Li Shichao, Gao Yuanfeng, Liu Ye, Li Jing, Yang Xiyan, Hu Roumu, Liu Jia, Zhang Yuan, Zuo Kun, Li Kuibao, Yin Xiandong, Chen Mulei, Zhong Jiuchang, Yang Xinchun
Heart Center and Beijing Key Laboratory of Hypertension, Beijing Chaoyang Hospital, Capital Medical University, Beijing, China.
Heart Center and Beijing Key Laboratory of Hypertension, Beijing Chaoyang Hospital, Capital Medical University, Beijing, China,
Cardiology. 2020;145(5):324-332. doi: 10.1159/000505641. Epub 2020 Apr 1.
Atrial fibrosis plays a critical role in atrial fibrillation (AF). A key event in the pathogenesis of fibrosis is the activation of fibroblasts (FBs) into myofibroblasts (MFBs). Paracrine factors released from MFBs lead to ion channel expression changes in cardiomyocytes (CMs). Downregulation of L-type calcium channel Cav1.2 expression is a hallmark of AF-associated ionic remodeling. However, whether exosome (Exo)-mediated crosstalk between MFBs and CMs regulates Cav1.2 expression remains unknown.
Atrial FBs and CMs were isolated and cultured from neonatal rats by enzymatic digestion. The activation of FBs into MFBs was induced by angiotensin II. Co-culture assay and in vitro Exo treatment were used to determine the effect of MFB-derived Exos on Cav1.2 expression. Confocal Ca2+ imaging was performed to examine the adrenergic stimulation-elicited Ca2+ influx signals. The levels of potential Cav1.2-inhibitory microRNAs (miRNAs) were measured by qRT-PCR.
Untreated FBs expressed limited amounts of alpha smooth muscle actin (α-SMA), while angiotensin II induced a significant upregulation of α-SMA-expressing MFBs. Co-cultures of MFBs and CMs resulted in downregulation of Cav1.2 expression in CMs, which was largely abolished by pretreatment of MFBs with exosomal inhibitor GW4869. More importantly, treatment with MFB-derived Exos caused repression of Cav1.2 expression in CMs. Additionally, the adrenergic receptor agonist-elicited Ca2+ influx signals in CMs were remarkably attenuated by pretreatment with MFB-derived Exos, corresponding to the paralleled change in Cav1.2 expression. Finally, miR-21-3p, a potential Cav1.2-inhibitory miRNA, was enriched in MFB-derived Exos and upregulated in CMs in response to MFB-derived Exos.
We uncover an Exo-mediated crosstalk between MFBs and CMs, contributing to increased vulnerability to AF by reducing the expression of Cav1.2 in CMs.
心房纤维化在心房颤动（AF）中起关键作用。纤维化发病机制中的一个关键事件是成纤维细胞（FBs）激活为肌成纤维细胞（MFBs）。MFBs释放的旁分泌因子导致心肌细胞（CMs）中离子通道表达发生变化。L型钙通道Cav1.2表达下调是AF相关离子重塑的一个标志。然而，外泌体（Exo）介导的MFBs与CMs之间的相互作用是否调节Cav1.2表达仍不清楚。
通过酶消化从新生大鼠中分离并培养心房FBs和CMs。用血管紧张素II诱导FBs激活为MFBs。采用共培养试验和体外Exo处理来确定MFB来源的外泌体对Cav1.2表达的影响。进行共聚焦钙成像以检测肾上腺素能刺激引发的钙内流信号。通过qRT-PCR测量潜在的Cav1.2抑制性微小RNA（miRNAs）水平。
未处理的FBs表达有限量的α平滑肌肌动蛋白（α-SMA），而血管紧张素II诱导表达α-SMA的MFBs显著上调。MFBs与CMs共培养导致CMs中Cav1.2表达下调，用外泌体抑制剂GW4869预处理MFBs可在很大程度上消除这种下调。更重要的是，用MFB来源的Exos处理导致CMs中Cav1.2表达受到抑制。此外，用MFB来源的Exos预处理可显著减弱肾上腺素能受体激动剂引发的CMs中的钙内流信号，这与Cav1.2表达的平行变化相对应。最后，miR-21-3p是一种潜在的Cav1.2抑制性miRNA，在MFB来源的Exos中富集，并在CMs中因MFB来源的Exos而上调。
我们发现了一种Exo介导的MFBs与CMs之间的相互作用，通过降低CMs中Cav1.2的表达，增加了对AF的易感性。
