Abstract 1529: Grp94 Overexpression Counteracts Ischemia-Induced Activation of Caspase-12 and Protects Cells After Transplantation in the Infarcted Myocardium.
Extensive apoptosis of grafted cells represents a major hindrance towards a successful cell therapy. We then compared in vitro and in vivo the extent of anti-apoptotic protection induced by different protocols of cell stress with that one due to the selective overexpression of the Endoplasmic-Reticulum (ER) stress-protein Grp94. H9c2 cell clones, stably trasfected with an empty vector (control clone) or containing Grp94 cDNA (overexpressing clone) were used. Cells from the Grp94 overexpressing clone had a double Grp94 amount and, after exposure to 8h simulated ischemia (anoxia in the presence of ischemic buffer) showed a 50% reduction in activated caspase-3 and complete inhibition of caspase-12 activation, compared to control clone cells. A comparable increase in Grp94 amount and in anti-apoptotic cytoprotection was obtained in control clone cells 24h after the induction of ER stress by a brief exposure to non-toxic amounts of SERCA inhibitors. Conversely, heat shock pretreatment of control clone cells did not increase Grp94 amount, nor counteracted caspase-12 activation, despite the presence of a 20% reduction in caspase-3 activation. We therefore assessed whether the increased Grp94 amount, either secondary to ER stress or to specific overexpression, provided a comparable anti-apoptotic protection of cells to be trasplanted in vivo. About 2.25x106 H9c2 clone cells pre-labelled with BrdU were injected 25min after occlusion of the left anterior descending coronary at the border of the ischemic region of 19 rat hearts. TUNEL analysis, performed on cryosections of 24h-infarcted ventricles, showed that BrdU-positive Grp94-overexpressing clone cells displayed a reduced percentage of apoptotic nuclei compared to control cells (mean and SE 7.53±1.54% and 17.46±0.70%, respectively, P<0.005). Comparable results were observed when control clone cells were pretreated with SERCA inhibitors before transplantation in the infarcted myocardium. In conclusion, this study suggests that the ER-stress protein Grp94 represents a major effector of anti-ischemic cytoprotection and proposes safe and effective protocols of ER stress to raise this protein amount before cell transplantation in an ischemic environment.