Abstract 849: Prolyl Hydroxylase Inhibition Attenuates Post-Ischemic Injury By Inducing Endoplasmic Reticulum Stress Genes
Background: Ischemia/reperfusion (I/R) unleashes a cascade of cellular and molecular events whose sequelae threaten organ survival. I/R affects endoplasmic reticulum (ER) integrity and activates the unfolded protein response (UPR). The adaptive arm of the UPR normally attenuates ER stress by decreasing protein synthesis and increased expression of chaperones that promote proper protein folding. Failure to resolve ER stress leads to initiation of apoptosis. We recently showed that prolyl hydroxylase inhibition using dimethyloxalylglycine (DMOG) or in vivo silencing of prolyl 4-hydroxylase 2 (P4HA2) significantly attenuated post-ischemic cardiac injury. We hypothesized that prolyl hydroxylase inhibition attenuated myocardial I/R damage through activation of the UPR resulting in enhanced expression of molecular chaperones.
Methods and Results: Adult ICR mice received saline, DMOG, P4HA2 siRNA or non-targeting control siRNA i.p. 24 hours prior to a 30 min left coronary artery occlusion and 2 hr reperfusion. Hearts were collected for cardiac UPR mRNA (QPCR) and protein (Western Blot) quantification. DMOG exposure alone activated all three regulatory arms of the UPR in intact hearts and in vitro in murine microvascular endothelial cells. DMOG induced PERK phosphorylation and subsequent ATF4 mRNA induction (2.5 fold, n=3, p<0.001). DMOG activated the two pathways involved in transcriptional regulation (ATF6, IRE-1), by inducing expression of Grp78 (3 fold, n=3, p<0.001) and EDEM (1.7 fold, n=3, p<0.001) as well as XBP-1 splicing (4.7 fold, n=3, p<0.001). In vivo cardiac I/R activated expression of pro-apoptotic CHOP (2.8 fold, n=3, p<0.01) and XBP-1 splicing (1.8 fold, n=3, p<0.005). P4HA2 gene silencing significantly decreased CHOP expression (50%, n=3, p<0.05) and XBP-1 splicing (55%, n=3, p<0.005) following I/R. P4HA2 gene silencing also induced ATF4 (3.5 fold, n=3, p<0.001), Grp78 (6 fold, n=3, p<0.001) and EDEM (2.8 fold, n=3, p<0.001) expression following I/R.
Conclusion: The pro-apoptotic components of the UPR (CHOP, XBP-1) are activated in post-ischemic heart. Prolyl hydroxylase inhibition induces protective ER stress proteins and attenuates myocardial damage following I/R by decreasing the pro-apoptotic components of the UPR.