Nieto-Marín Paloma, Tinaquero David, Utrilla Raquel G, Cebrián Jorge, González-Guerra Andrés, Crespo-García Teresa, Cámara-Checa Anabel, Rubio-Alarcón Marcos, Dago María, Alfayate Silvia, Filgueiras-Rama David, Peinado Rafael, López-Sendón José Luis, Jalife José, Tamargo Juan, Bernal Juan Antonio, Caballero Ricardo, Delpón Eva
Department of Pharmacology and Toxicology, School of Medicine, Universidad Complutense de Madrid. Instituto de Investigación Gregorio Marañón. CIBERCV, Madrid 28040, Spain.
Fundación Centro Nacional de Investigaciones Cardiovasculares. CIBERCV, Madrid 28029, Spain.
Cardiovasc Res. 2022 Mar 16;118(4):1046-1060. doi: 10.1093/cvr/cvab045.
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.
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 wild-type (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.
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.
转录因子Tbx5控制心脏发育并驱动小鼠Scn5a的表达。我们在两名结构正常心脏但被诊断为长QT综合征（LQTS）和Brugada综合征（BrS）的无关患者中，分别鉴定出TBX5编码p.D111Y和p.F206L Tbx5的两个变体。在此，我们对每个变体的后果进行了表征，以阐明潜在的疾病机制。
我们将临床分析与体内外电生理及分子技术相结合，应用于人类诱导多能干细胞衍生的心肌细胞（hiPSC-CMs）、HL-1细胞以及转染表达人类野生型（WT）或突变蛋白的小鼠心肌细胞。Tbx5增加了编码心脏Nav1.5通道的SCN5A的转录，同时分别抑制了编码钙/钙调蛋白激酶IIδ（CaMKIIδ）和βIV-血影蛋白的CAMK2D和SPTBN4基因。这些作用显著增加了hiPSC-CMs和转染表达Tbx5的小鼠心肌细胞中的钠电流（INa）。因此，小鼠心电图中的动作电位（AP）幅度增加，QRS间期变窄。与SCN5A启动子结合的p.F206L Tbx5无法激活它，从而排除了WT Tbx5的促转录作用。因此，p.F206L显著降低了hiPSC-CM、HL-1细胞和小鼠心肌细胞中的INa。转染表达p.F206L的小鼠中INa的降低转化为QRS增宽和对氟卡尼敏感性增加。p.D111Y Tbx5增加了SCN5A的表达，但未能抑制CAMK2D和SPTBN4。增加的CaMKIIδ和βIV-血影蛋白显著增加了INa的晚期成分（INaL），进而显著延长了hiPSC-CMs和小鼠心肌细胞中的AP持续时间。选择性INaL抑制剂雷诺嗪消除了转染表达p.D111Y的小鼠中出现的QT和QTc间期延长。
除了峰值INa外，Tbx5还对人类心肌细胞中的INaL和复极化持续时间起关键调节作用。我们的原始结果表明，TBX5变体与LQTS和BrS患者的电表型强度相关并对其进行调节。
