Tbx5 variants disrupt Nav1.5 function differently in patients diagnosed with Brugada or Long QT Syndrome

Abstract

AIMS The transcription factor Tbx5 controls cardiogenesis and drives Scn5a expression in mice. We have identified two variants in TBX5 encoding p.D111Y and p.F206L Tbx5, respectively, in two unrelated patients with structurally normal hearts diagnosed with Long QT (LQTS) and Brugada (BrS) Syndrome. Here we characterized the consequences of each variant to unravel the underlying disease mechanisms. METHODS AND RESULTS We combined clinical analysis with in vivo and in vitro electrophysiological and molecular techniques in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), HL-1 cells, and cardiomyocytes from mice trans-expressing human wildtype (WT) or mutant proteins. Tbx5 increased transcription of SCN5A encoding cardiac Nav1.5 channels, while repressing CAMK2D and SPTBN4 genes encoding Ca-calmodulin kinase IIδ (CaMKIIδ) and βIV-spectrin, respectively. These effects significantly increased Na current (INa) in hiPSC-CMs and in cardiomyocytes from mice trans-expressing Tbx5. Consequently, action potential (AP) amplitudes increased and QRS interval narrowed in the mouse electrocardiogram. p.F206L Tbx5 bound to the SCN5A promoter failed to transactivate it, thus precluding the pro-transcriptional effect of WT Tbx5. Therefore, p.F206L markedly decreased INa in hiPSC-CM, HL-1 cells, and mouse cardiomyocytes. The INa decrease in p.F206L trans-expressing mice translated into QRS widening and increased flecainide sensitivity. p.D111Y Tbx5 increased SCN5A expression but failed to repress CAMK2D and SPTBN4. The increased CaMKIIδ and βIV-spectrin significantly augmented the late component of INa (INaL) which, in turn, significantly prolonged AP duration in both hiPSC-CMs and mouse cardiomyocytes. Ranolazine, a selective INaL inhibitor, eliminated the QT and QTc intervals prolongation seen in p.D111Y trans-expressing mice. CONCLUSIONS In addition to peak INa, Tbx5 critically regulates INaL and the duration of repolarization in human cardiomyocytes. Our original results suggest that TBX5 variants associate with and modulate the intensity of the electrical phenotype in LQTS and BrS patients.

Zfhx3 Transcription Factor Represses the Expression of SCN5A Gene and Decreases Sodium Current Density (INa)

“…Time dependence of I Na activation was quantified by fitting a monoexponential function to the activation phase of the maximum I Na trace of each experiment [ 13 ]. The activation time constant (τ act ) averaged 0.19 ± 0.01 ( n = 43) and 0.22 ± 0.03 ms ( n = 19) in cells transfected/not transfected with Zfhx3, respectively, indicating that the TF did not modify the activation kinetics ( p > 0.05) ( Table 1 ).…”

“…Consistently with these findings, Zfhx3 silencing did not modify it either ( Figure 3 B and Table 1 ). The persistent component of I Na or late I Na (I Na,L ) was quantified as the percentage of the peak I Na [ 13 ] and plotted in Figure 3 C,D. These figures demonstrate that I Na,L magnitude was not different in HL-1 cells transfected/not transfected with Zfhx3 ( Figure 3 C) ( p > 0.05, n ≥ 14) or in cells in which Zfhx3 expression was silenced/not silenced ( Figure 3 D) ( p > 0.05, n ≥ 7).…”

“…Additionally, both Zfhx3 and Pitx2c regulate the expression of other cardio-specific TF such as Tbx5 and Nkx2.5 [ 4 ]. Thus, we decided to test the effect of Zfhx3 on the expression of these TFs, which also regulated the magnitude of cardiac I Na [ 13 ]. Figure 5 A,B shows that transfection with Zfhx3 significantly decreased the protein expression of Tbx5 as demonstrated by WB experiments.…”

“…Thus, it plays a critical role in excitability and intracardiac conduction velocity. Recently, it was functionally demonstrated that Tbx5, a TF belonging to the T-box family, promotes the expression of the SCN5A gene, and thus increased I Na in human cardiomyocytes derived from induced pluripotent stem cells (hiPSC-CM) [ 13 ]. Furthermore, the Brugada Syndrome (BrS)-associated variant p.F206L Tbx5, lacks this pro-transcriptional effect, thus markedly reducing I Na [ 13 ].…”

“…Recently, it was functionally demonstrated that Tbx5, a TF belonging to the T-box family, promotes the expression of the SCN5A gene, and thus increased I Na in human cardiomyocytes derived from induced pluripotent stem cells (hiPSC-CM) [ 13 ]. Furthermore, the Brugada Syndrome (BrS)-associated variant p.F206L Tbx5, lacks this pro-transcriptional effect, thus markedly reducing I Na [ 13 ]. On the other hand, Nav1.5 expression decreases in atrial chamber-specific Pitx2 conditional mutants, suggesting that Pitx2c positively modulates Scn5a expression [ 14 ].…”