Tbx5 double-conditional knockout mice, only one action potential upstroke and one dOAP/dt maximum are observed, which indicates that most of this region consists of the ventricular working myocardium (Figure 6C and D, respectively). In summary, the significant remodeling of electrical impulse propagation due to Tbx3:Tbx5 double-conditional knockout was observed by the loss of a distinct fast VCS impulse in the region of the His bundle, but not on the anterior epicardial surface of the ventricles or distal to the His bundle on the right ventricular (RV) septum (Figures 5 and 6).

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Our study investigated the impact of compound Tbx3:Tbx5 deficiency on mature VCS function and molecular identity. Using a double-conditional knockout strategy, both genes were targeted specifically in the adult VCS. Loss of Tbx3 and Tbx5 expression in the mature VCS led to profound conduction defects, characterized by prolonged PR interval and QRS duration, increased susceptibility to VT, and sudden death. These alterations were observed in the absence of discernible changes in cardiac contractility or histological morphology, indicating a primary conduction system defect. Molecular characterization of the adult VCS unveiled an altered gene expression profile in Tbx3:Tbx5 double-conditional knockout mice, suggesting a transition from the distinctive fast VCS transcriptional profile to that resembling ventricular working myocardium. Optical mapping demonstrated loss of the specialized fast VCS function in Tbx3:Tbx5 double-conditional knockout mice, further suggesting that this region acquired an electrophysiological phenotype similar to the ventricular working myocardium.

Our previous research (Arnolds et al., 2012; Moskowitz et al., 2007; Burnicka-Turek et al., 2020; Moskowitz et al., 2004) and published literature van Duijvenboden et al., 2014; Bakker et al., 2012; Bruneau et al., 2001; Frank et al., 2011; Mohan et al., 2020; van Eif et al., 2018 have suggested that the balance between Tbx3 and Tbx5 expression determines the regional specialization of the mature central CCS (Figure 7). Tbx3 expression dominates in nodal myocardium, imparting nodal physiological characteristics. Tbx5 expression dominates in fast VCS myocardium, where a T-box-dependent fast conduction system network drives physiologically dominant fast conduction physiology, overriding nodal physiology (Burnicka-Turek et al., 2020; Figure 7). This model accurately predicts the outcome of targeted manipulations, such as adult VCS-specific removal of TBX5 or overexpression of TBX3, which convert the fast VCS into a slow nodal-like system (Burnicka-Turek et al., 2020; Figure 7).

Figure 7

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The adult CCS organization model posits that Tbx3 and Tbx5 coordinately establish CCS characteristics and suggests their compound necessity to maintain specialized VCS identity (Figure 7), a hypothesis that remained untested. This model predicted that VCS-specific genetic ablation of both the TBX3 and TBX5 transcription factors would transform fast-conducting adult VCS into cells resembling working myocardium, eliminating specialized CCS identity (Figure 7). Testing this model necessitated the generation of a Tbx3:Tbx5 double-conditional knockout allele due to their proximal chromosomal location in cis on mouse Chr5. VCS-specific double-knockout mice showed a notable deceleration in VCS conduction, manifested by prolonged PR and QRS intervals, along with increased susceptibility to VT. A similar functional phenotype was observed in single deletion of Tbx5 from the adult VCS, leading to the transformation of the fast VCS into a nodal-like phenotype (Arnolds et al., 2012; Burnicka-Turek et al., 2020). However, we predicted that the nodal-like characteristics of the Tbx5-ablated VCS were due to retained expression of Tbx3. In fact, the autonomous beating and impulse initiation observed in the Tbx5-mutant VCS (Arnolds et al., 2012; Burnicka-Turek et al., 2020) were absent from the double Tbx3:Tbx5 mutant VCS, further suggesting the transformation from a nodal-like to an inert myocardial functionally and a shift from conduction toward simple working myocardium.

