Abstract 16238: Intracellular Targeting of Hsp70 for Myocardial Cytoprotection After an Infarction

Abstract

Background: Myocardial cells affected by an infarction endure oxidative stress during reperfusion. It has been suggested that the induction of a heat-shock protein 70 (Hsp72) in myocardial cells counteracts oxidative stress and improves post-ischemic contractile recovery, but clinically relevant methods of inducing Hsp70 in myocardium have yet to be successfully employed. Methods: Mab 3E10 binds extracellular nucleosides, a target that is quite accessible in damaged tissues, allowing 3E10 to penetrate still viable cells through an equilibrative nucleoside salvage pathway. We have developed the scFv fragment of the cell-penetrating 3E10 as an intracellular transporter to deliver exogenous Hsp70. Primary cardiomyocytes exposed to H 2 O 2 were incubated in vitro with a 3E10 scFv-Hsp70 fusion (Fv-Hsp) to demonstrate cytoprotection against oxidative damage. In addition, rabbits were subjected to occlusion of the left coronary artery followed by reperfusion of the heart and intravenous injection of Fv-Hsp (20 mg) or a molar equivalent of a sham control. SPECT imaging and tissue gamma counting of 99m Tc-labeled annexin V was used to visualize the infarct volume and quantify cell death, respectively. Results: In vitro , 30-40% fewer dead cells were observed with Fv-Hsp treatment 30 minutes after exposure, even when 3E10 scFv was added as a competitor (p<0.0003). Reduced to oxidized glutathione ratios were greater in treated cells suggesting less oxidative stress. Measurement of annexin uptake (% injected dose of 99m Tc /gram tissue) revealed Fv-Hsp-treated rabbits had less uptake compared to controls (ANOVA); 49% less in the entire area of risk (p<0.0001) and 43% less at the heart apex (p=0.01) where the maximum damage was expected/occurred. The annexin-verified damage was substantially higher in the control rabbits injected either with PBS or with a molar equivalent of 3E10 scFv. Echocardiographic left ventricular ejection fraction demonstrated recovery of significantly superior contractility in Fv-Hsp-treated rabbits 3 hours after reperfusion compared with no recovery in control rabbits. Conclusion: Targeted accumulation of Hsp70 at infarcted tissue is achievable and worth investigating further for clinical cytoprotection post-infarction.