PM393 Microrna-22 Protects Against Ischemia/Reperfusion-Induced Myocardial Injury Via Anti-Apoptosis Through Its Target Gene Cbp

Abstract

Introduction: MKK3 and MKK6 are the primary upstream signalling proteins of the p38-MAPK pathway involved in modulating ischaemic stress. Previous studies involving MKK3 and MKK6 in isolated cardiomyocyte and transgenic animal models have both been used to determine that MKK3 preferentially activates p38a while MKK6 activates both p38a and p38b, however, a comprehensive characterisation of the transcriptional response prior to and following ischaemic stress has yet to be carried out. Objectives: The objective of this study is to examine the effects of ischaemic stress in isolated cardiomyocytes following transient knockdown of MKK3, MKK6 and dual MKK3/ MKK6. Methods: Cell stress, as determined by LDH release, will be quantified in normoxic and ischaemically stressed MKK3, MKK6 and dual MKK3 and MKK6 miRNA transfected cardiomyocytes referenced to LacZ GFP transfection controls, after which transcriptional changes will be examined using RT-qPCR. Ischaemia will be simulated using a buffer exchange of ischaemia mimetic media in an oxygen poor environment in a hypoxic chamber for 2 hours followed by cell recovery in KH media at physiological oxygen levels for 6 hours, all at 37 degrees. Protein and activated protein will also be quantified and compared across all samples to characterise the post-transcriptional response concurrently. Results: Preliminary results obtained in previous gene knockdown studies of MAPK14 (p38a) and MAPK11 (p38b) determined that LDH release was reduced and MKK6 transcription was significantly increased when the p38 –MAPK pathway was partially silenced. To be more specific in MAPK14 (p38a) silenced cells MKK3 transcription levels remained unchanged compared to normoxic LacZ transfection control. In MAPK11 (p38b) knockdown cells however the transcription of MKK3 was impaired and LDH release was reduced to normoxic levels compared to normoxic LacZ transfection control. Conclusion: In Conclusion these preliminary results lend credence to previous findings that determined MKK6 transcription to be cardioprotective while MKK3 transcription reduced tolerance to ischaemic stress. What is yet to be determined is how loss of either MKK3 or MKK6 transcription will affect cell tolerance to ischaemic stress and what are the associated post-translational and transcriptional changes at the cell surface, the various intracellular signalling pathways, the nucleus and the mitochondria. Disclosure of Interest: None Declared