726 Paracrine effects of ultrasound-mediated survivin gene delivery ameliorates doxorubicin-induced cardiomyopathy

Abstract

Survivin (SURV), a member of the inhibitor of apoptosis protein family, is a potent suppressor of apoptosis. Recent developments indicate that in addition to their known intracellular localization, a new, extracellular compartmentalization of SURV may exist in cancer cells. We hypothesized that extracellular SURV is capable of promoting cell survival in doxorubicin (DOX)-stressed cells in vitro, and therefore may be an important mechanism whereby SURV gene therapy ameliorates left ventricular (LV) systolic dysfunction in DOX-induced cardiomyopathy. H9c2 cells were transfected with SURV plasmid using nucleofection. Cell supernate was collected at days 1, 2, and 4 and the level of SURV protein was measured by ELISA. Apoptosis rates in response to DOX treatment (1uM for 6hrs) were measured in control H9c2 cells, cells transfected with SURV, and non-transfected cells incubated with cell supernate. SURV gene therapy using ultrasound-mediated gene delivery was performed in a model of DOX-induced cardiomyopathy in male Fisher rats. Echocardiographic assessment was performed at week 0, 3, and 6. A subset of animals was treated with either the SURV gene (DOX+SURV, N = 30) or an empty vector (DOX+EMPTY, N = 8) using ultrasound-mediated gene delivery (UMGD) at week 3. Control animals (DOX, N = 24) did not receive any treatment at week 3. SURV was present in culture media supernate at all time-points after transfection (103.4 ± 22.2pg/ml, 221.9 ± 52.3pg/ml, 68.8 ± 94.4pg/ml, respectively, P< 0.001 for days 1 and 2, P = ns for day 4), whereas no survivin was detected in supernate from non-transfected cells. With DOX stress, non-transfected cells showed an increase in apoptosis (N = 5, 49.5 ± 7.2%, P< 0.001 vs control cells), and SURV-transfected cells showed a decrease in apoptosis (N = 4, 22.8 ± 2.2%, P< 0.01 vs non-transfected DOX cells). Cells cultured in conditioned media from SURV-transfected cells showed a modest decrease in apoptosis compared to that of the non-transfected cells (N = 5, 34.9 ± 13.3%, P = ns). For in vivo studies, LV fractional shortening (LVFS), measured by echocardiography at week 0 (pre-DOX), was comparable in all groups (DOX 47.38 ± 1.0%, DOX+SURV 47.9 ± 0.7%, DOX+EMPTY 50.2 ± 1.4%, P = ns). However, by week 6, LVFS in DOX and DOX+EMPTY animals declined significantly from baseline (38.59 ± 1.5% and 38.89 ± 2.3%, respectively, P< 0.0001 vs week 0) whereas LVFS was maintained in DOX+SURV animals (43.9 ± 1.1%, P = ns vs week 3). Survivin gene therapy by UMGD can prevent the progression of LV systolic dysfunction in doxorubicin-induced cardiomyopathy. This effect may be attributed to the decrease in apoptosis of functional cells within the myocardium, via both a direct transfection of cells and by paracrine mechanisms.