Response to mechanical stress in ageing tissue

The defining qualities of our cells - their physical features and function - can be determined by their surroundings, but the tissue environment can be demanding. Just as our muscles regenerate and are conditioned by exercise to maintain health, our cells have developed mechanisms to protect against damage induced by mechanical stress. Perhaps a predictable response to increased load would be to 'bulk up' the structural components within the cell. However, changes in the regulation of metabolism, the consumption of small molecules that fuel cells and form the building blocks of growth and regeneration, could be equally crucial in repairing damage. A fundamental response to stress is the production of 'chaperone' or 'heat shock' proteins within our cells. These molecular machines act to limit and reverse damage by refolding proteins unwound by stress back into functional structures. However, these mechanisms are thought to deteriorate in old age. This loss of function may be compounded by other factors affecting aging tissue: the matrix - the material in which cells are embedded - is known to stiffen with age and the tissue has a reduced potential to regenerate its cells through mechanisms such as the proliferation of stem cells.

This proposal describes work to examine the extent to which heat shock proteins are needed to protect the cytoskeleton, the network of structural proteins that give our cells the robustness necessary to withstand mechanical stressing. It will consider the consequences of an impaired stress response, as seen in our cells as they age, by blocking or removing the activity of heat shock proteins. The effects of this perturbation will be measured in model systems that reflect the conditions found in mechanically stressed tissues such as muscle and heart. These systems will also be tuned to model the changes in matrix composition and stiffness that manifest in aging tissue. I will also examine the effects of mechanical stress and impaired heat shock response on the ability of tissue to repair itself by looking at the effect on the regenerative capacity of stems cells. By better understanding the stress response, the proteins it is designed to protect and how they alter during stress and aging, I will be able to suggest better pathways to target in developing future therapies for heart and muscle wasting. Findings of this work may also caution against inhibiting the stress response machinery when treating other diseases such as cancer in patients particularly at risk of heart disease.

In recent years, scientists have developed powerful methods to characterize the genetic programing, protein contents and metabolic activities of cells. My proposal will combine these methods with new tools, developed during my post-doctoral work, to examine protein interactions and changes in 'fold' or shape. Determining which proteins interact is particularly important for this study in order to uncover the identities of the damaged 'client' proteins that heat shock proteins are targeting. Likewise, a measure of protein fold is necessary to identify which proteins have been stressed into the incorrect shape. I will look for mechanisms of regulation by searching for protein modifications in stressed systems: chemical alterations such as phosphorylation have potential as mechano-sensitive 'flags' for signaling, whereas modifications such as oxidation may be markers of an overloaded stress response machinery. A combination of these methods will give a complete picture of the cellular response to stress and thus give a new perspective on the stiff-tissue disorders associated with old age.

I will test the hypothesis that heat shock proteins (HSPs) are necessary for maintenance of the cytoskeleton and to explore the consequences of the degeneration of the stress management machinery during aging on the health of stiff tissues, such as muscle and heart. Cells in stiff tissue have a well developed cytoskeleton to maintain the structural integrity of the cell. However, the role of HSPs, which can account for a significant fraction of total cellular protein, in managing mechanically-induced damage to the cytoskeleton is poorly understood. Furthermore, the loss of heat shock response in aging tissue is accompanied by changes in matrix stiffness and composition, and a loss of regenerative capacity (e.g. through stem cells); I propose to investigate the links and coupling between these factors. Initial experiments will use cardiomyocytes and mesenchymal stem cell systems, with stress applied to model systems by varying the stiffness of the underlying matrix, by applying stain-cycling and by subjecting cultures to shear flow. Later work will be expanded to test lineages derived from pluripotent stem cells, thus exploring aspects of tissue regeneration. I will consider the effects of drug inhibitors of HSP activity, such as are currently in trials for cancer therapy, examining whether such treatments have deleterious side effects in aging tissue. The cellular response to stress will be examined by a combination of proteomic, transcriptomic and metabolomic profiling tools. I will apply new techniques developed during my post-doc to study in situ protein conformation and complex formation. The first of these methods uses a cysteine-reactive probe in combination with mass spectrometry (MS), used previously to analyze folding of the nuclear cytoskeleton protein, lamin. The second method uses cross-linking and immuno-precipitation, followed by MS, to identify interacting proteins - a tool that will be essential in determining the role of HSPs in stressed cells.

This project will bring potential benefits to a broad range of people in the UK and throughout the world. The work aims to deliver an understanding of how our cells deal with mechanical stress, why this is particularly important as we age and how exercise and activity may help to strengthen the resistance of our cells to damage. The need for a better understanding of these problems is particularly important in the UK, which has an aging population. It is imperative to maintain a high-quality of life for the elderly and a better knowledge of the aging process will help us manage the resources of state care. I am confident that my research can make an important and meaningful contribution to understanding stress-management and regeneration mechanisms in stiff tissue (e.g. heart and muscle) during the five year fellowship period. These findings might also suggest certain regulatory pathways as important targets for future drug developments, or warn against certain combinations of drugs (e.g. inhibitors of heat shock response in cancer treatment) in patients with predisposition towards heart disease. Results, findings and recommendations will be delivered in the final two years of the fellowship period through means of peer-reviewed publications and the presentation of conference papers. Additionally, the work will find significant interest as it seeks to develop new and emerging technologies and methodologies. Studies of proteomics, transcriptomics, metabolomics and variations on such technologies are becoming increasingly central to modern biological science and new developments are keenly anticipated.

It is important to engage the public to reinforce both the implications of this work and the continuing relevance of scientific research. I will achieve this aim by contributing to KCL's planned Science Gallery, scheduled to open on the Guy's campus in late 2015. This exciting development seeks to inspire interest from the public by bringing a combination of science, art and engineering to a projected audience of some quarter-of-a-million visitors annually. Further interaction with the public will be achieved through an active web presence and contribution to the CSCRM's planned patient and visitor outreach centre.