Introduction

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 (∼ 5cm/sec), containing the sinoatrial node (SAN) and atrioventricular node (AVN), and the rapidly propagating ventricular conduction system (VCS) (∼ 200cm/sec), including the AV (His) bundle (AVB) 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 (1–3). 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 (1, 3–6). 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 (1, 6–9). 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 1, 10). Tbx3 and Tbx5 play crucial roles in adult CCS development and function (1, 3, 11–20). Tbx5 encodes a T-box transcriptional activator required for structural and conduction system cardiac development (3, 12, 20, 21). 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 (22–24). The cardiac phenotype of HOS, including atrioventricular conduction delay, has been recapitulated in the Tbx5 heterozygous mice (20). Moreover, VCS-specific Tbx5 knockout caused slowed VCS function and ventricular tachycardia resulting in sudden death in mice (1), emphasizing the importance of Tbx5 in VCS conduction. Tbx5 is strongly expressed in the atria and VCS (1, 3, 12, 17), and directly regulates several targets required for VCS function (3, 20, 25), including Gja5 (Cx40) (20) and Scn5a (Nav1.5) (1). Tbx3 encodes a T-box transcriptional repressor which is critical for cardiac development (17, 26). Dominant mutations in human TBX3 cause Ulnar-Mammary syndrome (OMIM:181450), a developmental disorder (27, 28), that includes functional conduction system defects (29). In the heart, Tbx3 is specifically expressed within CCS (17, 30), and its deficiency below critical level leads to lethal arrhythmias (26). Furthermore, Tbx3 is required for the molecular identity but not the function of the VCS (17). In contrast, Tbx3 in SAN and AVN is required for their proper function (30), emphasizing its critical role in maintaining proper cardiac rhythm.

A model for regional CCS patterning suggests that the adult CCS is patterned 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 (11). Adult VCS-specific removal of TBX5 or overexpression of TBX3 re-patterned the fast VCS into a slow nodal-like system, indicating that the Tbx3/Tbx5 ratio determines nodal versus VCS function (11). 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 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, re-patterning them from conduction to non-conduction myocytes. These results demonstrated the coordinated requirements of both Tbx3 and Tbx5 for maintained specification of the mature ventricular conduction system.

Results

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 (26) using the CRISPR-Cas9 system (31, 32) (Figure 1A). We engineered a Tbx5 floxed allele mirroring a previously published allele (20). This design enabled us to utilize the previously published individual Tbx3 floxed allele (26) and individual Tbx5 floxed allele (20) to serve as controls (Figure 1A and Supplementary Figure 1 and 2).

Figure 1.

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 (MinK^CreERT2 [Tg(RP23-276I20-MinkCreERT2) (Figure 1A) (8). Individual Tbx3 floxed and Tbx5 floxed mouse lines combined with Mink^CreERT2 transgenic mouse lines (Tbx3^fl/fl;Tbx5^+/+; R26^EYFP/+;MinK^CreERT2/+ and Tbx3^+/+;Tbx5^fl/fl;R26^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;R26^EYFP/+;MinK^CreERT2/+) with control littermates (Tbx3^+/+;Tbx5^+/+; R26^EYFP/+;MinK^CreERT2/+) and VCS-specific Tbx3:Tbx5 double-conditional heterozygous littermates (Tbx3^fl/+;Tbx5^fl/+;R26^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 (1, 20) (Supplementary Figure 1 and 2, Supplementary Material 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;R26^EYFP/+;MinK^CreERT2/+) but not in their littermate controls (Tbx3^+/+;Tbx5^+/+;R26^EYFP/+;MinK^CreERT2/+) (Figure 1B and C). Partial loss of Tbx3 and Tbx5 expression in the adult VCS of Tbx3:Tbx5 double-conditional heterozygous mice (Tbx3^fl/+;Tbx5^fl/+;R26^EYFP/+;MinK^CreERT2/+) was observed compared to littermate controls (Tbx3^+/+;Tbx5^+/+;R26^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 (8), 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;R26^EYFP/+;MinK^CreERT2/+) (Figure 1C).

