Mechanisms of mitogen activated kinase kinase 6 mediated cardioprotection
Cardiovascular disease (CVD) in the leading cause of death in the industrial world and has a great financial impact on all aspects of society. Ischemia followed by reperfusion (IR), the major component of CVD, activates multiple cellular signaling pathways, some of which are protective and others are damaging. The p38 signaling cascade has been shown to be a mediator of cell fate. It was the hypothesis of this dissertation that increasing p38 activity by over-expression (OE) of the upstream activator MKK6 would be cardio-protective. Experimental in vitro results here show that OE of MKK6 in cultured ventricular myocytes leads to a reduction in hypoxia/reoxygenation induced apoptosis as well as blockade of caspase-3 activation induced by sorbitol. This protective phenotype was examined in vivo by generation of cardiac specific MKK6 OE mouse lines. When faced with IR stress, transgenic mouse hearts showed a 50% reduction in damage both in vivo and ex vivo. This led to a hypothesis-driven study that found increased expression of Bcl-2 and the small heat shock protein, αB-crystallin (αBC), as well as altered localization of αBC to the mitochondria, changes which would be expected to provide a protective phenotype.
A proteomic shotgun approach was used to identify novel protective alterations in MKK6 transgenic mouse hearts. The proteomics method was able to quantify the relative abundance of over 4000 proteins from whole heart extracts. Further proteomic analysis of a cytosolic/mitochondrial sub-cellular fraction confirmed decreased expression of mitochondrial electron transport chain (ETC) subunits seen in the whole heart. ETC decreases were verified by immunoblot, and mitochondria exhibited a reduction in O2 consumption. Since the ETC is a key site for ROS generation, a correlative decrease in ROS generation due to IR was predicted and experimentally observed in MKK6 transgenic hearts.
The effects of MKK6 were also seen to translate to an atrial endocrine context. In cultured atrial myocytes, MKK6 OE increased promoter activity for both ANF and BNP. Secretion of ANF into culture media was also elevated with increased MKK6 expression. Thus, potential protective mechanisms also were encountered in increased atrial endocrine production due to MKK6 OE.
0307: Molecular biology