IDENTIFYING CARDIAC SUBSTRATES OF p38alpha MITOGEN-ACTIVATED PROTEIN KINASE

During cardiac stress an enzyme known as p38alpha mitogen-activated protein kinase (p38) becomes active. The purpose of the p38 enzyme is to alter the function of proteins by adding phosphate to them. In many cases this helps cells respond to stress. However it is clear that under some circumstances p38 activation worsens injury. The fact that activation of p38 can have both good and bad consequences may explain why clinical trials of p38 inhibition have not yet shown benefit.
The purpose of this project is to create mutations within p38 that subtly alter its enzyme activity. These mutations mean that it is possible to trace the proteins to which p38 has added a phosphate. This will allow us to identify the individual proteins modified by p38 and work out which changes help cells respond to stress and which worsen injury. Ultimately this knowledge may enable refined therapies that help cells respond to stresses such as the myocardial infarction that follows coronary artery thrombosis.

The activation of the alpha isoform of p38 mitogen-activated protein kinase (p38) during myocardial infarction worsens injury. Under other circumstances its activation ensures cardiac adaptation to stress. Clinical trials of p38 inhibition have been unsuccessful due to a combination of toxicity and escape from inhibition with prolonged therapy. These observations suggest downstream targeting of p38 substrates may both enhance efficacy and reduce toxicity.
We plan to use chemical genetic approaches to identify proteins phosphorylated by p38 during myocardial ischaemia and other stresses. Firstly we have created an analogue-sensitive (AS) form of p38 that can use expanded ATP analogues to transfer a thiophosphate affinity tag to substrate proteins. Secondly we have characterised a targeted mouse line in which p38 is resistant to pharmacological inhibition (IR).
Recombinant active ASp38 will be used to tag proteins in heart homogenate. The tagged residues within proteins will be identified by affinity purification and mass spectrometry. These post-translational modifications will then be validated in cultured cells using ectopic expression of ASp38. Further validation will occur in whole heart by comparing IR and wild-type hearts subjected to pathological stresses in the presence of a pharmacological inhibitor of p38. Phosphopeptides from these heart samples will be identified based on inclusion lists generated using ASp38 and quantified by direct evaluation against matching peptides from stable isotope labelled heart. The relevance of dynamic changes in phosphorylation will then be determined in primary heart cells subjected to pathophysiologically-relevant stresses in culture following adenovirally-driven expression of prioritised proteins harbouring key substitutions. Based on this screen the most promising phosphoacceptor substitutions will be knocked into the relevant mouse allele for in-vivo characterisation and estimation of the spectrum of benefit.

The ultimate purpose of this application is to identify the substrates of p38 MAPK that contribute to myocardial infarction (MI) and myocardial contractile dysfunction (heart failure). MI is the most frequent cause of death in the UK. Collectively cardiovascular diseases were responsible for 35% of total UK mortality in 2007. Despite recent reductions in this high mortality, morbidity is increasing as more patients survive MI. For example, in 2007 there were about 1.4M people in the UK who had survived acute MI and 0.9M with heart failure.
The extraordinarily high impact of cardiovascular disease on the Nation's Health is reflected in its economic cost. Coronary artery disease alone costs the UK Healthcare system £3,200M with an estimated further £5,800M lost to the economy through premature death, illness and informal care. Even if our findings contributed modestly to this disease process the impact on quality and quantity of life would be considerable and be accompanied by substantial economic benefit.
The principal applicant is clinically-active and leads the Atherosclerosis theme within the NIHR Comprehensive Biomedical Research Centre at Guy's and St Thomas' NHS Foundation Trust which has GMP facilities and a dedicated phase 1 clinical trials centre. In addition, as discussed within the application, there is a formal collaboration with the Heart Failure Drug Discovery Unit of GlaxoSmithKline. These features will increase the timeliness and probability of clinical translation if tractable substrates are discovered within this programme of work.