Tbx3:Tbx5 double-conditional knockout mice (Tbx3^fl/fl;Tbx5^fl/fl;R26^EYFP/+; MinK^CreERT2/+) appeared morphologically and functionally normal and indistinguishable from control littermates (Tbx3^+/+;Tbx5^+/+;R26^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; R26^EYFP/+;MinK^CreERT2/+ mice had died suddenly (n=40). In contrast, no mortality was observed among the tamoxifen-treated Tbx3^+/+;Tbx5^+/+; R26^EYFP/+;MinK^CreERT2/+ or Tbx3^fl/+;Tbx5^fl/+;R26^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;R26^EYFP/+;MinK^CreERT2/+ mice by ambulatory telemetry ECG analysis compared to Tbx3:Tbx5 double-conditional heterozygous mice (Tbx3^fl/+;Tbx5^fl/+;R26^EYFP/+;MinK^CreERT2/+) and littermate controls (Tbx3^+/+;Tbx5^+/+;R26^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, and D; PR: Tbx3:Tbx5 double-conditional knockout mice vs control mice P<0.05, n=5, Welch t test; QRS: Tbx3:Tbx5 double-conditional knockout mice vs control mice P<0.05, n=5, Welch t test). Removal of both Tbx3 and Tbx5 from the adult VCS resulted in increased episodes of spontaneous ventricular tachycardia (VT). Ambulatory studies revealed episodes of spontaneous VT in 4 out of 7 Tbx3^fl/fl;Tbx5^fl/fl;R26^EYFP/+;MinK^CreERT2/+ mice, in contrast to none observed in 7 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;R26^EYFP/+;MinK^CreERT2/+) showed significantly increased susceptibility to ventricular tachycardia following burst stimulation in invasive electrophysiology (EP) studies (3 of 3 Tbx3^fl/fl;Tbx5^fl/fl;R26^EYFP/+;MinK^CreERT2/+ mice versus 0 of 7 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-Cre), no changes in the refractory/recovery periods of atrium, ventricle, or nodes (atrial effective refractory period, ventricular effective refractory period, atrioventricular nodal effective refractory period, or sinus node recovery time) were detected by intracardiac electrophysiology conducted on experimental and control mice (Figure 2I, P<0.05, Welch t test).

Figure 2.

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;R26^EYFP/+;MinK^CreERT2/+) and control (Tbx3^+/+;Tbx5^+/+;R26^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, 3B and 3D). Histological examination of all four-chambers demonstrated no discernible differences between VCS-specific Tbx3:Tbx5 double-knockout (Tbx3^fl/fl;Tbx5^fl/fl;R26^EYFP/+; MinK^CreERT2/+) and control (Tbx3^+/+;Tbx5^+/+;R26^EYFP/+; MinK^CreERT2/+) mice. Ventricular muscle appeared normal without hypertrophy or myofibrillar disarray and no fibrosis was present (Figure 3G and 3I). QRT-PCR analysis for fibrosis genes Col1a1 (33–35) and Postn (35–38) 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/+;R26^EYFP/+; MinK^CreERT2/+) (Figure 3A, 3C, 3H, and 3L). Taken together, these data indicate that the conduction defect and ventricular tachycardia observed in mice with VCS-specific Tbx3:Tbx5 deletion (Figure 2) occur prior to the onset of left ventricular dysfunction or evidence of remodeling (Figure 3), implying a primary origin.

Figure 3.

To assess the hypothesis that Tbx3 and Tbx5 cooperatively promote VCS versus working myocardium phenotype, we conducted a transcriptional characterization of the adult VCS in Tbx3^fl/fl;Tbx5^fl/fl;R26^EYFP/+;MinK^CreERT2/+ mutant mice compared to their Tbx3^+/+;Tbx5^+/+;R26^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) (15, 30, 39–46) (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) (39, 40, 46–50). The third set contained markers specifically present in the working myocardium but absent in the CCS, such as Gja1 (Cx43) and Smpx (Figure 4C) (30, 39, 51, 52). 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).

Figure 4.

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 (OAP) 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.

Figure 5.

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 and 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 (Figure 5C and 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 and 5F). 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 ventricular effective refractory period (VERP) observed in invasive electrophysiology (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 and B versus Figure 5A and 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 septum (Figure 5 and 6).

Figure 6.