Molecular studies support a transformation from conduction to working myocardium in the Tbx3:Tbx5 double VCS knockout. Previous studies investigating the roles of Tbx3 and Tbx5 in maintaining adult VCS identity demonstrated that Tbx3 deletion resulted in the silencing of Pan-CCS gene expression in the atrioventricular conduction system (van Duijvenboden et al., 2014; Bakker et al., 2012; Bruneau et al., 2001; Frank et al., 2011; Mohan et al., 2020). Alternately, specific deletion of Tbx5 from the adult VCS led to the decreased expression of VCS-specific markers while Pan-CCS markers remained unchanged, indicating a transformation toward a nodal-like transcriptional phenotype in the absence of Tbx5 (Arnolds et al., 2012; Burnicka-Turek et al., 2020). Consistently, a similar transformation was induced by Tbx3 overexpression in the adult VCS. These findings underscored the significance of the Tbx3:Tbx5 ratio in preserving the molecular and functional characteristics of the fast versus slow conduction system. In Tbx3:Tbx5 double VCS knockout, we observed a reduction in the expression of both fast VCS markers and Pan-CCS markers transcribed throughout the entire CCS. As expected, not all VCS markers were ablated in VCS-specific Tbx3:Tbx5 mutants. A significant portion of VCS fast conduction markers are also transcribed in ventricular working myocardium, albeit at lower levels than in the VCS, and are crucial for normal working myocardial function, for example, Ryr2, Scn5a, and Kcnj2 (van Eif et al., 2020; Schram et al., 2002; Remme et al., 2009). Consistent with a shift from fast VCS to working myocardium, their expression levels are present, but at significantly lower levels in the double-knockout mutants than in normal VCS. Furthermore, the expression of markers specific for working myocardium, which are normally excluded in the VCS, emerged in the VCS of Tbx3:Tbx5 double mutant. These observations are consistent with a transcriptional shift from VCS conduction cardiomyocytes to working myocardium-like characteristics in the absence of both Tbx3 and Tbx5.

Our study highlights the critical role of Tbx3 and Tbx5 in maintaining the specialized electrical properties of the VCS. Comparative analysis of the phenotypes observed in single Tbx3 or Tbx5 knockouts and the Tbx3:Tbx5 double-conditional knockout supports a coordinated function for these transcription factors in preserving VCS functionality. In a normal mammalian heart, electrical or optical recordings from the proximal part of the VCS typically display two distinct electrical excitations, reflected as two spikes in electrograms or two upstrokes in OAPs. These signals correspond to the sequential activation of the VCS, followed by ventricular excitation, a phenomenon well documented in both basic and clinical studies across species, including mice and humans. Previous whole-heart EP and optical mapping studies demonstrated that the single conditional knockout of Tbx5 in the adult VCS caused significant ventricular conduction slowing (Arnolds et al., 2012), accompanied by a phenotypic shift of VCS cells toward a pacemaker-like cell, evidenced by ectopic beats originating in the ventricles, retrograde activation, and inappropriate automaticity (Burnicka-Turek et al., 2020). Whole-cell patch clamp recordings of Tbx5-deficient VCS cells further supported these findings, revealing action potential characteristics resembling pacemaker cells, including a slower upstroke (phase 0), prolonged plateau (phase 2), delayed repolarization (phase 3), and enhanced phase 4 depolarization (Burnicka-Turek et al., 2020). In contrast, reduced expression of Tbx3 in the Tbx3 haploinsufficiency model resulted in AV bundle hypoplasia, PR interval shortening, and prolonged QRS duration (Mohan et al., 2020). Optical mapping in these mice showed delayed apical activation with multiple small breakthroughs separated by regions of delayed activation. This fragmented activation pattern suggests that the reduced Tbx3 expression impaired the homogeneity and efficiency of ventricular electrical impulse propagation due to AV bundle and bundle branch hypoplasia (Mohan et al., 2020). In the Tbx3:Tbx5 double-conditional knockout mice, we observed a complete loss of fast VCS conduction, similar to the phenotype of the Tbx5 single conditional knockouts. However, unlike in the Tbx5 single CKO, the VCS in Tbx3:Tbx5 double mutants did not acquire pacemaker-like activity. Instead, optical mapping revealed a single upstroke in the His bundle region of Tbx3:Tbx5 double-conditional knockout mice, indicating the absence of rapid AV bundle electrical impulse propagation. Functionally, this transformation resulted in significantly slowed ventricular activation, without the retrograde activation observed in the Tbx5 single conditional knockouts or the rapid electrical impulse propagation typically associated with an intact VCS. The homogenous morphological and molecular transformation of His bundle and VCS cells in the Tbx3:Tbx5 double-conditional knockout mice rendered the electrical properties of the VCS-located cells functionally indistinguishable from those of ventricular working myocardium. Our data, including gene expression analysis of the double mutant VCS, are most consistent with the transition of double mutant VCS cells toward a working myocardium phenotype.

