School of Engineering and Materials Science, Queen Mary, University of London, Mile End Road, London, E1 4NS, UK.
Biomech Model Mechanobiol. 2012 Mar;11(3-4):439-47. doi: 10.1007/s10237-011-0323-1. Epub 2011 Jun 25.
Gap junction communication is an essential component in the mechanosensitive response of tenocytes. However, little is known about direct mechanoregulation of gap junction turnover and permeability. The present study tests the hypothesis that mechanical loading alters gap junction communication between tenocyte within tendon fascicles. Viable tenocytes within rat tail tendon fasicles were labelled with calcein-AM and subjected to a fluorescent loss induced by photobleaching (FLIP) protocol. A designated target cell within a row of tenocytes was continuously photobleached at 100% laser power whilst recording the fluorescent intensity of neighbouring cells. A mathematical compartment model was developed to estimate the intercellular communication between tenocytes based upon the experimental FLIP data. This produced a permeability parameter, k, which quantifies the degree of functioning gap functions between cells as confirmed by the complete inhibition of FLIP by the inhibitor 18α-glycyrrhentic acid. The application of 1N static tensile load for 10 min had no effect on gap junction communication. However, when loading was increased to 1 h, there was a statistically significant reduction in gap junction permeability. This coincided with suppression of connexin 43 protein expression in loaded samples as determined by confocal immunofluorescence. However, there was an upregulation of connexin 43 mRNA. These findings demonstrate that tenocytes remodel their gap junctions in response to alterations in mechanical loading with a complex mechanosensitive mechanism of breakdown and remodelling. This is therefore the first study to show that tenocyte gap junctions are not only important in transmitting mechanically activated signals but that mechanical loading directly regulates gap junction permeability.
缝隙连接通讯是肌腱细胞机械敏感性反应的一个重要组成部分。然而，对于缝隙连接周转率和通透性的直接机械调节知之甚少。本研究检验了这样一个假设，即机械加载会改变肌腱束内肌腱细胞之间的缝隙连接通讯。用 calcein-AM 标记大鼠尾巴肌腱束内的存活肌腱细胞，并通过荧光损失诱导漂白（FLIP）方案使其荧光强度降低。在一排肌腱细胞中，用 100%激光功率连续漂白一个指定的靶细胞，同时记录相邻细胞的荧光强度。根据实验 FLIP 数据，开发了一个数学隔室模型来估计肌腱细胞之间的细胞间通讯。该模型产生了一个通透性参数 k，该参数定量了细胞之间功能性缝隙连接的程度，这一点通过抑制剂 18α-甘草次酸完全抑制 FLIP 得到了证实。施加 1N 的静态拉伸载荷 10 分钟对缝隙连接通讯没有影响。然而，当加载增加到 1 小时时，缝隙连接通透性呈统计学显著降低。这与加载样本中连接蛋白 43 蛋白表达的抑制相一致，这一点通过共聚焦免疫荧光确定。然而，连接蛋白 43 mRNA 表达上调。这些发现表明，肌腱细胞会根据机械加载的变化重塑它们的缝隙连接，这是一种复杂的机械敏感性破坏和重塑机制。因此，这是第一项表明肌腱细胞缝隙连接不仅在传递机械激活信号方面很重要，而且机械加载还直接调节缝隙连接通透性的研究。