Discussion

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 ventricular tachycardia, 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 (1, 3, 11, 12) and published literature (7, 15, 20, 26, 53, 54) have suggested that the balance between Tbx3 and Tbx5 expression determines the regional patterning of the mature central cardiac conduction system (Figure 7). Tbx3 expression dominates in nodal myocardium, imparting nodal physiologic 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 (11) (Figure 7). This model accurately predicts the outcome of targeted manipulations such as adult VCS-specific removal of TBX5 or overexpression of TBX3, in which fast VCS is re-patterning into a slow nodal-like system (11) (Figure 7).

Figure 7.

The CCS patterning model predicts that Tbx3 and Tbx5 coordinately pattern the CCS and suggests their compound necessity for specialized VCS fate (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 fate (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 ventricular tachycardia (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 (1, 11). 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 (1, 11) were absent from the double Tbx3:Tbx5 mutant VCS, further suggesting the transformation from a nodal-like to an inert myocardial functionally and a fate transformation from conduction to simple working myocardium.

Molecular studies support a transformation from conduction to working myocardium in the Tbx5:Tbx3 double VCS knockout. Previous studies investigating the roles of Tbx3 and Tbx5 in adult VCS specification demonstrated that Tbx3 deletion resulted in the silencing of Pan-CCS gene expression in the atrioventricular conduction system (7, 15, 20, 26, 53). Alternately, specific deletion of Tbx5 from the adult VCS led to the repression of VCS-specific markers while Pan-CCS markers remained unchanged, indicating a transformation towards a nodal-like transcriptional phenotype in the absence of Tbx5 (1, 11). Consistently, a similar transformation was induced by Tbx3 overexpression in the adult VCS. These findings underscored the significance of the Tbx3:Tbx5 ratio in the molecular and functional patterning of the fast versus slow conduction system. In Tbx3:Tbx5 double VCS knockout, we observed repression of fast VCS markers and also repression of 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, e.g., Ryr2, Scn5a, and Kcnj2 (39, 46, 49). 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.

Optical mapping provided further evidence in favor of the transformation from VCS to working myocardium in the absence of both Tbx3 and Tbx5. We observed the loss of fast VCS in the region of the His bundle in the Tbx3:Tbx5 double-conditional knockout mice, indicated by presence of only one upstroke in the OAP in the region of the His bundle and VCS. The homogenous morphological and molecular transformation of His bundle and VCS cells in the Tbx3:Tbx5 double-conditional knockout mice resulted in the electrical properties of the VCS-located cells being functionally indistinguishable from that of the ventricular cells. Thus, the electrical impulse propagation in the ventricles is slowed, now activating in a manner similar to exogenous pacing rather than rapid impulse propagation through the VCS.

Here, we have shown that Tbx3 and Tbx5 are cooperatively responsible for the patterning of the fast conduction VCS versus the non-specialized ventricular working myocardium (Figure 7). Deletion of both transcription factors from the VCS resulted in characteristics resembling ventricular working myocardium. This study combined with prior literature (1, 7, 11, 15, 26, 53, 54) indicates that the presence of both Tbx3 and Tbx5 is necessary for the specification of the adult VCS (Figure 7).

Methods

Data Availability

The authors declare that all supporting data and materials presented within this manuscript and its Online Supplemental Materials are available from the corresponding author upon reasonable request.

Experimental animals

All animal experiments were performed under the University of Chicago Institutional Animal Care and Use Committee (IACUC) approved protocol (ACUP no. 71737) and in compliance with the USA Public Health Service Policy on Humane Care and Use of Laboratory Animals. MinK^CreERT2 [Tg(RP23-276I20-MinkCreERT2)] and Tbx3^fl/fl mice have been reported previously (8, 26).

Tbx3:Tbx5 double floxed mouse line was generated by University of Utah Core Research Facility using CRISPR/Cas9. Guide RNA (sgRNA) constructs were designed with software tools (ZiFiT Targeter (55) and crispr.genome-engineering.org) predicting unique target sites throughout the mouse genome (Supplementary Figure 3 - 5). The sgRNA constructs were transcribed in vitro using MEGAshortscript T7 (Invitrogen AM1354) and mMessage Machine T7 transcription kit (Invitrogen AM1344) according to manufacturer instructions. The strategy to generate mouse founders involved a single-step microinjection into one-cell Tbx3 floxed zygotes (on a mixed background) with 10 ng/µL of each sgRNA (TBX5-I2-S22 and TBX5-I3-S31, Supplementary Figure 3 - 5), 30 ng/µL of Cas9 protein, and 10 ng/µL of a long ssDNA donor. The donor contained two lox2272 sites in cis, spanning partial intron 2, entire exon 3, and partial intron 3 of the mouse Tbx5 gene, along with 100/150 bp 5’/3’ homology arms (Supplementary Figure 4).Founders were validated by PCR, restriction enzyme digestion, and Sanger sequencing (Supplementary Figure 1B, C, and D). Founders were backcrossed with wild-type CD1 IGS mice (Charles River Lab, USA) to confirm germline transmission of the CRISPR/Cas9-generated compound Tbx3:Tbx5 double-floxed allele and obtain the F1 generation. F1 mice were then interbred to establish a stable Tbx3:Tbx5 double-floxed mouse line. Downstream experiments were performed on F4-F6 mice.

