目的:心房颤动(AF),最常见的心律失常,以心房电重构和结构重构为特征。先前的研究发现,钠-葡萄糖协同转运蛋白2抑制剂(SGLT2i)可以通过不依赖葡萄糖的机制保护心肌。但是SGLT2i在调节AF中的作用仍然未知。这项研究,我们旨在研究达格列净(DAPA)通过抑制电重构和结构重构降低房颤易感性的作用.
方法:通过血管紧张素II(2000ng/kg/min)输注3周建立小鼠模型,并通过用AngII(1μM)刺激HL-1和原代小鼠成纤维细胞24小时来产生体外模型。程序电刺激,心电图和全细胞膜片钳检测DAPA对AngⅡ诱导的心房电重构的影响。观察DAPA对AngⅡ诱导的心房结构重构的影响,我们用了超声心动图,H&E和Masson染色评价心房扩张。为进一步探讨DAPA的保护机制,我们采用计算机分子对接方法来研究AngII和CaMKII在Met-281位点的结合亲和力。Westernblot检测CaMKII的表达水平,ox-CaMKII,Nav1.5、Kv4.3、Kv4.2、Kchip2、Kir2.1和Cx40。
结果:AngII诱发房颤,心房扩张和纤维化,导致心房电重构和结构重构。然而,DAPA治疗明显消除了这些影响,一个特定的SGLT2i。我们对小鼠心房电活动的观察表明,DAPA可以挽救由AngII输注引发的延长的动作电位持续时间(APD)和IK1,Ito和INaL的异常电流。DAPA可以降低AngII和CaMKII在Met-281位点的结合亲和力,这表明DAPA可以直接减轻AngII引起的CaMKII的激活。DAPA可以减少AngII在心房组织中引起的ox-CaMKII的上调。此外,DAPA还改善了由AngII引起的电活性相关蛋白(Nav1.5,Kv4.3,Kv4.2,Kchip2,Kir2.1和Cx40)和纤维化相关信号通路(TGF-β1,p-smad/smad)的异常表达水平。此外,我们确认了DAPA,以及其他SGLT2i(EMPA,CANA),可以逆转由HL-1细胞和原代小鼠成纤维细胞中AngII孵育引起的这些异常,分别。
结论:总体而言,我们的研究确定了DAPA,广泛使用的SGLT2i,有助于抑制AngII诱导的ox-CaMKII上调和电和结构重塑,以降低AF易感性,提示DAPA可能是治疗房颤的潜在疗法。
OBJECTIVE: Atrial fibrillation (AF), the most common arrhythmia, is characterized by atrial electrical and structural remodeling. Previous studies have found that sodium-glucose cotransporter 2 inhibitor (
SGLT2i) can protect myocardium in a glucose independent mechanism. But the role of
SGLT2i in regulating AF remains largely unknown. This study, we aimed to investigate the effect of Dapagliflozin (DAPA) in reducing AF susceptibility via inhibiting electrical and structural remodeling.
METHODS: The mouse model was established by Angiotensin II (2000 ng/kg/min) infusion for 3 weeks, and an in vitro model was generated by stimulating HL-1 and primary mouse fibroblast with Ang II (1 μM) for 24 h. Programmed electrical stimulation, ECG and whole-cell patch clamp were used to detect DAPA effect on atrial electrical remodeling induced by Ang II. To observe DAPA effect on atrial structural remodeling induced by Ang II, we used echocardiographic, H&E and Masson staining to evaluate atrial dilation. To further explore the protective mechanism of DAPA, we adopt in silico molecular docking approaches to investigate the binding affinity of Ang II and CaMKII at Met-281 site. Western blot was to detect expression level of CaMKII, ox-CaMKII, Nav1.5, Kv4.3, Kv4.2, Kchip2, Kir2.1 and Cx40.
RESULTS: Ang II induced AF, atrial dilatation and fibrosis, led to atrial electrical and structural remodeling. However, these effects were markedly abrogated by DAPA treatment, a specific
SGLT2i. Our observation of atrial electrical activity in mice revealed that DAPA could rescue the prolonged action potential duration (APD) and the abnormal currents of IK1, Ito and INaL triggered by Ang II infusion. DAPA could reduce the binding affinity of Ang II and CaMKII at Met-281 site, which indicated that DAPA may directly alleviate the activation of CaMKII caused by Ang II. DAPA could reduce the upregulation of ox-CaMKII caused by Ang II infusion in atrial tissues. Moreover, DAPA also ameliorated the aberrant expression levels of electrical activity related proteins (Nav1.5, Kv4.3, Kv4.2, Kchip2, Kir2.1 and Cx40) and fibrosis related signal pathways (TGF-β1, p-smad/smad) caused by Ang II. Furthermore, we confirmed that DAPA, as well as other
SGLT2i (EMPA, CANA), could reverse these abnormalities caused by Ang II incubation in HL-1 cells and primary mouse fibroblasts, respectively.
CONCLUSIONS: Overall, our study identifies DAPA, a widely used
SGLT2i, contributes to inhibiting Ang II-induced ox-CaMKII upregulation and electrical and structural remodeling to reduce AF susceptibility, suggesting that DAPA may be a potential therapy of treating AF.