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有类似的问题。两人都经常损伤腿部的大肌腱，这些肌腱作为“生物弹簧”，在腿部承重时通过拉伸和储存能量来节省肌肉的力量，然后反弹推动运动员前进。在人类中，跟腱（AT）具有这种功能，而在马中，它是指浅屈肌腱（SDFT）。随着越来越多的人参加体育运动以改善健康状况并在衰老期间保持活动能力，越来越多的AT损伤正在发生。在苏格兰，发病率在过去15年中增加了90%，现在占软组织损伤的10%以上。多达30%的赛马遭受类似的问题。肌腱愈合缓慢，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，以及包括2005年切尔滕纳姆杯赢家“踢王”在内的许多著名赛马的职业生涯。肌腱损伤是在运动过程中对其物质（基质）的无痛性损伤积累的一段不确定的时期。肌腱细胞，称为腱细胞，不修复这种“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，导致恶性损伤循环。多年来的研究表明，这种退化代表了一个正常的与年龄有关的过程的加速。减缓或防止这种情况发生将对预防受伤产生更大的影响，从而提高运动员的表现和福利，而不是试图治疗。我们认为造成肌腱细胞损伤的一个主要因素是高温。当肌腱伸展和收缩时，一些储存的能量会以热量的形式损失，而这些热量不容易消散。在舞动过程中，SDFT核心的温度从正常的37- 38 oC至少达到45 oC。由于AT以类似的方式发挥作用，因此其核心也极有可能发生高热。在实验室中，大多数肌腱细胞在45 ℃下10分钟后死亡，但有趣的是，这涉及通过间隙连接（GJ）在细胞之间传递“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不太有效的基质蛋白的上调。这是令人兴奋的，因为GJ可以被操纵。我们将使用来自SDFT的细胞和组织来确定相对于加热，腱细胞何时“决定”死亡或改变其活性。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些什么？其次，我们将探索一种无药物的干预方法：运动后冷却肌腱，例如使用冰浴通常被用作“预防伤害”的措施。冷却可以关闭其他细胞类型的GJ，这可以防止运动后关键时期死亡信号的传播。轻度低温（即32 ℃）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温差下应用，以减少肌腱微损伤。在这个项目的最后一部分，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，即防止其在腱细胞内产生。这将使我们能够更好地了解GJ如何影响热休克后的细胞死亡/损伤，但也可能促进药物的开发，以调节它可以在局部，例如在凝胶或软膏中递送。随着即将到来的伦敦奥运会和格拉斯哥英联邦运动会以及越来越多的人将受到启发参加体育活动的可能性，这是一个及时的机会，以制定预防受伤的方法，同时也提高了马匹的福利。

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.