To simultaneously conditionally delate the Tbx3 and Tbx5 genes specifically from the adult VCS, we crossed our Tbx3:Tbx5 double-floxed mouse line (Tbx3^fl/fl;Tbx5^fl/fl) with a VCS-specific tamoxifen (TM) inducible Cre transgenic mouse line (MinK^CreERT2 [Tg(RP23-276I20-MinkCreERT2; (8)) (Figure 1A). All mice were maintained on a mixed genetic background. Tamoxifen (MP Biomedical) was administered at a dose of 0.167 mg/g body weight for 5 consecutive days by oral gavage at 6 weeks of age and then mice were evaluated at 9 weeks of age, as previously described (1, 8, 11). Age-, gender-, and genetic strain-matched controls were used in all experiments to account for any variations in data sets across experiments. Mice were bred and housed in specific pathogen–free conditions in a 12-hour light/12-hour dark cycle and allowed ad libitum access to standard mouse chow and water. Mice requiring medical attention were provided with appropriate veterinary care by a licensed veterinarian and were excluded from the experiments described. No other exclusion criteria were applied.

All experiments and subsequent analysis were conducted in a blinded fashion, with animals randomly assigned to experimental groups. Following genotyping, mice were randomly allocated to the studies based on their genotypes. Subsequently, their identities were anonymized using a numerical code to ensure that all experiments and analyses were performed in a blinded manner. Both, male and female animals have been used in our studies in the ratio of 41%/59%, based on availability of relatively rare compound genotypes, respectively.

Echocardiography studies

Transthoracic echocardiography in mice was conducted under inhaled isoflurane anesthesia administered through a nose cone. Prior to imaging, chest hairs were removed using a topical depilatory agent. Limb leads were affixed for electrocardiogram gating, and animals were imaged in the left lateral decubitus position with a VisualSonics Vevo 770 machine using a 30-MHz high-frequency transducer. To ensure stability, body temperature was carefully maintained using a heated imaging platform and warming lamps. Two-dimensional images were meticulously recorded in parasternal long- and short-axis projections, accompanied by guided M-mode recordings at the midventricular level in both views. Left ventricular (LV) cavity size and percent fractional shortening were measured in at least 3 beats from each projection and averaged. M-mode measurements were employed to ascertain LV chamber dimensions and percent LV fractional shortening, calculated as ([LVIDd – LVIDs]/LVIDd), where LVIDd and LVIDs represent LV internal diameter in diastole and systole, respectively.

Surface electrocardiography (ECG)

9 weeks old, tamoxifen treated control and mutant mice were anesthetized with a mixture of 2-3% isoflurane in 100% oxygen. Anesthetized mice were secured in a supine position on a regulated heat pad while lead I and lead II ECGs were recorded using platinum subdermal needle electrodes in a 3-limb configuration. Core temperature was continuously monitored using a rectal probe and maintained at 36–37 °C throughout the procedure. ECG data were collected and analyzed using Ponemah Physiology Platform (DSI) software and an ACQ-7700 acquisition interface unit (Gould Instruments, Valley View, OH, USA). Key parameters derived from the ECG measurements included: Heart rate (HR), PR interval (from the beginning of the P wave to the beginning of the QRS complex), and QRS complex duration.

Telemetry ECG analysis

9 weeks old, tamoxifen treated control and mutant mice were anesthetized with 2-3% isoflurane in 100% oxygen, and wireless telemetry transmitters (ETA-F10; DSI) were surgically implanted in the back with leads tunneled to the right upper and left lower thorax, as previously described (1, 56). Following 24-hour recovery period after surgical instrumentation, heart rate and PR and QRS intervals were calculated using Ponemah Physiology Platform (DSI) from 48-hour recordings.

