Discovery and Translational Science Department, Leeds Institute of Cardiovascular and Metabolic Medicine, School of Medicine, University of Leeds, Leeds LS2 9JT, United Kingdom.
Multidisciplinary Cardiovascular Research Centre, University of Leeds, Leeds LS2 9JT, United Kingdom.
J Biol Chem. 2019 Nov 15;294(46):17395-17408. doi: 10.1074/jbc.RA119.009167. Epub 2019 Oct 4.
Piezo1 is a mechanosensitive cation channel with widespread physiological importance; however, its role in the heart is poorly understood. Cardiac fibroblasts help preserve myocardial integrity and play a key role in regulating its repair and remodeling following stress or injury. Here we investigated Piezo1 expression and function in cultured human and mouse cardiac fibroblasts. RT-PCR experiments confirmed that mRNA in cardiac fibroblasts is expressed at levels similar to those in endothelial cells. The results of a Fura-2 intracellular Ca assay validated Piezo1 as a functional ion channel that is activated by its agonist, Yoda1. Yoda1-induced Ca entry was inhibited by Piezo1 blockers (gadolinium and ruthenium red) and was reduced proportionally by siRNA-mediated Piezo1 knockdown or in murine cells. Results from cell-attached patch clamp recordings on human cardiac fibroblasts established that they contain mechanically activated ion channels and that their pressure responses are reduced by Piezo1 knockdown. Investigation of Yoda1 effects on selected remodeling genes indicated that Piezo1 activation increases both mRNA levels and protein secretion of IL-6, a pro-hypertrophic and profibrotic cytokine, in a Piezo1-dependent manner. Moreover, Piezo1 knockdown reduced basal IL-6 expression from cells cultured on softer collagen-coated substrates. Multiplex kinase activity profiling combined with kinase inhibitor experiments and phosphospecific immunoblotting established that Piezo1 activation stimulates IL-6 secretion via the p38 mitogen-activated protein kinase downstream of Ca entry. In summary, cardiac fibroblasts express mechanically activated Piezo1 channels coupled to secretion of the paracrine signaling molecule IL-6. Piezo1 may therefore be important in regulating cardiac remodeling.
Piezo1 是一种机械敏感阳离子通道，具有广泛的生理重要性；然而，其在心脏中的作用知之甚少。心肌成纤维细胞有助于保持心肌完整性，并在应激或损伤后调节其修复和重塑中发挥关键作用。在这里，我们研究了 Piezo1 在培养的人和鼠心肌成纤维细胞中的表达和功能。RT-PCR 实验证实 在心机成纤维细胞中的 表达水平与内皮细胞相似。Fura-2 细胞内 Ca 测定的结果验证了 Piezo1 是一种功能性离子通道，其激动剂 Yoda1 可激活该通道。Piezo1 阻断剂（钆和钌红）和通过 siRNA 介导的 Piezo1 敲低或在鼠 细胞中减少了 Yoda1 诱导的 Ca 内流，从而抑制了 Yoda1 诱导的 Ca 内流。人源心肌成纤维细胞细胞贴附式膜片钳记录的结果表明，它们含有机械激活的离子通道，并且其压力反应通过 Piezo1 敲低而降低。对 Yoda1 对选定的重塑基因的影响进行了研究，结果表明 Piezo1 的激活以 Piezo1 依赖的方式增加了 IL-6 的 mRNA 水平和蛋白分泌，IL-6 是一种促肥大和促纤维化的细胞因子。此外，Piezo1 敲低降低了在较软的胶原蛋白包被的基质上培养的细胞的基础 IL-6 表达。多重激酶活性谱分析结合激酶抑制剂实验和磷酸化特异性免疫印迹表明，Piezo1 激活通过 Ca 内流下游的 p38 丝裂原活化蛋白激酶刺激 IL-6 分泌。总之，心肌成纤维细胞表达与旁分泌信号分子 IL-6 分泌偶联的机械激活的 Piezo1 通道。因此，Piezo1 可能在调节心脏重塑中起重要作用。