These findings emphasize the interdependent roles of Tbx3 and Tbx5 in maintaining the specialized electrical properties and identity of the VCS, distinguishing it from non-specialized ventricular working myocardium (Figure 7). While Tbx5 alone is critical for suppressing pacemaker-like characteristics and preserving fast conduction properties, Tbx3 plays a pivotal role in ensuring the structural and functional integrity of the AV and BB conduction pathways. The simultaneous deletion of both Tbx3 and Tbx5 disrupts these regulatory mechanisms, leading to a loss of VCS identity and a functional shift toward a working myocardium phenotype. Our study combined with prior literature (Arnolds et al., 2012; van Duijvenboden et al., 2014; Burnicka-Turek et al., 2020; Bakker et al., 2012; Frank et al., 2011; Mohan et al., 2020; van Eif et al., 2018) indicates that the concurrent presence of both Tbx3 and Tbx5 is necessary for maintaining VCS identity in the adult heart, ensuring efficient electrical conduction and preventing the transition of VCS cells into a working myocardium-like state (Figure 7).

All data generated and analyzed during this study are included in the main manuscript and its supplementary materials. The source data for statistical analyses are provided in Source Data 1. Uncropped gels and blots have been supplied for Figure 1-figure supplements 1 and 2, as well as for Figure 4. No additional data are required to interpret the findings.

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Author details

1. Ozanna Burnicka-Turek

   Departments of Pediatrics, Pathology, and Human Genetics, University of Chicago, Chicago, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Validation, Investigation, Visualization, Methodology, Writing – original draft, Project administration, Writing – review and editing

   For correspondence

   burnickatureko@uchicago.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3770-4314

2. Katy A Trampel

   Departments of Biomedical Engineering, Northwestern University, Chicago, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-5735-1037

3. Brigitte Laforest

   Departments of Pediatrics, Pathology, and Human Genetics, University of Chicago, Chicago, United States

   Contribution

   Data curation, Formal analysis, Validation, Investigation, Visualization

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-6919-8922

4. Michael T Broman

   Department of Medicine, Section of Cardiology, University of Chicago, Chicago, United States

   Contribution

   Resources, Data curation, Formal analysis, Supervision, Validation, Investigation, Visualization, Methodology

   Competing interests

   No competing interests declared

5. Xinan H Yang

   Departments of Pediatrics, Pathology, and Human Genetics, University of Chicago, Chicago, United States

   Contribution

   Data curation, Formal analysis, Visualization, Writing – review and editing

   Competing interests

   No competing interests declared

6. Zoheb Khan

   Departments of Pediatrics, Pathology, and Human Genetics, University of Chicago, Chicago, United States

   Contribution

   Data curation, Validation, Investigation

   Competing interests

   No competing interests declared

7. Eric Rytkin

   Departments of Biomedical Engineering, Northwestern University, Chicago, United States

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

8. Binjie Li

   Departments of Biomedical Engineering, Northwestern University, Chicago, United States

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

9. Ella Schaffer

   Departments of Pediatrics, Pathology, and Human Genetics, University of Chicago, Chicago, United States

   Contribution

   Data curation, Investigation, Visualization

   Competing interests

   No competing interests declared

10. Margaret Gadek

    Departments of Pediatrics, Pathology, and Human Genetics, University of Chicago, Chicago, United States

    Contribution

    Data curation, Validation, Investigation

    Competing interests

    No competing interests declared

11. Kaitlyn M Shen

    Departments of Pediatrics, Pathology, and Human Genetics, University of Chicago, Chicago, United States

    Contribution

    Data curation, Validation, Investigation

    Competing interests

    No competing interests declared

12. Igor R Efimov

    Departments of Biomedical Engineering, Northwestern University, Chicago, United States

    Contribution

    Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Methodology, Writing – original draft, Project administration, Writing – review and editing

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-1483-5039

13. Ivan P Moskowitz

    Departments of Pediatrics, Pathology, and Human Genetics, University of Chicago, Chicago, United States

    Contribution

    Conceptualization, Resources, Supervision, Funding acquisition, Writing – original draft, Project administration, Writing – review and editing

    For correspondence

    imoskowitz@peds.bsd.uchicago.edu

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-0014-4963

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