Catheter-based intracardiac electrophysiology

Detailed protocols for invasive electrophysiology studies (EP) have been previously described (57, 58). Briefly, 9 weeks old, tamoxifen treated control and mutant mice were anesthetized using 2-3%isoflurane in 100% oxygen. Then, a 1.1-Fr octapolar electrode catheter (EPR-800; Millar Instruments) was advanced via a right jugular venous cut-down to record right atrial (RA), His bundle, and right ventricular (RV) potentials, as well as to perform programmed electrical stimulation. Signals were identified through alignment with simultaneous surface electrocardiography (ECG) using subcutaneous needle electrodes in a Lead II configuration. “Near-Field” and “Far-Field” signals were identified based on ECG alignment, signal deflection upstroke speed and total signal duration. Standard tachycardia induction protocols included an 8-beat drive train with beats 80-120ms apart (S1), followed by 5 beats (S2) at 50ms apart (penta-extrastimulus, PES). Two attempts at this PES protocol were carried out. Mice also underwent single extrastimulus testing (SES) with 8 beats 80-120ms apart (S1) followed by a single S2 at 50ms. This SES protocol was carried out 5 separate times per mouse. If these two protocols in the right atrium and right ventricle (separately) failed to initiate their respective tachycardias, the study was deemed negative. S1 drive train intervals varied slightly due to the presence of the AV Wenckebach block at faster pacing rates in some mice; the S1 interval was lengthened to prevent this during the drive train.

Additional atrial and ventricular pacing protocols were carried out to obtain atrial, atrio-ventricular, and ventricular effective refractory periods (AERP, AVERP, VERP) as well as sinus node recovery time, as described previously (57–60). Effective refractory periods were measured using 8-beat S1 drive trains of 100 ms followed by single extra-stimulus.

ECG and Optical mapping

Electrocardiogram (ECG) Acquisition and Analysis

ECGs were recorded in conscious Tbx3:Tbx5 double-conditional knockout mice and control littermates using the ecgTUNNEL device (emka Technologies) 2 weeks after tamoxifen treatment. Mice were positioned in the tunnel and ECGs were recorded for 5 minutes at a sampling rate of 1 kHz using lead I. ECGs were analyzed using a custom MATLAB program to measure P, and QRS durations, as well as PR, QT and RR intervals (61, 62) (Supplementary Figure 6).

Langendorff Perfusion

Tbx3:Tbx5 double-conditional knockout mice (n=8) and control littermates (n=10) were deeply anesthetized with isoflurane. The heart was quickly excised following cervical dislocation and thoracotomy. The aorta was cannulated, and the heart was retrogradely perfused with warmed (37°C) and oxygenated (95% O2 and 5% CO2) modified Tyrode’s solution (in mM, NaCl 130, NaHCO3 24, NaH2PO4 1.2, MgCl2 1, Glucose 5.6, KCl 4, and CaCl2 1.8) at a pH of 7.4. The heart was placed in a constant-flow (1.0 - 2.0 ml/min) Langendorff Perfusion system, laying horizontally in a tissue bath.

Ventricular Optical Mapping

The Langendorff Perfused heart was electromechanically uncoupled by 15 μM of blebbistatin (Cayman Chemicals 13186) perfusion. A voltage-sensitive fluorescent dye, 80 µM di-4-ANEPPs (ThermoFisher Scientific D1199), was administered through the dye injection port. The heart was illuminated using a 520 ± 5 nm (Prizmatix, UHP-Mic-LED-520) wavelength light source to excite di-4-ANEPPs. Emitted photons were captured using complementary metal-oxide semiconductor (CMOS) cameras (MiCAM, SciMedia). The stimulation threshold, where the heart would capture the stimuli and action potential 1:1, was determined using a point source platinum electrode placed on the anterior surface of the heart on the anterior epicardial surface of the ventricles. Pacing was applied at 1.5x the threshold amplitude to maintain 1:1 capture over the duration of the experiment. Optical recordings were analyzed using Rhythm 1.2 to analyze transmembrane potential (63). Action potential duration (APD) at 50% and 80% repolarization were calculated for the ventricles of the intact whole heart preparation.

