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

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.

The methodologies needed to fulfil our objectives are detailed beneath the objectives: 1. Mechanism: to determine hyperthermic injury thresholds that damage gap junction (GJ)-linked tendon fibroblast (tenocyte) networks in energy-storing tendons. 2. Prevention: to engineer a drug-free adaptive response by tenocytes to heat shock that can be easily adopted by athletes i.e. application of temperature differentials. 3. Therapy: to validate the GJ component Cx43 as a molecular target for protection of energy-storing tendons from cumulative stress-induced microdamage. Methodology: Objectives 1 and 2 require a heating and cooling apparatus and use of cyto/histo chemical analyses or biochemical techniques that are routinely used in the laboratory. Rapid heating will be achieved using a controllable peltier system with integrated thermocouple (WATronix). Longer incubations at low temperature will be with a gas-perfused humidified incubator to better preserve cell viability. Objective 3 requires us to genetically manipulate tenocytes. We will use antisense oligodeoxynucleotide technology with which our collaborator, Prof David Becker, is expert. MicroRNA silencing of Cx43 will be by transfection of a microRNA precursor, which is processed to form the small RNA that interferes with Cx43 translation. pSilencer-mir-206 (Ambion) will be used to express our microRNA; the vector is called pSilencer as it is also has siRNA applications. Transfection will be with TransIT-1 (Mirus) which is non toxic and effective in transfecting primary equine fibroblasts. In-vitro models: Primary tenocytes will be cultured at 2% O2 (~the oxygen tension in the tendon), on matrix-coated surfaces including micro-thin tendon tissue as a base material, with the inclusion of agonists to stimulate 'stretch' pathways (developed by TR/JPK). A tissue explant model; tendon strips will be adapted to culture conditions for 18h, then cultured at the liquid air interface (developed by JD).

Athletes Beneficiaries: Elite and recreational athletes, trainers, coaches, physiotherapists, UK Sport, Sport England, The Institute of Sport and Recreation Management, Sports Coach UK. Benefits: Health: Improved training regimens, extension of high-quality performance/career, implementation of preventative rather than therapeutic methods, increased focus on an under-recognised musculoskeletal condition. Wealth: Cost-effective intervention, extension of athletic careers, improved professional team management. Culture: Increased participation in national sports, reduced injuries to high-profile athletes. Ageing population Beneficiaries: Middle-aged to older athletes (including leisure activities), inclusive of younger individuals with premature ageing changes in the AT and individuals with other conditions that result in deterioration of tendon structure and function e.g. diabetics, Age UK (amalgamation of Age Concern and Help the Aged). Benefits: Health: Slowing or prevention of premature ageing of soft connective tissues, promotion of mobility into old age with associated other health and mental benefits. Wealth: Reduced impact on health services due to maintenance of health in old age, matching increased health span to increasing life spans. Culture: Promoting a culture of healthy exercise and ageing. Equine industry Beneficiaries: Horses (1.35 million in the UK), horse owners and trainers (elite competition and recreational use), horse riders (4.3 million) equine veterinarians (British Equine Veterinary Association, American Association of Equine Practitioners), British Horseracing Authority, British Horseracing Education and Standards Trust, The Jockey Club, (UK) National Association of Veterinary Physiotherapists. Benefits: Health: Slowing or prevention of premature ageing of tendons, reduced incidence of clinical injury, reduced time out of work, reduced wastage, maintenance of high performance levels, implementation of preventative rather than therapeutic strategies at low cost, use of preventative methods easily accepted and adopted by the industry, improved horse welfare. Wealth: Reduced economic loss to the industry/horse owners in terms of time out of work, costs of treatments (most without scientific basis), recurrence of injuries, loss of use and reduced performance (the economic impact of British Racing is 2.8 billion per annum). Culture: Improved public image of equine industries, reduced distress to horse owners and public. Government (Health policy) Beneficiaries: Department for Culture, Media and Sport, Department of Health, The Scottish Government (Active Nation, Commonwealth Games Legacy Initiative). Benefits: Health: Reduced burden on NHS by improving healthy life spans in an ageing population, improved health of athletes, assistance in policy development for athletes of all ages (premature ageing prevention). Wealth: Reduced economic burden of tendon injuries and loss of mobility. Culture: Improved attitude to healthy exercise. Health providers Beneficiaries: The Chartered Society of Physiotherapy, National Health Service, British Association of Sports and Exercise Medicine. Benefits: Health: Scientifically based preventative strategies, improved understanding of tendon pathology. Wealth: Reduced economic burden of tendon injuries and loss of mobility. Culture: Improved basis for recommendation of sustaining exercise activity throughout life. Time scale: Should effective timing, magnitude and duration of temperature changes be determined to reduce tendon cell damage, we are well placed to implement an immediate follow-up epidemiological study that will involve cooperation of human athletes and horse owners. Such a study will provide the basis for improvements in tendon health within the next 5 years.