Department of Medical Physiology, Division of Heart & Lungs, University Medical Center Utrecht, Utrecht, the Netherlands.
Department of Medical Physiology, Division of Heart & Lungs, University Medical Center Utrecht, Utrecht, the Netherlands.
J Mol Cell Cardiol. 2019 Jan;126:86-95. doi: 10.1016/j.yjmcc.2018.11.007. Epub 2018 Nov 17.
The intercalated disc (ID) is important for cardiac remodeling and has become a subject of intensive research efforts. However, as yet the composition of the ID has still not been conclusively resolved and the role of many proteins identified in the ID, like Flotillin-2, is often unknown. The Flotillin proteins are known to be involved in the stabilization of cadherins and desmosomes in the epidermis and upon cancer development. However, their role in the heart has so far not been investigated. Therefore, in this study, we aimed at identifying the role of Flotillin-1 and Flotillin-2 in the cardiac ID.
Location of Flotillins in human and murine cardiac tissue was evaluated by fluorescent immunolabeling and co-immunoprecipitation. In addition, the effect of Flotillin knockout (KO) on proteins of the ID and in electrical excitation and conduction was investigated in cardiac samples of wildtype (WT), Flotillin-1 KO, Flotilin-2 KO and Flotilin-1/2 double KO mice. Consequences of Flotillin knockdown (KD) on cardiac function were studied (patch clamp and Multi Electrode Array (MEA)) in neonatal rat cardiomyocytes (NRCMs) transfected with siRNAs against Flotillin-1 and/or Flotillin-2.
First, we confirmed presence in the ID and mutual binding of Flotillin-1 and Flotillin-2 in murine and human cardiac tissue. Flotillin KO mice did not show cardiac fibrosis, nor hypertrophy or changes in expression of the desmosomal ID proteins. However, protein expression of the cardiac sodium channel Na1.5 was significantly decreased in Flotillin-1 and Flotillin-1/2 KO mice compared to WT mice. In addition, sodium current density showed a significant decrease upon Flotillin-1/2 KD in NRCMs as compared to scrambled siRNA-transfected NRCMs. MEA recordings of Flotillin-2 KD NRCM cultures showed a significantly decreased spike amplitude and a tendency of a reduced spike slope when compared to control and scrambled siRNA-transfected cultures.
In this study, we demonstrate the presence of Flotillin-1, in addition to Flotillin-2 in the cardiac ID. Our findings indicate a modulatory role of Flotillins on Na1.5 expression at the ID, with potential consequences for cardiac excitation.
闰盘对于心脏重构十分重要，已成为研究热点。然而，闰盘的组成尚未完全明确，许多在闰盘中鉴定到的蛋白，如 Flotillin-2 的作用也并不清楚。Flotillin 蛋白已知参与稳定表皮的黏附连接和桥粒，在癌症发生时也发挥作用。然而，其在心脏中的作用目前尚未研究。因此，本研究旨在鉴定 Flotillin-1 和 Flotillin-2 在心脏闰盘中的作用。
通过荧光免疫标记和共免疫沉淀评估 Flotillin 在人类和鼠类心脏组织中的位置。此外，在野生型（WT）、Flotillin-1 KO、Flotillin-2 KO 和 Flotillin-1/2 双 KO 鼠的心脏样本中，研究 Flotillin 敲除（KO）对闰盘蛋白以及电兴奋和传导的影响。通过转染针对 Flotillin-1 和/或 Flotillin-2 的 siRNA 的新生大鼠心肌细胞（NRCM）进行心脏功能的 Flotillin 敲低（KD）研究（膜片钳和多电极阵列（MEA））。
首先，我们在鼠类和人类心脏组织中证实了 Flotillin-1 和 Flotillin-2 的存在及其在闰盘中的相互结合。Flotillin KO 鼠没有表现出心脏纤维化、肥厚或桥粒 ID 蛋白表达的变化。然而，与 WT 鼠相比，Flotillin-1 和 Flotillin-1/2 KO 鼠的心脏钠通道 Na1.5 蛋白表达显著降低。此外，与对照和 scrambled siRNA 转染的 NRCM 相比，NRCM 中的 Flotillin-1/2 KD 导致钠电流密度显著降低。MEA 记录显示，Flotillin-2 KD NRCM 培养物的尖峰幅度显著降低，与对照和 scrambled siRNA 转染的培养物相比，尖峰斜率有降低的趋势。
在这项研究中，我们证明了 Flotillin-1 除了 Flotillin-2 之外，还存在于心脏闰盘中。我们的发现表明 Flotillins 对 ID 处 Na1.5 表达具有调节作用，可能对心脏兴奋产生影响。