Right Septal Preparation Optical Mapping Experiments

Following intact whole heart ex vivo optical mapping, the heart was removed from the tissue bath for the dissection of the right ventricular (RV) free wall to expose the RV septal surface (64). The heart was quickly returned to the tissue bath and the RV septal surface was focused into the field of view. Sinus rhythm optical recordings were acquired.

A custom MATLAB program was written to plot individual pixels in a 100×100-pixel image stack. A Butterworth filter of order 5 was applied to provide temporal filtering (65). The location of the His bundle was identified as the region at the base of the interventricular septum (a red 10×10 pixel area in Figure 5B). Signals from the working ventricular myocardium were recorded from the 10×10 pixel region plotted in the working ventricular myocardium distal to the His bundle (Figure 6B).

Isolation of adult VCS cardiomyocytes and cell sorting

To isolate EYFP-positive VCS cardiomyocytes, a tip of ventricular septum below the AV annulus was microdissected out from 9 weeks old TM-treated control and Tbx3:Tbx5 double-conditional mutant mice (1, 2, 66) under a fluorescence dissecting scope. Single-cell fluorescent-activated cell sorting (FACS) samples were prepared as previously described (67). Propidium iodide (ThermoFisher Scientific) was added immediately before FACS to facilitate live/dead discrimination. Cells were sorted on a FacsAria flow cytometer (BD Biosciences) located at the University of Chicago Flow Cytometery Core using Influx software. Samples from wild-type age-matched hearts were used for gating. Samples were gated to exclude debris and cell clumps. Fluorescent cells were collected into ice-cold RNase-free PBS and processed for RNA extraction.

RNA isolation and QRT-PCR

Total RNA was extracted from EYFP-positive VCS cardiomyocytes obtained by sorting from 9 weeks old control and VCS-specific Tbx3:Tbx5-deficient mice using RNeasy Mini Kit (Qiagen), followed by DNase treatment according to the manufacturer’s instructions. Reverse transcription reaction was carried out using the SuperScript III First-Strand Synthesis SuperMix for quantitative RT-PCR (Invitrogen) as per the manufacturer’s recommendations. QRT-PCR was performed using the POWER SYBR Green PCR master mix from Applied Biosystems and run on an Applied Biosystems AB7500 machine in 96 well plates. The relative gene expression level was calculated by the ΔΔCt method (68) using glyceraldehyde-3-phosphate dehydrogenase (Gapdh) gene expression level as internal control. The data presented are the average of three independent experiments.

Protein isolation and Western blotting

Atria and ventricles were dissected from Tbx3:Tbx5 mutant mice (Tbx3^fl/fl;Tbx5^fl/fl; R26^EYFP/+;MinK^CreERT2/+)and their littermate controls Tbx3^+/+;Tbx5^+/+;R26^EYFP/+; MinK^CreERT2/+) littermates that had been administered tamoxifen at 6-7 weeks of age and studied 4-5 weeks following tamoxifen administration. The tissues were snap-frozen in liquid nitrogen, pulverized, and homogenized in RIPA buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 1% Triton-X, 0.5% sodium deoxycholate, 0.1% SDS, 5 mM EDTA) with 1 Roche EDTA-Free complete protease inhibitor tablet per 50 mL of buffer. Samples were tumbled for 1 hour at 4°C and then centrifuged for 10 minutes at 13,200 x g. Protein concentration was determined using the BCA assay (Pierce) with BSA as a standard. For Western blot analysis, 25 µg of protein was diluted in Laemmli buffer, heated at 70°C for 10 minutes, and subjected to SDS-PAGE on 4-20% TGX gels (Bio-Rad). Proteins were then transferred to nitrocellulose membranes, blocked with 5% milk in TBS-T, and incubated overnight at 4°C with primary antibodies diluted in 2.5% milk in TBS-T. The primary antibodies used were: goat polyclonal anti-TBX3 (Santa Cruz Biotechnology, sc-31656, 1:250), rabbit polyclonal anti-HCN4 (Millipore, AB5808, 1:500), rabbit polyclonal anti-CAV1.3/CACNA1D (Alomone, ACC-005, 1:200), rabbit polyclonal anti-Cx45/GJC1 (Thermo Fisher, PA5-77357, 1:250), sheep polyclonal anti-TBX5 (R&D, AF5918, 1:200), rabbit polyclonal anti-CX40/GJA5 (Zymed/Invitrogen, 36-4900, 1:500), rabbit polyclonal anti-NAV1.5/SCN5A (Alomone, ASC-005, 1:200), mouse monoclonal anti-KCNK3/TASK1 (Abcam, ab186352, 1:1000), rabbit polyclonal anti-CX43/GJA1 (Cell Signaling Technology, 3512, 1:1000), and mouse monoclonal anti-GAPDH (Abcam, ab8245, 1:1000). After rinsing in TBS-T, membranes were incubated for one hour at RT with secondary antibodies diluted in 2.5% milk in TBS-T, rinsed again, and visualized using enhanced chemiluminescence reagents (Pierce ECL/ECL Plus, Thermo Fisher Scientific) and Kodak X-OMAT film. Results were normalized to GAPDH loading control and then quantified using ImageJ software (69, 70). Secondary antibodies used were donkey anti-goat IgG AlexaFluor-594 (Invitrogen, A-11058, 1:250 dilution) and donkey anti-goat IgG AlexaFluor-488 (Invitrogen, A-11055, 1:250 dilution) for experiments involving co-staining for goat primary antibodies. Secondary antibodies were as follows: rabbit anti-goat-HRP (Jackson ImmunoResearch, 305-035-003, 1:10 000), goat anti-rabbit-HRP (Jackson Immuno120 Research, 111-035-144, 1:3000), donkey anti-sheep-HRP (Ab cam, ab6900, 1:5000), and sheep anti-mouse-HRP (Amersham GE, NA931, 1:2500).

