Protein and gene expression analyses in bone marrow stem cells mediated restoration of myocardium after ischemic insult

Abstract

Myocardial Infarction (MI) is caused by occlusion of the coronary artery following atheromatous plaque rupture, the subsequent ischemia in the myocardium leads to myocyte necrosis unless treated quickly. Bone marrow derived stem cell treatment is a promising therapy for improving the outcome of patients with MI. The aim of this thesis was to study myocardial protein and gene expression changes in a rat ischemia/reperfusion (I/R) model in order to look for potential repair mechanisms of the myocardium triggered by endogenous bone marrow mononuclear cells (BMMNCs). Rat myocardial samples were obtained from three experimental groups: one group had a sham operation, the other two groups had undergone myocardial I/R injury induced by left anterior descending (LAD) coronary artery ligation followed by treatment with either a BMMNC preparation or PBS. Comparative proteomic analyses were carried out using 2D electrophoresis; differentially expressed proteins were identified using LC-MS/MS. Western blotting was used to confirm the most significant findings including expression of 14-3-3 epsilon protein. Global comparative gene expression profiling was performed using Illumina RatRef12 BeadChips and QPCR was used to validate the top results. Bioinformatic tools were used to assess the biology of the differentially expressed genes and proteins. Thirty-seven proteins were found to be differentially expressed in I/R injury compared to sham. These were primarily sarcomeric, energy production or stress response proteins and most were down-regulated. Expression levels were ‘corrected’ by BMMNC treatment for many of these proteins. Over 1500 genes were affected by I/R injury, 20 were affected by BMMNC treatment, and many of these were related to inflammation and apoptosis signalling and responses. The 14-3-3 epsilon protein was chosen for follow-up work as it presented as a good candidate for mechanistic involvement. This protein has many roles including interactions with the proapoptotic BCL2-associated agonist of cell death (Bad) protein. Western blotting was used to look at Bad expression and found it to be significantly increased in the treatment group, although I could not reliably measure the expression of phosphorylated (serine 136) form of Bad. A preliminary pull-down assay was performed to look for binding partners of 14-3-3 epsilon. Two ATP synthase subunits, one of which is known to bind 14-3-3 epsilon, a protein involved in fatty acid β-oxidation and a protein of unknown function were found to bind. Further work will be required to follow up these findings and ascertain the exact role of 14-3- 3 epsilon in cardioprotection. In summary, my data supports the power of profiling methods to derive new candidates for a role in repair mechanisms in this therapeutic model.