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

Lead Research Organisation: University of Glasgow
Department Name: College of Medical, Veterinary &Life Sci


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.

Technical Summary

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).

Planned Impact

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.
Description We had three main objectives.
The first objective was to determine hyperthermic injury thresholds that damage gap junction (GJ)-linked tendon fibroblast (tenocyte) networks of energy-storing tendons.

We determined the hyperthermic injury threshold for our monolayer cultures of tendon-derived tenocytes. Tenocytes were extracted from the injury prone superficial-digital flexor tendon (SDFT) which is functionally equivalent to the Achilles tendon and compared with tenocytes extracted from the deep digital flexor tendon (DDFT) that is anatomically close to the SDFT but is very rarely injured. The injury threshold is surprisingly high; a heating profile that incrementally increased temperature to 49?C over 15 minutes was the tipping point for viability. This experiment turned out to be a surprisingly difficult task as the apparatus recommended were not sensitive enough and could not provide a sufficiently uniform heat table - this was evident from the altered viability across the culture dishes in which we grew our cells. We therefore purchased a resistive heater from Cell MicroControls (Norfolk, VA). A sensitive wire thermistor was attached to a coated glass heating plate and a second was threaded through the lid of a plastic dish such that it made contact with the floor of the dish as it sat on the heated glass surface; i.e. we could directly measure the temperature of the cell monolayer as well as the temperature required at the glass plate in order to create that temperature in the dish. The glass plate that we used to heat the cells is coated with indium tin oxide (ITO) which allows a current to be transmitted to generate heat. Both thermistors were attached to a mTCII micro-Temperature Controller (firmware version V49H) and information from the controller was relayed to the computer every 5-6 secs. This allowed heat recording by the software program RealTerm version The resultant txt file format could be downloaded into Microsoft Excel 2003. This software program had the advantages of allowing the laboratory group to calibrate the heating rig and determine the accuracy of the temperature at the cell monolayer surface. Dr Hannah Cornell, the RA employed on this project, created a one-touch computer code that activated a heating profile which included a cool-down step back to 37?C (using a micro fan). Reference temperatures for both the dish and the plate were collected in experiments bracketing our experimental test runs such that we could complete these without using the second thermistor which would contaminate the cell monolayer.

Our experiments have confirmed that the heat-induced relocalisation of proteins from nuclear structures called PML bodies is intact in equine tenocytes and that this relocalization is not trainable (i.e. it recurs with each shock without adaptation) but is not provoked by mild cold-shock irrespective of its duration. These data are of importance as the movement of proteins such as Daxx from PML bodies is thought to occur when these structures detect hyperthermic cell stress and this response allows the release of Daxx protein which then lowers the threshold of activation for the heat shock induced transcription factor, HSF.

Objective 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.

Using the apparatus detailed above we determined that preconditioning by heat shock (47?C) and not cold shock (26?C) was effective in protecting against an absolute lethal temperature of 52?C for both DDFT and SDFT cells. Continuous cold shock at 26?C for either SDFT or DDFT derived tenocytes could only induce protective heat shock chaperones after 48-72h of cooling. The magnitude of induction following this long term and continuous cooling was identical to that achieved in:
1. SDFT derived tenocytes after one hour of cooling followed by recovery to 37?C for 4h.
2. DDFT derived tenocytes cooled for 16h followed by a 1h recovery at 37?C.

Our data suggested that a similar sluggish induction of HSP70 protein was detected in DDFT versus SDFT following heat shocks. These data, if confirmed, suggest that the response to temperature differentials in the injury prone SDFT tendon may be different to that in the non-injury prone tendon.

We also discovered that DNA damage (evident by double strand break detection or COMET assay) could be generated in tendon populations if extraction protocols were too harsh. We have also found that the age of the tendon and the time taken to extract the tenocytes following euthanasia of the horse may alter the health of the ex-vivo tendon cultures. Data describing these effects have been published from this project. We are therefore repeating our earlier experiments using cells extracted in such a way as to minimize injury. If confirmed, our data showing a marked conservation of phenotypic difference between tenocytes derived from a tendon susceptible to damage versus a tendon that is ordinarily not damaged are novel and would suggest that temperature differentials may more rapidly induce protective responses in the SDFT. Moderate cold shock induced both heat and cold shock chaperones although their kinetics of induction were slow and clearly inadequate in terms of providing any protection against injurious heat shock. Therefore moderate cold shock will probably not protect the tendon. However, a more radical temperature drop may be more effective and we are currently evaluating this possibility.

Objective 3. Therapy: to validate the GJ component connexin 43 as a molecular target for protection of energy-storing tendons from cumulative stress-induced microdamage.

We have found that cell tension (stretching) may regulate Cx43 turnover. We have therefore decided to improve this experiment by conducting the knockdown experiments on stretched monolayers generated using a Flexcel apparatus. We have been awarded a grant for this apparatus from arthritis research UK and have used collagen coated dishes carrying a deformable membrane with which to complete this objective. Cx43 knockdown siRNAs have been generated with transfection using TransIT-TKO (Mirus). However, thus far results have been inconclusive. We have however determined that 5% stretch of the tenocytes (equivalent to that achieved by walking) can increase resistance to heat stress.
Exploitation Route An aligned project is currently running at the RVC to take these findings forward into tissue.
Sectors Leisure Activities, including Sports, Recreation and Tourism

URL http://www.horsework.net/
Description Our findings have not yet been used as we have not completed our assessment of the objectives. A second, BBSRC linked project, using tissue explants rather than tissue-derived cells will help with the final evaluation of how useful our findings may be. In the meantime we have tried to increase the engagement of end users and basic scientists by holding a workshop in which issues relating to horse training, injury and training regimes that could be influenced using our findings were discussed. We found that there is very little scope to alter training regimes in most stable yards. However, there are practical issues surrounding tissue availability for pathologists and issues of the cost and efficacy of stem cell therapeutics that have been discussed. This conversations are ongoing on the twitterfeed (twitter.com/Horsework_) linked to our webpage - www.horsework.net
First Year Of Impact 2013
Sector Leisure Activities, including Sports, Recreation and Tourism
Impact Types Societal,Economic

Description Basic Research
Amount £140,397 (GBP)
Funding ID T9/2012 
Organisation Horserace Betting Levy Board 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2013 
End 09/2015
Description Equipment grant
Amount £35,494 (GBP)
Organisation Versus Arthritis 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2012 
End 01/2013
Description A website and associated twitterfeed 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Online discussion - high levels of on-line engagement

Increased dissemination of scientific material - discussion amongst interested groups - enthusiastic take-up of twitterfeed
Year(s) Of Engagement Activity 2013,2014
URL http://www.horsework.net/
Description Online news story 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Media (as a channel to the public)
Results and Impact Online article

Year(s) Of Engagement Activity 2014
URL http://www.gla.ac.uk/news/headline_360001_en.html
Description Workshop at Glasgow for vets, horse trainers and scientists 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Talks, discussion, working paper

A published document discussing the need for preventative therapy
Year(s) Of Engagement Activity 2013
URL http://onlinelibrary.wiley.com/doi/10.1111/evj.12269/pdf