Histology

Hearts from 9-week-old control and mutant mice were carefully dissected, flushed with ice-cold PBS, and then fixed for 48 hours at 4 °C in a 4% paraformaldehyde solution (Sigma-Aldrich). Following fixation, the hearts were processed for paraffin-embedded sections and subjected to analysis through H&E staining, following the manufacturer’s protocol provided by Sigma-Aldrich.

Immunofluorescence

Hearts from 9 weeks old control and mutant mice were dissected out and placed in ice-cold PBS, followed by freezing in OCT (Fisher) within the gas phase of liquid nitrogen. Cryosections of 7 μm thickness were mounted onto Superfrost Plus glass slides (Fisher Scientific), air-dried, and fixed for 10 minutes in ice-cold 4% paraformaldehyde (Sigma-Aldrich). Subsequently, sections were permeabilized in 1% Triton-X100 (Sigma-Aldrich) in PBS for 10 min, then blocked in 10% normal goat serum (Invitrogen) in PBS-T (PBS+0.1% Tween-20) for 30 min at room temperature (RT). Sections were then incubated overnight at 4°C in primary antibody diluted in blocking buffer, rinsed in PBS, then subsequently incubated for 60 min at RT in secondary antibody diluted in blocking buffer. Slides were mounted in VectaShield+DAPI (Vector Laboratories) or counterstained with DAPI and mounted in ProLog Gold (Invitrogen) priori to visualizing fluorescence. Primary antibodies were as follows: goat polyclonal anti-TBX3 (Santa Cruz, sc-31656, 1:250 dilution) and goat polyclonal anti-TBX5 (Santa Cruz, sc-17866, 1:250 dilution). Secondary antibodies used were donkey anti-goat IgG AlexaFluor-594 (Invitrogen, A-11058, 1:250 dilution) and donkey anti-goat IgG AlexaFluor-488 (Invitrogen, A-11055, 1:250 dilution) for experiments involving co-staining for goat primary antibodies. A secondary antibody-only control was employed in each case to ensure the specificity of immunostaining.

Statistics

The numbers of independent experiments are specified in the relevant figure legends. Quantitative data are presented as mean ± SD. Statistical analysis was performed using R version 3.6.2. or the GraphPad Prism statistical package version 10.2.1 (GraphPad Software, Boston, MA, USA). All data sets were analyzed independently for normality using Shapiro-Wilk normality test. If samples presented a normal distribution, the two-sample Welch t-test was performed. If normal distribution was not present, the non-parametric Mann-Whitney U test or Kruskal-Wallis H test was used, as indicated in the text. Statistical significance was assumed if P reached a value ≤ 0.05. The multiple comparison correction tests were performed on VCS, atrial and ventricular qRT-PCR data by calculation the false discovery rate (FDR) using the Benjamini & Hochberg procedure (71). A significant group-mean was considered at FDR<0.05.

