Division of Cardiology, Department of Internal Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Jiefang Avenue NO.1095, Qiaokou District, Wuhan, 430030, China.
Hubei Key Laboratory of Genetics and Molecular Mechanisms of Cardiological Disorders, Jiefang Avenue NO.1095, Qiaokou District, Wuhan, 430030, China.
Cardiovasc Res. 2022 Oct 21;118(13):2805-2818. doi: 10.1093/cvr/cvab325.
Abnormal intracellular calcium (Ca2+) handling contributes to the progressive nature of atrial fibrillation (AF), the most common sustained cardiac arrhythmia. Evidence in mouse models suggests that activation of the nuclear factor of activated T-cell (NFAT) signalling pathway contributes to atrial remodelling. Our aim was to determine the role of NFATc2 in AF in humans and mouse models.
Expression levels of NFATc1-c4 isoforms were assessed by quantitative reverse transcription-polymerase chain reaction in right atrial appendages from patients with chronic AF (cAF). NFATc1 and NFATc2 mRNA levels were elevated in cAF patients compared with those in normal sinus rhythm (NSR). Western blotting revealed increased cytosolic and nuclear levels of NFATc2 in AF patients. Similar findings were obtained in CREM-IbΔC-X transgenic (CREM) mice, a model of progressive AF. Telemetry ECG recordings revealed age-dependent spontaneous AF in CREM mice, which was prevented by NFATc2 knockout in CREM:NFATc2-/- mice. Programmed electrical stimulation revealed that CREM:NFATc2-/- mice lacked an AF substrate. Morphometric analysis and histology revealed increased atrial weight and atrial fibrosis in CREM mice compared with wild-type controls, which was reversed in CREM:NFATc2-/- mice. Confocal microscopy showed an increased Ca2+ spark frequency despite a reduced sarcoplasmic reticulum (SR) Ca2+ load in CREM mice compared with controls, whereas these abnormalities were normalized in CREM:NFATc2-/- mice. Western blotting revealed that genetic inhibition of Ca2+/calmodulin-dependent protein kinase II-mediated phosphorylation of S2814 on ryanodine receptor type 2 (RyR2) in CREM:RyR2-S2814A mice suppressed NFATc2 activation observed in CREM mice, suggesting that NFATc2 is activated by excessive SR Ca2+ leak via RyR2. Finally, chromatin immunoprecipitation sequencing from AF patients identified Ras and EF-hand domain-containing protein (Rasef) as a direct target of NFATc2-mediated transcription.
Our findings reveal activation of the NFAT signalling pathway in patients of Chinese and European descent. NFATc2 knockout prevents the progression of AF in the CREM mouse model.
异常的细胞内钙（Ca2+）处理导致心房颤动（AF）的进行性发展，AF 是最常见的持续性心律失常。在小鼠模型中的证据表明，激活 T 细胞激活核因子（NFAT）信号通路有助于心房重构。我们的目的是确定 NFATc2 在人类和小鼠模型中的 AF 中的作用。
通过定量逆转录聚合酶链反应（qRT-PCR）评估慢性 AF（cAF）患者右心耳中 NFATc1-c4 同工型的表达水平。与窦性心律（NSR）相比，cAF 患者的 NFATc1 和 NFATc2 mRNA 水平升高。Western blot 显示 AF 患者的 NFATc2 细胞质和核水平升高。在 CREM-IbΔC-X 转基因（CREM）小鼠中也获得了类似的发现，这是一种进行性 AF 的模型。遥测心电图记录显示，CREM 小鼠存在年龄依赖性自发性 AF，NFATc2 敲除可预防 CREM：NFATc2-/- 小鼠的 AF。程控电刺激显示 CREM：NFATc2-/- 小鼠缺乏 AF 底物。形态计量分析和组织学显示，与野生型对照相比，CREM 小鼠的心房重量和心房纤维化增加，而 CREM：NFATc2-/- 小鼠则逆转。共聚焦显微镜显示，与对照相比，尽管肌浆网（SR）Ca2+负荷减少，但 CREM 小鼠的 Ca2+火花频率增加，而 CREM：NFATc2-/- 小鼠中的这些异常则正常化。Western blot 显示，通过抑制钙/钙调蛋白依赖性蛋白激酶 II 介导的肌浆网 2 型钙释放通道（RyR2）上 S2814 的磷酸化，抑制 CREM：RyR2-S2814A 小鼠中观察到的 NFATc2 激活，这表明 NFATc2 通过 RyR2 过度 SR Ca2+渗漏激活。最后，从 AF 患者的染色质免疫沉淀测序中发现 Ras 和 EF 手域蛋白（Rasef）是 NFATc2 介导转录的直接靶标。
我们的发现揭示了 NFAT 信号通路在中、欧裔患者中的激活。NFATc2 敲除可预防 CREM 小鼠模型中 AF 的进展。
