Abstract 1670: Extracellular Hsp72 and the Systemic Inflammatory Response to Cardiopulmonary Bypass
Introduction: Myocardial expression of Hsp72 is protective following cardiopulmonary bypass (CPB). However, recent data suggest that extracellular Hsp72 (eHsp72) modulates the host inflammatory response.
Hypothesis: We hypothesized that (1) eHsp72 levels increase after CPB; (2) eHsp72 contributes to inflammation and myocardial depression following CPB.
Methods: Plasma samples were obtained at baseline and at 0, 4, and 24 h after CPB in 49 children (median age 5 mo) with congenital heart disease. Plasma Hsp72 was measured via ELISA. We treated neonatal rat cardiomyoblasts (H9C2) with recombinant Hsp72 (dose, 10–1000 ng/mL) and measured expression of the murine IL-8 ortholog, KC via ELISA and NF-κB activation via Western blot and EMSA. We next transfected H9C2 cells with Toll-like receptor (TLR)-4 siRNA prior to treatment with Hsp72 (1000ng/mL) and then measured KC expression.
Results: CPB (mean CPB 119 mins; cross-clamp, 71 mins) increased IL-6, IL-8, and IL-10 expression. Plasma Hsp72 levels increased significantly immediately following CPB (7 ng/mL vs 0 ng/mL; p<0.05), remained elevated at 6 h (5 ng/mL vs 0 ng/mL; p<0.05), and returned to baseline at 24 h. Plasma Hsp72 after CPB correlated significantly with age (r=−0.34; p=0.03), IL-8 (r=0.45; p=0.02), IL-6 (r=0.42; p=0.03), and aortic cross-clamp time (r=0.3; p=0.05). These correlations remained significant at 6 h after CPB. There was a weak correlation between eHsp72 and troponin-I. HSF1 EMSA, Western blot, and in vitro experiments with H9C2 and human PBMC suggest that monocytes are the source of eHsp72 following CPB. eHsp72 induced expression of KC in a dose- and time-dependent manner (1000 ng/mL dose: 1028±153 pg/mL at 24 h; p<0.05). These effects were due to increased activation of NF-κB, as shown by luciferase assay, Western blot, and EMSA. Additional experiments showed that these effects were not due to LPS contamination of the eHsp72 preparation and occurred in a TLR-4-dependent manner.
Conclusions: CPB produces a systemic inflammatory response associated with increased plasma Hsp72 levels. Recombinant Hsp72 increases KC expression in a TLR-4-and NF-κB-dependent manner, suggesting that extracellular Hsp72 may contribute to myocardial inflammation and depression following CPB.