Temperature-driven premature ageing of cellular populations in energy-storing tendons: the gap junction connection.

参加跑步和跳跃的运动员与赛马场上的马匹同行有类似的问题。两人都经常损伤腿部的大肌腱，这些肌腱起着生物弹簧的作用，当腿部承受重量时，通过拉伸和储存能量来节省肌肉力量，然后反弹来推动运动员前进。人的跟腱(AT)具有这种功能，而马的则是指浅屈肌腱(SDFT)。随着越来越多的人参加体育运动以改善他们的健康并在老龄化期间保持活动能力，更多的AT伤害正在发生。在苏格兰，软组织损伤的发病率在过去的15年里增加了90%，现在占软组织损伤的10%。多达30%的赛马都有类似的问题。肌腱愈合缓慢，有疤痕组织，永远不会恢复原来的力量。这样的伤病中断了大卫·贝克汉姆和凯利·霍姆斯的国际职业生涯，以及许多著名赛马的职业生涯，包括2005年切尔滕纳姆杯冠军踢人金。肌腱损伤是指在运动过程中，肌腱的物质(基质)有一段不确定的无痛性损伤累积。被称为腱细胞的肌腱细胞不会修复这种微损伤，可能会被杀死，开始产生错误类型的胶原蛋白，和/或降解周围的基质，导致恶性循环。多年来的研究表明，这种退化代表着通常与年龄相关的过程的加速。减缓或预防这种情况将在预防受伤方面产生更大的影响，从而比试图治疗更能改善运动员的表现和福利。我们认为造成肌腱细胞损伤的一个主要因素是高温。随着肌腱的伸展和收缩，一些储存的能量会以不容易散失的热量的形式损失。在疾驰过程中，SDFT核心至少达到45摄氏度，而正常温度为37-38摄氏度。由于AT的功能类似，其核心也极有可能发生体温过高。在实验室中，大多数腱细胞在45℃下10分钟后死亡，但有趣的是，这涉及到细胞之间通过缝隙连接(GJ)传递“死亡信号”。GJ是直接将相邻细胞连接成网络的小孔。当GJ在加热后被化学阻断时，可以防止大部分腱细胞死亡和低效基质蛋白的上调。这是令人兴奋的，因为GJ可以被操纵。我们将使用SDFT中的细胞和组织来确定，相对于加热，腱细胞何时“决定”死亡或改变它们的活动。我们将测量一系列生物标志物，以确定我们可能逆转这些变化的点，例如，我们需要在几分钟或几小时内做一些事情吗？其次，我们将探索一种无药物干预的方法：运动后对肌腱进行降温，例如，经常使用冰浴作为一种‘预防损伤’的措施。降温可以关闭其他类型细胞中的GJ，这可以防止在运动后关键时期死亡信号的传播。轻度低温(即32oC)也可能上调保护细胞的“冷应激蛋白”。我们的目标是了解运动员应该在什么时候、多长时间、在什么水平和多少次应用温差来减少肌腱的微损伤。在这个项目的最后部分，我们将研究组成GJ的主要蛋白质之一，称为连接蛋白43(Cx43)。我们将研究靶向Cx43分子的方法，即防止它在腱细胞内生成。这将使我们更好地了解GJ如何影响热休克后的细胞死亡/损伤，但也可能促进调节它的药物的开发，这些药物可以在局部释放，例如在凝胶或软膏中。随着伦敦奥运会和格拉斯哥英联邦运动会的即将到来，越来越多的人可能会受到激励参加体育活动，这是一个及时的机会，可以开发防止受伤的方法，同时也提高马匹的福利。

Athletes who participate in running and jumping have a similar problem to their equine counterparts on the racetrack. Both frequently injure large tendons in their legs that function as 'biological springs' to save muscular effort by stretching and storing energy when the leg bears weight, then rebounding to propel the athlete forward. In people the Achilles tendon (AT) has this function, while in horses it is the superficial digital flexor tendon (SDFT). As more people participate in sports to improve their health and maintain mobility during ageing, more AT injuries are occurring. In Scotland the incidence has increased by 90% over the last 15 years, now comprising >10% of soft tissue injuries. Up to 30% of racehorses suffer similar problems. Tendons heal slowly with scar tissue and never regain their original strength. Such injuries interrupted the international careers of David Beckham and Kelly Holmes, and those of many well-known racehorses including Kicking King, the 2005 Cheltenham Cup winner. Tendon injuries follow an undefined period of accumulation of painless damage to their substance (matrix) during exercise. Tendon cells, called tenocytes, do not repair this 'microdamage' and may be killed, begin to produce the wrong type of collagen, and/or degrade the surrounding matrix, causing a vicious injury cycle. Research over many years has suggested that this degeneration represents acceleration of a normally age-related process. Slowing or preventing this would have a greater impact on preventing injuries, and so improving performance and welfare of athletes than attempting treatment. We think a major factor causing tenocyte injury is high temperature. As tendons stretch and contract, some stored energy is lost as heat that cannot be easily dissipated. The SDFT core reaches at least 45oC during galloping, from its normal temperature of 37-38oC. As the AT functions in a similar way, hyperthermia is also highly likely to occur in its core. In the laboratory, most tenocytes die after 10 min at 45oC, but interestingly this involves transmission of 'death signals' between cells through gap junctions (GJ). GJ are small pores directly connecting neighbouring cells into networks. When GJ are chemically blocked following heating much of the tenocyte death and upregulation of less effective matrix proteins can be prevented. This is exciting, as GJ can be manipulated. We will use cells and tissue from SDFTs to determine when, relative to heating, the tenocytes 'decide' to die or alter their activity. We will measure an array of biomarkers to identify points at which we might reverse these changes e.g. do we need to do something within minutes or hours? Secondly we will explore a drug-free method of intervention: cooling of tendons after exercise e.g. using ice baths is often used as an 'injury prevention' measure. Cooling can close GJ in other cell types, which could prevent spread of death signals during critical post-exercise periods. Mild hypothermia (i.e. 32oC) may also upregulate 'cold-stress proteins' that protect cells. We aim to understand when, for how long, at what levels and how many times temperature differentials should be applied by athletes to reduce tendon microdamage. In the final part of this project we will look at one of the major proteins making up the GJ, called connexin 43 (Cx43). We will investigate methods of targeting the Cx43 molecule i.e. preventing it from being made within tenocytes. This will allow us to better understand how GJ influence cell death/damage following heat shock, but may also facilitate development of drugs to regulate it that could be delivered locally e.g. in gels or ointments. With the upcoming London Olympic Games and Glasgow Commonwealth Games and the likelihood that increasing numbers of people will be inspired to participate in athletic activity, this is a timely opportunity to develop methods of preventing injuries while also improving the welfare of horses.