Additional information

Sources of Funding

This work was supported by National Institutes of Health (R01 HL126509, R01 HL148719, R01 HL147571, and R33 HL123857 to I.P. Moskowitz), Foundation Leducq Transatlantic Networks of Excellence (to I.P. Moskowitz), and American Heart Association (7CSA33610126 to I.P. Moskowitz and 13POST17290028 to O. Burnicka-Turek).

Conflict of Interest Statement

None declared.

Contributions

IPM and OBT conceived and designed the study; OBT, KAT, BL, MTB, ZK, ER, BL, ES, MG, KMS, and DEA performed wet lab experiments; OBT, KAT, BL, MTB, MG, KMS, IRE, and IPM participated in data analysis. IPM directed the project; OBT and IPM drafted the manuscript. OBT and IPM serve as co-corresponding authors. All authors contributed to the final manuscript.

Nonstandard Abbreviations and Acronyms

• ACUP: animal care and use protocol

• AERP: atrial effective refractory period

• A-H: atrio-hisian interval

• APD50, 80: action potential duration at 50 and 80% of repolarization

• AVB: atrioventricular bundle

• AVERP: atrioventricular nodal effective refractory period

• AVN: atrioventricular node

• BBs: bundle branches

• Cacna1d/Cav1.3: calcium channel, voltage-dependent, L type, alpha 1D subunit

• Cacna1g/Cav3.1d: calcium channel, voltage-dependent, T type, alpha 1G subunit

• Cacna1h/Cav3.2: calcium channel, voltage-dependent, T type, alpha 1H subunit

• CCS: cardiac conduction system

• Chr5: chromosome 5

• Col1a1: collagen, type I, alpha 1

• CREs: cis-regulatory elements

• CRISPR: clustered regularly interspaced short palindromic repeats

• EP studies: electrophysiology studies

• ECG: electrocardiography

• Eyfp: enhanced yellow fluorescent protein

• FACS: fluorescent-activated cell sorting

• FDR: False Discovery Rate

• FS: fractional shortening

• Gja1/Cx43: gap junction protein, alpha 1

• Gja5/Cx40: gap junction protein, alpha 5

• Gjc1/Cx45: gap junction protein, gamma 1

• Gjd3/Cx30.2: gap junction protein, delta 3

• GRN: gene regulatory network

• GWAS: genome wide association studies

• Hcn1: hyperpolarization activated cyclic nucleotide gated potassium channel 1

• Hcn4: hyperpolarization-activated, cyclic nucleotide-gated K+ 4

• Hd: his-duration

• HOS: Holt-Oram syndrome

• H-V: hisio-ventricular interval

• IACUC: institutional animal care and use committee

• Kcne1/MinK: potassium voltage-gated channel, Isk-related subfamily, member 1

• Kcnj2/Kir2.1: potassium inwardly-rectifying channel, subfamily J, member 2

• Kcnj3/Kir3.1: potassium inwardly-rectifying channel, subfamily J, member 3

• Kcnj4/IRK3: potassium inwardly-rectifying channel, subfamily J, member 4

• Kcnj12/Kir2.2: potassium inwardly-rectifying channel, subfamily J, member 12

• Kcnk3/Task-1: potassium channel, subfamily K, member 3

• lssDNA donor: long single-stranded DNA donor

• LVEF: left ventricular ejection fraction

• OAP: optical action potential

• OE: overexpression

• OMIM: online Mendelian inheritance in man

• QRS: QRS complex

• QT: QT-interval duration

• PAM sequence: protospacer-adjacent motif sequence

• Postn: periostin, osteoblast specific factor

• PR: PR-interval duration

• RFLP: Restriction fragment length polymorphism

• RR: RR-interval duration

• Ryr2: ryanodine receptor 2, cardiac

• SAN: sinoatrial node

• Scn5a/Nav1.5: sodium channel, voltage-gated, type V, alpha

• sgRNA: single guide RNA

• Smpx: small muscle protein, X-linked

• Tbx3: T-box 3

• Tbx5: T-box 5

• TM: tamoxifen

• VCS: ventricular conduction system

• VERP: ventricular effective refractory period

• VT: ventricular tachycardia